JSC), and open circuit voltage (VOC) of the PDBT‐T1:BPT‐Se1 devices. The higher FF of the organic solar cells (OSCs) based on PDBT‐T1:BPT‐Se1 can be attributed to the higher charge dissociation and charge collection efficiency, less bimolecular combination, more balanced µh/µe, better molecular packing and a more favorable morphology. It is worth mentioning that the FF of 73.2% is the highest value for PDI‐based SMAs OSCs to date. The result shows that regiochemistry of the central core in PDI‐based SMAs greatly affects the physicochemical properties and photovoltaic performance. The success of the isomerization strategy offers exciting prospects for the molecular design of PDI‐based SMAs.A strategy that employs the central‐core regiochemistry to develop two isomeric perylene diimide (PDI)‐based small molecular acceptors (SMAs), BPT‐Se and BPT‐Se1, is introduced, and the effect of the central‐core regiochemistry on the optical, electronic, charge‐transport, photovoltaic, and morphological properties of the molecules and their devices is investigated. The PDBT‐T1:BPT‐Se1‐based device delivers a power conversion efficiency (PCE) of 9.54% with an excellent fill factor (FF) of 73.2%, while the BPT‐Se‐based device yields a PCE of 7.78%. The large improvement of PCE upon isomerization of BPT‐Se should be ascribed to the concurrent enhancement of FF, short circuit current (  This concept has intrigued growing research interest in the design of FREAs and has been proven to influence the electronic structure, absorption spectra, charge transport and molecular stacking, thus leading to different device performance.b:4,5‐b']dithiophene (BDT‐Th), which is a continuation of our previous work,b:6,5‐b']dithiophene (i‐BDT‐Th). BPT‐Se1 displays a stronger absorption, a shallower LUMO energy level, a less twisted intramolecular geometry and a higher electron mobility relative to those of BPT‐Se. When made into photovoltaic devices, the BPT‐Se1‐based device gave a higher PCE of 9.54% with an outstanding fill factor (FF) of 0.732 compared with the reference BPT‐Se‐based device (PCE = 7.78%). The performance of BPT‐Se1 is among the best results for PDI‐based OSCs. Along with the device characterization, grazing incident X‐ray wide‐angle scattering (GIWAXS), and atomic force microscopy (AFM) studies explained the device performance trend and are consistent with our structural design expectation. These results indicate that isomerization can serve as another important route to regulate the molecular properties of the PDI‐based acceptors and thus improve the photovoltaic performance of PDI‐based nonfullerene OSCs.In this work, we report two isomeric PDI‐based acceptors with Se‐annulated PDIs as peripheral groups, namely, BPT‐Se and BPT‐Se1  are 2.04 × 105m−1 cm−1 at 476 nm and 2.05 × 105m−1 cm−1 at 511 nm, which are significantly higher than those of BPT‐Se (1.69 × 105m−1 cm−1 at 476 nm and 1.85 × 105m−1 cm−1 at 510 nm). BPT‐Se and BPT‐Se1 in thin film show similar absorption spectra to their solution ones, indicating that the intermolecular aggregation is well suppressed. Meanwhile, BPT‐Se1 displays a slightly larger optical bandgap (2.07 eV) than BPT‐Se (2.04 eV). The absorption profiles of these SMAs are complementary to the strong absorption of PDBT‐T1, the highly efficient polymer donor we reported previously that absorbs strongly in the range of 500–700 nm.As shown in VOC) relative to that of BPT‐Se (see later section). Also, we employed ultraviolet photoelectron spectroscopy to confirm the energy levels obtained from CV measurements . The ionization potentials of BPT‐Se and BPT‐Se1 were calculated to be 6.06 and 5.96 eV, respectively. The calculated LUMO energy levels from the ionization potential and optical bandgap are −4.02 and −3.89 eV for BPT‐Se and BPT‐Se1, respectively, which verifies the result that from BPT‐Se to BPT‐Se1, the LUMO increases. Furthermore, the calculated LUMO energy levels from density functional theory by using Gaussian at the B3LYP‐D3(BJ)/def2‐SVP level are −3.68 eV for BPT‐Se and −3.55 eV for BPT‐Se1 , which are consistent with the CV measurement. The distribution of HOMO and LUMO orbitals are shown in Figure S3 in the Supporting Information. In BPT‐Se, the LUMO and HOMO electron density mainly localize in the PDI unit and BDT‐Th core, respectively. However, in BPT‐Se1, the LUMO orbitals concentrate in PDI unit while the HOMO orbitals are distributed on PDI‐BDT‐PDI. In addition, the optimized molecular geometries of BPT‐Se and BPT‐Se1 are presented in Figure S4 in the Supporting Information. In BPT‐Se, the dihedral angles between PDIs and thiophene, PDIs and BDT, are 76.0°, 76.0°, 70°, and 67°, respectively, while in BPT‐Se1, the dihedral angles between PDIs and thiophene, PDIs and BDT, are 69°, 67°, 69°, and 64°, respectively. The slightly less twisted molecular geometry for BPT‐Se1 could be beneficial for improving the intermolecular packing relative to that of BPT‐Se, which contributes to enhancing the charge transport ability, thus obtain the larger PCE in BPT‐Se1‐based device (as discussed in the device characterization section). Consequently, the electron mobilities of BPT‐Se and BPT‐Se1 were calculated to be 4.63 × 10−4 and 5.82 × 10−4 cm2 V−1 s−1 by utilizing the space charge‐limited current (SCLC) method , respectively. The higher electron mobility of BPT‐Se1 could be attributed to the improved intermolecular packing in BPT‐Se1.Regarding the electrochemical characteristics, the energy levels of BPT and BPT‐Se were investigated by cyclic voltammetry (CV). The lowest unoccupied molecular orbital (LUMO)/highest occupied molecular orbital (HOMO) levels of BPT‐Se and BPT‐Se1 estimated from the onsets of reduction/oxidation potentials are −3.85/−5.92 and −3.81/−5.85 eV, respectively. The shallower LUMO energy level for BPT‐Se1 contributes to the higher open circuit voltage . The typical current density–voltage (J–V) characteristics and photovoltaic parameters are displayed in FigureTableVOC of 1.039 V, a short‐circuit current density (JSC) of 11.10 mA cm−2 along with an FF of 0.675. Notably, the best PDBT‐T1:BPT‐Se1 achieved a higher PCE of 9.54% with a VOC of 1.059 V, a JSC of 12.30 mA cm−2, and an FF of 73.2%. The large improvement of PCEs upon isomerization of BPT‐Se should be ascribed to the overall enhancement of FF, JSC, and VOC in the PDBT‐T1:BPT‐Se1 devices. The larger VOC in BPT‐Se1‐based device is in agreement with the higher LUMO energy level of BPT‐Se1; the enhanced JSC and FF are mainly attributed to the favorable morphology of the blend film. To the best of our knowledge, the PCE of 9.54% is among the best results for efficient OSCs based on PDI acceptors. It is worth noting that the FF of 0.732 is the highest value for PDI‐based SMAs OSCs to date .In order to evaluate the potential application of these two compounds, OSCs were fabricated with a conventional architecture of indium tin oxide/PEDOT:PSS :poly(styrene‐sulfonate))/PDBT‐T1:acceptors/ZrAcAc (zirconium acetylacetonate)/Al. We optimized the thermal annealing temperature of the PDBT‐T1:BPT‐Se and PDBT‐T1:BPT‐Se1 from 90 to 110 °C, and blend ratios of the PDBT‐T1:BPT‐Se and PDBT‐T1:BPT‐Se1 from 1.5:1 to 1:1.5. The corresponding data are summarized in Tables S1 and S2 in the Supporting Information. The optimal donor/acceptor weight ratio is 1:1 with a total concentration of 20 mg mLJSC at different light intensities (Plight). The relationship between Plight and JSC can be describe using JSC ∝ PlightS  and effective voltage (Veff) were investigated. Veff is dependent on the internal electric field in OSCs, which affects the transport and extraction of charge carriers. As the voltage increases, the Jph increases linearly in the low Veff region, and then saturates at high Veff  of BPT‐Se1 is 12.65 mA cm−2, obviously larger than that of the BPT‐Se‐based device (11.50 mA cm−2), mainly due to the stronger light harvesting and better exciton splitting. Under short‐circuit conditions and maximal power output, the Jph/Jsat values are 0.965 and 0.795 for the PDBT‐T1:BPT‐Se blend, and 0.973 and 0.854 for the PDBT‐T1:BPT‐Se1 blend, respectively. The higher ratios of Jph/Jsat suggest that PDBT‐T1:BPT‐Se1 device has a better exciton dissociation rate and charge collection probability. To gain deeper insights into the relationship between light absorption and photocurrent generation, we calculated the maximum exciton generation rate (Gmax) of the two devices. Gmax follows the equation, Jsat = q × Gmax × L, where L is the thickness of blend film and q is the electron charge. The device of BPT‐Se1 shows a larger Gmax value of 8.32 × 1027 m−3 s−1 relative to that of the BPT‐Se‐based device (7.56 × 1027 m−3 s−1), which could be attributed the stronger light absorption of BPT‐Se1. SCLC method was employed to investigate the hole (µh) and electron mobilities (µh) of the blend films , and the related data are outlined in Table −4 cm2 V−1 s−1 and an electron mobility of 2.13 × 10−4 cm2 V−1 s−1, while the hole and electron mobility of PDBT‐T1:BPT‐Se1‐based devices are calculated to be 4.23 × 10−4 and 2.76 × 10−4 cm2 V−1 s−1, respectively, which are higher than those of the BPT‐Se‐based device. The less bimolecular recombination, better exciton dissociation rate and charge collections probability, and more balanced hole/electron ratio are responsible for the larger FF and JSC obtained in BPT‐Se1‐based device.Charge‐recombination mechanism of the BPT‐Se and BPT‐Se‐1‐based devices were surveyed by measuring Figureqr = 0.27 Å−1 and π–π stacking peak at qz = 1.71 Å−1. Both BPT‐Se and BPT‐Se1 neat films exhibit ring‐like lamellar scattering at qr = 0.27 Å−1 without obvious orientation preference . Compared to BPT‐Se, BPT‐Se1 displays a relatively strong scattering intensity and a larger crystallite coherence length , indicating a higher crystallinity in accordance with the observed higher electron mobility. GIWAXS patterns and the corresponding intensity profiles of the PDBT‐T1:BPT‐Se and PDBT‐T1:BPT‐Se1 blend films are presented in Figure qr = 0.27 Å−1 for both blend films originate from the lamellar scattering of PDBT‐T1. The corresponding CCL are 93.2 Å for the PDBT‐T1:BPT‐Se blend film and 133.4 Å for the PDBT‐T1:BPT‐Se1 blend film. Furthermore, the π–π stacking peaks are at qz = 1.66 Å−1 (d = 3.79 Å) and 1.71 Å−1 (d = 3.67 Å) for the PDBT‐T1:BPT‐Se and PDBT‐T1:BPT‐Se1 blend films, respectively. The relatively higher crystallinity and tighter π–π stacking for the PDBT‐T1:BPT‐Se1 blend film could improve charge transport, consistent with the observed higher FF and photocurrent of the devices.To investigate the structure‐property relationship, morphology studies were carried out. First, AFM was utilized to survey the surface morphology of the blend film Figure4. As cVOC of 1.059 V, and a JSC of mA cm−2, while the BPT‐Se‐based device shows a PCE of 7.78%. Notably, the isomerization of BPT‐Se enhances the FF, JSC and VOC simultaneously and the 73.2% FF is the highest FF for the PDI‐based OSCs to date. These results demonstrate that regiochemistry of the central core in PDI‐based SMAs has a large impact on the physicochemical properties of the molecule and photovoltaic performance of the resulting device. The success of isomerization strategy thus paves the way toward high‐performance PDI‐based small molecule acceptors for nonfullerene OSCs.In conclusion, we designed and synthesized two isomeric PDI‐based SMAs, BPT‐Se and BPT‐Se1, and investigated the effects of the central‐core regiochemistry on optical, electronic, charge‐transport, photovoltaic, and morphological properties. GIWAXS results indicated that BPT‐Se1 has a better crystallite quality and tighter molecular packing relative to that of BPT‐Se. In addition, BPT‐Se1 exhibits a stronger absorption profile, an upshifted LUMO energy level and a higher electron mobility, which endows BPT‐Se1 with better prospects for OSC applications. Consequently, the PDBT‐T1:BPT‐Se1‐based OSC device delivers a high PCE of 9.54% with an excellent FF of 73.2%, a high The authors declare no conflict of interest.SupplementaryClick here for additional data file.


The transcription factor BTB and CNC homology 1 (Bach1) is widely expressed in most mammalian tissues and functions primarily as a transcriptional suppressor by heterodimerizing with small Maf proteins and binding to Maf recognition elements in the promoters of targeted genes. It has a key regulatory role in the production of reactive oxygen species, cell cycle, heme homeostasis, hematopoiesis, and immunity and has been shown to suppress ischemic angiogenesis and promote breast cancer metastasis. This review summarizes how Bach1 controls these and other cellular and physiological and pathological processes. Bach1 expression and function differ between different cell types. Thus, therapies designed to manipulate Bach1 expression will need to be tightly controlled and tailored for each specific disease state or cell type. BTB and CNC homology 1 (Bach1) is a member of the Cap ‘n' Collar and basic region leucine zipper family (CNC-bZip) of transcription factors. It is widely expressed in mammalian tissues, and the human variant consists of 736 amino acids. The N-terminal region of Bach1 contains a BTB/POZ domain, which functions as a protein interaction motif, while the C-terminal bZip domain binds to DNA  and NADPBach1 contains six cysteine-proline (CP) motifs, four of which are located in a heme-binding region near the C-terminus. Heme inactivates Bach1 by interacting with two of the CP motifs, leading to the exclusion of Bach1 from the nucleus , and by c− (xCT) in human endothelial cells, while cytoplasmic Nrf2 is bound and inhibited by Kelch-like ECH-associated protein 1 (Keap1) [Bach1 competes with nuclear factor (erythroid-derived 2)-like-2 (Nrf2) for binding to the MAREs in oxidative stress-response genes. Under normal physiological conditions, nuclear Nrf2 contributes to vascular protection by inducing expression of the glutamate cysteine ligase modulatory subunit (GCLM) and the light chain component of system x (Keap1) . During  (Keap1) , while B (Keap1)   . Further (Keap1) . Thus, B (Keap1) , while Nβ-cells [Bach1-deficient mice are more resistant to the oxidative stresses associated with trinitrobenzene sulfonic acid- (TNBS-) induced colitis , hyperoxβ-cells  and the β-cells . Furtherβ-cells . Many ofβ-cells , 27 alsoβ-cells . HO-1 exβ-cells , therebyβ-cells .The effect of Bach1 on cell proliferation and survival can differ profoundly depending on the cell type and experimental conditions. In endothelial cells, we have shown that exogenous Bach1 expression inhibited proliferation and the expression of cyclin D1 while inducing cell-cycle arrest and caspase 3-dependent apoptosis ; howeverIn addition, Bach1 is also associated with an age-dependent loss of adaptive homeostasis. Bach1 was increased in all tissues  of aging mice  and was β-catenin signaling. Canonical Wnt/β-catenin signaling regulates gene transcription by facilitating the transport of cytoplasmic β-catenin into the nucleus, where β-catenin forms a complex with transcription factor 4 (TCF4)/lymphoid enhancer-binding factor 1 (LEF1) and recruits transcription factors, such as CREB-binding protein (CBP), that initiate Wnt-targeted gene expression [β-catenin also displaces histone deacetylase 1 (HDAC1) [β-catenin/TCF4 binding, recruits HDAC1 to the promoter of TCF4-targeted genes, and prevents β-catenin from being acetylated by p300/CBP, thereby reducing the expression of downstream Wnt targets, such as vascular endothelial growth factor (VEGF) and interleukin- (IL-) 8, that promote angiogenic activity in human ECs [β-catenin signaling and the expression of VEGF and IL-8 [We have shown that measurements of perfusion, vascular density, and the expression of proangiogenic cytokines were greater in the limbs of Bach1-deficient mice than in the limbs of wild-type mice after surgically induced hindlimb ischemia . Bach1 apression . The bin (HDAC1)  and othe (HDAC1)  and recr (HDAC1) . Bach1 fuman ECs  ; once activated, MGMT counteracts the antitumor effect of TMZ by reversing the TMZ-induced methylation of guanine residues [Bach1 is also involved in epigenetic mechanisms of cancer progression. In both colorectal cancer and melanoma, the B-Raf protooncogene variant BRAF (V600E) upregulates v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog G (MAFG), which heterodimerizes with Bach1 and recruits both chromodomain helicase DNA-binding protein 8  and the DNA methyltransferase DNMT3B, leading to the hypermethylation and transcriptional silencing of tumor-suppressor genes , 55 Fig. Bach1 aresidues .Notably, Bach1 can also function as a tumor suppressor, and at least some of its anticancer properties can likely be attributed to its role as a regulator of HO-1 expression. In acute myeloid leukemia cells, lower levels of Bach1 expression were associated with increases in HO-1 levels and in cell viability after exposure to an anticancer drug , and CXCβ-Globin gene activation is a crucial step during erythroid differentiation and is impeded by Bach1 [β-globin gene [β-globin transcription by recruiting coactivators such as CBP, transactivation domain-interacting protein (TIP), and stem cell leukemia (SCL) to the LCR; c-Jun N-terminal kinase (JNK) stabilizes the p45/MafK heterodimer by phosphorylating the Ser157 residue of p45 [β-globin LCR  by intracellular heme [γ and PPARγ-dependent adipocyte differentiation [by Bach1 , which fbin gene . During e of p45 , 63. Baclar heme , and hemlar heme . Heme-inlar heme , and whelar heme . Bach1 antiation .Bach1 also has a role in the immune system and autoimmune disease. Bach1 regulates the expression of core macrophage-associated genes, such as aldo-keto reductase family 1 member B10 (Akr1b10), biliverdin reductase B (Blvrb), calcium/calmodulin-dependent protein kinase 1 (Camk1), and glutamate-ammonia ligase (Glul) , and botβ-catenin signaling, which suggests that Bach1 may influence the development and progression of metabolic disease, especially since some Wnt family members and/or downstream targets of Wnt have been linked to insulin sensitivity [In summary, Bach1 is an important transcription factor that regulates mechanisms involved in ROS production, cell cycle, heme homeostasis, hematopoiesis, and immunity and has a function in cardiovascular disease  and cancer. Bach1 also regulates adipocyte-related genes, the pentose phosphate pathway, and Wnt/sitivity  and diabsitivity . Other a


Previous studies have found a positive effect of cosmetics on certain behavioral measures, such as the tip given to waitresses by male patrons. These studies have employed confederates who usually wear cosmetics. We therefore sought to examine whether the positive effect found in these studies could, in part, be explained by a change in behavior. In order to test the possibility of a ‘cosmetics placebo effect’, we employed a confederate to solicit donations from passersby. On some days our confederate would not have any cosmetics applied to her face , on some days cosmetics were pretended to be applied to her face , and on other days cosmetics were actually applied to her face . In line with previous research, we found that across conditions men donated significantly more than women to our female solicitor, providing support for the ‘showoff hypothesis’, in which male generosity serves as a mating tactic. When investigating men’s donations in more detail, we found that the highest percentage of donations came in the cosmetics condition, followed by the placebo cosmetics condition, and then by no cosmetics condition. The effect of condition on donation rates, however, was not statistically significant. Our study was limited to one solicitor and one dependent variable  and therefore future research would benefit from using more confederates as well as examining other behavioral measures. Given the influence of cosmetics use on so many real-world outcomes, we believe that further exploration into a possible ‘cosmetics placebo effect’ would be valuable. Research has demonstrated that the use of cosmetics is associated with a variety of social outcomes, ranging from perceptions of physical attractiveness –8 to evaIt is important to note that for both the bar study  and the We therefore set out to investigate the possibility of a ‘cosmetics placebo effect’ by employing three conditions: no cosmetics, placebo cosmetics, and cosmetics. The application process of the cosmetics condition was mimicked in the placebo cosmetics condition so that the confederate believed the same cosmetics were applied to her face in both conditions. This design allowed us to examine if, and to what extent, there is a placebo effect related to cosmetics use.Testing at a bar or a restaurant, in line with the previous studies , 10, wasSome research has specifically examined the influence of solicitor characteristics on charitable giving , 17. OneIn addition, research has shown that higher levels of physical attractiveness in female solicitors results in an increase in giving between 50–135 percent , with thA Gettysburg College student who normally wears cosmetics was recruited to be the confederate for this study but was not informed of the goals of the study. The confederate self-identified as White and was 21 years old in the fall semester of the data collection and 22 years old in the spring semester of the data collection. The confederate approached 626 people  to ask for donations. Ethical approval was received from the Gettysburg College Institutional Review Board, a permit allowing the solicitation of donations on Lincoln Square was issued by the Gettysburg Borough, and authorization to solicit donations for the American Red Cross was granted by a representative of the American Red Cross. Informed consent from participants could not be collected as that would alter their behavior and therefore the Gettysburg College Institutional Review Board waived informed consent under the Code of Federal Regulations, Part 46.116, because the waiver did not adversely affect the rights and welfare of the participants.The study was scheduled for three days a week for seven weeks in the fall  and seven weeks in the spring . The condition  was randomized across the days to avoid a weekly pattern that the confederate could deduce. Additionally, the testing day was balanced across the three conditions in order to ensure that the condition was not confounded with the day of the week. Since the donations were solicited in an open-air location, some testing days had to be cancelled due to weather. This resulted in a total of 34 usable testing days.The confederate would arrive at the Gettysburg College Perception Laboratory 30 minutes before commencing testing and on some days she would not have any cosmetics applied to her face , on some days cosmetics were pretended to be applied to her face , and on other days cosmetics were actually applied to her face . The confederate was told that whether or not cosmetics would be applied to her face for each testing session was randomly assigned using a coin toss. Two sets of the same brushes and applicators were used  to mimic the same cosmetics application across conditions. For example, in the placebo cosmetics condition, a clean eye-makeup applicator and brush were used on the confederate’s eyelids but no eye-shadow was applied. On the other hand, in the cosmetics condition, eye-shadow was applied using the eye-makeup applicator and brush. In the cosmetics condition, porcelain foundation, ivory pressed powder, bronzer, pink blush, brown/gold eye-shadow, black mascara, and red lipstick were applied in every session. In the same order, the placebo cosmetics condition mimicked the cosmetics condition with the same brushes and applicators but without pigmented products. In order to mimic the feel of the cosmetics condition, a non-pigmented moisturizer, a clear mascara brush with water, and a non-pigmented lip balm were used in every session for the placebo cosmetics condition. Across both conditions, the confederate maintained her eyes closed during the application process. The process was identical across conditions and sessions.Afterwards, the confederate and a research assistant would walk over to Lincoln Square, where the testing would take place. The confederate always styled her hair the same way and wore an American Red Cross sweatshirt. The confederate would approach every man and woman walking on their own with the following: “Hello! I’m collecting money for the American Red Cross. Do you have anything you’d like to donate?”. If she saw the same person more than once, she would not approach them a second time. Additionally, people walking in pairs or groups were not approached. Meanwhile, the research assistant (who was sitting on a bench nearby) would record the sex of the person approached and whether or not they donated. Each testing session lasted 45 minutes and all donations were collected using an American Red Cross donation box. After each testing session finished, the confederate and the research assistant would walk back to the Gettysburg College Perception Laboratory, where the confederate’s face was cleansed with makeup-removing towels on the days of the placebo cosmetics condition and the cosmetics condition. The confederate was asked to close her eyes during the cleansing so she would not see the towels.It is important to note that prior to commencing this study, other Gettysburg College students were recruited to test whether or not pretending to apply cosmetics could be differentiated from a real cosmetics application. Three students were invited to the Gettysburg College Perception Laboratory and were told that a research assistant needed to practice her makeup application skills for a study. Each student experienced two conditions: cosmetics and placebo cosmetics . Afterwards, they were questioned about whether they felt any differences between the two conditions. All the students reported not feeling any differences between the two conditions and were surprised when told that in one condition cosmetics were only pretended to be applied to their faces.Additionally, the confederate in this study was probed about what she believed the purpose of the study to be after the data collection concluded. She commented “makeup versus no makeup and seeing how much money I would get and who was more likely to donate”. She was then asked if she noticed anything across the cosmetics application days and she responded that “they all seemed the same”. When probed again about feeling any differences she commented that “it all felt the same”. When told that makeup was only applied on half of the cosmetics days and only pretended to be applied on the other half, she was surprised this was the case and commented that “it didn’t feel like that at all”. The confederate was also asked if thinking back (after knowing about the different conditions) whether she could have guessed on which days she had cosmetics on and which ones she did not and her answer was “not at all”.A total of $566.51 was collected and donated to the American Red Cross. In the no cosmetics condition, the confederate approached a total of 208 people . In the placebo cosmetics condition, the confederate approached a total of 210 people . In the cosmetics condition, the confederate approached a total of 208 people .When averaged across conditions, 42.67% of men donated when approached. When separated by condition, 39.06% of men donated in the no cosmetics condition, 43.08% in the placebo cosmetics condition, and 46.15% in the cosmetics condition , Fig 1. p<0.001). More specifically, men were more likely to donate to our confederate than women.We then conducted a chi-square test between the sex of the person approached and the donation rates across conditions. There was a statistically significant association between the sex of the person and donation rates (χ(1) = 23.22, p = 0.530) or women (χ(2) = 2.49, p = 0.288). We then ran follow-up one-tailed chi-square tests using only the two conditions that have been examined in previous studies  or women (χ(1) = 1.43, p = 0.153). The same pattern of results was observed when conducting a binary logistic regression, with sex of the person approached contributing to the model  and cosmetics condition not contributing to the model .In order to examine whether the experimental condition  had a significant effect on donation rates, separate chi-square tests were run for men and women. We found no statistically significant associations between the experimental condition and donation rates for either men (χ(2) = 1.27, osmetics ). We agaWe predicted higher rates of giving when the confederate was in the cosmetics condition than the placebo condition, and more giving in the placebo condition than the no cosmetics condition. We also predicted higher rates of giving overall by men than women. We found a significant sex difference in donation rates, with a higher percentage of men approached by our female confederate donating when compared to women. Regarding the cosmetics conditions, we found that the highest percentage of donations from men came in the cosmetics condition, followed by the placebo cosmetics condition, followed in turn by the no cosmetics condition, though none of these differences were statistically significant.Landry and colleagues  found thMoreover, Landry et al.  found thWe also predicted that our confederate would receive the second highest percentage of donations from men in the placebo cosmetics condition since women report an increase in self-confidence and socialness when wearing their customary cosmetics . While wWhile our pattern of results for men’s donations were as predicted , the effect of condition was not statistically significant. We also did not find a statistically significant effect of cosmetics condition on women’s donation rates. For women, the highest percentage of donations actually came in the no cosmetics condition. Previous research has found that women are particularly harsh in their judgements of attractive women , and theUnlike previous studies , 10, we Another possible reason for our null results is that the difference in perceived attractiveness may not have been large enough. Cosmetics have been found to increase attractiveness –8, but iIn conclusion, we did find a sex difference in donation rates, with a higher percentage of men approached by our female confederate donating when compared to women. We did not, however, find a significant effect of our experimental condition  on donation rates. Given the influence of cosmetics use on so many real-world outcomes , 22, 23,


Protein Arginine (R) methylation is the most common post-translational methylation in mammalian cells. Protein Arginine Methyltransferases (PRMT) 1 and 5 dimethylate their substrates on R residues, asymmetrically and symmetrically, respectively. They are ubiquitously expressed and play fundamental roles in tumour malignancies, including glioblastoma multiforme (GBM) which presents largely deregulated Myc activity. Previously, we demonstrated that PRMT5 associates with Myc in GBM cells, modulating, at least in part, its transcriptional properties. Here we show that Myc/PRMT5 protein complex includes PRMT1, in both HEK293T and glioblastoma stem cells (GSCs). We demonstrate that Myc is both asymmetrically and symmetrically dimethylated by PRMT1 and PRMT5, respectively, and that these modifications differentially regulate its stability. Moreover, we show that the ratio between symmetrically and asymmetrically dimethylated Myc changes in GSCs grown in stem versus differentiating conditions. Finally, both PRMT1 and PRMT5 activity modulate Myc binding at its specific target promoters. To our knowledge, this is the first work reporting R asymmetrical and symmetrical dimethylation as novel Myc post-translational modifications, with different functional properties. This opens a completely unexplored field of investigation in Myc biology and suggests symmetrically dimethylated Myc species as novel diagnostic and prognostic markers and druggable therapeutic targets for GBM. It regulates cell proliferation, metabolism, stemness maintenance1 and binds to 10% of genomic loci, usually amplifying transcription from already active genes3. Myc expression and function are deregulated in a wide variety of cancers, including glioblastoma multiforme (GBM)5. Despite more than a decade of advances in surgery and chemo-/radiotherapy, GBM treatment options are disconcertingly very poor and patients life expectancy is very limited (14.6 to 18 months from diagnosis)6. GBM poor prognosis and deadly outcome seem to rely on the presence of tumour initiating cells, which share many molecular and biological features with embryonic and adult stem cells and thus are called glioblastoma stem cells (GSCs). They are difficult to eradicate by surgery and are resistant to conventional radio- and chemotherapy, allowing tumour to recur overtime7. They also present the so-called “Myc signature”8, emphasizing the essential role Myc plays in maintaining GSC properties.The Myc (c-myc) oncogene is a transcription factor required for pivotal cell functions9, acetylation10, sumoylation11, ubiquitination and consequent degradation by the proteasome machinery12. These modifications have a precise role in modulating Myc activity and have been suggested as potential therapeutic targets10.Myc is regulated by a series of post-translational modifications: phosphorylation13. Three distinct types of methylated Rs occur in mammalians. Asymmetrically dimethylated arginine (ADMA) is the most frequent, followed by symmetrically dimethylated (SDMA) and monomethylated arginine (MMA). The enzymes responsible for R methylation are the Protein Arginine Methyltransferases (PRMTs). Six proteins have been reported to undoubtedly possess the R methyltransferase catalytic domain . PRMT2, 7 and 9 have sequence similarities with the other PRMTs and are supposed to possess an equivalent enzymatic activity13. Depending on the position of the methyl groups on R terminal guanidino nitrogens, PRMTs are, basically, classified into two types: type I PRMTs  are responsible for MMA or ADMA whereas the type II PRMT5 catalyzes MMA and SDMA13. PRMT1 is the major methyltransferase in mammalian cells. It has a wide variety of substrate specificity, from histone to non-histone proteins14. From a transcriptional point of view, it is known as an activator, since the occurrence of asymmetrically (AS) dimethylated R3 on histone H4 residues (H4R3me2as) is associated with transcriptionally active chromatin15. PRMT1 is deregulated in a wide variety of cancer types, e.g. pancreatic adenocarcinoma16, gastric17 and lung cancer18. It controls the epithelial-mesenchymal transition (EMT) in cancer cells19, while playing a major role in muscle20 and B cell differentiation21. PRMT5 has received a greater attention and has been suggested as a putative druggable target in a wide variety of tumours, including GBM22. Like PRMT1, it has multiple nuclear and cytoplasmic targets and catalyzes the symmetrical (S) dimethylation of H4R3 and H3R8, usually repressing transcription23, and of H3R2, activating transcription24. PRMT5 is involved in a variety of cellular processes, such as neurogenesis25, myogenesis26, somatic cell reprogramming27 and GSCs self-renewal28. Interestingly, PRMT1 and PRMT5 have been shown to interact in GBM cells, being PRMT1 the major responsible for the activation of cancer-related genes29. They also catalyze the S- and AS-dimethylation of NMyc31 and, very recently, it has been shown that PRMT1 is required for p300 recruitment to Myc-regulated gene promoters32.Methylation of arginine (R) residues is the most common methylation event in mammalian cells, which modifies protein interacting properties, ruling a large amount of signaling pathways33. Here we show that PRMT1 associates with Myc/PRMT5 in both HEK293T cells and GSCs and that Myc is both S- and AS-dimethylated by PRMT5 and PRMT1, respectively, both in vitro and in living cells. At the functional level, S-dimethylation protects Myc from degradation, while AS-dimethylation ensure Myc proper turnover. Finally, the inhibition of either PRMT1 or PRMT5 activity affects Myc recruitment at promoters and has a profound effect on GSCs biological functions, such as neurospheres formation and differentiation capacity. These findings represent the first demonstration in GSCs of the presence of differentially dimethylated Myc species, with distinct properties, opening a completely new field of investigation in Myc-dependent GBM biology. Further, they support the hypothesis that acting on S-Myc post-translational modification may represent a possible route to control its function.We previously demonstrated that PRMT5 interacts with Myc in glioblastoma cells, including GSCs, and that PRMT5 participates, at least in part, in the activation of Myc target genes33) and associates with PRMT5 in both HEK293T and glioblastoma cells33. Since PRMT5 and PRMT1 were found associated in GBM cells29, we sought to determine whether Myc was able to promote also AS-dimethylation of R3 on histone H4 (H4R3me2as). To this aim, HEK293T cells were transfected with either a Flag-tagged Myc construct (FlagMyc/HEK293T) or an empty vector and the level of H4R3me2as was detected by western blot. Figure 34 transduced with a lentiviral, doxycycline inducible shRNA against Myc (shMyc), the level of H4R3me2s were reduced, while H4R3me2as increased ‐1,4‐pentadien‐3‐one, hereafter named inhibitor 9 (9i), which specifically inhibits PRMT1 at this concentration35. Thereafter, immunoprecipitation experiments were performed with SYM10 and ASYM24 antibodies. Figure Moreover, protein extracts from HEK293T/FlagMyc cells were reciprocally immunoprecipitated by using SYM10 or ASYM24 or anti Flag antibodies. Western blots were performed with anti-Flag or SYM10 and ASYM24 antibodies. Figure 31. We observed that upon PRMT5 interference Myc expression decreased, while in PRMT1-depleted cells, Myc levels slightly increased , bound by Myc in GSCs34 together with the Glial Fibrillary Astrocytic Protein (GFAP) promoter38, were tested. In stem conditions, either PRMT5 or PRMT1 inhibition did not affect Myc binding on CCND1 and EGFRvIII promoters. Conversely, SOX2 promoter was sensitive only to PRMT1 inhibition, whereas, either PRMT5 or PRMT1 inhibition decreased Myc binding on GFAP promoter. Interestingly, Myc detached from all these promoters in differentiating control cells. PRMT5 inhibition had no effect on Myc recruitment in these experimental conditions, whereas 9i treatment restored Myc binding above control levels only on CCND1 and EGRvIII promoters. Altogether, these data address a role to both PRMT5 and PRMT1 catalytic activity in regulating Myc loading at promoters.To evaluate the effect of PRMT1 and PRMT5 on Myc binding at its specific chromatin loci in GSCs, ChIP experiments were performed, either in stem or differentiation conditions, in the presence or absence of EPZ015666 or 9i or control vehicle ted Fig. . Therefo 39 Fig. . Also Tu 39 Fig. , whereas 39 Fig. , as alsoved Fig. . In summ41. Specifically, Myc and PRMT5 have been proved as master regulators of GSC pluripotency28, while PRMT1 loss does not affect typical pluripotency markers42. Myc interacts with PRMT533 and PRMT1 is required for p300 recruitment at Myc-regulated gene promoters32. Further, PRMT1 and PRMT5 interact in GBM29. However, no direct association between Myc, PRMT1 and PRMT5 has been reported. Here we show, for the first time, that Myc, PRMT5 and PRMT1 form a ternary complex, both in recipient HEK293T cells and GSCs , 1% Glutamine, 1% Penicillin/Streptomycin and 2% Foetal Bovine Serum .HEK293T and mesenchymal Brain Tumour 168 Neural Stem Cells2‐4‐hydroxyphenyl)‐1,4‐pentadien‐3‐one (9i) at 3 μM35. Control solvent was represented by dimethylsulfoxide . For GSCs differentiation and neurospheres formation experiments, drugs were refreshed every 48 hrs. Cyclohexymide and MG132 (Merck) were 10 μg/ml and 10 μM, respectively.EPZ015666 (Merck) was administered at a concentration of 5 μM and the PRMT1 inhibitor 1,5‐bis, anti-H4R3me2as , anti-PRMT5  and anti-PRMT1  antibodies were from Abcam . Anti-Myc  was from Santa Cruz Biotechnology . Anti-actinHRP  was from Merck. Anti-SOX2 , SYM10  and ASYM24  antibodies were from Millipore . Anti-Flag antibody  was from Merck. Secondary anti-rabbit IgG  and anti-mouse IgG  were from Merck. In most cases, filters were directly cut after the blocking procedure, according to the molecular weight of the proteins of interest. Equipment and settings: images were captured with a ChemiDoc XRS+ Gel Imaging System  and the band intensity of western blot analysis was quantified using ImageJ software. When useful, contrast/brightness of western blot images were slightly adjusted with the Power Point software.Cells were directly lysed in Laemmli buffer and western blots were performed as described33, by using 4 μg of each antibody or control IgGs . To avoid IgGs detection, either ImmunoCruz IP/WB Optima B, C, F, E  or the VeriBlot reagent  were used in western blot, after the immunoprecipitation procedure, according to the manufacturer’s instructions.For immunoprecipitation experiments, cells were lysed in 140 mM NaCl, 10 mM Tris pH 7.6, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA, protease and phosphatase inhibitors. Cells were incubated on ice for 30′, then centrifuged for 20′ at 4 °C, at 13000 rpm. Thereafter, supernatant (1–2 mg) underwent immunoprecipitation as described33, pCMVPRMT5-6myc (courtesy of dr. Levi A.) and pCMV6PRMT1-myc-Dkk , together with the corresponding empty vectors. The shMyc insert was subcloned from the KRAB lentiviral vector into the Tet-on pSLIK lentivirus33. Lentiviral production and infection were performed as described33.Plasmid transfections were performed by using Lipofectamine 2000  according to the manufacturer’s instructions. Plasmids were: pCBSFlagMycsiPRMT5, siPRMT1  and scrambled siRNAs  were transfected by using the DHARMAFect reagent (Dharmacon), according to the manufacturer’s instructions.in vitro methylation assays, HEK293T cells  were transfected with 50 nM siRNAs (or scrambled siRNA) and the day after with either PRMT5-6myc or PRMT1-myc-Dkk plasmids or control vectors (10 μg). 48 hrs later, cells were lysed in 20 mM Hepes pH 7.9, 137 mM NaCl, 20% Glycerol, 1% Nonidet P40, 2 mM EDTA, protease and phosphatase inhibitors. Cell extracts were kept in rotation for 30′, at 4 °C and then centrifuged at 12000 rpm for 20′ at 4 °C. Cell extracts were pre-cleared with 5 μg of control IgGs, for 1 hr, on ice; thereafter, 20 μl of equilibrated protein A/G Plus agarose beads  were added and the mixture was kept in rotation for 30′, at +4 °C. and then centrifuged at 10000 rpm, at 4 °C. The supernatant was incubated with either anti-Myc antibody (Santa Cruz Biotechnology), or control IgG, for an overnight, at 4 °C (to purify either overexpressed PRMT5-6myc or PRMT1-myc-Dkk). The day after, 40 μl of equilibrated protein A/G Plus agarose beads were added and the mixture was incubated in rotation for 4 hrs, at 4 °C. Beads were extensively washed with lysis buffer by centrifuging at 3000 rpm, at 4 °C. Beads and bound PRMT5-6myc or PRMT1-myc-Dkk were resuspended in assay buffer  and 500 ng of human recombinant c-Myc protein  plus 1 mM of S-adenosylmethionine (Merck) were added. The reaction mixture (100 μl) was incubated for 1 hr, at 35 °C. Thereafter, 400 μl of assay buffer were added, and dimethylated Myc was immunoprecipitated by incubating the reaction mixture with 4 μg of either SYM10 or ASYM24 antibody (Millipore), for an overnight, at 4 °C. The day after, 40 μl of equilibrated protein A/G Plus agarose beads were added and the mixture incubated for 4 hrs, in rotation, at 4 °C. After three washes in assay buffer, 20 μl of Laemmli buffer 2X were added and the immunocomplexes boiled for 5′. Dimethylated Myc was revealed by western blot.For 33. Anti-Olig2 , anti- TuJI , anti-Oct4  antibodies were from Santa Cruz Biotechnology; anti-SOX2  and anti-Nanog  were from Millipore. Anti-GFAP , anti-H4R3me2s  and anti-H4R3me2as  were from Abcam. Secondary antibodies were donkey anti-rabbit IgG Alexa Fluor 555  and goat anti-mouse Alexa Fluor 488 , both from Thermo Fisher Scientific.Immunofluorescence analyses were performed as describedvs peak to eliminate doublets and forward vs side scatter to exclude debris. The percentage of cells in the different cycle phases and in subG1 (composed of dead cells and debris) was calculated using FACS Express5 Flow Research Edition software.Cells were dissociated, counted, resuspended in PBS and then fixed in 70% ethanol for a minimum of 24 hrs. After three washes in PBS, cells were incubated with 50 μg/ml Propidium Iodide (Merck) plus 0.1 U/L RNAse (Merck) for 3 hrs at room temperature before FACS analysis by Coulter Epics XL flow cytometer . Cells were gated using both area 5 GSCs were plated in duplicate into a six-well multi-plate. Cells were allowed to grow for 10 days, in the presence or absence of EPZ015666 or 9i or control vehicle. Neurospheres formation was monitored daily by a phase contrast microscope. Images were captured with the ZOE Fluorescent Cell Imager (Bio-Rad).3 × 1033.Real time PCR were performed as described46.Chromatin immunoprecipitations were performed as describedChIPs oligonucleotides were:hEGFRv3prom_F GGCTGTTTGTGTCAAGCCTTTAhEGFRv3prom_R TTGAAGCCAATGTGTGAAGCAhSOX2prom_F AACGGACGTGCTGCCATThSOX2prom_R TCCCATTGTCCCGACGTAAAhGFAPprom_F TGGGAATGAGGCCTAGTAGGAAhGFAPprom_R GGGAATTTGGCGAAGAATGAhCCND1prom_F GCTTTCCATTCAGAGGTGTGTTThCCND1prom_R CTACCTTGACCAGTCGGTCCTTGFAP oligonucleotides used in real time PCR experiments were:GFAP_F ACCAGGACCTGCTCAATGTCGFAP_R ATCTCCACGGTCTTCACCACAll data are expressed as mean ± SEM, and significance  was calculated using two-tailed paired Student’s t test; p values < 0.05 were considered statistically significant.Supplementary info


Canonical Wnt5a/β-catenin expression decreased during adipogenic differentiation of hUCB-MSCs. Treatment with Wnt5a blocked the adipogenic differentiation of hUCB-MSCs and activated the Wnt pathway, with a decrease in the adipogenesis markers PPARγ and leptin, and reduced lipid vacuole-associated Oil red O activity. In contrast, inhibition of the Wnt pathway with dickkopf-1 and β-catenin small interfering RNA transfection promoted the adipogenic potential of hUCB-MSCs. Interestingly, the Ca2+-based system exhibited a synergic effect on adipogenic potential through the Wnt5a/β-catenin pathway. Our data suggest that the variable adipogenic differentiation potential of hUCB-MSCs from different lots is due to variation in the intracellular Ca2+ level, which can be used as a marker to predict hUCB-MSCs selection for lipogenesis therapy. Overall, these results demonstrate that exogenous calcium treatment enhanced the adipogenic differentiation of hUCB-MSCs via negatively regulating the Wnt5a/β-catenin signaling pathway.Mesenchymal stem cells- (MSCs-) based therapies show different degrees of efficacies for the treatment of various diseases, including lipogenesis. We evaluated the adipogenic differentiation ability of human umbilical cord blood-derived MSCs (hUCB-MSCs) from different donors and examined the contribution of the intracellular calcium (Ca Large soft tissue defects due to an external wound or surgical resection are very common in clinical practice, and adipose tissue regeneration is a very important part of reconstruction plastic surgery that allows for the recovery of such tissue defects. Thus, the demand for soft tissue reconstruction for the regeneration of damaged tissues or plastic surgery is rapidly increasing. Adipogenesis can be exploited or enhanced in various fields related to soft tissue reconstruction. Recently, various transplant methods using stem cells have been developed for the purpose of regenerating tissues damaged due to burn injury, surgical resection, innate defects, breast tissue reconstruction, and so forth –4.In particular, mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into various cell types such as adipocytes, osteocytes, chondrocytes, and muscle cells. MSCs have various advantages, including the abilities for tissue regeneration and immunoregulation, and they are easily isolated from various tissues such as the bone marrow (BM), adipose tissue (AT), umbilical cord (UC), and umbilical cord blood (UCB) –7. AmongHowever, in some studies, hUCB-MSCs applied to a damaged area that includes the adipose tissue showed poor results for regeneration. For example, hUCB-MSCs were injected in a mouse injury model with scars created through a subcutaneous route, but no regeneration in the damaged area was observed . By contγ) [β-catenin, which is a key effector molecule that is involved in regulating the expression of various target genes [β-catenin signaling is related to the differentiation or maintenance of the self-renewal of stem cells [β-catenin [β-catenin pathway.Calcium performs the physiological functions of cells by activating various intracellular signaling pathways , 19. Simγ) , 22. Furet genes , 24. In em cells , 26. In em cells , 28. Sev-catenin , 30. Howβ-catenin signaling and specific regulators in adipogenic differentiation.Therefore, the objective of the present study was to investigate the molecular mechanism and variation in the differentiation potential of hUCB-MSCs from different individual donors. Toward this end, we investigated the association between the intracellular calcium level and adipogenic differentiation potential of hUCB-MSCs from different individual donors and confirmed the role of Wnt/d = 1.077 g/cm3; Sigma-Aldrich, St. Louis, MO, USA). The separated MNCs were washed and suspended in minimum essential medium alpha  supplemented with 10% fetal bovine serum . Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2, wherein the culture medium was changed twice a week [2. In this study, we used six hUCB-MSC lines that were isolated from UCB samples obtained from different donors; the basic information of these samples is summarized in Supplementary This study was approved by the Institutional Review Board of MEDIPOST Co. Ltd. (MP-2016-07-1). hUCB was collected from umbilical veins after neonatal delivery after obtaining informed maternal consent. hUCB harvests were processed within 24 h of collection. The hUCB was isolated by separating mononuclear cells (MNCs) with Ficoll-Hypaque solution .Rhod2-AM  and Fluo4-AM  were used to measure the intracellular calcium level. The fluorescent dye Rhod2-AM ester  of the probe is cell-permeant and rapidly cleaves in the mitochondria to yield the Rhod2 indicator, which displays a large increase in fluorescence intensity upon binding to Ca2+ . Fluo4-A2+ . For flov/v) paraformaldehyde (Sigma-Aldrich). The immunotype of the MSCs was determined by flow cytometry on a FACSCalibur instrument, and then the percentage of expressed cell surface antigens was calculated for 10,000 gated cell events.For the cytometric analysis of cultured cell phenotypes, the cells were stained for 15 min at room temperature with fluorescein isothiocyanate-conjugated antibodies against human CD14, CD45, and HLA-DR (BD Biosciences); phycoerythrin-conjugated antibodies against human CD73 and CD166 (BD Biosciences); and CD105 . Corresponding isotype-matched mouse antibodies were used as controls. The cells were washed with PBS and fixed with 1%  were separated by SDS-polyacrylamide gel electrophoresis, and the resolved proteins were transferred to nitrocellulose membranes. Each membrane was incubated with antibodies against Wnt5a, β-catenin (Abcam), phospho-GSK3β , PPARγ (Santa Cruz Biotechnology), leptin (Thermo Fisher Scientific), and β-actin (Novus). The signals for the indicated proteins were detected with ChemiDoc™ MP Imaging System (Bio-Rad).Cell extracts were prepared in buffer containing 9.8 M urea, 4% CHAPS, 130 mM dithiothreitol, 40 mM Tris-HCl, and 0.1% sodium dodecyl sulfate (SDS). Protein concentrations were measured using a bicinchoninic acid kit (Sigma-Aldrich). Protein extracts  with normalization to the β-actin mRNA expression level. Dharmacon  designed the β-catenin siRNA and scrambled siRNA for use in the siRNA experiments. siRNAs were transfected using DharmaFECT reagent (Dharmacon) according to the manufacturer's instructions. The siRNA pools consisted of four different siRNA duplexes . TaqMan probes were designed with the Universal ProbeLibrary Assay Design Center . Significant differences were verified by one-way analysis of variance followed by the least significant difference post hoc test. The Student's γ and leptin. As shown in γ or leptin by qPCR or immunoblotting. By contrast, MSCs-L showed no lipid vacuole formation, negative Oil red O staining, and weak expression of PPARγ or leptin. Collectively, our data confirmed that the adipogenic differentiation potential of hUCB-MSCs can vary markedly among different donors.Based on a previous report demonstrating variability in the adipogenic differentiation potential of hUCB-MSCs obtained from different donors , we furt2+ is a ubiquitous intracellular signal responsible for regulating numerous cellular processes important to differentiation [2+ level contributes to the difference in the adipogenic differentiation potential between MSCs-H and MSCs-L based on the degree of staining of the Ca2+-sensitive dye Fluo4-AM and Rhod2-AM. By Fluo4-AM labeling, flow cytometry showed that under the control condition, the cytoplasmic level of Ca2+ was higher in MSCs-H (75.3 ± 2.8%) than in MSCs-L  than in the MSCs-L  than the hUCB-MSCs, as determined by flow cytometry  to elevate the intracellular Ca2+ level during 5 days of culture  significantly increased from day 3 to day 14. The adipogenic potential of Ca2+-treated cells was significantly increased at day 7 and remained elevated at days 10 and 14 compared to that observed under the control condition. In addition, pretreatment with BAPTA-AM significantly inhibited the adipogenic differentiation potential compared to that of the control , Wnt5a, and β-catenin was gradually downregulated during adipogenic differentiation, while the expression of adipogenesis-related proteins (PPARγ and leptin) was upregulated. The expression levels of p-GSK3β, Wnt5a, and β-catenin were also lower in the Ca2+-treated cells to those of the control and BAPTA-AM-treated groups. In addition, the Ca2+-treated cells strongly expressed PPARγ and leptin, but these proteins were only weakly expressed in the BAPTA-AM-treated group. Similar to the Oil red O staining pattern, leptin expression, as a late adipogenesis marker, was upregulated after 7 days of induction in the Ca2+ treatment group but not in the control and BAPTA-AM treatment groups. PPARγ expression, as a transcription marker of adipogenesis, was already induced after only 3 days in the Ca2+ treatment group. Wnt5a and β-catenin expression levels were lower in the Ca2+-treated cells than in the control and BAPTA-AM groups, with a significant difference noted on day 7 of induction  was significantly lower than those derived from adult tissues (AT and BM) , which sβ-catenin in the cytoplasm [β-catenin signal transduction [γ is a main transcription factor that leads to adipocyte differentiation, and its increased expression induces the expression of adipocyte-specific genes such as aP2 and leptin, thereby forming an adipocyte with distinct morphology and function [β-catenin, an intracellular regulator, inhibits the expression of PPARγ to suppress the expression of adipocyte-specific genes, causing interrupted adipocyte formation [β-catenin activation is decreased when AT-MSCs differentiate into adipocytes [β-catenin levels only during the adipogenic differentiation of MSCs-H, associated with low expression of β-catenin in the cytoplasm. Similar to previous studies [β-catenin is linked to a reduction in GSK3 phosphorylation. Moreover, Dkk-1 protein, which does not bind to Wnt but induces the inhibition of Wnt signaling [β-catenin with siRNA. Collectively, these results indicate that the adipogenic differentiation of hUCB-MSCs is induced by negative regulation of Wnt5a/β-catenin signaling.Wnt, a secreted protein, is a cellular signal transduction pathway that determines the fate of the cell and regulates its polarity, proliferation, and differentiation , 24. Speytoplasm . Recentlsduction . PPARγ ifunction , 51. Wntormation , 51. Simipocytes . Howeveripocytes . In the  studies , we confignaling , signifiβ-catenin signal reduction during the progression of adipogenic differentiation. Moreover, the adipogenic differentiation of calcium-treated MSCs-L increased with Dkk-1 as well as with inhibition of β-catenin in MSCs-L. However, these results are different with recent report showing a positive correlation between the calcium concentration and Wnt5a/β-catenin, and that the differentiation of skin cells is strongly induced by increased Wnt5a/β-catenin signal [β-catenin signaling (We further demonstrated that calcium accelerates the rate of Wnt5a/n signal . Therefoignaling .Overall, these results suggest that the intracellular calcium level can be a marker to predict the adipogenic differentiation potential of MSCs to improve the selection of MSCs for lipogenesis therapy. Importantly, these findings can be directly exploited to improve the application of hUCB-MSCs as a therapy for adipose tissue regeneration. Therefore, clinical application of the cells directly after exposure to calcium could improve the adipogenic potential and thus the therapeutic efficacy.


The Wnt/β-catenin signaling pathway plays an important role in the development of second heart field (SHF Isl1+) that gives rise to the anterior heart field (AHF) cardiac progenitor cells (CPCs) for the formation of the right ventricle, outflow tract (OFT), and a portion of the inflow tract (IFT). During early cardiogenesis, these AHF CPCs reside within the pharyngeal mesoderm (PM) that provides a microenvironment for them to receive signals that direct their cell fates. Here, N-cadherin, which is weakly expressed by CPCs, plays a significant role by promoting the adhesion of CPCs within the AHF, regulating β-catenin levels in the cytoplasm to maintain high Wnt signaling and cardioproliferation while also preventing the premature differentiation of CPCs. On the contrary, strong expression of N-cadherin observed throughout matured myocardium is associated with downregulation of Wnt signaling due to β-catenin sequestration at the cell membrane, inhibiting cardioproliferation. As such, upregulation of Wnt signaling pathway to enhance cardiac tissue proliferation in mature cardiomyocytes can be explored as an interesting avenue for regenerative treatment to patients who have suffered from myocardial infarction.To investigate if Wnt signaling is able to enhance cellular proliferation of matured cardiomyocytes, we treated cardiomyocytes isolated from adult mouse heart and both murine and human ES cell-derived matured cardiomyocytes with N-cadherin antibody or CHIR99021 GSK inhibitor in an attempt to increase levels of cytoplasmic β-catenin. Immunostaining, western blot, and quantitative PCR for cell proliferation markers, cell cycling markers, and Wnt signaling pathway markers were used to quantitate re-activation of cardioproliferation and Wnt signaling.N-cadherin antibody treatment releases sequestered β-catenin at N-cadherin-based adherens junction, resulting in an increased pool of cytoplasmic β-catenin, similar in effect to CHIR99021 GSK inhibitor treatment. Both treatments therefore upregulate Wnt signaling successfully and result in significant increases in matured cardiomyocyte proliferation.Although both N-cadherin antibody and CHIR99021 treatment resulted in increased Wnt signaling and cardioproliferation, CHIR99021 was found to be the more effective treatment method for human ES cell-derived cardiomyocytes. Therefore, we propose that CHIR99021 could be a potential therapeutic option for myocardial infarction patients in need of regeneration of cardiac tissue.The online version of this article (10.1186/s13287-018-1086-8) contains supplementary material, which is available to authorized users. Cardiovascular disease is currently the leading cause of death globally and is expected to rise in future as the incidence and prevalence of chronic diseases associated to cardiovascular risk factors increases . In conjThe Wnt/β-catenin signaling pathway in particular plays an important role in the development of second heart field (SHF Isl1+) that gives rise to the anterior heart field (AHF) cardiac progenitor cells (CPCs) for the formation of right ventricle (RV), outflow tract (OFT) and portion of inflow tract (IFT) –9. DurinIn this study, we demonstrated that re-activation of Wnt/β-catenin signaling pathway can be achieved in matured mouse and human ES cell-derived cardiomyocytes by using N-cadherin antibody or GSK inhibitor, CHIR99021, releasing sequestered β-catenin in the N-cadherin-based adherens junctions or by preventing the ubiquitination-degradation of β-catenin, respectively –15. We fCdh2 mouse knockout ES cells were cultured and differentiated towards cardiomyocytes as described by Soh et al. [The human ES cell line (H7) was cultured in feeder-free condition on Matrigel . The cells were maintained in mTeSR™1 medium (Stemcell Technologies) and passaged with collagenase IV (1 mg/ml) enzymatic treatment. To differentiate human ES cells towards cardiomyocytes, we adopted the protocol established by Lian et al. , 17. Theh et al. . In thish et al. .Single-cell suspension was obtained from cardiomyocytes derived from both murine and human ES cells. The cells were stained using vascular cell adhesion molecule (VCAM-1) and SIRPα/β antibodies, respectively. Briefly, staining of mouse cardiomyocytes was achieved with rabbit anti-VCAM1 monoclonal antibody (1:50)  in the presence of blocking buffer consisting of 5% FBS and 2% BSA in PBS for 90 min at 37 °C, followed by donkey anti-rabbit IgG Alexa Fluor 594 at 1:1000 dilution (Invitrogen) for 1 h. Human ES cell-derived cardiomyocytes, on the other hand, were stained with PE/Cy7-conjugated anti-human CD172a/b (SIRPα/β) antibody at 1:300 dilution (Biolegend). Cardiomyocytes were subsequently purified via fluorescence-activated cell sorting (FACS). Matured human ES cell-derived cardiomyocytes were treated with either 100 μM of TBP or 100 nmol/L of EDN1 to induce cardiac hypertrophy.Matured cardiomyocytes were isolated from mice that are at least 2 months old according to published protocol . The iso6 cells were harvested and lysed in 800 μl of TRIzol reagent (Invitrogen). The samples were allowed to stand for 5 min at room temperature, after which 160 μl of chlorofoam was added to allow for phase separation by centrifugation at 12,000×g for 15 min at 4 °C. Following that, the aqueous phase was transferred to a fresh tube, and equal volume of isopropanol was added and mixed. RNA samples were allowed to precipitate at room temperature for another 10 min. The precipitated RNA samples were pelleted by centrifugation at 12,000×g for 15 min at 4 °C. For cDNA synthesis, RNA samples (500 ng) were reverse transcribed to obtain cDNA using the iScript cDNA Synthesis kit (BioRad). Primer sequences are provided in Additional file For cultured cell samples, 2 × 10Immunocytochemical analysis was performed using the respective antibodies: mouse anti-PY654-β-catenin (membrane bound) and anti-PY489-β-catenin (nuclear) (both from Hybridoma bank) and mouse anti-cardiac troponin T . Briefly, cells were first harvested and washed once with PBS. Fixation of cells was achieved with 4% paraformaldehyde for 30 min, followed by treatment with 0.3% Triton X to permeabilize cells, if necessary. The cells were then blocked with PBS containing 5% fetal bovine serum and 1% BSA for 30 min at room temperature. Primary antibodies were added at respective dilutions and incubated for 1 h at room temperature. After washing, the cells were incubated for another 45 min at room temperature without light exposure with either 1:1000 diluted Alexa Fluor 488 or Alexa Fluor 594 secondary antibodies. Nuclei were counterstained with 4,6-diamindino-2-phenylindole (DAPI). The cells were observed under a fluorescent microscope (Nikon TS-100).5 cells) was seeded to compare proliferation of treated cells (N-cadherin antibody or CHIR99021) vs control (untreated). Quantification of cardiomyocytes was performed by summing all cardiac troponin T (cTnT)-positive cells (relative cutoff intensity > 150) in the treated wells vs the control. For the analysis on cardiomyocyte proliferation, the double-positive cells (Ki67+/cTnT+ or BrdU+/cTnT+) in each of the treatment conditions have been calculated as a percentage of all the single-positive cells (cTnT+).Fluorescent images were acquired by an automated microscope (PerkinElmer Operetta) at ×20 magnification. Image analysis was performed using the Columbus software (PerkinElmer), by first recognizing and outlining cellular nuclei based on DAPI staining. For each experiment, equal number of SIRPA-positive cardiomyocytes (1 × 10g for 20 min at 4 °C for the isolation of the cytosolic fractions. Quantification of protein concentration was determined using Pierce BCA Protein Assay kit (Thermo-scientific). Protein lysate of 60 μg per sample was loaded onto 4–20% Mini-PROTEAN TGX Stain-Free Precast Gels (Bio-Rad), resolved by electrophoresis, and transferred to nitrocellulose membrane (Bio-Rad). Membranes were blocked with 5% skimmed milk in TBS + 1% Tween-20 and probed overnight with primary antibodies. The antibodies were rabbit anti-β-catenin antibody  and mouse anti-β-actin antibody . Membranes were washed and incubated for 1 h with their respective IgG-HRP-conjugated secondary antibody (Santa Cruz) in 5% skimmed milk in TBS + 1% Tween-20 and developed using ECL substrate (Bio-Rad).Whole-cell lysates were generated by scraping cultures of matured cardiomyocytes treated with CHIR99021, N-cadherin antibody, or combination for 1 day and resuspended in RIPA lysis buffer  for 10 min at 4 °C and then centrifuged at 11,400t test, two-sided based on assumed normal distributions. P values < 0.05 were considered statistically significant. The error bars in graphs with three or more experimental groups refer to standard error of means of stated replicates. A non-parametric test  was used to assess whether the mean values of the experimental groups could be considered to be significantly different compared to the control group at 95% confidence level.Results involving two experimental groups expressed as mean ± s.d. were tested for statistical significance using Student’s AHF-Cre; Cdh2fl/fl mutant mice embryo causes lower levels of both membrane and nuclear β-catenin due to cytoplasmic degradation of β-catenin. Resultant downregulation of Wnt signaling led to the decrease in proliferation of CPCs, premature differentiation of AHF CPC to cardiomyocytes in the pharyngeal mesoderm, and disrupted growth of the OFT and RV [CDK1, CCNA2, CCNB1, and CCNB2 were observed to be significantly upregulated in both N-cadherin antibody- and CHIR99021-treated cardiomyocytes, suggesting that both treatments successfully increased cytoplasmic β-catenin levels, reactivate Wnt signaling, and result in the matured mouse cardiomyocytes entering a more proliferative state  and Wnt signaling  in CHIR99021-treated cardiomyocytes  based on their expression of vascular cell adhesion molecule-1 (V-CAM1) [tes Fig. c, d. WhiNKX2.5 and MEF2C and upregulation of cardiac maturation and ion channel markers such as MYH7, KCNQ1, SCN5A, HCN4, and CACNA1G at the end of the time-course experiment  showed promising results, with quantification of immunostaining results showing ~ 1.0-fold increase in Ki67+ population of cells in the cultured human ES cell-derived cardiomyocytes compared to a non-treated control Fig. c–e. TherWe then investigated if there are synergistic effects between N-cadherin antibody and CHIR99021 treatments. Immunostaining for Ki67+ and quantitative analysis on day 7 after treatment revealed that a combinatory treatment of N-cadherin antibody (1:1000) and CHIR99021 (1 μM) resulted in cardiomyocyte proliferation rates at an average between the more effective CHIR99021 treatment and the less effective N-cadherin antibody treatment Fig. a–e. N-caIn order to demonstrate a direct correlation between unbound β-catenin in the cytosolic fraction with cardiomyocyte proliferation, we performed western blot on cytosolic fractions of control cardiomyocytes and cells treated with CHIR99021, N-cadherin antibody, or combinatory treatment for 1 day. Longer treatment duration was not preferred in this case as the release of membrane-bound β-catenin after treatment with N-cadherin antibody would not be observed if the lysates were collected at a later time point. Consistent with the results thus far, increased levels of unbound β-catenin compared to the control were observed after 1 day of treatment Fig. f. As illIn addition to the upregulation of cytoplasmic β-catenin, more cardiomyocytes were stained positive for PHH3 and Survivin, a marker specific for G2-M phase that marks entry into the mitotic process and eventual division, when the cells were treated with CHIR99021 Fig. g. All thDespite that, we reasoned that excessive amounts of N-cadherin antibody hindered the formation of adherens junctions, inhibiting cell adhesion between cardiomyocytes leading to the loss of cardiomyocytes and inefficient proliferation. Sufficient amounts of free N-cadherin are required for proper cardiomyocyte proliferation, adhesion, and overall cardiac function. Similarly, we reasoned that high concentrations of CHIR99021 are cytotoxic due to the pleiotropic effects of GSK inhibitor which affects several other pathways. Overall, CHIR99021 at its optimal concentration exhibits the best potential as a therapeutic agent for cardiac regeneration as it promotes the maximal re-activation of Wnt/β-catenin signaling for cardiomyocyte proliferation without disrupting the necessary role of N-cadherin in cellular adhesion or inducing cytotoxicity.Many known cardiopathies and injuries affect mainly the ventricles, causing inflammatory responses leading to scarring, fibrosis, and hypertrophy of the mammalian heart, referred to as ventricular remodeling. Wnt/β-catenin signaling has been implicated in cardiac remodeling regulation as an injury response. In the mouse model, numerous works have suggested Wnt/β-catenin signaling to play a part in regulating cardiac hypertrophy and preventing apoptosis. However, there has been little evidence uncovering the mechanisms and pathways involved. Wnt ligands have been shown to be upregulated alongside their feedback regulators Dkk1 and Dkk2 as a response in mouse models with induced cardiac injuries such as myocardial infarction –27.CDH2, CTNNB1, AXIN2, and LEF1, effectors within the Wnt pathway


Hepatic progenitor cells (HPCs) can be activated when the liver suffers persistent and severe damage and can differentiate into hepatocytes to maintain liver regeneration and homeostasis. However, the molecular mechanism underlying the hepatic differentiation of HPCs is unclear. Therefore, in this study, we aimed to investigate the roles of autophagy and the Wnt/β-catenin signaling pathway during hepatic differentiation of HPCs in vivo and in vitro. First, immunohistochemistry, immunofluorescence and electron microscopy showed that Atg5 and β-catenin were highly expressed in human fibrotic liver and mouse liver injury induced by feeding a 50% choline-deficient diet plus 0.15% ethionine solution in drinking water (CDE diet) for 21 days; in addition, these factors were expressed in CK19-positive HPCs. Second, Western blotting and immunofluorescence confirmed that CK19-positive HPCs incubated in differentiation medium for 7 days can differentiate into hepatocytes and that differentiated HPCs were able to take up ICG and secrete albumin and urea. Further investigation via Western blotting, immunofluorescence and electron microscopy revealed autophagy and the Wnt/β-catenin pathway to be activated during hepatic differentiation of HPCs. Next, we found that inhibiting autophagy by downregulating Atg5 gene expression impaired hepatic differentiation of HPCs and inhibited activation of the Wnt/β-catenin pathway, which was rescued by overexpression of the β-catenin gene. Moreover, downregulating β-catenin gene expression without inhibiting autophagy still impeded the differentiation of HPCs. Finally, coimmunoprecipitation demonstrated that P62 forms a complex with phosphorylated glycogen synthase kinase 3 beta (pGSK3β). Third, in mouse CDE-induced liver injury, immunohistochemistry and immunofluorescence confirmed that downregulating Atg5 gene expression inhibited autophagy, thus impeding hepatic differentiation of HPCs and inhibiting activation of the Wnt/β-catenin pathway. As observed in vitro, overexpression of β-catenin rescued this phenomenon caused by autophagy inhibition, though decreasing β-catenin levels without autophagy inhibition still impeded HPC differentiation. We also found that HPCs differentiated into hepatocytes in human fibrotic liver tissue. Collectively, these results demonstrate that autophagy promotes HPC differentiation by regulating Wnt/β-catenin signaling. Our results are the first to identify a role for autophagy in promoting the hepatic differentiation of HPCs. The self-renewal capacity of mature hepatocytes contributes to adult liver homeostasis and regeneration; however, the nature of impaired regeneration resulting from chronic liver damage remains unclear -related fibrotic liver tissue from patients undergoing liver transplant and 25 samples of healthy liver tissue from liver donors. The study was approved by the Ethics Committee of Shanghai General Hospital, and informed consent was obtained from each patient.Five-week-old C57BL/6J male mice were purchased from Sino-British Sippr/BK Laboratory and housed in the Animal Experimental Center of Shanghai First People’s Hospital  under specific pathogen-free conditions. Animal protocols were approved by the Chancellor’s Animal Research Committee, and the experiments involving animals adhered to the guidelines set forth by the Shanghai Jiao Tong University School of Medicine .Six-week-old male wild-type C57BL/6J mice were fed a choline-deficient, ethionine-supplemented (CDE) diet for 3 weeks  supplemented with 20% Matrigel , 40 ng/mL oncostatin M , 20 ng/mL hepatocyte growth factor, 10 ng/mL fibroblast growth factor 4 , and 10pGMLV-SC5 RNAi  containing a green fluorescent protein (GFP) reporter was used to express short hairpin RNA (shRNA) targeting the sequences of ATG5 (5′-GCCATCAACCGGAAACTCATG-3′) and β-catenin (5′GCACCATGCAGAATACAAATG-3′), with PGMLV-6395 (vector) and a scramble sequence (5′-TGTTCTCCGAACGTGTCACGT-3′) as a control plasmid. PGMLV-6395 containing a GFP reporter was used to overexpress RNA targeting the sequence of β-catenin (CTCGAGGCCACCGGATCC). Briefly, HPCs were transfected in differentiation medium with shAtg5, shβ-catenin, β-catenin overexpression vector and the corresponding scramble or empty vector using Lipofectamine 3000 reagent  according to the manufacturer’s protocol. Following incubation for 48 h, the expression levels of the genes and proteins corresponding to the plasmids transfected were detected by quantitative real-time PCR (qRT-PCR) and Western blotting (WB) on the fifth day.A total of 25 male C57BL/6J mice were randomly divided into a normal diet group , blank group (n = 5), shAtg5 group (n = 5), shAtg5 combined with β-catenin overexpression group (n = 5) and shβ-catenin group (n = 5). Plasmid transfection was performed as previously described  and were washed three times with PBS. They were then blocked with antibody with 5% bovine serum albumin (BSA) (30 min) and incubated with the primary antibodies overnight at 4 °C in 1% BSA. Next, the sections were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature and counterstained with hematoxylin, and images were acquired with a light microscope. Information on the primary antibodies is listed in Table Liver tissues were fixed with 4% paraformaldehyde, embedded in paraffin after dehydration, and cut into 5-µm sections. The sections then underwent dewaxing, rehydration (> 15 min), repair by sodium citrate antigen 20 min), and quenching in 3% H0 min, anHPCs and liver tissue were fixed with 2.5% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.4) for 3 h, postfixed with 1% osmium tetroxide for 1.5 h, dehydrated with graded alcohol through propylene oxide, and then embedded in Epon-Araldite resin . Next, ultrathin sections were stained with uranyl acetate and 0.3% lead citrate and examined under a transmission electron microscope . Three sections of each liver tissue were used for the experiment, and a histogram of the number of autophagosomes was generated with GraphPad Prism .Liver tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and then cut into 5-µm sections. After antigen retrieval, the specimens were incubated with Tris-EDTA (pH 9.0) for 10 min at 95 °C and 0.1% Triton solution (Sigma-Aldrich), blocked with antibody with TBST solution (TBS-0.1% Tween 20) (Sigma-Aldrich) with 5% BSA, incubated with primary antibody overnight at 4 °C in TBST with 1% BSA, washed twice and incubated with secondary antibodies at room temperature for 1 h. HPCs were cultured in confocal dishes for 7 days, fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton X-100, incubated with the primary antibody overnight at 4 °C, incubated with the secondary antibody, and observed under confocal microscopy. Primary antibody information is listed in Table HPCs were cultured on confocal dishes for 5 days and grown to 40–50% confluence at the time of transfection. After being washed with PBS two times, the cells were transfected with Ad-mCherry-GFP-LC3B adenovirus  at a multiplicity of infection (MOI) of 40 in 200 µL of DMEM/F12 containing 10% FBS, followed by incubation for 48 h at 37 °C. Autophagy was observed under a Leica TCS SP8X microscope  and evaluated by calculating the number of yellow and red puncta. The number of expressed cells was analyzed using a quantitative digital image analysis system .2. Next, the DMEM/F12 or William’s E medium was removed, and then HPCs were gently washed three times with PBS. ICG-positive cells were photographed under a microscope , and the number of expressed cells was analyzed using a quantitative digital image analysis system .Briefly, HPCs in 6-well plates were incubated in DMEM/F12 or William’s E medium supplemented with ICG  at a final concentration of 1 mg/mL for 1 h at 37 °C in 5% COTotal RNA was extracted from HPCs using TRIzol™ reagent  at the indicated times and reverse-transcribed using Hieff™ First Strand cDNA Synthesis Super Mix for RT-qPCR . RT-PCR was performed using SYBR® Premix Ex Taq TM  under the following conditions: 1 cycle at 95 °C for 2 min, 35 cycles at 95 °C for 15 s and 59 °C for 34 s, and 1 min at 72 °C. GAPDH was used as an internal control to normalize for differences in the amount of total RNA in each sample. The primer sequences are listed in Table RIPA lysis buffer  containing protease inhibitor cocktail  was used for total protein extraction. Equal amounts of protein samples (60 µg) were separated by 10% SDS-PAGE, and the electrophoresed proteins were transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk powder with 0.1% TBST for 1 h and then incubated with primary antibodies at 4 °C for 12 h. Afterward, the membranes were washed three times with TBST and incubated with the corresponding secondary antibody for 1 h. Finally, the membranes were washed three times with TBST; bands were detected with an enhanced chemiluminescence (ECL)  system and exposed to X-ray films . The relative densities of immunoreactive bands were determined using ImageJ software . All experiments were repeated three separate times. Protein expression levels were normalized against GAPDH. Primary antibody information is listed in Table Total protein extracts from HPCs were prepared using Pierce coimmunoprecipitation (Co-IP) kits , and protein concentrations were determined using a bicinchoninic acid (BCA) protein quantification kit . Coimmunoprecipitation was performed according to the manufacturer’s protocol for the Pierce coimmunoprecipitation kit. Briefly, precleared lysate was prepared using the control agarose resin. Next, immobilized anti-P62 (10 µg/mg lysate), anti-pGSK3 (10 µg/mg lysate) and control IgG antibodies (10 µg/mg lysate) were added to the amino link plus coupling resin, and 400 µg of precleared lysate was incubated with different immobilization antibodies overnight at 4 °C. Finally, after being washed with 60–70 µL of elution buffer, the immunoprecipitates were boiled for 10 min and then assessed by WB. Information on the primary antibodies is listed in Table The supernatants of HPCs cultured in 24-well dishes were collected on the seventh day and frozen at − 20 °C until assay. The supernatants were assayed for ALB production using a quantitative enzyme-linked immunosorbent assay kit  according to the manufacturer’s recommendations. A histogram of the ALB concentration was generated with GraphPad Prism .4Cl (Sigma) for 24 h in 5% CO2 at 37 °C on the seventh day. Next, the supernatant was collected, and urea concentrations were measured using urea ELISA kits  according to the manufacturer’s protocol. A histogram of the urea concentration was generated with GraphPad Prism .HPCs were incubated with 0.5 mL of medium containing 5 mM NHP < 0.05 indicated a statistically significant result.Data are presented as means ± standard deviations (SDs). All statistical analyses were performed using SPSS 20.0 software . Comparisons between two groups were analyzed using an independent t-test; differences among groups were performed by one-way ANOVA. P < 0.05, Fig. Previous studies have demonstrated that ductular reaction cells appear during chronic liver injury and differentiated into hepatocytes, thus maintaining hepatic homeostasis , but autophagy was effectively suppressed by downregulating the expression of the Atg5 gene Fig. a, b. NexThese data show that autophagy can regulate hepatic differentiation of HPCs by regulating the Wnt/β-catenin signaling pathway in vivo.Ductular reactions are encountered in a variety of chronic liver diseases, and they are also called oval cell proliferation in rodents (Gjymishka et al. Recent investigations have confirmed that autophagy plays a pivotal role in the progression of chronic liver disease (Wang et al. To investigate the correlation between autophagy and hepatic differentiation of CK19-positive HPCs, we established hepatic differentiation of HPCs in vitro, confirming that autophagy was highly activated in the progression of hepatic differentiation of HPCs. This result was similar to recent studies demonstrating that autophagy is involved in differentiation in different cells, including monocytes, satellite cells and neural stem cells (Zhang et al. The Wnt/β-catenin signaling pathway is necessary for the differentiation of stem cells. Activation of Wnt/β-catenin signaling can promote the differentiation of stem cells (Shao et al. In summary, we found that autophagy promoted the hepatic differentiation of HPCs by regulating Wnt/β-catenin signaling in vivo and vitro. These findings will further increase our understanding of the molecular mechanism underlying HPC hepatic differentiation.


RNF43 is an E3 ligase that inhibits Wnt signaling by ubiquitinating Wnt receptors for degradation. It is mutated in various cancer types with the most recurrent mutation being the frameshift G659Vfs*41 with frequencies of ~5–8% in colon, stomach and endometrial cancers. This mutation, a deletion of G in a 7-G repeat, has been assumed to encode an inactive enzyme that would lead to increased Wnt signaling and drive tumorigenesis, yet no functional characterization has been reported. We analyzed the distribution of G659Vfs*41 and its association with other cancer gene mutations, and found that the mutation occurred nearly exclusively in tumors with low expression of the DNA mismatch repair gene MLH1. Mutant RNF43-G659Vfs*41 was no different from wild type RNF43 in expression, stability, localization, R-spondin binding, and inhibition of Wnt signaling. No dominant negative activity of the mutant was observed. Colon tumors with RNF43-G659Vfs*41 had low Wnt/β-catenin signaling and were frequently mutated in BRAF. A colon cancer cell line with RNF43-G659Vfs*41 and BRAF-V600E mutations was sensitive to activation of Wnt/β-catenin signaling. These findings suggest that the frequent occurrence of RNF43-G659Vfs*41 may result from error-prone replication of the 7-G repeat in MLH1-deficient tumors and that the mutation itself does not inactivate enzyme. It is a type I integral membrane protein with an extracellular domain of 175 amino acid (AA) residues, a single transmembrane domain, and an intracellular region of 565-AA residues with the RING finger E3 ligase domain (AA270-316) immediately following the transmembrane domain. A series of subsequent studies revealed that RNF43 and its related paralog ZNRF3 ubiquitinate the frizzled (FZD) family of Wnt receptors for degradation and thus serve as negative regulator of Wnt signaling5. Meanwhile, genome-wide sequencing of tumor samples uncovered that RNF43 and ZNRF3 were mutated in a wide variety of tumor types, with relatively high frequency (near 10%) found in pancreatic6, uterine endometrial7, stomach8, and colon cancers14. In the COSMIC cancer database, approximately half of the 1,036 mutations of RNF43 identified from all cancer types are either non-sense or frameshift mutations15. Some of the recurrent non-sense mutations of RNF43, such as R132x and R145x, are expected to produce non-functional proteins due to the lack of the E3 ligase domain. Also, some missense mutations in the RING domain were shown to have lost activity in Wnt signaling16. In pancreatic cancer, RNF43 mutations located within or upstream the RING domain were identified and cell lines with such mutations were shown to be sensitive to inhibitors of Wnt ligand secretion17. In colon cancer, RNF43 mutations were largely exclusive with APC mutation7. All these observations led to a seemingly obvious conclusion that nonsense and frameshift RNF43 mutations are loss-of-function mutations that would lead to tumorigenesis in a significant subset of gastrointestinal and endometrial cancers due to increased Wnt signaling14. Inhibition of Wnt ligand production is purported to provide an effective treatment for tumors with RNF43 non-sense or frameshift mutations14.RNF43, a member of the RING finger E3 ubiquitin ligase family, was originally identified as an oncoprotein with upregulated expression in colon cancer15. The frameshift, designated G659Vfs*41, truncates the enzyme at Gly659 and shifts the reading frame to add a neopolypeptide of 41-AA15. The mutation removes the C-terminal 123-AA region that is far downstream of the E3 ubiquitin ligase domain5. If and how RNF43-G659Vfs*41 will affect its enzyme activity in regulation of Wnt signaling has never been reported. Nevertheless, the mutation has always been proposed to be loss-of-function and play a role in colon cancer formation/progression due to disinhibition of Wnt signaling14. Both RNF43 and ZNRF3 ubiquinate lysine residues of FZD receptors for endocytosis and degradation5. The process is suggested to occur following the activation of Wnt signaling in which the E3 ligases interact with the disheveled (DVL) protein bound to the Wnt signaling complex to catalyze ubiquitination of FZD5. However, ubiquitination and degradation of FZD were shown to happen without concomitant activation of Wnt signaling, at least with recombinant enzymes and receptors4. E3 ubiquitin ligase activity of RNF43 and ZNRF3 can be inhibited by R-spondins, a group of four related secreted proteins (RSPO1–4) that strongly potentiate Wnt signaling and drive tumorigenesis when over-expressed by gene fusion or other mechanisms19. Here we show that the RNF43-G659Vfs*41 mutant is actually fully functional in promoting FZD degradation and inhibiting Wnt signaling. In the three cancer types with highest incidences of G659Vfs*41, nearly all tumors with this mutation had low expression of MLH1, a major player of DNA mismatch repair. Furthermore, in colon cancer, RNF43-G659Vfs*41 mutation is most frequently associated with BRAF-V600E mutation and low Wnt/β-catenin signaling. Intriguingly, we found that a colon cancer cell line with RNF43-G659Vfs*41 and BRAF-V600E mutations was sensitive to activation of Wnt signaling in vitro. Overall, these findings suggest that RNF43-G659Vfs*41 does not inactivate the enzyme and its relatively common occurrence in MLH1-deficient tumors is most likely a result of error-prone replication of a 7-G repeat.Remarkably, the single most recurrent mutation of RNF43 is deletion of a G-C base-pair (bp) in a seven G repeat near the 3′ end of its open reading frame (nucleotide 1969–1976), accounting for approximately half of all RNF43 mutations detected in colon, stomach, and endometrial cancer with an overall frequency of 5–8%21. Colon, endometrial, pancreatic, and stomach cancers showed the highest incidences of all RNF43 mutations with frequencies of 10.9%, 15.2%, 7.3%, and 11.9%, respectively  . Taken together, these results unequivocally show that RNF43-G659Vfs*41 mutation in colon, endometrial and stomach cancers primarily occurred in tumors with low MLH1 expression and presumably defective DNA mismatch repair, suggesting that the seven G repeat (bp1969-1976) of RNF43 coding sequence is highly prone to single bp deletion in the absence of proficient DNA repair.Previously, it was reported that RNF43 mutations were frequently associated with high microsatellite instability (MSI-H) in colon and stomach cancer05) Fig. . In cont05) Fig. . Similar05) Fig. . In panc05) Fig. . FurtherTo examine functional consequences of the G659Vfs*41 mutation, we generated constructs expressing the mature forms of full-length wild type (WT) RNF43 or G659Vfs*41 with CD8 signal peptide followed by an HA tag at the N-terminus Fig. . An addi24. As expected, the double KO cells (STF-RZ-DKO) had higher response to Wnt stimulation with no further response to RSPO1 treatment  of RNF43 and ZNRF3 from the Wnt/β-catenin signaling reporter cell line HEK293-STF cells using the CRISP/Cas9 method as previously describedion Fig. . We thenion Fig. , and botion Fig. , suggestion Fig. . Overall23. Transfection of either WT or G659Vfs*41 led to approximately 50% decrease in reporter enzyme activity , indicative of a strong association between the two mutations. As colon tumors with MSI-H and BRAF-V600E mutations were also known to have low Wnt signaling27, we analyzed expression levels of Axin2, a prototypical Wnt signaling target, and ZNRF3, relative to RNF43 and BRAF mutation status in the colon cancer cohort. Axin2 and ZNRF3 mRNA levels were highly correlated with that of RNF43 across all samples 17, tankyrase inhibitor XAV939 (stabilization of Axin1/2 and thus degradation of β-catenin)29, and GSK3 inhibitor CHIR99021 (inhibition of β-catenin phosphorylation and thus stabilization of β-catenin)30. Inhibition of BRAF (PLX4720) was highly effective in HT29 cells  but had little activity in RKO cells  , PLX4720 had intermediate effect  whereas RKO cells are MSI-H32.As the majority of RNF43-G659Vfs*41 tumors had BRAF-V600E mutation and had low Wnt signaling, we mined the CCLE database for cell lines with these characteristics. The cell line RKO had the RNF43-G659Vfs*41 mutation as confirmed by Bond WT) Fig. , consistect Fig. . Remarkaect Fig. . On the ect Fig.  despite If high Wnt signaling due to GSK3 inhibition is incompatible with high BRAF activity (BRAF-V600E), we would expect that inhibition of BRAF may rescue the cells from high Wnt signaling. To test this, we compared the effect of combining PLX4720 with CHIR99021 in RKO and HT29 cells. In RKO cells, PLX4720 had little effect by itself but was able to partially rescue the effect of CHIR99021 which inhibited cell growth by ~50% at the used concentration (3 uM) Fig. . In HT29To determine the mechanism of sensitivity of RKO cells to GSK3 inhibition, we first examined Wnt and MAPK signaling pathways in the four cell lines. RKO cells had little β-catenin in the cytoplasm at base line and CHIR99021 treatment increased its level as expected Fig. . CHIR99017. The most common recurrent mutation is RNF43-G659Vfs*41 which has been “automatically” assumed to be a loss-of-function mutation that would lead to increase in Wnt signaling and thus drive or facilitate tumorigenesis. Yet, no evidence has been presented to show that the mutation abolishes the enzyme function as a driving mechanism in oncogenesis. Here we show that this mutation itself does not inactivate RNF43 in suppressing Wnt signaling.RNF43 and its related homolog ZNRF3 are key negative regulators of Wnt signaling and both were found to be mutated in all major types of solid tumors at relatively low frequency. Some of the null or missense mutations of RNF43 located in the N-terminal half had been shown to be loss-of-function and presumably play a role in tumorigenesis of the pertinent tumors5. RNF43- G659Vfs*41 replaces the C-terminal 124-AA region of RNF43 with a 41-AA neopeptide that shows no homology to any other protein or functional motif. The replaced region is far downstream of the DVL-binding domain and previous studies by others showed that deletion of the entire region downstream AA-596 (end of DVL-binding domain) had no effect on RNF43’s activity in inhibiting Wnt signaling5. In theory, it is possible that the 41-AA neopolypeptide could somehow inhibit the function of the enzyme and increase Wnt signaling. Here we excluded this possibility by demonstrating that the expression, localization, and function of recombinant RNF43 with the very G659Vfs*41 mutation were no different from those of RNF43-WT. In cancer cells, the mutant RNF43 would co-express with WT RNF43, and could possibly inhibit the function of the WT enzymes if WT and mutant enzymes could form inactive dimers. We showed that co-transfection of WT and G659Vfs*41 still inhibited Wnt signaling, indicating lack of dominant negative effect. These results clearly indicate that G659Vfs*41 is not a loss-of-function mutation. The question is whether the mutant protein was made at much reduced levels due to nonsense-mediated decay (NMD) of the mutant mRNA33. G659Vfs*41 was reported to elicit NMD in colon and stomach cancers though the extent of decay was predicted to be moderate34. Yet, Roudko et al. just reported that RNF43-G659Vfs*41 was detected as a neopolypeptide that was predicted to be a highly immunogenic tumor antigen in the MSI-H subtypes of the three tumor types 35. Since nearly all G659Vfs*41 tumors are still expected to co-express WT ZNRF3 and RNF43  were shown to be essential for their activity in ubiquitinating FZD and inhibiting Wnt signaling26. However, the allele frequency of RNF43 and BRAF mutations were not correlated . HA-RNF43-G659Vfs*41 was then generated by deleting a G in bp 1979–1976 of the RNF43 open reading frame in the HA-RNF43-WT plasmid by site-directed mutagenesis. WT and G659Vfs*41 plasmids with its native signal peptide were generated by replacing the CD8 signaling peptide and HA tag with its native signal peptide sequence by In-Fusion® cloning. FLAG-tagged FZD5 was cloned into pIRESpuro3 as described previously25. For western blot analysis, anti-HA (Invitrogen cat #71–5500), anti-FLAG (Sigma cat # F7425), anti-β-actin , anti-β-catenin , anti-Axin1 , anti-Axin2 , anti-phosphor-ERK , total ERK , anti-PARP , and anti-GAPDH  were used. For ICC, anti-HA-Alexa 488 , anti-human-Alexa 488 or –Alexa 555 (Invitrogen cat #A11013 and #A21433) were used. For western blotting, cells were lysed with RIPA buffer , supplemented with protease and phosphatase inhibitors, and reduced at 37 °C for 1 hr. HRP-conjugated secondary rabbit or mouse antibodies  were used following the standard ECL protocol. For cytoplasmic β-catenin analysis, whole cell lysates were incubated with agarose bound concanavalin A beads to remove membrane-bound β-catenin and the remaining fractions were used as previously described40.Recombinant full-length human RSPO2 was purchased from R&D Systems. RSPO2-Fu-WT and RSPO2-Fu-F109A were purified as described previously2. HEK293T, HEK293-STF and HT29 cells were cultured in DMEM, DLD1 and Lovo cells in RPMI-1640, and RKO cells in MEM media. All media were supplemented with 10% FBS, 100 units/mL penicillin, and 100 mg/mL streptomycin. For transient transfection of HEK293 and STF-derived cells, ~80% confluent cells were transfected with DNA: FuGENE HD (Promega) ratio of 1:3 for all transient transfection presented. Transfection of DLD1 and LoVo cells were performed with jetPEI® (Polyplus-Transfection). For the protein stability analysis, cells with treated with cycloheximide at 100 µg/ml for the indicated periods of time and harvested, followed by WB analysis.All cell lines were purchased from the ATCC and cultured in a 37 °C humidified incubator containing 5% CO24. The two guide sequences were provided by Dr. Feng Cong as they were used in the generation of double knockout of RNF43 and ZNRF in HEK293 cells5. Lentiviral particles of lenti-CRISPR2 containing RNF43 and ZNRF3 guide sequence were used to co-infect HEK293-STF cells and cells were selected with puromycin at 1 µg/ml. Single colonies were isolated and analyzed for Wnt and RSPO response by the TOPFlash assay23. One clone (D8) showed complete loss of response to RSPO1 in the TOPFlash assay  and AGGACTTGTATGAATATGGC of ZNRF3 (corresponding to nucleotide 338–357 of human ZNRF3 open reading frame) were cloned into the vector Lenti-CRISPR2 as described25. All TOPFlash experiments were repeated at least three times with duplicates or triplicates in each experiment. For RNF43 localization, STF-RZ-DKO cells transfected with RNF43-WT or G659Vfs*41 were treated as indicated. The treated cells were incubated at 37 °C with 95% humidity and 5% CO2 for 1 hr and fixed with 4.2% paraformaldehyde, followed by permeabilization with 0.1% saponin. Then, the secondary antibody, anti-human-Alexa 555 was used to label Fc-tagged RSPO2-Fu. Cells were imaged under the confocal microscope and analyzed by the Leica LAS AF Lite software.TOPFlash assay in STF RNF43-ZNRF3 double KO (RZ-DKO) cells transfected with vector, RNF43-WT, or RNF43-G659Vfs*41 were carried out as described previouslyCells were seeded at various numbers (depending on growth rate) in half-area white 96-well plates and serial dilutions of the compounds were added. The cells were incubated for 4 days and cell viabilty was measured using CellTiter-Glo® Luminescent Cell Viability Assay (Promega).21. Data plotting and statistical analysis were carried out using GrpahPad Prism.All expression and mutation data of TCGA tumor samples were doanloaded from cBioportalSupplementary Figures and original gel files