NUTR and NUTR + EX followed a dietary intervention targeting an increase in protein-rich meals at breakfast, lunch, and dinner. Exercise training in EX and NUTR + EX consisted of 24 min sessions of concurrent aerobic and resistance exercise performed three times per week. Daily protein intake increased in NUTR and NUTR + EX, but not EX. The increase in 1RM leg press strength was greater  in NUTR + EX [29.6  kg] than increases observed in NUTR [11.1  kg] and EX [12.3  kg]. The increase in 1RM chest press strength was greater  in NUTR + EX [6.3  kg] than the increase observed in NUTR [2.9  kg], but not EX [6.3  kg]. Hand-grip strength and sit-to-stand performance were each improved in all three groups, with no differences observed between groups . An increase in percentage body fat was observed in NUTR, but not in EX or NUTR + EX . No between-group differences were observed for change in lean body mass . Concurrent aerobic and resistance exercise training improves strength and physical function in older adults, but combining this training with an increase in daily protein intake through whole foods may be advantageous to increase lower limb strength.Most studies in older adults have utilized powdered protein supplements or oral nutrition solutions as a source of additional dietary protein, but whole foods may provide a greater anabolic stimulus than protein isolated from food matrices. Therefore, the present study investigated a concurrent aerobic and resistance exercise training program in older adults, in the absence or presence of a high protein whole food-based dietary intervention, for effects on strength, physical function, and body composition. Community-dwelling older adults ( The inc = 0.70) .P < 0.001) in all groups, i.e., NUTR , EX , and NUTR+EX  (P = 0.031) than the increases observed in NUTR by 3.4  kg , but the greater increase in EX by 3.3  kg compared to NUTR did not reach statistical significance , and no differences were observed between groups   .P < 0.001; d = 1.01) and NUTR+EX  , but a directional improvement in NUTR did not reach statistical significance  (P < 0.001), and no differences were observed between groups  (Gait speed improved in EX ( = 0.35) . Sit-to-= 0.671) .P = 0.020; d = 0.11], but not in EX  or NUTR+EX   for fat mass and the directional increase in fat mass in NUTR [0.56  kg] did not reach statistical significance  (P = 0.018), with the increase in percentage body fat in NUTR being greater than changes observed in EX by 0.99 %  and in NUTR+EX by 0.92 %   kg;  = 0.05) , an inte = 0.94) .P = 0.402) indicating the absence of between-group differences in LBM in response to the interventions  kg; P = 0.977; d = 0.01] or EX   kg; P = 0.641; d = 0.05] or EX   resulted in improvements in several measures of physical function, i.e., chest press strength, hand-grip strength, and sit-to-stand. These outcomes are not unexpected as several studies of protein supplementation in the absence of exercise have demonstrated similar improvements in physical function in older adults  compared to other groups  is notable as an augmentation of the response to exercise training when additional dietary protein is consumed. This positive effect is, however, discordant with the conclusions of recent meta-analyses that conclude that providing additional protein does not augment improvements in strength after resistance exercise training in community-dwelling, non-frail older adults  exclusively through whole food sources, as opposed to supplementing with powdered protein supplements and oral nutrition solutions. While concurrent aerobic and resistance exercise training alone improved strength and physical function in older adults, combining an increase in dietary protein intake from whole foods with this type of exercise training was more advantageous for increasing lower limb strength and may support an increase in lean body mass, primarily in the form of appendicular lean mass. Such outcomes may be valuable in contexts such as during a period of rehabilitation after an adverse event that resulted in declines in muscle size and/or strength, or may inform clinicians and practitioners working in the field of exercise prescription, rehabilitation, and nutritional care of older adults.The present study is novel in its methodology in view of participants achieving high protein intakes  in accordance with the Declaration of Helsinki. The patients/participants provided their written informed consent to participate in this study.JT and BE designed the study. JT, MH, and KC performed the assessments. JT, MH, and OD were responsible for performing the interventions. JT, MH, and BE analyzed the data. JT, MH, and BE wrote the first draft of the manuscript. KC and BE edited the manuscript. BE had the primary responsibility for final content. All authors contributed to editing and approved the final version of the manuscript.BE is formerly (previous 36 months) or presently funded under the Health, and Glycemic Management pillars, respectively, of the Enterprise Ireland-funded technology centres Meat Technology Ireland, and Food for Health Ireland, whose focus is on the potential benefits of meat and dairy ingredients for human health. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


At adherens junctions (AJs), α-catenin forms heterodimers with β-catenin that are believed to resist force on F-actin. Outside AJs, α-catenin forms homodimers that regulates F-actin organization and directly connect the cell membrane to the actin cytoskeleton, but their mechanosensitive properties are inherently unknown. By using ultra-fast laser tweezers we found that a single α-β-catenin heterodimer does not resist force but instead slips along F-actin in the direction of force. Conversely, the action of 5 to 10 α-β-catenin heterodimers together with force applied toward F-actin pointed end engaged a molecular switch in α-catenin, which unfolded and strongly bound F-actin as a cooperative catch bond. Similarly, an α-catenin homodimer formed an asymmetric catch bond with F-actin triggered by protein unfolding under force. Our data suggest that α-catenin clustering together with intracellular tension engage a fluid-to-solid phase transition at the membrane-cytoskeleton interface. By using laser tweezers, the authors show that a single α-catenin molecule does not resist force on F-actin. However, clustering of multiple molecules and force applied toward F-actin pointed end engage a molecular switch in α-catenin, which unfolds and strongly binds F-actin. Among these proteins, α-catenin has been reported to act as a mechanosensor that regulates adherens junctions (AJ) in response to mechanical cues from neighboring cells in a tissue2. It is widely accepted that α-catenin in AJ binds β-catenin, which, in turn, connects to the cytoplasmatic portion of E-cadherin to constitute the cadherin-catenin complex (CCC). Since α-catenin also binds F-actin, α-catenin has been indicated as a major candidate for providing a link between the CCC and the actin cytoskeleton. This molecular link is required to provide mechanical stability to AJ. According to the most accepted model of α-catenin mechanosensitivity, tension on the actin cytoskeleton is transmitted to bound α-catenin inducing a conformational change that opens up (i.e. unfolds) its central M-domain and increases the bond strength2. α-catenin unfolding is predicted to expose cryptic binding sites for vinculin and other actin-binding proteins, which further reinforce the connection to the actin cytoskeleton. However, the role of α-catenin as the mechanical link between the CCC and F-actin has been challenged by the finding that the binding of mammalian α-catenin to F-actin and β-catenin is mutually exclusive3.In any living cell, an array of mechanotransduction proteins responds to mechanical cues to trigger complex molecular signaling, driving cell morphology and gene expression profilesDanio Rerio α-catenin4, Buckley et al. recently demonstrated that the bond between CCC and F-actin is reinforced by increasing force 5. However, it remains to be determined whether mammalian α-β-catenin heterodimers show similar catch-bond properties. Moreover, it is still unclear whether a single α-β-catenin heterodimer can bind to F-actin and bear force on it. CCCs have been recently shown to be organized in clusters in cells6. This finding raises the intriguing question whether CCC clustering and the interaction of F-actin with multiple α-catenins might be important in defining the mechanosensitivity of AJ.Using a 7 and promotes cell adhesion and migration by providing a direct link between the cell membrane and the actin cytoskeleton8. While α-catenin forms α-β-catenin heterodimers in AJ, outside AJ α-catenin binds itself and is prevalently found as homodimers. Despite its biological relevance, no data is currently available on the force-dependence of the interaction between α-catenin homodimers and F-actin.Recent reports revealed the importance of α-catenin outside AJ as well, where it regulates F-actin organization9. However, there is no direct evidence that forces applied to α-β-catenin heterodimers or α-catenin homodimers through F-actin are sufficient to induce α-catenin unfolding.Finally, it is still unclear whether α-catenin unfolds during its interaction with F-actin. Using a truncated α-catenin that lacked the actin and β-catenin binding domains, Yao et al. showed that forces of about 5 pN trigger reversible unfolding of the α-catenin M-domain and allow vinculin binding13, to dissect the mechanosensitivity of single mammalian α-catenin homodimers and α-β-catenin heterodimers. We show that a single mammalian α-β-catenin heterodimer does not bear force on F-actin, but instead slips along F-actin in the direction of force. Conversely, the cooperative action of multiple molecules together with force applied toward F-actin pointed end triggers α-catenin unfolding and engages a catch bond with actin. Similarly, a single α-catenin homodimer forms a directionally asymmetric catch bond with F-actin triggered by α-catenin unfolding, which resists forces directed toward F-actin pointed end.Here, we use ultrafast force-clamp spectroscopy, a technique with sub-millisecond and sub-nanometer resolution based on laser tweezersWe first analyzed single purified recombinant α-catenin homodimers (named as α-catenin below). A single actin filament was suspended between two optically trapped beads and brought in close proximity to a single α-catenin that was attached to a micron-sized glass bead on the coverslip surface  , the negative step is largely reduced  Fig. . Likewisnm) Fig. . Analysinm) Fig. . This inced Fig.  or disapced Fig.  and in tced Fig.  in the dF > 7pN. We could detect such rapid interactions thanks to the unprecedented time resolution of our technique10. The lifetime vs force plot was well fitted by the Bell slip-bond model, in which the lifetime decreases exponentially with force  of a biotinylated actin filament was specifically linked to a gelsolin-coated bead. In the other trap, we caught a neutravidin-coated bead. Actin on the gelsolin-coated beads was rhodamine-labelled, whereas neutravidin-coated beads were labelled with Alexa647 to clearly distinguish the two beads and assess the actin filament orientation Fig. . Using t5, whereas they could possibly detect the action of multiple molecules, which behave as a catch bond and produce much longer interactions than single molecules under force.Here, we provided direct evidence that a single mammalian α-catenin bound to β-catenin forms a slip bond with F-actin. The weak and rapid interactions that we observed could be detected thanks to the 10–100 μs time resolution of our setup, which is about two orders of magnitude better than conventional optical trapping assays. The limited-time resolution is likely the reason why previous reports could not detect the slip bond between a single α-β-catenin heterodimer and F-actinr, i.e., the fraction of time that a molecule spends attached to the actin filament: 16. When the number of α-catenin molecules that can interact with the actin filament is above r < 0.2 for low forces (F < 5 pN) to r = 0.8–0.9 in the force range where also the lifetime is maximized  cause unidirectional flow of CCC clusters in cell-cell junctions21 and (ii) drive cluster formation through increased diffusional trapping mediated by α-catenin cooperativity and tension applied by myosin motors on the actin cytoskeleton6.Our results indicate that α-catenin binds F-actin as an unconventional slip bond that cooperatively switches into an asymmetric catch bond. A cooperative action of multiple α-catenin molecules is required because a mere sum of multiple slip bonds would result again in a slip bond. Further insight into this mechanism can be gained by analysis of the duty ratio rce Fig. , whereasrce Fig. . The r ∼An indication of the average number of α-catenins that were simultaneously bound to actin in the different experiments can be obtained from a measurement of the average stiffness of the molecules bound to the dumbbell during the interactions (see methods). The single α-β-catenin heterodimer gave an average stiffness of 0.263 ± 0.006 pN/nm (error s.e.m), about half of the α-catenin homodimer (0.46 ± 0.03 pN/nm). Assuming that the stiffness of an α-catenin monomer in the homodimer and in the heterodimer is the same, this result indicates that both α-catenins in the homodimer participate to the interaction with actin for most of the time (~87% from the ratio of the homodimer stiffness to double the heterodimer stiffness). In the experiments with multiple α-β-catenin heterodimers, the average stiffness was in between the single heterodimer and the single homodimer (0.336 ± 0.007 pN/nm), suggesting that simultaneous binding of two α-catenin molecules occurred for about 64% of the time in these experiments. This result reinforces our conclusion that the catch bond behavior is a consequence of the cooperative binding of multiple α-catenin molecules to actin.9. The unique ultra-fast force-clamp configuration used here allowed us to directly observe under which conditions folding/unfolding transitions of the α-catenin molecule actually occur when force is applied to the interacting actin filament, as previously hypothesized9.Notably, our data show that the catch-bond behavior is accompanied by a large conformational change (unfolding) of the α-catenin molecule, both in a single α-catenin homodimer and in multiple α-β-catenin heterodimers. Unfolding is not observed in a single α-β-catenin heterodimer slip bond, indicating that α-catenin unfolding provides the molecular switch that converts the link to F-actin into a catch bond. The measured unfolding step size is compatible with unfolding of the bundled α-helices in the vinculin binding domain into a linear chain of extended α-helices22.Finally, the bond between actin and both α-catenin homodimers and multiple α-β-catenin heterodimers showed a strong asymmetry. The lifetime was much longer when force was applied toward the actin pointed end Fig. . This reF), as a function of the α-catenin duty ratio (r) (see methods). When a constant force F is applied to an actin filament immersed in a viscous fluid, the filament slides at constant velocity Based on our results, we developed a simple chemo-mechanical model to relate the sliding velocity 23 and in-vivo24. Notably, disruption of E-cadherin clusters in living cells has been shown to increase cell-cell junction fluidity25. We thus propose that, as a consequence of α-catenin cooperative mechanosensitivity, AJ might switch from a fluid phase in the absence of CCC clustering to a solid phase when CCC become organized into clusters and tension applied by non-muscle myosin or generated by the actin retrograde flow builds up on the actin cytoskeleton.This equation tells us that when the duty ratio is significantly smaller than one and independent of force, as in the case of a single α-β-catenin heterodimer Fig. , the α-cmer Fig.  or to mumer Fig. , the vel26. The plasmid encodes the protein fused with a 6x His-tag at the N-terminus with a linker sequence containing a thrombin cleavage site and restriction sites for NdeI and NheI (Amino acid sequence of the His tag and linker: “MGSSHHHHHHSSGLVPRGHMAS”). The plasmid was transformed in BL21 DE3 pLysS strains (Agilent). Recombinant αE-catenin was expressed via isopropyl 1-thio-β-d-galactopyranoside  induction. Cultures were grown to an OD of 0.7 and induced with 0.5 mM IPTG for 3 h at 37 °C. Protein was purified via FPLC  using HisTrap HP columns and eluted in 25 mM Tris, pH 7.5, 300 mM NaCl, 300 mM Imidazole. Size exclusion chromatography was performed in 25 mM Tris, pH 7.5, 150 mM NaCl, 1 mM DTT through with a HiLoad Superdex 200  as a further purification step. Monomeric and homodimeric fractions were pooled separately and purity was evaluated by SDS-PAGE gel electrophoresis. Protein concentration was measured using absorbance at 280 nm  for an hour. F-actin filaments were labeled overnight with 10 μM rhodamine-phalloidin and 20 mM DL-Dithiothreitol.The pET28a(+) vector (Novagen) containing the full-length mouse αE-catenin was an offering from Dr. Noboru Ishiyama27. For reactions containing β-catenin, 80 μg/ml of protein was allowed to react with the surface for 5 minutes. αE-catenin was then incubated for 5 minutes to allow binding. Unbound protein was washed away with a solution containing 20 mM MOPS pH 7.2, 50 mM KCl, 1 mM MgCl2, 0.1 mM EGTA and 1 mg/ml bovine serum albumin, following incubation with a solution containing 100 nM rhodamine phalloidin F-Actin in 20 mM MOPS pH 7.2, 50 mM KCl, 1 mM MgCl2, 0.1 mM EGTA, 1.2 µM glucose-oxidase, 0.2 µM catalase, 17 mM glucose and 20 mM DTT. Flow-cell assays were performed on an inverted fluorescence microscope (Nikon ECLIPSE TE300) equipped with a 532 nm laser (Coherent Sapphire) for rhodamine excitation (~3 mW on the sample). Images were acquired in total internal reflection configuration, through Nikon Plan Apo TIRF, 1.45 oil immersion objective on an EMCCD camera  with 82 nm pixel size, and 40 × 40 μm2 field of view. Integration time was 0.2 s, and EM gain 40.For the flow-cell assay, a 1% nitrocellulose-pentyl acetate solution was smeared on a coverslip and assembled to make a flow chamber2. Binding reactions were performed at room temperature for 20 min and spun at 14000 rpm for 2 h at 4 °C. The supernatant was collected and the pellet resuspended in the original volume of assay buffer. Laemmli Sample Buffer (Sigma-Aldrich) was added to the samples and pellet and supernatant fractions were run on 4–12% BIS-Tris gels (Thermo Fisher Scientific). The gels were stained with Brilliant Blue reagent (Sigma-Aldrich) and scanned with a ChemiDocTM MP Imaging system (Bio-Rad) and analyzed by densitometry with ImageJ.For the pull-down assay of αE-catenin with F-actin, 3 µM F-actin was incubated with increasing concentrations of αE-catenin (0.05 µM–8 µM) in 20 mM MOPS pH 7.2, 50 mM KCl, 0.1 mM EGTA, 1 mM MgCl28 with the following modifications. β-catenin was conjugated to 1 μm diameter carboxyl-polystyrene beads 10% v/w (sigma-Aldrich) using the method established by Pertici et al.29. At the end of the first day of preparation, for the β-catenin conjugation, 40 µl of 200 µg/ml β-catenin was added to 4.8 µl functionalized beads and incubated overnight at 4 °C. In the second day of preparation, during the blocking step, 10 mg/ml of BSA (sigma-Aldrich) was added to 4.8 µl of β-catenin conjugated polystyrene beads. For control, we prepared BSA coated beads following Lapetina et al.28. β-catenin and BSA conjugated beads were incubated with increasing concentrations of αE-catenin (2 nM–150 nM) in 400 μl of binding buffer . Beads concentration was adjusted to have β-catenin at a fixed concentration of 21.7 nM. Binding reactions were performed at room temperature for 30 min and the sample spun at 19400 rcf for 10 min at 4 °C. The supernatant was collected and the pellet resuspended in 30 μl of binding buffer. 7.5 μl Laemmli Sample Buffer (Thermo Fisher Scientific) was added to 22.5 μl of the supernatant and pellet fractions, which were run on 4–12% BIS-Tris gels (Thermo Fisher Scientific). The gels were stained with Brilliant Blue reagent (Sigma-Aldrich) and scanned with a ChemiDocTM MP Imaging system (Bio-Rad) and analyzed using ImageJ.For the pull-down assay of αE-catenin with β-catenin, we followed the protocol by Lapetina et al.30. In the latter experiments, the correct orientation of the biotinylated actin filament was achieved by using the Bead Tailed Actin (BTA) method established by Pertici et al.31. The method takes advantage of the property of Gelsolin TL40 to cap the barbed (+end) of the actin filament. Gelsolin TL40  was covalently bound to carboxylated microspheres  with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in order to bind the +end of a single actin filament. TL40-coated beads were stored in the stock solution  glycerol, and 3 mM NaN3) at 0 °C for about 6 months.Neutravidin beads were prepared as follows: 0.5% carboxylated microspheres  were incubated with 7.4 mg/ml EDC (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, Sigma-Aldrich) and 0.2 mg/ml biotin-x-cadaverine  for 30 minutes at room temperature. Biotinylated beads were then incubated with 0.1 M glycine, 0.5 mg/ml neutravidin and 0.02 mg/ml streptavidin-alexa532 or, alternatively, with 0.02 mg/ml streptavidin-alexa647 (Thermo Fisher Scientific) in the experiments with oriented dumbbells30. For reactions containing β-catenin, 80 μg/ml of protein was allowed to react with the surface for 5 min. αE-catenin (4 μg/ml for single molecule experiments or 40 μg/ml for multiple molecule experiments) was incubated for 5 min to form the α/β catenin complex, or in the absence of β-catenin to bind non-specifically the nitrocellulose surface. The flow chamber was then incubated with 1 mg/ml bovine serum albumin (Sigma-Aldrich) for 5 min to minimize non-specific interactions. In the experiments with non-oriented dumbbells, a solution containing the neutravidin coated-beads and the biotinylated actin was loaded into the chamber. The final reaction was composed by 0.005% neutravidin functionalized beads, 1 nM rhodamine phalloidin F-Actin in 20 mM MOPS pH 7.2, 50 mM KCl, 1 mM MgCl2, 0.1 mM EGTA, 1.2 µM glucose-oxidase, 0.2 µM catalase, 17 mM glucose and 20 mM DTT. Measurements were carried out after sealing the flow chamber with high vacuum grease. A single biotinylated actin filament was anchored through specific binding to the trapped neutravidin-coated microspheres and pre-tensioned to ∼3 pN. At low concentration of αE-catenin, approximately 1 in 4 silica beads showed interaction with the actin filament, providing evidence that the large majority of the beads contained at most a single catenin molecule.Silica beads , dispersed in 1% nitrocellulose-pentyl acetate solution, were smeared on a coverslip and allowed to dry before the flow chamber was assembledControl experiments to probe non-specific interactions were performed without αE-catenin. Few tens of beads were tested in different slides and negligible non-specific interactions were detected in these conditions . Moreover, the experimental conditions assure the minimization of possible artifactual contributions to our measurements which might derive by the presence of a mixture of homodimers and heterodimers in the experiments .2, 1 mM DTT, 3 mM NaN3) and neutravidin-coated beads were loaded into the chamber, in the same final reaction mix, and discriminated under fluorescence microscopy from the different emission spectra of the rhodamine phalloidin and alexa647, respectively. First, a BTA with an anchored actin filament was detected under 532 nm laser illumination and trapped. Second, switching to the 635 nm laser, a neutravidin-coated bead was identified and captured in the second trap. The biotinylated actin filament linked to the TL40 gelsolin coated bead was anchored through specific binding to the trapped neutravidin-coated bead and pre-tensioned to ∼3 pN. The dumbbell orientation with respect to the two traps was changed from one experiment to another to exclude that trap asymmetries might influence the results. The experimental conditions and control experiments for the oriented dumbbell were otherwise the same described above for the non-oriented dumbbell experiments.In the experiments with oriented dumbbells, BTA . The forward scatter signal from the tweezers was filtered by two interferential filters before reaching the two quadrant detector photodiodes (QDPs) for particle position measurements. The applied force was calculated from the displacement of the bead (x) and the trap stiffness (k). The trap stiffness was always calibrated prior experiments for the entire range of the trap positions using a power spectrum method32. A high-magnification camera (CCD 2000X) monitored the coordinates of the pedestal bead and with the aid of a 3D piezo translator system nm-stabilization against thermal drifting and low-frequency noise was achieved33. Additionally, a 532 nm laser (Coherent Sapphire) was used for the excitation of rhodamine-labelled F-actin and streptavidin-alexa532 coated beads. A 635 nm laser  was used for the excitation of streptavidin-alexa647 coated beads. Both data acquisition and force-clamp feedback were controlled at 200 kHz sample rate through an FPGA board (NI-PCI-7830R) by a custom LabVIEW software.Ultra-fast force-clamp spectroscopy and the experimental apparatus are described in detail elsewhere30. Step detection was performed as in Gardini et al. with small modifications12. Steps were detected from velocity changes due to proteins experiencing conformational changes or rapid detachment and reattachment to the filament. In this case, step length (>5 ms) and absolute amplitude (>5 nm) thresholds were set to limit detection of false steps due to thermal fluctuations, while step amplitude sign switch was allowed to account for both forward and backward steps. In order to distinguish if the steps were within an interaction or belonged to new interactions, maximum time interval (<10 ms) and step size (<75 nm) were set. The average stiffness of the molecule (or molecules) bound to the actin filament during the interactions was evaluated from the position variance T is the absolute temperature. Here, we assume that (i) the dumbbell is rigid and (ii) the force applied on the trapped beads is constant, i.e. the stiffness of the traps is negligible under force-clamp. The stiffness of the molecules was evaluated as the average stiffness from the experiments reported in Figs. 27, we evaluate the stiffness underestimation to be below 20%.The analysis of the ultra-fast-force-clamp data was carried out by a custom MATLAB algorithm which detects catenin-actin interactions by identifying changes in the derivative of the position signal i.e., dumbbell velocity which drops to zero upon binding. Limits were imposed to ensure that false events were kept at a maximum of 1% of the total event numberB is the Boltzmann constant, T the absolute temperature, F is the absolute value of the force, τ0± and dαβ± are the unloaded lifetime and the distance parameter under positive (+) and negative (-) force respectively. The force sign is arbitrarily chosen. Fitting parameters are reported in the caption of Fig. We fitted the lifetime vs force plot of a single α-β-catenin heterodimer with a Bell-bond model in which the lifetime τ decreases exponentially with force: kWS and kSW, respectively. Detachment can occur from both states W and S with rates kWU and kSU, respectively, with kWU ≫ kSU. All rates depend exponentially on force dWS, dWU, dSU > 0 and dSW < 0 when F > 0. With F < 0 signs of distance parameters are changed. Therefore, if k0SW > k0WS, α-catenin spends most of its time in state W at zero force. As the force increases, kWS increases and kSW decreases, so that, above a threshold force, state S becomes more populated than state W. The kinetic model can be solved following Nolting34 to get the measured lifetime τ(F), which depends on the detachment rates kWU and kSU from both bound states W and S and on the transition rates kWS and kSW between them. The probability density function of the bound state ([W]+[S] = 1-[U]) is:C2 and C4 are calculated with initial conditions [U] = [S] = 0, [W] = 1. From the probability density function, we can calculate the average lifetime of the bound state:We divided the plot of the lifetime vs force for the α-catenin homodimer interaction with actin into a region of low force and high force to separate the lifetime peaks (Supplementary Fig. mentclass2pt{minimN α-catenin molecules and a single actin filament. We hypothesize that the α-catenin molecules interact independently with the actin filament with an attachment rate τoff being the average lifetime of the unbound state, and a detachment rate τon being the average lifetime of the bound state (see Supplementary Fig. 16. We develop a simple mechano-kinetic model of the interaction between F is applied to the actin filament and no molecule is attached to it, the filament slides at constant velocity v* against viscous drag: When a constant force Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The standard treatment of locally advanced esophageal cancer comprises multimodal treatment concepts including preoperative chemoradiotherapy (CRT) followed by radical surgical resection. However, despite intensified treatment approaches, 5-year survival rates are still low. Therefore, new strategies are required to overcome treatment resistance, and to improve patients’ outcome. In this study, we investigated the impact of Wnt/β-catenin signaling on CRT resistance in esophageal cancer cells. Experiments were conducted in adenocarcinoma and squamous cell carcinoma cell lines with varying expression levels of Wnt proteins and Wnt/β-catenin signaling activities. To investigate the effect of Wnt/β-catenin signaling on CRT responsiveness, we genetically or pharmacologically inhibited Wnt/β-catenin signaling. Our experiments revealed that inhibition of Wnt/β-catenin signaling sensitizes cell lines with robust pathway activity to CRT. In conclusion, Wnt/β-catenin activity may guide precision therapies in esophageal carcinoma patients. Esophageal cancer is a leading cause of cancer-related morbidity and mortality, with 19,260 estimated new cases and 15,530 estimated deaths in the US in 2021, and 5-year overall survival rates of around 20% ,5,6. A pUnfortunately, the molecular basis of the heterogeneous response largely remains unknown. Accordingly, there is a strong need to elucidate the molecular characteristics underlying treatment resistance in esophageal cancer, and to identify alternative strategies to increase the fraction of patients who respond to multimodal treatment. Depending on the histological subtype, esophageal cancer frequently harbors copy number alterations or mutations in oncogenes or tumor suppressor genes like TP53, SMAD4, ERBB2, NOTCH1, GATA4, KLF5, PIK3CA, and CTNNB1 ,13,14. FIncreasing evidence indicates that Wnt/β-catenin signaling plays an important role for the development and progression of esophageal cancer ,20,21,22Flash/FOPFlash reporter assays to assess the Wnt/β-catenin activity represented by the transcriptional TCF/LEF reporter activity, as a standard in the field [We and others have demonstrated that aberrant Wnt/β-catenin signaling plays an important role in mediating resistance to different treatment modalities, including CRT ,24,25,26he field . Due to he field . As dispTo assess responsiveness to RT, either alone or in combination with 5-FU, we next performed colony formation assays (CFA), as standard in the field. This assay measures the ability of a single cell to form a colony, while one colony is defined as at least 50 living cells . TowardssiRNA#1 = 1.37, RERsiRNA#2 = 1.24 at 4 Gy), and in OE-33 cells . In contrast, there was no effect in OAC-P4C, FLO-1, Kyse-150, and Kyse-180 cells , Axin, glycogen synthase kinase 3 beta (GSK3β), and casein kinase 1 alpha (CK1α) ,35. Path80 cells C–F. ThessiRNA#1 = 1.6, RERsiRNA#2 = 1.41 at 6 Gy). However, the surviving fraction of Kyse-150 cells remains high with a fractionated irradiation protocol after β-catenin inhibition, which limits the value of targeting the Wnt/β-catenin pathway in this model. At this point, it is important to note that Kyse-150 originates from a patient who was treated with RT [In the clinical setting, irradiation is delivered in fractionated doses . Therefo with RT . Therefo2.5 µM XAV-939 = 1.61 and an RER5 µM XAV-939 = 1.56 at 4 Gy  different response rates to RT and 5-FU-based CRT, measured by clonogenic survival. Comparing CRT responses of these cell lines F,G to ouhe field . In thisThe potential relevance of Wnt/β-catenin signaling in mediating RT/CRT resistance has been demonstrated in other tumor entities, including CRC ,24,48,49Until today, extensive efforts have been made in the development of small-molecule inhibitors that target the Wnt/β-catenin pathway, but none of them have yet reached clinical application as an FDA approved drug ,35,49,58From our data, only a subset of esophageal cancer cell lines showed basal Wnt/β-catenin activity and could be re-sensitized to CRT after pathway inhibition. Other pathways, such as IL-6/JAK/STAT signaling, were similarly shown to mediate CRT resistance in a subset of esophageal adenocarcinomas . Here, m® Mycoplasma Detection Kit .Human esophageal cancer cell lines FLO-1, OAC-P4C, OE-19, OE-33, SK-GT-4 , and Kyse-70, Kyse-150, Kyse-180, and Kyse-270  were obtained in 2013 directly from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH . The DSMZ ensures authenticity of these cell lines using short tandem repeat profiling . Kyse ceWestern blot analysis was performed as previously described ,24,62. BFlash, SuperFOPFlash , and Renilla  using X-tremeGENE HP DNA Transfection Reagent . To measure the inducibility of the pathway, mutated pCl-neo-β-catenin-S33Y was co-transfected. pCI-neo-β-catenin-S33Y was a gift from Bert Vogelstein . Twenty-four hours after transfection, cells were lysed by passive lysis buffer (Promega), and both firefly and Renilla luciferase activity was measured in a microplate reader . Relative basal transcriptional activation was calculated by dividing Renilla-normalized values of SuperTOPFlash and SuperFOPFlash, whereas inducible activity was calculated by dividing samples that were co-transfected with pCl-neo-β-catenin-S33Y. Detailed experimental conditions are shown in Plasmid transfections were performed as described before . Briefly® reagent (Promega). Reduction of resazurin to resorufin was measured at various time points after the respective treatment using a plate reader  according to the manufacturer’s instructions. Cellular viability of treated or siRNA-transfected cells was compared to untreated cells, or cells transfected with a non-silencing control siRNA (siCtrl.). Detailed information can be found in Cellular viability following 5-FU treatment, synthetic small interfering RNA (siRNA)-transfections, or inhibitor treatment  was assessed using the CellTiter-BlueTransfections with siRNA duplexes were performed as previously described . BrieflyTo test the sensitivity to CRT, standard CFA were conducted as previously described ,24,62. Bt-test in Microsoft Excel and visualized in Grapher (version 8.2.460). p-values < 0.05 were scored as significant. For analyses of the irradiation data, analysis of variance (ANOVA) was used to calculate significant differences between the control group and treatment group. All analyses were performed using Microsoft Excel software Add-in “Data Analysis” (ANOVA: Two-Factor with Replication). For visualization, irradiation data are presented as mean and standard error of the mean (SEM) from at least three independent experiments using the software KaleidaGraph (version 4.1.0). Again, p-values < 0.05 were considered significant, suggesting an influence of the treatment on the dose response. All p-values determined in this study are provided in Statistical analyses of SF6 levels for RT and CRT, cellular viability, and luciferase reporter activity experiments were performed using an unpaired two-tailed Student’s


Astragalus membranaceus (Huangqi) and Rhizoma curcumae (Ezhu) has been reported as a potential antitumor agent for colorectal cancer (CRC) in experimental and clinical studies, but its underlying mechanism is still unclear.The decoction of Astragalus membranaceus (Huangqi) and Rhizoma curcumae (Ezhu) decoction (AR decoction) in the treatment of CRC and explore the underlying mechanism. SW620 cells were transient transfection to overexpress or knock down wnt 5 or β-Catenin. Astragalus membranaceus (Huangqi) and Rhizoma curcumae (Ezhu) -containing serum (AR-CS) was used to interfere with SW620 cells. Additional AR-CS, Wnt5 inhibitor (IWP-4), and β-Catenin inhibitor (JW55) were used to intervene in SW620 cells. Furthermore, subcutaneously injection of SW620 cells into the right flank of nude mice replicated xenograft mice, which were treated with AR decoction for 21 days.The current research aims to explore the potential of AR-CS significantly reduced the mRNA and protein expression levels of Wnt5, β-Catenin, ARF6, and N-Cadherin in SW620 cells, while inhibiting the proliferation and migration of SW620 cells. In cells overexpressing Wnt5 or β-Catenin, these effects of AR-CS were significantly suppressed. On the contrary, the inhibitory effect of AR-CS on the mRNA and protein levels of ARF6 and N-Cadherin and cell proliferation and migration of SW620 was enhanced, when Wnt5 or β-Catenin were knocked down or suppressed by the inhibitors. Moreover, in the mouse model of xenograft tumors, AR decoction not only reduced the tumor volume and inhibited the mRNA levels and protein levels of Wnt5, β-Catenin, ARF6, and N-Cadherin in the tumor, but also inhibit the protein levels of LRP5, LRP6, TCF-4, and LEF1.The histopathology of mice also showed increased apoptosis in tumor tissues, and AR decoction treatment did not cause pathological damage to the kidney and liver.Our results provide evidence that AR decoction inhibits Wnt5/β-catenin signaling and inhibits the development of CRC, which is a promising traditional medicine in the clinical treatment of CRC. Colorectal cancer (CRC), including colon cancer and rectal cancer, is the most common human digestive tract malignancy. In 2015, the incidence and mortality of CRC ranked fifth among all malignant tumors in China .Astragalus membranaceus (Huangqi) [Rhizoma curcumae (Ezhu) [Astragalus membranaceus (Huangqi) or Rhizoma curcumae (Ezhu) have been used for cancer patients, and the survival and quality of life of patients with colorectal cancer was significantly improved after treatment [Astragalus membranaceus (Huangqi) or Rhizoma curcumae (Ezhu) has also shown its potential in the treatment of colorectal cancer in experimental studies [Astragalus membranaceus (Huangqi) in combination with Rhizoma curcumae (Ezhu) on colorectal cancer needs to be confirmed in more studies.In China, traditional Chinese medicine combined with chemotherapy is part of the most important methods for the comprehensive treatment of colorectal cancer . More imHuangqi)  and Rhize (Ezhu)  have ante (Ezhu) , 10 or be (Ezhu) , 12 etc.reatment . The dec studies , 15, butDisorders of the Wnt/β-catenin signaling pathway are widely present in different types of malignant tumors, and are particularly significant in digestive system tumors . In coloAstragalus membranaceus (Huangqi) in combination with Rhizoma curcumae (Ezhu) decoction (AR decoction) in the treatment of CRC. First, the rat Astragalus membranaceus (Huangqi) and Rhizoma curcumae (Ezhu)–containing serum (AR-CS) was used in vitro to evaluate the inhibitory effects of Astragalus membranaceus (Huangqi) and Rhizoma curcumae (Ezhu) on the proliferation and migration of colorectal cancer cells. Further, we provided the evidence for its treatment of CRC by inhibiting Wnt/β-catenin signaling in vitro. Importantly, AR decoction could inhibit tumor growth and inhibit Wnt/β-catenin signaling in mouse models of CRC xenograft tumors.In this study, we explored the potential of IWP-4 (#HY-12879) and JW55 (#HY-13968) were purchased from MedChemExpress . CCK8 kit (#CA1210), 4% paraformaldehyde (#P1110), hematoxylin–eosin staining kit (#G1120), Trizol(#R1100), BCA Protein Quantitative Kit (#PC0020), and 30% acrylamide (29:1) (#A1010) were purchased from Solarbio life sciences . HiFiScript gDNA Removal RT MasterMix (#CW2020) and SuperRT One Step RT-PCR Kit (#CW0742) were purchased from cwbiosciences . Protein loading buffer (#P0015L), protein pre-staining Marker(#P0069), and BeyoECL Star Luminescent Liquid (#P0018AS) were purchased from Beyotime . Cr(C011-2-1), BUN (C013-2-1), AST (C010-2-1), and ALT(C009-2–1), detection kits were purchased from Nanjing Jiancheng Bioengineering Institue . Antibody for Wnt5 (#55184-1-AP), β-Catenin (#51067-2-AP), ARF6 (#20225-1-AP), and N-Cadherin (#22018-1-AP), and GAPDH (#60004-1-Ig), HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (#SA00001-2), and FITC-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (#SA00003-2) were purchased from Proteintech . Antibody for LRP5 (ab223203), LRP6 (ab134146), TCF-4 (ab130014), and LEF1 (ab137872) were purchased from abcam .Astragalus membranaceus (Huangqi) and Rhizoma curcumae (Ezhu) were purchased from China Traditional Chinese Medicine Co., Ltd. AR decoction are obtained by soaking the traditional Chinese medicine Astragalus membranaceus (Huangqi) and Rhizoma (Ezhu) curcumae decoction with traditional water decoction [Astragalus membranaceus (Huangqi) (500 g) and Rhizoma curcumae (Ezhu) (500 g) were weighed and added to a stainless pot. After 2 l water added and boiled for 1 h, the liquid was collected. Then 2 l water is added and boiled for another 1 h to collect liquid again. The solution that was combined twice, a rotary evaporator was used to concentrate the solution and the final volume was concentrated to 500 ml. The concentration of Astragalus membranaceus (Huangqi) and Rhizoma curcumae (Ezhu) is  + 1 g Rhizoma curcumae (Ezhu))/ml. The clinical dosage of Astragalus membranaceus and Rhizoma curcumae is (25 g + 25 g)/day. Based on the conversion method drove from body surface coefficient of mice (Body surface area ratio of mice and humans = 0.0026) or rat (Body surface area ratio of rat and humans = 0.018) and human. The dose for mice is 6.5 g/kg, the dose for rats is 4.5 g/kg. They were calculated as follows: Mice: 50 g/day (human dosage)*0.0026/0.02 kg(weight of mice) = 6.5 g/kg/day. Rat: 50 g/day (human dosage)*0.018/0.2 kg(weight of rat) = 4.5 g/kg/day.ecoction . AstragaAll procedures of animals described in this study were performed in accordance to the guidelines of the Institutional Animal Ethical Committee of Nanjing University of Chinese Medicine and in accordance with the ethical standards established by Ethics Committee of Nanjing University of Chinese Medicine. Forty male SD rats with SPF grade, 12 weeks old, weighing 250–280 g, were housed in a standard SPF animal feeding center . After a week of adaptation, 20 of the rats were intragastric administration with AR decoction (4.5 g/kg) for seven consecutive days. Other 20 rats were intragastric administration with water. After seven days, these rats were deeply anesthetized and blood was taken from the common intracranial artery into the pro-coagulation tube [The content of representative substances in AR decoction and AR-CS was determined by Agilent 1260 high performance liquid chromatograph HPLC . The chromatographic column is a Hedera ODS-2 C18 chromatographic column . Methanol-0.1% phosphoric acid aqueous solution (A: B) was used as mobile phase, gradient elution, elution time and mobile phase ratio are shown in Table Sample pretreatment: For AR decoction, 1 ml of extract was accurately drawn and add 1 ml of absolute ethanol. After mixing, it was standed for 24 h, centrifuged at 13,000 rpm for 10 min. Then the supernatant was taken, and passed through 0.22 μm filter membrane. And the sample was injected.For AR-CS: 400 μl of serum was precisely aspirated, precisely added 1.6 ml of methanol, extracted with ultrasound for 10 min, centrifuged at 13,000 rpm for 10 min. Then 1.8 ml of the supernatant was precisely aspirated, blowed dry with N2, and added 100 μl of methanol-0.1% phosphoric acid aqueous solution (20:80) for redissolve, centrifuged at 13,000 rpm for 10 min, and the supernatant was taken for injection.Nine main components in AR extract were quantitatively detected by HPLC, among which the maximum absorption wavelength of Calycosin-7-glucoside CAS: 20633-67-4), Ononin (CAS: 486-62-4), Calycosin (CAS: 20575-57-9), Kaempferol CAS: 520-18-3), Formononetin (CAS: 485-72-3), Curdion (CAS: 13657-68-6), Curzerene (CAS: 17910-09-7), and Isocurcumenol (CAS: 24063-71-6) were 215 nm. The contents in the extract were 0.08, 0.05, 0.04, 0.03, 0.02, 0.03, 0.02 mg/ml, and the contents in the serum of rats were 0.79, 2.09, 1.01, 1.26, 1.07, 2.06, 0.77, 0.81 μg/ml, respectively. The maximum absorption wavelength of curcumin was 420 nm, and the content of curcumin in extract and serum after administration were 0.01 mg/ml and 0.68 μg/ml, respectively. The results are shown in Fig. 8-3, Form0633-67-42, at 37 °C.SW620 cell line was purchased from ATCC. SW620 cells were cultured with DMEM  containing 10% fetal bovine serum , 100 U/ml penicillin and 100 mg/mL streptomycin . SW620 cells were incubated in a cell incubator  containing 5% CO5 cells in 6-well plates. According to the manufacturer's instructions, lipofectamine 3000 reagent , pcDNA-Wnt 5 (2 μg), pcDNA-β-catenin (2 μg), control vector (2 μg) or wnt 5 siRNA, β-catenin siRNA (50 nM) and control siRNA (50 nM) were used to transiently transfect into cells. After 6 h of transfection, complete culture medium was added to the transfection medium, continue culturing for 12 h. Then the cells are collected for inoculation.Wnt 5 (#sc-41112) and β-catenin (#sc-29209) siRNA and control (#sc-37007) siRNA were purchased from Santa Cruz Biotechnology . pcDNA-Wnt 5 and pcDNA-β-catenin and control vector were purchased from Addgene . SW620 cells were cultured in DMEM medium at a density of 10Cell Count Kit-8 (CCK-8) was used to test cell viability. The current research conducted multiple cell viability tests, including Sw620 cells were treated with different concentrations of AR solution for 12 h, and SW620 cells were treated with different concentrations of FBS or rat serum for 12 h. Different concentrations of AR-CS  were used to incubate SW620 cells for 12 h, or 20% AR-CS treated SW620 cells for different administration time  were another two independent experiments. When different concentrations of AR-CS  were used on SW620 cells for 24 h, diffferent concentrations of blank rat serum were added to make the serum concentration to 20% in all groups.For cell viability detection, SW620 cells were seeded at a density of 5000 cells/well in 96-well plates. After the cells adhered, different concentrations of drug-containing serum were added to SW620 cells for different time. 10 μl of CCK-8 reagent was added to the 96-well plate 2 h before the termination of the experiment. After two hours, microplate reader was used to determine the absorbance (OD) value at a wavelength of 450 nm. The cell viability of the control group was defined as 100%, and the cell viability was calculated according to the formula: Cell viability = (OD of test group/Everage OD of control group)*l00%.The current study performed four Transwell assays. The treatment of SW620 cells in these four cell experiments is different. In the first experiment, different concentrations of AR-CS  treated SW620 cells for 24 h. In the second experiment, WT, Wnt5 over-expressed, and β-Catenin over-expressed SW620 cells with or without 20% AR-CS treated for 12 h. In the third experiment, WT, Wnt5 knock-down, and β-Catenin knock-down SW620 cells with or without 20% AR-CS treated for 24 h. In the fourth test, SW620 cells were treated with or without 20% AR-CS, Wnt5 inhibitor (IWP-4), and β-Catenin inhibitor (JW55) treated for 24 h.Briefly, WT, Wnt5 over-expressed, β-Catenin over-expressed, Wnt5 knock-down, or β-Catenin knock-down cells are digested and seeded into the upper chamber of the Transwell chamber. After adherent, cells were incubated with AR-CS and tool drugs for the corresponding time. Subsequently, the cells on the upper chamber were fixed and stained with crystal violet. The cells inside the inner membrane of the chamber were erased, and the number of cells on the underside of the membrane placed on the glass slide was observed with an inverted optical microscope. Then the pictures were taken. The cell migration rate of the normal group was defined as 100%, and the overall migration rate of the other groups was calculated. The calculation formula for the migration rate is: migration rate = (number of test group/Average number of control group)*l00%.Like the Transwell experiment, the wound scratch assay also tested the wound healing rate under four different conditions. Briefly, WT, Wnt5 over-expressed, β-Catenin over-expressed, Wnt5 knock-down, or β-Catenin knock-down cells were seeded into 24-well plates after digestion. After the cells adhere to the wall, a scratch needle was used to slightly scratch along the blank center axis. After cleaning once, pictures were taken by an inverted optical microscope, and the scratch area (Area 1) was calculated. The cells were incubated with AR-CS and tool drugs for the corresponding time. Then the cells were fixed and washed. The healing of the scratches was observed under an inverted optical microscope, and the area of the scratches (Area 2) was calculated. The wound healing rate is calculated by the following formula: wound healing rate = (Area 1 − Area2)/Area1*100%.Briefly, WT, Wnt5 over-expressed, β-Catenin over-expressed, Wnt5 knock-down, or β-Catenin knock-down cells were seeded into 24-well plates with glass coverslips. After the cells adhere to the coverslips, different concentrations of drug-containing serum were added to SW620 cells for different time. After that, the cells were fixed with 4% paraformaldehyde. PBS was used to clean and β-Catenin (1:100) was used to incubate the cells for overnight. FITC-conjugated goat anti-rabbit IgG (1:100) was used to incubate the cells for 1 h, and then DAPI was used to stain the cells for 5 min. Photographs were taken with a fluorescence microscope after glycerin gelatin was used to seal the coverslips on glass slides.The treatment of cells was the same as in the Transwell assay. A total of four different cell treatments were carried out. For collected SW620 cells or tumor tissues derived from xenograft mice, after using Trzol reagent to extract total RNA, the mRNA of SW620 cells or tumor tissues was reverse transcribed into cDNA, referring to the instruction. After referring to the PCR reaction kit instructions, the mRNA expression levels of Wnt5, β-Catenin, ARF6, and N-Cadherin were measured by the ABI7500 system. GAPDH(#B661104) primer was purchased from Sangon Biotech . The other primers were synthesized by Sangon Biotech , the sequences were as follows:Wnt5: forward: CCTGAAGGAGAAGTACGACAG, reverse: GATGTAGACCAGGTCTTGTGTG, product size: 112 bp.β-Catenin: forward: TGGATTGATTCGAAATCTTGCC, reverse: GAACAAGCAACTGAACTAGTCG, product size: 92 bp.ARF6: forward: GCTCTGGCGGCATTACTACACTG,reverse: AGGATTATGGCGTCCCTCATCTCC, product size: 138 bp.N-Cadherin: forward: CGATAAGGATCAACCCCATACA, reverse: TTCAAAGTCGATTGGTTTGACC, product size: 142 bp.−ΔΔ method was used to calculate the relative expression levels of Wnt5, β-Catenin, ARF6, and N-Cadherin mRNA in SW620 cells or tumor tissues.The 2The treatment of cells was the same as in the Transwell assay. A total of four different cell treatments were carried out. For the collected SW620 cells or tumor tissues derived from xenograft mice, after using the REPI lysate to extract the total protein, the total protein level of SW620 cells or tumor tissues was determined, referring to the BCA protein determination kit instruction. After each sample was boiled with the loading buffer, the protein was separated by SDS-PAGE gel electrophoresis. After transferring the protein in the gel to the PVDF membrane, the PVDF membrane was incubated with Wnt5, β-Catenin, ARF6, N-Cadherin, LRP5, LRP6, TCF-4, and LEF1 and GAPDH overnight at 4 °C. After that, the PVDF membrane was incubated with HRP-modified goat anti-rabbit Igg for 2 h. After washing, the high-sensitivity chemiluminescence solution combined with the gel imaging system was used to visualize the blots of Wnt5, β-Catenin, ARF6, N-Cadherin, LRP5, LRP6, TCF-4, LEF1, and GAPDH in SW620 cells or tumor tissues.6 SW620 cells were subcutaneously injected into the right flank of nude mice. After the cells were injected. The animals were divided into three groups: the model group, the AR treatment group and the oxaliplatin treatment group, each group contained 5 mice. AR decoction (6.5 mg/kg) was given by gavage daily for 21 days. Oxaliplatin (1 mg/kg) was used as a positive drug and was administered by intrapulmonary injection every day for 21 consecutive days. As a model control, mice were given water by gavage every day. The mice are weighed every 2 days. The length and width of the tumor were measured every three days and the volume of the tumor was calculated as length × width2. After 21 days, the mice were sacrificed and the tumor tissues were taken out, and the tumors were photographed and weighed.Fifteen nude mice with SPF grade were 6 weeks old, weighing 18–22 g. And 2 × 10After the mice were treated, they were sacrificed and tumor tissues were obtained. The tumor mass of each mouse was incised from the middle. Half was used for the above-mentioned WB experiment, and the other half was immersed in paraformaldehyde. At the same time, the liver and kidney of mice were fixed in paraformaldehyde. Tumors, livers and kidneys from mice were coated into wax blocks after routine dehydration, transparency and wax immersion treatments. Then HE staining was performed and photographed by an ordinary light microscope. The experimental procedures of tissue embedding, sectioning and HE staining refer to the previous report .After the mice were treated, they were sacrificed and serum was obtained. The levels of Cr, BUN, AST, and ALT in serum were detected according to the kit manufacturer's instructions.Statistical analysis was carried out using ANOVA. Results are expressed as mean ± SD. p < 0.05 was considered as a significant difference. All analyses were performed with GraphPad Prism Version 5.01 .Different concentrations of AR decoction  were used to incubate SW620 cells for 12 h, the cell viability results showed that AR decoction (0.1–6.4 g/ml) had a significant inhibitory effect in SW620 cells cultured with 10%FBS- containing medium  for 12 h, and the cell viability was detected. After 20% AR-CS was applied to SW620 cells for 12 h, the cell viability was approximately 60–70% , or β-Catenin inhibitor (JW55) alone or together with AR-CS verified the above results. After incubating SW620 cells with IWP-4 or JW55 for 24 h, the mRNA and protein levels of Wnt 5 or β-Catenin were significantly reduced. At the same time, the mRNA and protein levels of ARF6 and N-Cadherin, and the proliferation and migration of SW620 cells were also reduced (Fig. After confirming that AR-CS can reduce proliferation and migration of SW620 cells by inhibiting Wnt5/β-Catenin, we further verified the effect of AR decoction on tumor growth in vivo. Nude mice were injected subcutaneously with SW620 cells to replicate xenograft tumor mouse models, and AR decoction was given daily for 21 days after inoculation. Continuous weight data showed that neither AR decoction nor the positive control drug oxaliplatin had a significant effect on the body weight of xenograft mice Fig. A. ContinHere we report a traditional Chinese medicine AR decoction to treat colorectal cancer by inhibiting Wnt5/β-Catenin signaling. In fact, traditional Chinese medicine have been used in the treatment of clinical tumors for many years , not onlThe Wnt protein family plays a key role in tumor development through the classic Wnt/β-catenin pathway, or the non-canonical pathway independent of β-catenin . The WntIn the current study, first, we used AR-CS to interfere with SW620 cell line. After obtaining the appropriate intervention concentration and intervention time, it was confirmed that AR-CS could significantly inhibit the proliferation and migration of SW620 cells. We also confirmed that AR-CS inhibited the expression of Wnt 5 and β-catenin in SW620 cells in a concentration-dependent manner. In SW620 cells overexpressing Wnt5 or β-catenin, the inhibitory effect of AR-CS on the proliferation and migration of SW620 cells disappeared. Our research also showed that in SW620 cells, either knockdown of Wnt5 alone, β-catenin alone, or use of Wnt5 or β-catenin inhibitors directly inhibited the proliferation and migration of SW620 cells. As previously reported, inhibition of Wnt5/β-catenin is an effective treatment for CRC . When ARIn the process of AR-CS reducing the proliferation and migration of SW620 cells by inhibiting Wnt5/β-catenin, our data also showed that the expression of ARF6 and N-Cadnerin in SW620 cells was significantly reduced by AR-CS. The small GTPase protein ARF6 has been proved to play a non-negligible role in promoting the proliferation, invasion and migration of a variety of tumor cells. By stimulating invasion ability, disrupting E-cadherin-mediated cell adhesion, and inducing the recycling of β-catenin, the high expression of ARF6 promotes the development of breast cancer, renal cell carcinoma, lung adenocarcinoma and colorectal cancer –42. N-caWhen we applied AR-CS on SW620 cells overexpressing Wnt5 or β-catenin, the inhibitory effect of AR-CS on the expression of ARF6 and N-Cadnerin disappeared, indicating that the effect of AR-CS depended on Wnt5/β-catenin signal. In SW620 cells, either knock-down of Wnt 5 alone, β-catenin alone, or use of Wnt5 or β-catenin inhibitors directly inhibited the expression of ARF6 and N-Cadnerin in SW620. And when we use AR-CS on Wnt 5 or β-catenin knock-down SW620 cells, or when AR-CS was combined with Wnt 5 or β-catenin inhibitors, the inhibitory effect of AR-CS on the expression of ARF6 and N-Cadnerin in SW620 cells was enhanced, compared with wild-type SW620 cells or compared with SW620 cells treated with AR-CS alone. Overall, the current results provide evidence that AR decoction inhibits the expression of ARF6 and N-Cadnerin in colorectal cancer cells by inhibiting Wnt5/β-catenin.In this study, we also provided evidence that AR decoction inhibited tumor proliferation in CRC mice, and inhibited Wnt5/β-catenin signaling and the expression of ARF6 and N-Cadnerin in tumors of CRC mice. More importantly, in vivo we performed protein expression detections of LRP5 and LRP6 which were associated with Wnt assembly , and proSome deficiencies of this research are still worthy of in-depth exploration, including the failure to explain in depth how AR decoction interacts with Wnt5/β-catenin signals to treat CRC; and the failure to collect solid tumor samples from CRC patients receiving AR decoction treatment in China to verify the changes in Wnt5/β-catenin signal and the expression of ARF6 and N-Cadnerin in patients. Some studies have shown that Huangqi or Ezhu could promote the efficacy of chemotherapeutic drugs or reduce drug resistance in cancer treatment , 49. WheThe present study provided evidence that AR decoction inhibits Wnt5/β-catenin signaling and inhibits the development of CRC in vitro and in vivo. AR decoction is a promising traditional medicine in the clinical treatment of CRC.


Protein arginine methyltransferase 5 (PRMT5) is the type II arginine methyltransferase that catalyzes the mono‐ and symmetrical dimethylation of protein substrates at the arginine residues. Emerging evidence reveals that PRMT5 is involved in the regulation of tumor cell proliferation and cancer development. However, the exact role of PRMT5 in human lung cancer cell proliferation and the underlying molecular mechanism remain largely elusive. Here, it is shown that PRMT5 promotes lung cancer cell proliferation through the Smad7‐STAT3 axis. Depletion or inhibition of PRMT5 dramatically dampens STAT3 activation and thus suppresses the proliferation of human lung cancer cells. Furthermore, depletion of Smad7 blocks PRMT5‐mediated STAT3 activation. Mechanistically, PRMT5 binds to and methylates Smad7 on Arg‐57, enhances Smad7 binding to IL‐6 co‐receptor gp130, and consequently ensures robust STAT3 activation. The findings position PRMT5 as a critical regulator of STAT3 activation, and suggest it as a potential therapeutic target for the treatment of human lung cancer. β (TGF‐β) independent manner. This finding positions PRMT5 as a promising therapeutic target for lung cancer treatment.The role of protein arginine methyltransferase 5 (PRMT5) in Janus kinase (JAK)‐signal transducer and activator of transcription 3 (STAT3) signaling pathway and lung cancer cell proliferation is unveiled. PRMT5 symmetrically dimethylates Smad7 on Arginine‐57, promotes Smad7 binding to interleukin‐6 (IL‐6) co‐receptor glycoprotein 130 (gp130), and consequently ensures robust STAT3 activation in a transforming growth factor‐ PRMT5 methylates arginine residues in histones and epigenetically controls the expression of an array of target genes. In addition, PRMT5 also modifies non‐histone proteins to control their functions, including transcription factors p53, KLF4, E2F‐1, NF‐κB/p65, ribosomal protein S10, and RAF kinases. PRMT5 is ubiquitously expressed in all eukaryotes and is involved in a wide range of biological processes, such as germ cell survival, cell cycle progression, muscle stem cell expansion, and myogenesis. In addition to these functions, PRMT5 plays a pivotal role in cancer cell proliferation and transformation. Increased expression of PRMT5 has been observed in a variety of carcinomas, including ovarian, lung, lymphoma, melanoma, colon, gastric, bladder cancer, and germ cell tumors.. Methylosome protein 50  is a critical cofactor of PRMT5, both with similar expression pattern. MEP50 binds to PRMT5 and greatly enhances its catalytic ability. PRMT5 also directly associates with a range of other protein factors, including pICln, Menin, CoPR5, and RioK1, which may alter its subcellular localization and protein substrate selection.Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that catalyzes the mono‐ and symmetrical dimethylation of protein substrates at the arginine residues.1 It w Elevated levels of IL‐6 are observed in a large number of patients with hematopoietic malignancies or solid tumors. Aberrantly increased IL‐6 stimulates hyperactivation of JAK/STAT3 signaling, which is often associated with poor patient outcomes. In the tumor microenvironment, IL‐6/JAK/STAT3 signaling acts to drive the proliferation, survival, invasiveness, and metastasis of tumor cells.  In the classic IL‐6/JAK/STAT3 signaling, IL‐6 binds to the membrane‐bound IL‐6 receptor‐α (IL‐6Rα), thus inducing the formation of a heterohexameric complex with gp130 . Formation of this complex results in JAK2 activation and STAT3 phosphorylation. Then phosphorylated STAT3 becomes dimerized, transports into the nucleus and binds to STAT‐binding elements in the promoter regions of target genes. Although PRMT5 is first identified as a JAK2 interacting protein, until now PRMT5 substrates have not been identified in the JAK complex. Moreover, it still remains unknown whether or how PRMT5 affects JAK/STAT signaling.The IL‐6/JAK/STAT3 pathway has a key role in the growth and development of many human cancers.24 EleIn this study, we identified and characterized PRMT5 as an essential mediator of STAT3 activation. Depletion or inhibition of PRMT5 dramatically dampens STAT3 activation and suppresses proliferation and migration of human lung cancer cells. Moreover, we uncovered that PRMT5 promotes STAT3 activation through methylation of Smad7 on Arg‐57. We previously reported that Smad7 directly binds to the intracellular domain of gp130, thereafter disrupting the SHP2‐ or SOCS3‐gp130 complex and amplifying STAT3 activation. We now provide evidence that methylated Smad7 exhibits a stronger interaction with gp130. Our findings identified the oncogenic role of PRMT5 in human lung cancer and elucidated a novel mechanism underlying the modulation of the Smad7‐gp130‐STAT3 axis by PRMT5 in those cancer types with aberrantly hyperactivation of STAT3.22.1 implicating that PRMT5 may regulate STAT3 signaling. However, it remains unknown whether PRMT5 acts through STAT3 signaling in growth regulation and tumorigenesis. In this study, we sought to address this long‐standing question on the potential connection between PRMT5 and STAT3 signaling. To this end, we first utilized siRNAs/shRNAs against two different target sequences to knockdown the expression of PRMT5 and then examined the status of STAT3 activation, as indicated by Tyr‐705 phosphorylation. Stable depletion of PRMT5 profoundly reduced endogenous STAT3 phosphorylation and expression of two STAT3 targets, e.g., survivin and c‐Myc, in lung carcinoma A549 cells  that recognized a subset of cellular proteins with symmetrical dimethylation at arginine residues . These results suggest that the methyltransferase activity strongly support a direct role of PRMT5 in STAT3 activation.PRMT5 is a protein methyltransferase that is responsible for the vast majority of cellular symmetric dimethylation of arginine (SDMA) modification. We then examined whether the methyltransferase activity of PRMT5 is essential for STAT3 activation by using chemical inhibitors of PRMT5. Treatment of H358 and A549 cells with EPZ015666 or GSK591, two recently developed potent and specific PRMT5 chemical inhibitors,31 leas Figure  and A549 we reasoned that Smad7 might be involved in PRMT5‐regulated STAT3 activation in epithelial cells. We first determined if Smad7 could promote STAT3 signaling in non‐mESC cells and/or in response to a non‐LIF cytokine. Like in mESC, overexpression of Smad7 could enhance STAT3 activation in lung carcinoma A549 cells ,32 we s Figure . Converss Figure  and MCF1s Figure , when SmβRI‐null HaCaT cells could still inhibit the STAT3 activation . Taken together, these results suggest that PRMT5 stimulates JAK/STAT3 signaling dependent on Smad7, but independent of TGF‐β signaling.To connect Smad7 to PRMT5 on STAT3 activation, we determined whether Smad7 mediates PRMT5‐mediated STAT3 activation. In control cells, ectopic expression of PRMT5, but not the methyltransferase‐dead mutant G367A/R368A, enhanced IL‐6‐induced STAT3 activation Figure . Notably2.2Figure Having established that PRMT5 activated STAT3 signaling through Smad7, we assessed the potential interaction between PRMT5 and Smad7 by using coimmunoprecipitation (co‐IP) in HEK293T cells. We found that although PRMT5 is barely bound to Smad7, the PRMT5 cofactor MEP50 enabled a significantly higher PRMT5‐Smad7 interaction Figure 2A. To evTo further investigate the PRMT5–Smad7 interaction under physiological conditions, we examined the binding of endogenous PRMT5 to Smad7. In the absence of a good anti‐Smad7 antibody for IP, we established stable tet‐on cell lines to inducibly express SFB‐Smad7 in MCF10A cells. In the Smad7‐tet‐on cells, doxycycline (Dox) treatment induced expression of SFB‐Smad7 . Dox‐induced Smad7 could interact with endogenous PRMT5, whereas no Smad7‐bound PRMT5 was detected in the absence of Dox Figure . Further2.3 We next attempted to determine whether PRMT5 could methylate any other components in the canonical JAK/STAT3 signaling pathway. We examined PRMT5‐mediated methylation in cells cotransfected with MYC‐PRMT5/MEP50 together with key components of the STAT3 signaling pathway, including SFB‐tagged JAK2, gp130, STAT3, SHP2, and SOCS3 as well as Smad7. Interestingly, PRMT5 could only methylate Smad7, but not others, as detected by sdme‐RG antibody . Smad6 is another inhibitory Smad in the TGF‐β superfamily with a C terminus similar to that of Smad7. We found that only Smad7 and Smad7/6 chimera, but not Smad6 or Smad6/7 chimera can be methylated by PRMT5 . These data point out that PRMT5 methylates the MH1 domain of Smad7.We next mapped which domain of Smad7 is methylated by PRMT5. In cells transfected with PRMT5/MEP50 and a Smad7 variant, we found that PRMT5 methylated Smad7 in the MH1 region that spans amino acids 1‐228 . To further delineate this, we constructed two molecular chimeras between Smad6 and Smad7, which contained the MH1 (or N) domain of one Smad and the MH2 (or C) domain of the other as previously described34  in the MH1 domain of Smad7. A GR motif represents putative preferred methylation sites of PRMT5.36 Eac0 Figure . In addi0 Figure . Immunop0 Figure .In an in vitro methylation assay, immuno‐purified MYC‐PRMT5/MEP50 proteins were incubated with bacterially expressed Smad7 Figure  or immun2.4Figure As Smad7 potentiates STAT3 activation through direct binding to gp130, we speculated that methylation modification of Smad7 might affect this interaction. Indeed, methylated Smad7 exhibited a stronger binding ability to gp130 than unmethylated wildtype Smad7 or unmethylable R57K mutant Figure 4A. In adTo further investigate the role of R57 methylation in Smad7 binding to gp130, we immuno‐purified SFB‐Smad7 or SFB‐R57K that was coexpressed with MYC‐PRMT5 or MYC‐G367/R368A together with MYC‐MEP50 in HEK293T cells. As shown in Figure 2.5CDC25C . In RNA‐seq experiments, gene set enrichment analysis (GSEA) revealed that PRMT5 deficiency disabled IL‐6/STAT3 responsiveness as downregulated genes in PRMT5 knockdown A549 cells were significantly enriched in the IL‐6/STAT3 signaling gene set   Figure . These r could largely reverse the inhibitory effect of PRMT5 deficiency on cell growth  and invasion . Taken together, these results demonstrate that PRMT5 promotes cell growth and migration through STAT3 signaling.To link PRMT5 to the well‐established growth‐promoting role of STAT3, we examined the functions of PRMT5 in cellular functions using cell‐based assays. STAT3 inhibitor stattic could significantly block STAT3 phosphorylation  and inhibit the growth of A549 cells . Knockdown of PRMT5 profoundly suppressed proliferative activities in A549 cells, as analyzed by using EdU staining Figure ,F or CCK37 cou2.6Figure To further investigate the function of PRMT5 in tumor formation, a Lewis lung carcinoma (LLC) xenograft mouse model was used. In comparison to tumors derived from parental LLC cells, those with stable PRMT5 depletion using mouse‐specific shRNAs exhibited significantly reduced tumor growth and weight Figure . Accordihttp://www.kmplot.com) .Immunohistochemical (IHC) analysis of tissue microarray of nonsmall cell lung cancer (NSCLC) showed that PRMT5 is markedly overexpressed in cancer cells compared to adjacent normal tissues Figure ,E. FurthTaken all together, our results suggest that PRMT5‐mediated methylation of Smad7 enables its interaction with gp130, thus potentiating STAT3 activation, which consequently promotes cell proliferation and tumorigenesis Figure .3 Besides controlling specific gene expression through methylation of histones, PRMT5 also targets certain transcription factors involving in cell signaling. Although PRMT5 was discovered as a JAK2‐binding protein, the role of PRMT5 in the JAK/STAT3 signaling pathway still remains unknown. Here we show that PRMT5 is critical for IL6‐induced STAT3 activation through the recently recognized Smad7‐gp130‐STAT3 axis. PRMT5 methylates Smad7, enhances the Smad7‐gp130 interaction, and promotes STAT3 signaling, whereas depletion or inhibition of PRMT5 consequently abolishes STAT3 activation and inhibits cell proliferation.PRMT5 is overexpressed in a large number of cancer types and promotes cell proliferation, migration, and survival.16 Bes3.1 A variety of cytokines like IL‐6, epidermal growth factor (EGF), interferon (IFN) could induce STAT3 Tyr705 phosphorylation, a hallmark of JAK‐induced activation of STAT3. Some cells may have high levels of autocrine STAT3 inducers, while others can only trigger STAT3 activation in response to exogenous or paracrine cytokines/growth factors. We found that STAT3 is constitutively activated in human NSCLC A549 and H358 cells, which exhibit a high level of secreted IL‐6 . Addition of tocilizumab, a monoclonal antibody against IL‐6 receptor, to the growth medium of A549 cells could largely inhibit the endogenous STAT3 phosphorylation . In addition, mammary epithelial cell lines MCF10A and MCF7 exhibit STAT3 activation only when stimulated with IL‐6 in cell culture. Conditioned medium of A549 cells strongly stimulated STAT3 phosphorylation in MCF10A cells and this induction could be completely blocked by Tocilizumab . A variety of compounds against STAT3 signaling such as alantolactone, isocryptotanshinone, sinomenine suppress proliferation and invasion of A549 cells through disrupting STAT3 signaling pathway. Similar results were also observed for A549 cell proliferation in the presence of stattic (inhibitor specific to STAT3) . Thus, IL‐6/STAT3 signaling plays an important role in proliferation in A549 cells.Numerous evidence has clarified the importance of IL6/STAT3 signaling for cancer growth, metastasis, and maintenance of cancer stem cells.38 A v rescued the defect in cell proliferation and migration caused by PRMT5 inhibition . In addition, PRMT5 inhibition also blocks STAT3 activation induced by exogenous IL‐6 in MCF10A cells . These data support the notion that PRMT5 promotes cell growth and migration through maintaining or potentiating STAT3 activation.The critical evidence that connects PRMT5 to STAT3 activation comes from our observation that knockdown or inhibition of PRMT5 can significantly block STAT3 activation and cell responses to autocrine or exogenous IL‐6 . These observations suggest that PRMT5 methylates the MH1 of Smad7 although it binds to the MH2 domain. Interestingly, methylation of Smad7 at Arg57 possesses stronger binding to gp130 , which may explain why no methylation of Smad7 was previously reported by PRMT5.42 Mor7 Figure . In shar0 Figure , which mβ signaling. However, Smad7‐triggered STAT3 activation does not require functional TGF‐β receptors. Similarly, PRMT5 retains the ability to promote STAT3 activation in TβRI/ALK5‐null HaCaT cells . Although this further supports the non‐canonical function of Smad7, it does not exclude the possibility that Smad7 methylation also regulates TGF‐β signaling. A previous study has reported that Smad7 methylation by PRMT1 prevents its binding to TβRI/ALK5 and thus enables Smad3 activation, which is essential for TGF‐β‐induced epithelial‐mesenchymal transition. Since PRMT5 mediates symmetric di‐methyl modification on Smad7, which differs from PRMT1‐mediated mono‐methylation and asymmetric dimethylation, it is still worthy of future examination whether symmetric dimethylation also regulates Smad7 binding to TβRI/ALK5. If so, symmetric dimethylation serves as an important switch to facilitating TGF‐β signaling and gp130/STAT3 signaling.Smad7 is a well‐known negative feedback regulator in TGF‐eceptors.32 Simhttps://clinicaltrials.gov/). It would be of great significance to investigate the effects of PRMT5 inhibitors in cancer types with aberrantly hyperactivated STAT3 signaling. Moreover, PRMT5‐mediated Smad7 methylation may also impact inflammation and cell pluripotency where gp130/STAT3 signaling is involved. It is conceivable that manipulating PRMT5 activity may also be used in therapies against inflammatory diseases and stem cell‐based regenerative medicine.Finally, our findings position PRMT5 as a critical regulator of gp130/STAT3 signaling to control the growth and development of human cancer, which provides an important mechanism for the oncogenic role of PRMT5. Given its enzymatic activity, PRMT5 is a druggable target. Indeed, PRMT5 inhibitors are in a number of clinical trials, for instance, to treat myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and solid tumors (including NSCLC)  and pRK3HA , respectively. All mammalian vectors are derived from pRK5 (Genentech). All mutants, including PRMT5 G367A/R368A, PRMT5r and Smad7 R57K were made by mutagenesis and confirmed by DNA sequencing. GST‐Smad7 was generated by PCR and cloned into pGEX4T1 (GST tag).The following mammalian expression plasmids have been previously described: FLAG‐Smad7,34 Smaβ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β (TGFB1‐100) from StemRD.The following chemical compounds and recombinant proteins were commercially obtained: EPZ015666 (S7748) and GSK591 (S8111) from Selleck; Human IL‐6 (96‐200‐06‐20) from Peprotech and TGF‐2. A549 and H358 cells were cultured in PRMI 1640 (Corning) supplemented with 10% FBS. MCF10A were cultured in DMEM/F12 media (Corning) supplemented with 5% horse serum (Invitrogen), insulin (10 µg mL−1), EGF (20 ng mL−1), cholera toxin (100 ng mL−1) (Sigma‐Aldrich) and hydrocortisone (0.5 µg mL−1) (Sigma‐Aldrich). HEK293T cells were transfected with PEI  and MCF7 cells with X‐treme GENE HP DNA . Stable cell lines were obtained by using lentiviral infection and selected in the presence puromycin/G418 at appropriate concentrations.HEK293T, LLC, and MCF7 cells were grown in DMEM (Corning) supplemented with 10% FBS (Gibco) at 37 °C in a humidified incubator with 5% COsiRNAs were commercially synthesized (Ribo‐Bio Co) and transfected into cells using Lipofectamine RNAiMAX . siRNA sequences targeting human genes were as below: siPRMT5‐1, GCCCAGTTTGAGATGCCTTAT; siPRMT5‐2, CCGCTATTGCACCTTGGAA; siSmad7, CCGTGCAGATCAGCTTTGT.−1) and added to the target cells for infection of 8 h. Cells were subject to puromycin (1 µg mL−1) selection for 3 d.To generate stable knockdown of endogenous human PRMT5, mouse PRMT5 or Smad7, a lentiviral shRNA expression system was used. Two DNA fragments containing the target sequence of the corresponding target gene were cloned into the pLKO.1 lentivirus vector (Addgene #24150). Sequences are as below: human shPRMT5‐1, GCCATCACTCTTCCATGTTCT; human shPRMT5‐2, GCTATTGCACCTTGGAATTTC; mouse sh‐mPRMT5‐1, GGATGTGGTGGCATAACTTTC; mouse sh‐mPRMT5‐2, GCTAGAGAACTGGCAGTTTGA; human shSmad7, GTGCAGATCAGCTTTGTGA; nonspecific shCtrl, GGATAATGGTGATTGAGATGG. The pLKO.1‐shRNA plasmid together with the packaging plasmids was then transfected into HEK293T cells. After 48 h, viral supernatants were collected and filtered through a 0.45 µm filter, mixed with polybrene  were isolated from cells using TRIzol reagent (Sigma) and reverse‐transcribed to cDNA using transcriptor reverse transcriptase (Roche). cDNA was diluted and subject to qRT‐PCR analysis using SYBR green PCR Master Mix (Applied Biosystems). qRT‐PCR primers were listed as follows: h‐PRMT5‐Forward, 5’‐CTGTCTTCCATCCGCGTTTCA‐3’ and h‐PRMT5‐Reverse, 5’‐GCAGTAGGTCTGATCGTGTCTG‐3’; h‐Smad7‐Forward, 5’‐TTCCTCCGCTGAAACAGGG‐3’ and h‐Smad7‐Reverse, 5’‐CCTCCCAGTATGCCACCAC‐3’; h‐CDC25B‐Forward, 5’‐GCATGGAGAGTCTCATTAGTGC‐3’ and h‐CDC25B‐Reverse, 5’‐CTCCGCCTCCGCTTATTCT‐3’; h‐CDC25C‐Forward, 5’‐ATGACAATGGAAACTTGGTGGAC‐3’ and h‐CDC25C‐Reverse 5’‐GGAGCGATATAGGCCACTTCTG‐3’; h‐CyclinB1‐Forward, 5’‐AAGAGCTTTAAACTTTGGTCTGGG‐3’ and h‐CyclinB1‐Reverse, 5’‐CTTTGTAAGTCCTTGATTTACCATG‐3’.Co‐immunoprecipitation (co‐IP) was carried out using antibodies and protein A sepharose  or streptavidin beads. After several times washes, precipitated proteins were eluted in SDS loading buffer and separated by SDS‐PAGE, transferred onto PVDF membranes (Millipore), and detected in Western blotting with appropriate antibodies.−3m NH4HCO3/25% methanol, pH 8.0). Gel pieces were reduced with 10 × 10−3m DTT for 60 min at 56 °C and alkylation with 55 × 10−3m iodoacetamide for 45 min. Gel pieces were washed with digestion buffer  twice, dehydrated with acetonitrile, and then dried in a speed‐vac. Gel pieces were rehydrated with trypsin solution  and incubated overnight at 37 °C. Digested peptides were extracted from gel pieces with elution buffer 1 , elution buffer 2 , sequentially. Gel pieces were dehydrated with acetonitrile twice, and all of the supernatants were combined. Peptides solution was dried in a speed vac, digested peptides were resuspended with 5% formic acid and desalted with StageTip.Protein gel bands were cut into small pieces and destained with destaining buffer , followed by twenty HCD MS/MS scans at R = 15 000, NCE = 27, with an isolation width of 1.6 m/z. The AGC targets for MS1 and MS2 scans were 1 × 106 and 5 × 104, respectively, and the maximum injection time for MS1 and MS2 were 20 and 45 ms, respectively. Precursors of +1, +8 or above, or unassigned charge states were rejected; exclusion of isotopes was disabled; dynamic exclusion was set to 45 s. Mass spectrometry data were searched by MaxQuant.Digested peptides were loaded on the analytical column  with Easy‐nLC 1200 system. Samples were analyzed with a 60 min gradient at a flow rate 300 nL min In short, GST and GST‐fused Smad7 were prepared from Escherichia coli strain DE3 and purified using glutathione‐sepharose beads, then incubated with HEK293T cells expressing MYC‐tagged PRMT5/MEP50. PRMT5/MEP50 proteins associated with purified GST and GST‐fused Smad7 retrieved on the beads were analyzed by using Western blotting.GST pull‐down assays were carried out as described previously.45 In −3m tris–HCl pH8, 0.01% Triton X‐100 (Sigma), 5 × 10−3m DTT and 100 × 10−6m SAM (Shenggong) at room temperature for 2 h. SDS loading buffer was added to end the reaction, and followed by Western blotting analysis.HEK293T cells cultured in 10 cm dishes were transfected with SFB‐tagged Smad7 or MYC‐tagged PRMT5/MEP50 for 36 h. Cell lysates were immunoprecipitated by streptavidin beads or MYC antibody. The immunoprecipitated protein was incubated in the reaction buffer containing 20 × 103 cells, 100 µL per well, three replicates). 10 µL of the CCK‐8 solution was added to each well of the plate and continued to incubate in a humidified incubator for 2 h. Absorbance at 450 nm was measured using a microplate reader.A549 cells were split into 96‐well plates (1 × 103 cells each well). Cell culture medium was refreshed every other day. After 2 weeks, cells were stained with 0.1% crystal violet and recorded.A549 cells were plated in a six‐well plate  was used to test cell migration. A549 cells were serum‐starved overnight. 1 × 10A549 stable cells were harvested for total RNA extraction with TRIzol (Invitrogen). RNA samples were then subject to mRNA‐Seq library preparation using the VAHTS mRNA‐seq V3 Library Prep Kit for Illumina (Vazyme). Barcoded libraries were pooled and sequenced on a HiSeq X Ten system (Illumina) to generate 150 bp paired‐end reads. Sequencing reads were mapped to the human genome (hg19) using Tophat v2.1.1. Only uniquely mapped reads  were kept for subsequent analyses using Cufflinks v2.2.1, and gene expression levels were quantified as normalized FPKM (fragments per kilobase of exon per million mapped fragments). Genes with FPKM < 1 in all samples were excluded from the analyses. For the remaining genes, all FPKM values that are less than 1 were set to 1. Heatmaps were generated using the “pheatmap” package in R. GSEA was performed using the GSEA software (v3.0) with the hallmark gene sets (v7.0).296 cells were suspended in a mixture of 100 µL phosphate‐buffered saline (PBS) and 100 µL Matrigel (BD Bioscience) and then injected subcutaneously into 6 week old female nude mice in the right flank. Ten days later, mice were sacrificed and tumors were excised and measured.All animal studies were approved by Zhejiang University Committee for Experimental Animal Studies and Ethics. A total of 2 × 10t‐test. Data are shown as mean ± SD.Statistical analysis was performed using a two‐tailed Student's The authors declare no conflict of interest.Supporting InformationClick here for additional data file.