Recently, various immunosuppressant drugs have been shown to induce hair growth in normal hair as well as in alopecia areata and androgenic alopecia; however, the responsible mechanism has not yet been fully elucidated. In this study, we investigate the influence of mycophenolate (MPA), an immunosuppressant, on the proliferation of human dermal papilla cells (hDPCs) and on the growth of human hair follicles following catagen induction with interferon (IFN)-γ. IFN-γ was found to reduce β-catenin, an activator of hair follicle growth, and activate glycogen synthase kinase (GSK)-3β, and enhance expression of the Wnt inhibitor DKK-1 and catagen inducer transforming growth factor (TGF)-β2. IFN-γ inhibited expression of ALP and other dermal papillar cells (DPCs) markers such as Axin2, IGF-1, and FGF 7 and 10. MPA increased β-catenin in IFN-γ-treated hDPCs leading to its nuclear accumulation via inhibition of GSK3β and reduction of DKK-1. Furthermore, MPA significantly increased expression of ALP and other DPC marker genes but inhibited expression of TGF-β2. Therefore, we demonstrate for the first time that IFN-γ induces catagen-like changes in hDPCs and in hair follicles via inhibition of Wnt/β-catenin signaling, and that MPA stabilizes β-catenin by inhibiting GSK3β leading to increased β-catenin target gene and DP signature gene expression, which may, in part, counteract IFN-γ-induced catagen in hDPCs. A hair follicle (HF) is an organ composed of epithelial and mesenchymal tissues. The post-natal hair follicle undergoes a cycle of growth (anagen), regression (catagen) and rest (telogen) . The recin vitro.Interferon (IFN)-γ is among the growing number of cytokines involved in the regulation of HFs cycling ,6,7,8. FThe Wnt/β-catenin pathway is one of the most important elements of hair growth regulation. β-catenin is markedly activated in DPCs during anagen ,13 and iMycophenolate (MPA) is an immunosuppressant drug used to prevent rejection in organ transplantation. Recent studies have demonstrated stimulation of hair growth by immunosuppressants such as cyclosporin A (CsA) and tacrolimus ,18. Repein vitro using cultured hDPCs and organ cultures of human hair follicles with catagen induced by IFN-γ treatment in both. We investigated which signaling pathways mediate IFN-γ catagen induction in hDPCs. We then studied how MPA antagonizes the IFN-γ-induced catagen-like changes in these cells. Since β-catenin activity in DPCs is implicated in mediating anagen duration, we investigated whether IFN-γ inhibits the β-catenin pathway and whether MPA activates the β-catenin pathway after catagen induction by IFN-γ in hDPCs. Finally, we investigated the effect of MPA on human HFs cultured ex vivo that were treated with IFN-γ to induce catagen-like changes.The second aim of this study was to investigate the effects of MPA on human hair growth In order to determine the effect of MPA on proliferation of hDPCs we performed the MTT assay 3 days after culture in the presence or absence of IFN-γ. As shown in We examined whether β-catenin signaling is regulated by MPA in hDPCs. As shown in Next, cellular localization of β-catenin was examined after treating hDPCs for 24 h with MPA alone, IFN-γ alone, or MPA and IFN-γ together, with MNX as a positive control. In IFN-γ-treated cells, β-catenin was very weakly detected in the nuclei. However, MPA (100 nM) induced nuclear translocation of β-catenin in hDPCs to a similar extent as the positive control 1 µM MNX c.GSK3β is an upstream effector of β-catenin. As shown in Since Dickkopf (DKK)-1 promotes regression of hair follicles by blocking Wnt/β-catenin signaling , we inveWe examined changes in the expression of β-catenin target genes and DP signature genes by real-time PCR using hDPCs treated with MPA and/or IFN-γ for 24 h. Expression of Axin-2 was used as an indicator of β-catenin signaling pathway activity, which promotes the phosphorylation and degradation of β-catenin . In hDPCTGF-β2 gene and protein expression in human HFs suggesting that IFN-γ induces catagen, at least in part, via up-regulation of TGF-β2 [Since IFN-γ significantly enhances f TGF-β2 , we examex vivo culture of whole human scalp HFs. In this study, 350 HFs from three different individuals were cultured with MPA, IFN-γ or MPA plus IFN-γ for 10 days. Elongation of each hair shaft was measured every third day. MNX and vehicle served as positive and negative controls, respectively. As shown in ex vivo model of HFs catagen induction. With co-incubation of MPA (100 nM) and IFN-γ (100 ng/mL), MPA significantly abrogated IFN-γ-induced growth inhibition of cultured HFs ex vivo.In order to examine the effect of MPA in the presence or absence of IFN-γ at the organ level, we performed et al. showed that CsA at high doses induces anagen in resting  follicles [et al. reported that calcineurin activity is associated with hair keratinocyte differentiation in vivo, affecting nuclear factor of activated T cells (NFAT1) activity [ in vivo by CsA enhances hair growth [Various immunosuppressant medications have been shown to stimulate hair growth in normal hair and in conditions of abnormal hair growth such as alopecia areata and androgenic alopecia. Paus ollicles , and inhollicles  and chemollicles . High doollicles . Furtheractivity . Treatmer growth . Howeverr growth ,36,37,38 et al. previously reported that IFN-γ up-regulates TGF-β2 in the lower hair bulb, suggesting that the catagen induction by IFN-γ may, at least in part, be due to up-regulation of other potent catagen inducers such as TGF-β2 [In this study, we demonstrated that MPA increases the expression of β-catenin target genes and some DP signature genes such as ALP, Axin2, IGF-1, and FGF7. We also demonstrated that MPA suppresses TGF-β2, which has been known to be a potent catagen promoting factor in murine  and humas TGF-β2 . In this et al. reported that deletion of the β-catenin gene in DPCs results in reduced proliferation of matrix keratinocytes surrounding the DP, thus fewer progeny cells that generate the hair shaft, and also reported that inactivation of β-catenin within the DPCs of fully developed HFs causes premature induction of the catagen phase of the hair cycle in mice [Enshell-Seijffers in mice . Wnt/β-c in mice ,44. In t2 activate the Wnt/β-catenin pathway in vitro and in vivo to promote hair growth [In the absence of Wnt, β-catenin is ordinarily phosphorylated in the cytoplasm by glycogen synthase kinase (GSK)-3, a serine/threonine protein kinase encoded by GSK-3α and β, and the resulting phospho-β-catenin is tagged by ubiquitin and then destroyed by the proteasome. However, when the Wnt protein binds to cell surface receptors of the Frizzled family, GSK3β is inhibited and a pool of cytoplasmic β-catenin stabilizes leading to its translocation into the nucleus resulting in transcription of target genes. Recent studies have shown that the GSK3β inhibitor BIO restores the expression of ALP and IGF-1, which are markers for hair follicle induction in DPCs, leading to support of the hair bulb and promotion of hair growth . Since tr growth ,48,49. T et al. reported that mice expressing high levels of DKK-1 in the skin display an early and complete block in the development of HFs, suggesting an inhibitory role of DKK-1 on hair growth [et al. reported that DKK-1 is inducible by dihydrotestosterone (DHT) and that the level of DKK-1 is elevated in the scalp of patients with male pattern baldness compared to normal [Andlr growth . Kwack eo normal , suggesto normal . InteresZ,3'E)-6-bromoindirubin-3'-oxime) were purchased from Sigma Chemical Co. . Human IFN-γ was purchased from PEPERTECH .MPA was synthesized at IKSU CO., LTD. . 3--2,5-diphenyltetrazolium bromide, dimethyl sulfixide (DMSO), MNX, and GSK3 inhibitor BIO . Cultured hDPCs of early passage were used for experiments, and were maintained at 37 °C in a humidified atmosphere with 5% COet al [4 cells/well into 96-well plates and cultured for 24 h. The cells were then treated with MPA, IFN-γ, or MNX for 24 or 96 h. The samples were assessed by measuring absorbance at 540 nm with an ELISA plate reader .Cell viability was determined using a MTT assay that was performed by a slight modification of the method described by Wasserman et al . BrieflyEach cell lysate containing 20 µg of protein was analyzed by Western blotting using the appropriate antibodies to detect protein expression. Antibodies specific to GAPDH and ALP were purchased from Santa Cruz Biotechnology, Inc. . DKK-1 and p-GSK3β were purchased from Abcam Inc. . Antibodies against phospho-β-catenin and β-catenin were purchased from Cell Signaling Technology . Western blot was detected by chemoluminescence .Real-time PCR was performed with a C1000™ Thermal Cycler  using SYBR Green . β-catenin, Axin-2, BMP-4, BMP-6, IGF-I, IGFBP-5, FGF-7, FGF-10, TGFβ2, ALP and GAPDH oligonucleotide primers were obtained from Bioneer  and Cosmo Genetech Co, Ltd. . The primer sets used were as follow: Axin-2 sense, GCAACTCAGTAACAGCCCGA; Axin-2 antisense, AAGTCAGCAGGGGCTCATCT; BMP-4 sense, CACTGGCTGACCACCTCAAC; BMP-4 antisense, GGCACCCACA-TCCCTCTACT; BMP-6 sense, AACCAACCACGCGATTGTG; BMP-6 antisense, AAGTCTCATCGTCCCACCTC; IGF-I sense, CTCTTCTACCTGGCGCTGTG; IGF-I antisense, CATACCCTGTGGGCTTGTTG; IGFBP-5 sense, AGCAAGTCAAGATCGA-GAGAGA; IGFBP-5 antisense, TTCTTTCTGCGGTCCTTCTTCA; FGF-7 sense, TTGTGGCAATCAAAGGGGTG; FGF-7 antisense, CCTCCGTTGTGTGTCC-ATTTAGC; FGF-10 sense, TTCAAGGAGATGTCCGCTGG; FGF-10 antisense, GATGCTGTACGG-GCAGTTCT; ALP sense, CAAACCGAGATACAAGCACTCCC; ALP antisense, CGAAGAGACCCAATAGGTAGTCCAC; GAPDH sense, TGGGTGTGAACCATGAGAAG; and GAPDH antisense, GCT-AAGCAGTTGGTGGTGC.4 cells/500 µL were seeded into chamber slides, serum-starved for 24 h, and then treated with MPA, IFN-γ, and MNX for 48 h. These cells were then fixed in 4% paraformaldehyde for 30 min. After washing with DPBS, the cells were permeabilized with 0.1% Triton-X100 in PBS for 10 min at room temperature and then blocked with 5% BSA in 0.05% Triton X-100 for 30 min at room temperature. The samples were incubated with β-catenin antibody (1:200 dilution) at 4 °C overnight. They were then washed two times with PBS and four times with distilled water followed by incubation with a FITC-conjugated secondary goat anti-mouse IgG (1:200 dilution) in 5% BSA blocking solution for 2 h at room temperature. All samples were counterstained with 4'6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. Representative images were taken with a microscope  at 400×.hDPCs at 1 × 102 in 500 μL of basal medium supplemented with insulin 10 μg/mL, hydrocortisone 10 ng/mL, streptomycin 10 μg/mL, and penicillin 100 U/mL according to the Philpott’s method as described [Anagen HFs from human scalp skin specimens was obtained. The medical ethical committee of the Chung-Ang University approved specimen collection . Four hair follicles per well in 24-well plates were cultured in Williams E medium at 37 °C in a humidified atmosphere with 5% COescribed . Each exp), indicated by asterisks. p < 0.05 was considered to be significant.Results are expressed as mean ± standard deviation (SD). For statistical analyses, ANOVA with probabilities was performed for both the overall significance and the pairwise comparison (In conclusion, we demonstrate that IFN-γ induces catagen-like changes in hDPCs and in hair follicles via inhibition of Wnt/β-catenin signaling, and that MPA stabilizes β-catenin by inhibiting GSK3β leading to increased β-catenin target gene and DP signature gene expression, which may, in part, counteract IFN-γ-induced catagen in hDPCs.


Small-molecule antagonists of Wnt/β-catenin signaling are attractive candidates for developing effective therapeutics for CRC. In this study, we have identified a novel Wnt signaling inhibitor, isopropyl 9-ethyl-1- -9H-pyridoindole-3- carboxylate (Z86). Z86 inhibited Wnt reporter activities and the expression of endogenous Wnt signaling target genes in mammalian cells and antagonized the second axis formation of Xenopus embryos induced by Wnt8. We showed that Z86 treatment inhibits GSK3β (Ser9) phosphorylation, leading to its overactivation and promoting the phosphorylation and degradation of β-catenin. In vitro, Z86 selectively inhibited the growth of CRC cells with constitutive Wnt signaling and caused obvious G1-phase arrest of the cell cycle. Notably, in a nude mouse model, Z86 inhibited dramatically the xenografted tumor growth of CRC. Daily intraperitoneal injection of Z86 at 5 mg/kg resulted in >70% reduction in the tumor weight of HCT116 cell origin that was associated with decreased GSK3β (Ser9) phosphorylation and increased β-catenin phosphorylation. Taken together, our findings provide a novel promising chemotype for CRC therapeutics development targeting the canonical Wnt signaling.Wnt signaling pathway is aberrantly activated in a variety of cancers, especially in colorectal cancer (CRC), because of mutations in the genes encoding adenomatous polyposis coli (APC),  In the absence of Wnt ligands, β-catenin is phosphorylated by a destruction complex including adenomatous polyposis coli (APC)/Dsh/Axin/GSK3β that is then ubiquitinated and degraded through the proteasome pathway, keeping β-catenin at a low level in the cytoplasm. Once Wnt signaling pathway is activated, glycogen synthase kinase-3β (GSK3β) is inhibited and cytosolic β-catenin becomes stabilized. The accumulated β-catenin translocates to the nucleus and interacts with T-cell factor (TCF)/lymphoid enhancer factor (LEF) that activates transcription of Wnt target genes involved in cell proliferation and cell-fate determination.2 Numerous studies suggest that aberrant activation of the Wnt/β-catenin signaling pathway plays an important role in human tumorigenesis.3 In colon cancer, mutant APC and β-catenin highlighted the implications of aberrant Wnt/β-catenin signaling in the development and progression of this malignant phenotype.4β-Catenin mutations and elevated levels of nuclear β-catenin, both hallmarks of active Wnt signaling, have also been reported in colorectal cancer (CRC).6 The abnormal activation of Wnt signaling increased the expression of Wnt target genes such as cyclin D1,8 c-MYC 9 and survivin10 that are involved in proliferation, apoptosis and cell cycle deregulation in many human malignancies. Accordingly, small-molecule antagonists of Wnt/β-catenin signaling have become attractive candidates for developing effective therapies for CRC.12The Wnt signaling transduction pathway plays a critical role in embryonic developmental processes, tissue homeostasis and tumorigensis.β-catenin-mediated axis duplication in Xenopus embryos.14 Through cell-based transcriptional reporter assay screening, additional Wnt inhibitors were identified that works through inhibiting the interaction between β-catenin and TCF/LEF transcriptional complexes in CRC.15–18 Recently, several small-molecule Wnt inhibitors targeting the destruction complex composed of APC, Axin, GSK3β and other proteins were discovered. Huang et al.19 found that small-molecule compound XAV939 promoted the phosphorylation and subsequent degradation of β-catenin by stabilizing of Axin, thus causing inhibition of Wnt signaling and colorectal tumorigenesis. Several other studies have also confirmed the effectiveness of suppression of APC mutation-driven colorectal tumor growth through inhibition of Wnt signaling pathway by stabilizing Axin.20–22Based on assays of the transcriptional complexes, several small molecules were first identified as Wnt inhibitors that inhibited CRC cell proliferation and interfered with β-carboline structure-type compound. This type of compounds was first derived from natural alkaloids. In recent several decades, plenty of novel β-carboline-type compounds were designed and synthesized based on significant antitumor activity in vitro.24 Our study found that Z86 inhibited Wnt signaling through activation of GSK3β, leading to increased phosphorylation and degradation of β-catenin. Z86 reduced the secondary body axis formation induced by Wnt8 in Xenopus embryos. In vitro, Z86 exhibited selective inhibitory effects on the proliferation of colon cancer cells, and dramatically suppressed tumor growth of CRC HCT116 xenografts in vivo, both of which were mediated by inhibition of Wnt signaling. The identified chemotype Z86 may serve as a promising start point for the development of novel CRC therapeutics and deserve further evaluation.In the present study, we have identified a novel Wnt signaling inhibitor, isopropyl 9-ethyl-1--9H-pyrido-indole-3-carboxylate (Z86), that belongs to β-carboline structure type as a new class of inhibitors of Wnt signaling. Followed by this discovery, a library of β-carboline derivatives (∼150 compounds) were synthesized (50) values of 6.0, 7.5 and 2.8 μM, respectively. However, Z83 without substituents at indole N-9 position only exhibited weak activity with IC50 values of >20 μM. These results suggested that the N-9 substituents of β-carboline played an important role in the Wnt inhibition activity. Furthermore, compounds with a bulky group possess a better activity than with a smaller group like methyl or ethyl ester substituent at C-3 position. To test the specificity of the inhibitory activity of Z86 on Wnt signaling, we performed nuclear factor (NF)-κB reporter (NF-κB-Luc) activity assays. As shown in κB signaling at the concentration that significantly inhibited the SuperTopflash activity in HEK293T cells.To identify compounds that inhibit the canonical Wnt signaling pathway, a chemical library consisting of 4000 chemically diverse compounds was screened. Our screening led to the identification of thesized . The inhWnt1 transiently transfected HEK293T cells (β-catenin), in which the Wnt/β-catenin signaling pathway is constitutively activated. As in Wnt1-transfected HEK293T cells, Z86 efficiently inhibited the Topflash activity in both cancer cell lines in a dose-dependent manner after 24 h of treatment , Z86 treatment reduced β-catenin protein levels time dependently, with the phosphorylation of β-catenin  increased markedly. Consistently, similar effects were observed in SW480 and HCT116 CRC cells , which can no longer be phosphorylated by GSK3β and becomes stabilized,27–29 was transfected into HCT116 cells, the inhibition of Wnt signaling by Z86 was efficiently rescued, in a dose-dependent manner, suggesting that Z86 likely works upstream of β-catenin  that controls the phosphorylation and degradation of β-catenin, and functions as a negative regulator of Wnt signaling pathway.30–32 Therefore, we further investigated whether GSK3β is involved in Z86-promoted β-catenin phosphorylation and degradation in colon cancer cell lines. Interestingly, phosphorylated GSK3β (Ser9), the inactive status, significantly reduced upon Z86 treatment in HEK293W cells as well as in HCT116 and SW480 cells, with slight increase of the total level of GSK3β protein  in our study (data not shown), consistent with previous study.33 We then tested whether LiCl or dominant-negative kinase-inactive GSK3β (DNGSK3β)30 could rescue the inhibitory activity of Z86 on the Wnt signaling. HEK293T cells were transiently transfected with Wnt1 and DNGSK3β or pretreated with LiCl, respectively, before the cells were treated with Z86, and then Wnt reporter activities were monitored. Z86 efficiently antagonized Wnt1-induced activation of the canonical Wnt signaling but exhibited much weaker inhibitory activity on the Topflash activity stimulated by LiCl and, furthermore, lost the inhibitory activity completely when the Wnt signaling was stimulated with DNGSK3β -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. As shown in β-catenin/TCF and the dominant-negative TCF causes cells to arrest in the G1 phase of the cell cycle. Previous works provided evidences that mutant β-catenin produces high levels of cyclin D1 messenger RNA and protein constitutively in colon cancer cells.8 Z86 reduced the expression of cyclin D1 at both the mRNA and protein levels in a dose- and time-dependent manner in HCT116 and other colon cells  and vehicle daily for 21 days. The results showed that 5 mg/kg of Z86 dramatically reduced the growth of HCT116 xenografts in both tumor volume  and increased the levels of phospho-β-catenin (Ser33/Ser37/Thr41) in the tumor tissues compared with the vehicle-treated group. The total level of GSK3β increased slightly and that of β-catenin decreased as compared with the control groups . Other reagents unless otherwise indicated were purchased from Sigma-Aldrich . Lipofectamine 2000 was from Invitrogen . Dual-luciferase reporter assay system was purchased from Promega . Tumor necrosis factor-α (TNFα) was from PeproTech  and LiCl was purchased from Sigma-Aldrich. QuantiTect SYBR Green PCR Kit was obtained from QIAGEN .RPMI-1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotics–antimycotics solution and trypsin–EDTA were purchased from HyClone . Complete protease inhibitor cocktail was purchased from Roche Applied Science . Mouse monoclonal anti-Renilla) was from Promega. The SuperTopflash, pCS-mWnt1, human β-catenin and β-catenin (S37A) plasmids were gifted by Dr. Wei Wu . Xenopus Wnt8 and dominant-negative GSK3β (K85R) expression vectors were gifted by Dr. Aaron Zorn . NF-κB-luc was from Beyotime Biotechnology .The pF9A CMV hRluc-neoFIexi reporter plasmid (μg/ml streptomycin) . Normal colonic epithelial cell line (CCD-841-CoN) was kindly gifted by Professor Lin Li  and was maintained according to the recommendations of ATCC. All the cells were incubated at 37 °C, 5% CO2 in a humidified atmosphere, and were passaged once or twice a week.Colon cancer cell lines , human normal bronchus epithelial cell line (Beas-2B) and HEK293T were purchased from ATCC . Cells were cultured in medium , supplemented with 10% FBS and 1% antibiotics  was generated in HEK293 (named HEK293W). This cell-based screening strategy was sensitive to Wnt modulators as previously reported.49 Briefly, HEK293W cells were maintained in DMEM medium containing 200 μg/ml G418 and 100 μg/ml Hygromycin B (Sigma-Aldrich), respectively. Luciferase assay was carried out 24 h after compounds addition using SteadyGlo reagent (Promega). For the primary screen, compounds with the inhibition rate of >70% with concentration at 20 μM in 0.1% dimethylsulfoxide (DMSO) were identified as positive hits. Secondary screenings were carried out further with successive concentrations to confirm inhibitory activity with IC50 of the positive hits.To identify novel small-molecule inhibitors of the Wnt signaling pathway, cell line with stable expression of Wnt3a and dual luciferase reporters  and 10 ng of pF9A CMV hRluc-neoFIexi using Lipofectamine 2000 according to the manufacturer’s instructions. Each well of HEK293T cells was transfected with 200 ng of plasmids in total, including 80 ng of Topflash, 10 ng of Renilla and 10 ng of β-catenin or 64 ng of Wnt1 and other plasmids as indicated. At 3 h after transfection, cells were incubated with test compounds for 24 h. Cells were lysed and luciferase activity was measured with Dual-Luciferase Reporter Assay System (Promega). Transfection efficiency was normalized by the values of Renilla luciferase activity as previously described.17 All experiments were done independently in triplicate and the results reported are the means and S.D.s.HEK293T, SW480 and HCT116 cells were seeded in 96-well plates containing 100 17 For quantitative, all gene transcripts were done by real-time PCR and two-step RT-PCR using SYBR Green I (QIAGEN). The values of Axin2 and cyclin D1 were shown against the value of β-actin that was used as the control. Primer sequences are provided in Total RNAs were extracted from cultured cells with TRIzol according to the manufacturer’s instructions (Invitrogen). cDNA synthesis, reverse transcription and PCR were performed as previously described.β-catenin in SW480 cells was performed as previously described.49 Western blots were performed as described previously.17 Primary antibodies used include: phospho-β-catenin (Ser33/37/Thr41), phospho-GSK3β (Ser9), GSK3β, Axin2 , Survivin, β-catenin (BD Biosciences), cyclin D1  and β-actin (Sigma-Aldrich).Cells seeded in six-well plates were subjected to different treatments and the immunofluorescence staining of 17 GI50 values, the concentrations for 50% cell growth inhibition, were determined by nonlinear regression analysis using Table curve software .MTS assays were performed as previously described.5 cells/dish in 60 mm dishes) were incubated with test samples for 12, 24 and 48 h, respectively. All adherent cells were collected and washed twice with PBS. Cells were fixed with 70% ethanol overnight. Fixed cells were washed with PBS, and then stained with 50 μg/ml propidium iodide (PI) solution containing 50 μg/ml RNase A for 30 min in dark at room temperature. Fluorescence intensity was analyzed by FACSCalibur flow cytometer (BD Biosciences). The percentages of the distributions in distinct phases of cell cycle were determined using ModFIT LT V2.0 software .HCT116 cells . The in vitro fertilization, embryo culture, staging and microinjection were carried out as previously described.50 For microinjection, capped Wnt8 and DNGSK3β mRNAs were synthesized using SP6 mMessagemMachine kit  with their expression vectors linearized with NotI as templates. For secondary axis induction, 5 pg Wnt8 mRNA was injected into the ventral blastomeres of four-cell-stage embryos. For animal cap RT-PCR assays, 5 pg Wnt8 or 100 pg DNGSK3β were injected into the dorsal blastomeres of two-cell-stage embryos, and animal caps were excised from late blastula-stage embryos and then treated with test compounds for 6–8 h or not before being harvested. Primer sequences are provided in Adult 6) in 200 μl of PBS were implanted into the right flank of each nude mouse. Tumor size was measured in two dimensions with calipers and calculated using the formula (L×W2)/2, where L is length and W is width. After the tumor size reached ∼80 mm3, which occurred 7 days after cell injection, the mice were randomly assigned into three Z86 treatment groups with dose of 1, 2 and 5 mg/kg and a vehicle control group. Z86 in 100 μl PBS (with 1% DMSO and 5% Tween-80) was injected intraperitoneally into each mouse daily for 21 days. Mice were intraperitoneally injected with 100 μl PBS (with 1% DMSO and 5% Tween-80) as a vehicle control daily for 21 days. The mice body weight and tumor size was measured every 2 days and every 3 days, respectively. Tumors were removed from mice 23 days after treatment. Tumor weight was measured and tumor tissue was flash frozen at −80 °C for subsequent western blotting assay.Male nu/nu mice (3 weeks old) were purchased from Vital River Laboratory Animal Technology  and acclimated for 4 days. HCT116 cells . The difference was considered to be statistically significant when P<0.05.Data were presented as means and S.D.s for the indicated number of independently performed experiments. Figure data are shown as one representative of at least two independent experiments. Nonlinear regression analysis for calculation of GI


AbstractAlphitobius Stephens, 1829  from Africa and adjacent islands are revised. New species: Alphitobius capitaneussp. n. from Kenya. New synonyms: Cryptops ulomoides Solier, 1851, syn. n. of Alphitobius diaperinus ; Alphitobius rufus Ardoin, 1976, syn. n. of Alphitobius hobohmi Koch, 1953); Peltoides (Micropeltoides) crypticoides Pic, 1916, syn. n. of Peltoides (Micropeltoides) opacus , comb. n. Homonym: Alphitobius ulomoides Koch, 1953 = Alphitobius arnoldinom. n. New combinations from Alphitobius: Ulomoides basilewskyi , comb. n.; Peltoides (Micropeltoides) opacus , comb. n. Figures of all examined species are added and a species key is compiled.All species of the genus  Alphitobius Stephens, 1829 , namely Alphitobius diaperinus  and Alphitobius laevigatus , have a cosmopolitan synanthropic distribution. All the other species were described from mature habitats in Africa south of the Sahara, so probably this is also the native area of both synanthropic species. Diaclina Jacquelin du Val, 1861. Additional species were added by Alphitobius to Ulomoides and Peltoides respectively, providing also figures of all examined species, and compiling of a species key as well. Unfortunately, two taxa  from Madagascar remained unknown to the authors.Two species of the genus Alphitobius from Diaclina was doubtful for a long time. Alphitobius, narrower or as wide as eyes in Diaclina). However, some taxa described under Alphitobius have the canthus not broader than eyes  (Madagascar), Diaclina parallela  (Guinea), Micropedinus pullulus  (Hongkong), Menimus nitidulus  (Sri Lanka), Menimus punctulatus  (Sri Lanka), Ulomoides suffusus  (Cape Verde), Uloma sulcipennis  (Gabon), and Ulomoides xamiaphilus  . Alphitobius distinguendus Fairmaire, 1869 turned out to be a synonym of Cenoscelis pulla . Herein, we transfer one additional species from Alphitobius to Ulomoides: Ulomoides basilewskyi , comb. n., and one from Alphitobius to Peltoides (Micropeltoides): Peltoides opacus , comb. n.Some additional taxa were originally described under  Canadian National Collection of Insects, Ottawa, CanadaCNC Collection Dr. Rolf Aalbu, Dorado Hills, USA/CaliforniaCRA Collection Dr. Roland Grimm, Neuenbürg, GermanyCRG Museum für Naturkunde, Berlin, GermanyMNBPageBreak Muséum National d’Histoire Naturelle, Paris, FranceMNHN Museé Royal de l’Afrique Centrale, Tervuren, BelgiumMRAC Naturhistorisches Museum, Basel, SwitzerlandNHMB National Museum, Department Entomology, Prague, Czech RepublicNMP Staatliches Museum für Naturkunde, Stuttgart, GermanySMNS Ditsong National Museum of Natural History, Pretoria, South AfricaTMSA Zoologische Staatssammlung, Munich, GermanyZSMGebien, 1921http://species-id.net/wiki/Alphitobius_acutangulusSenegal, no further data, holotype NHMB (sex not examined).Sudan, Dilling, 20.–22.III.1914, leg. Ebner, 1 ex. NHMB. – Sudan, N Darfur Prov., El Geneina, 4.–18.VI.1979, leg. I. Abuzinid, 8 ex. TMSA, 1 ex. CRG, 1 ex. MNB, 1 ex. SMNS (det. Bremer). – Burkina Faso (labelled as Ob. Volta), Pundu, Olsufiew, no further data, 4 ex. TMSA. – Chad, Massaguet, without date, leg. H. Franz, 1 ex. NHMB. – Chad, Deressia, near Lai, without date, leg. H. Franz, 1 ex. NHMB.“Senegal”.Senegal ; Sudan ; BurkinaAlphitobius ulomoidesCryptops ulomoides Solier, 1851, syn. n.) Koch, 1953 , Bulawayo, leg. G. Arnold, holotype TMSA (sex not examined).Somalia, Car-Car Mts., IX.1959, leg. C. Koch, 2 ex. TMSA (det. Ferrer). – Somalia, Gardo, 810 m, 22.X.1957, leg. G. Scortecci, 1 ex. TMSA. – Kenya, Witu, Lamu, Wangi, without date, leg. G. Denhardt, 1 ex. ZSM, 3 ex. MNB (det. Bremer). – Tanzania, Mgorogoro Prov. 10 km N Mikumi, 11.I.2007, leg. F. Kantner, 1 ex. SMNS.“Bulawayo”.Cryptops ulomoides Solier, 1851 from Chile is a junior synonym of Alphitobius diaperinus  (see below). Thus Alphitobius ulomoides Koch, 1953 is a homonym and must have a new name, arnoldi nom. n.PageBreakHymenoptera, and collector of the holotype.The new name is derived in honor of George Arnold (1881–1963), former curator in the “Rhodesia Museum” , specialist of African Zimbabwe, Congo ; SomaliaPageBreakhttp://zoobank.org/C18D8875-7C0A-46B4-8195-313B8DE1A2BEhttp://species-id.net/wiki/Alphitobius_capitaneusHolotype male: Kenya, Samburu Nature Reserve, Samburu River Lodge, 28.III.1988, leg. R. Grimm, CRG.Alphitobius capitaneus sp. n. is distinguished from its congeners by size and shape of body. A similar body shape, especially the shape of pronotum has Alphitobius lamottei Ardoin, 1963, but this species is smaller (body length ≤ 7.0 mm) and differs by the reddish colour, by much finer punctation of dorsal surface, by longer and less distinct serrate antennae with basal antennomeres more elongated, and by the somewhat more stretched and apically narrowed apicale of aedeagus.nd antennomere wider than long; 3rd elongate, one and a half times as long as wide; 4th only slightly, 5th to 10th distinctly wider than long and distinctly serrate; distal antennomere rounded, as wide as long. Pronotum transverse, width/length ratio 1.7; transverse convex, widest at base, shallowly arcuate narrowing to apex. Anterior margin shallowly emarginate, basal margin bisinuate, all margins finely bordered; lateral margins separated from discal convexity by a narrow submarginal depression. Anterior and posterior corners rectangular. Propleura densely covered with small seta bearing tubercles, only along outer margins nearly smooth. Prosternum rugosely punctured, prosternal apophysis bent down behind procoxae. Elytra convex, elongate oval with subparallel sides and densely punctured striae; scutellar striole absent; intervals much broader than striae, nearly flat on disc, becoming more and more convex laterally and distally; lateral margins in dorsal view visible nearly over entire length, only concealed around apex; base as wide as base of pronotum; humeral angles obtuse, distinct. Scutellum large, triangular. Mesoventrite roughly punctured, with shiny median carina in basal part; triangular apophysis raised upwards and excavate. Metaventrite shiny, with fine median sulcus, finely punctured on disc, somewhat more coarsely punctured laterally. Abdominal ventrites with very dense and fine punctation throughout, basally and laterally longitudinally wrinkled. Tibiae gradually and faintly dilated towards apex, without modifications. Aedeagus as in Body length 8.8 mm, width at widest point behind middle of elytra 3.6 mm. Elongate, blackish brown, matt; borders of pronotum, lateral borders of elytra, sutural interval, and scutellum paler reddish brown. Complete dorsal surface with very fine and dense punctation, punctures bearing a minute seta. Head sub-trapezoidal; outline continuous, not interrupted between clypeus and frons; apical margin of epistome shallowly emarginate in the middle; fronto-clypeal suture complete and linearly impressed. Eyes large, constricted by genal canthus, dorsal part smaller than ventral part. Genal canthus hardly projecting beyond contours of eyes, with the outlines nearly continuous with the outlines of the latter. Tempora strongly narrowed towards neck. Antennae thickened, not reaching the base of pronotum, with the seven distal antennomeres forming a kind of club; 2Capitaneus (Latin) means conspicuous by greatness, refers in this case to the body size.PageBreakhttp://species-id.net/wiki/Alphitobius_crenatusPhaleria crenata Klug, 1834Cataphronetis luctuosa Fairmaire, 1869, syn.Madagascar, no further data, 2 ex. TMSA. – Madagascar, Ambaton, no further data, 1 ex. SMNS. – S Madagascar, Midongy, no further data, 3 ex. SMNS. – C Madagascar, Katsepy (Majunga), 24.–31.XII.1997, leg. P. Pacholátko, 1 ex. SMNS. – Madagascar, Antananarivo Distr., Moramanga, 12.II.1995, leg. I. Jeniš, 1 ex. ZSM. – Madagascar, Toliaro Prov., Andohahela NP, Forêt d’Ambohibory, 300 m, 16.–20.I.2002, leg. Fisher, Grifswold et al., 1 ex. CRA. – Madagascar, Toliaro Prov., Cap Sainte Marie, 200 m, 11.–15.II.2002, leg. Fisher, Grifswold et al., 3 ex. CRA. – Madagascar, Toliara Prov., Forêt de Tsinjoriaky, 70 m, 6.–10.III.2002, leg. Fisher, Grifswold et al., 1 ex. CRA. – Madagascar, Toliara Prov., Tsimanampetsotsa NP, 25 m, 18.–22.III.2002, leg. Fisher, Grifswold et al., 1 ex. CRA.crenata), “Nossi-Bé” (luctuosa).“Madagascar” , eastern Africa, Comores, Seychelles, Aldabra Islands .http://species-id.net/wiki/Alphitobius_diaperinusTenebrio diaperinus Panzer, 1796Tenebrio ovatus Herbst, 1799, syn.Uloma opatroides Brullé, 1838, syn.Cryptops ulomoidesAlphitobius ulomoides Koch, 1953, arnoldi nom. n.) Solier, 1851, syn. n. , Valparaiso, Gay 15-43, 1 syntype of PageBreakF.-T. Krell, 3 ex. SMNS. – Ivory Coast, Adiopodoumé, 11.V.1988, leg. F.-T. Krell, 8 ex. SMNS. – Cameroon, Buea, 11.II.1980, leg. H. Schmalfuss & M. Schlegel, 6 ex. SMNS. – Guinea, Kouroussa, 16.XI.1995, leg. U. Lange, 1 ex. SMNS. – Congo, Tshuapa, Flandria, 1946/1947, leg. P. Hulstaert, 3 ex. TMSA. – Uganda, Kampala, V.1927, leg. H. Hargreaves, 1 ex. TMSA (det. Bryant). – Kenya, no further data, leg. Škulina, 1 ex. NMP. – Kenya, Voi (Tsavo), 8.–10.XI.1996, leg. M. Snížek, 1 ex. CRG. – Zambia, 30 km NE Livingstone, 1.XI.2002, leg. F. Wachtel,1 ex. CRG. – Zambia, 30 km NW Sesheke, 13.I.2010, leg. P. Schüle, 1 ex. SMNS. – Zambia, Western Prov., 7.6 km S Ushaa, 1050 m, 2.XII.2010, leg. F. Génier, 1 ex. CNC. – Angola, Cunene Prov., 10 km N Humbe, 3.XI.2011, leg. P. Schüle, 1 ex. SMNS. – Angola, Oshikoto Prov., 9 km S Ondangwa Nakambale Museum, 25.XI.2012, leg. P. Schüle, 1 ex. SMNS. – Namibia, Abachaus, XII.1949, leg. G. Hobohm, 1 ex. TMSA. – Namibia, Kaokoveld, 13 km W Ehombe Mt., 11.II.1975, leg. S. Endrödy-Younga & Schulze, 1 ex. TMSA. – Namibia, Etosha NP, Halali, 16.–17.XII.1993, leg. M. Uhlig, 1 ex. MNB. – NW Namibia, Epupa Falls, 660 m, 11.–12,.IV.2005, leg. W. Schawaller, 1 ex. SMNS. – S Namibia, Naukluft Park East, 1500 m, 7.–10.II.2010, leg. W. Schawaller, 1 ex. SMNS. – Botswana, Okavango, Maxwee Lagoon, VIII.1976, leg. Russel-Smith, 1 ex. TMSA. – South Africa, Northern Cape, Pofadder, 760 m, 4.X.1990, leg. W. Wittmer, 1 ex. NHMB. – South Africa, Limpopo, Krüger NP, 18.VI.1990, leg. L. Braack, 3 ex. TMSA. – South Africa, Limpopo, Amatola Farm NE Vivo, 1000 m, 15.–17.XII.2003, leg. W. Schawaller, 4 ex. SMNS. – South Africa, Gauteng, Pretoria Distr, Roodeplat, 8.–10.X.1960, leg. Neubecker, 1 ex. TMSA. – South Africa, Gauteng, Ezemvelo NR, 26.I.2004, leg. TMSA staff, 3 ex. TMSA. – South Africa, KwaZulu-Natal, Hluhluwe, 29.XI.1992, leg. S. Endrödy-Younga, 1 ex. TMSA. – South Africa, KwaZuluNatal, Ndumo, 21.XI.2002, leg. J. Harrison & R. Müller, 1 ex. TMSA. – South Africa, KwaZulu-Natal, Kosi Bay NR, 11.–17.XI.2002, leg. W. Schawaller, 1 ex. SMNS. – South Africa, Free State, Farm Abel 52, 4 km E Parys, 12.–13.I.1992, leg. M. Krüger, 6 ex. TMSA. – South Africa, Northern Cape, 70 km S Olifantshoek, Witsand NR, 1200 m, 4.–7.II.2012, leg. W. Schawaller, 2 ex. SMNS. – Madagascar, Kirindy Forest, 21.XI.1998, leg. R. Müller, 2 ex. TMSA. – Madagascar, Tsaratanana, Antsirasira, Morwato West, 26.XI.–3.XII.2001, leg. V. Dolin, 1 ex. SMNS. – Rodrigues Island, Anse aux Anglais, Port Mathurin, 22.IX.1995, leg. R. Fricke, 4 ex. SMNS.Somalia, Genale, 1935, leg. R. Ciferri, 3 ex. TMSA. – Sudan, Mt. Sangha, no further data, leg. Škulina, 1 ex. NMP. – Sudan, Wad Medani, 21.XII.1952, leg. W. Büttiker, 1 ex. TMSA. – Ghana, Ashanti Region, Kumasi, Nhiasu, 16.V.1987, leg. S. Endrödy-Younga, 1 ex. TMSA. – Nigeria, Ile-Ife, 7.VII.1988, leg. F.-T. Krell, 10 ex. SMNS. – Liberia, Bong Town, 23.III.1988, leg. diaperinus); “Valparaiso” (ulomoides).“Germanica” .Examination of the syntype of Cosmopolitan.PageBreakFairmaire, 1897http://species-id.net/wiki/Alphitobius_grandisMaterial of this taxon is unknown to the authors. The type is said to be 9 mm long , the ant“Madagascar”.Madagascar.Koch, 1953http://species-id.net/wiki/Alphitobius_hobohmiAlphitobius rufus Ardoin, 1976, syn. n.hobohmi TMSA (sex not examined). – Tanzania, Mts. Uluguru, Morogoro Campus Fac. Agriculture, 600 m, V./VI.1971, leg. J. Debecker, ♀ holotype rufus MRAC.Namibia (labelled as SWA), Abachaus, XII.1946, leg. G. Hobohm, holotype, 1 paratype rufus det. Bremer). – Kenya, Meru Distr., Mojwa, 1300 m, 3.IV.1987, leg. R. Mourglia, 1 ex. ZSM (rufus det. Bremer). – Tanzania, Moschi, no further data, 1 ex. ZSM. – Tanzania, Manyara Lake, XII.1961, leg. H. & B. Frey, 1 ex. NHMB. – Namibia, Windhuk, 1906, leg. F. Kunze, 1 ex. MNB (det. Ferrer). – Angola, Blé Prov., Chissamba Mission Station, 1440 m, 9.XI.2011, leg. R. Müller, 1 ex. TMSA. – Angola, Huila Prov., 10 km SW Cacula, 1550 m, 4.–6.XI.2011, leg. R. Müller & P. Schüle, 1 ex. TMSA, 3 ex. SMNS. – Angola, Huila Prov., 15 km S Caluquembe, 1620 m, 6.XI.2011, leg. R. Müller, 1 ex. TMSA. – Angola, Huila Prov., 3.5 km SW Negola, 8.XII.2012, leg. P. Schüle, 2 ex. SMNS, 2 ex. CRG. – South Africa, Limpopo, Naboomspruit, Torino Ranche, 15.I.1990, leg. S. Endrödy-Younga, 1 ex. TMSA. – South Africa, Limpopo, 10 km N Modimolle (Nylstroom), 1300 m, 10.XII.2008, leg. R. Müller, 1 ex. TMSA. – South Africa, Free State, Bothaville, Vaal River, 16.I.2003, leg. M. Snižek, 1 ex. SMNS. – South Africa, KwaZulu-Natal, SW Magudu, 4.–5.I.2009, leg. R. Müller & P. Schüle, 2 ex. SMNS, 2 ex. TMSA. – South Africa, KwaZulu-Natal, Ndumo NR, 100 m, 10.XII.2010, leg. R. Müller, 1 ex. TMSA.Ethiopia, Hararge Prov., Bisidimo, 1500 m, V.–VII.1984, leg. V. Meyer, 2 ex. ZSM. – Kenya, Meru Distr., Materi (Mitunguu), 800 m, 8.IV.1987, leg. R. Mourglia, 1 ex. SMNS (hobohmi), “Morogoro” (rufus).“Abachaus, Otjiwarongo” , and from Tanzania and Kenya  show no distinct external differences. The aedeagi of type specimens can not be compared, because the holotype of rufus is a female. Nevertheless, Alphitobius rufus is considered as a junior synonym of Alphitobius hobohmi.The Type specimens examined of PageBreakPageBreakAlphitobius hobohmi was collected together with Alphitobius viator.In some localities (for example Modimolle and Magudu) Namibia ; TanzaniKoch, 1953http://species-id.net/wiki/Alphitobius_karrooensisEastern Cape, Willowmore, 20.III.1919, leg. H. Brauns, holotype, 3 paratypes TMSA.Alphitobius leleupi). – Zambia, Western Prov., 3.8 km E Namushakende, 1100 m, 29.XI.2010, leg. F. Génier, 1 ex. CNC. – Zambia, Western Prov., 7 km S Mukokwa, 1100 m, 1.XII.2010, leg. F. Génier, 1 ex. CNC, 1 ex. SMNS. – Botswana, Okavango, Thamalakane, XII.1973, leg. P. Reavel, 1 ex. CRG. – Botswana, Chizwina, Francistown-Mosetse, 5.V.1995, leg. J. Harrison, 1 ex. SMNS. – Namibia (labelled as S. W. Africa), 3 miles NE Waterberg, 1420 m, 21.XII.1966, leg. E. S. Ross & K. Lorenzen, 1 ex. CRA. – Namibia (labelled as S. W. Africa), Okahandja, Farm Okaundua, 21.–29.XI.1933, leg. W. Krieg, 1 ex. NHMB. – South Africa, Northwest Prov., Bloemhof, 24.IV.1961, leg. F. Zumpt, 2 ex. NHMB. – South Africa, Kalahari Gemsbok Park, Nossob River, V.1956, TMSA Expedition, 12 ex. TMSA, 2 ex. SMNS. – South Africa, Kalahari Gemsbock Park, Mata-Mata, 18.XII.1974, leg. S. Endrödy-Younga, 1 ex. TMSA. – South Africa, Limpopo, Waterberg, Geelhoutbush Farm, 3.X.1995, leg. S. Endrödy-Younga & C. Bellamy, 2 ex. TMSA. – South Africa, Northern Cape, Garies, 14.XI.1948, leg. C. Koch, 1 ex. TMSA. – South Africa, Northern Cape, Witsand NR, 1160 m, 5.II.2012, leg. R. Müller, 1 ex. TMSA. – South Africa, Eastern Cape, Willowmore, I.1954, leg. F. Zumpt, 7 ex. NHMB, 2 ex. SMNS.Tanzania, Tabora, leg. Holtz, 1 ex. MNB , Ivory Coast ; LiberiaPageBreakhttp://species-id.net/wiki/Alphitobius_laevigatusOpatrum laevigatus Fabricius, 1781Tenebrio mauritanicus Fabricius, 1792, syn.Helops picipes Panzer, 1794, syn.Helops piceus Olivier, 1795, syn.Alphitobius granivorus Mulsant & Godart, 1868, syn.Cataphronetis striatulus Fairmaire, 1869, syn.Microphyes rufipes MacLeay, 1873, syn.Alphitobius ruficolor Pic, 1925, syn.Somalia, Eil , III./IV.1938, leg. S. Venzo, 1 ex. TMSA. – Niger, Maradi, 5.–7.VIII.1981, leg. R. Grimm, 1 ex. CRG. – Niger, Niamey, 9. –14.VIII.1989, leg. R. Grimm, 2 ex CRG. – Ghana, Ashanti Region, Kumasi, Nhiasu, 16.V.1967, leg. S. Endrödy-Younga, 1 ex. TMSA. – Cameroon, Longji, 1905, leg. H. Paschen, 1 ex. MNB. – Cameroon, Sasse-Buea, IV.–V.1951, collector unknown, 1 ex. CRA. – Congo, Oubangui-Chari, no further data, leg. Škulina, 1 ex. NMP. – Kenya, no further data, leg. Škulina, 1 ex. NMP. – Tanzania, Daressalam, Pangani, about 1930, leg. R. Regner, 6 ex. MNB. – Tanzania, Pangani to Tanga, VI.1891, leg. Conradt, 2 ex. MNB. – Tanzania, Massa to Tanga, 14.VII.–6.VIII.1891, leg. Conradt, 3 ex. MNB. – Madagascar, Fianarantsoa Prov., Ranamafona, 29.XI.–2.XII.1995, leg. I. Jeniš, 1 ex. CRG. – Madagascar, Ambovombe Distr., Faux Camp, I.1956, leg. C. Koch, 2 ex. TMSA. – Namibia, Fish River Canyon, Ai-Ais, 250 m, 19.XI.1993, leg. M. Uhlig, 1 ex. MNB. – South Africa, Limpopo, Krüger NP, Skukuza, 29.I.1994, leg. S. Endrödy-Younga, 1 ex. TMSA. – South Africa, Limpopo, Krüger NP, Skukuza, 8.–22.XII.2009, leg. J. Harrison, 2 ex. TMSA. – South Africa, Limpopo, Krüger NP, Skukuza, VIII.1992, leg. L. Braack, 10 ex. TMSA. – South Africa, Gauteng, Pretoria, 28.XI.1999, leg. B. Dombrowsky, 1 ex. TMSA. – South Africa, Cape Town, 1965, leg. Dickson, 2 ex. TMSA.“Noua Zelandia”.Cosmopolitan.Ardoin, 1963http://species-id.net/wiki/Alphitobius_lamotteiIvory Coast, Bingerville, V.1962, leg. J. Decelle, 1 ex. NHMB. – Guinea, Gbakoré, XII.1983, leg. C. Girard, 2 ex. TMSA (det. Bremer). – Guinea, Mt. Nimba, Keoulenta, 12.I.1984, leg. C. Girard & M. Lamotte, 1 ex. TMSA (det. Bremer). – The Gambia, Kabafita Forest Park, 19.VII.2000, leg. R. Grimm, 1 ex. CRG. – Central African Republic, 35 km E Ndele, 450 m, 18.V.2009, leg. J. Halada, 1 ex. SMNS.PageBreakPageBreak“Mt. Nimba”.Guinea ; Senegal ; Ivory CKoch, 1953http://species-id.net/wiki/Alphitobius_leleupiCongo, Massif de Kundelungu, 14.XII.1949, leg. N. Leleup, 29 paratypes TMSA, 2 paratypes SMNS, 1 paratype CRG, 1 paratype ZSM.Congo, 57 miles N Popokabaka, 3.VIII.1957, leg. E. S. Ross & R. E. Leech, 2 ex. CRA, 1 ex. SMNS.“Kundelungu”.Alphitobius parallelipennis was found, see remarks under that species.Among the type series in TMSA, the missing holotype of Congo, Kenya .PageBreakFairmaire, 1901http://species-id.net/wiki/Alphitobius_limbalisWithout any data, 1 female “cotype” NHMB (Gebien collection).Alphitobius luctuosus   without any data is really a type specimen. Other material of this taxon is unknown to the authors. The species is said to be similar to g, 1833) , but is “Bélumbé”.Madagascar.Bremer, 1985http://species-id.net/wiki/Alphitobius_lucasorumSudan, N Darfur Prov., El Geneina, 7.–15.VI.1978, leg. I. Abuzinid, 1 paratype SMNS, 1 paratype TMSA, 1 paratype ZSM. – Ghana, Northern Prov., Nyankpala, 15 km W Tamale, 1.–30.IV.1970, leg. S. Endrödy-Younga, 2 paratypes TMSA.“El Geneina”.Cameroon, Nigeria, Ghana, Senegal, Sudan  ; Chad B.Ferrer, 1983http://species-id.net/wiki/Alphitobius_nigerNone, see remarks.Alphitobius ulomoides, for diagnosis and figures see Unfortunately, this species was overlooked during the study, and was added here only during the review process. The species is said to be similar to “Lake Manyara”.Tanzania .PageBreakKoch, 1953http://species-id.net/wiki/Alphitobius_parallelipennisCongo, Lulua, Tshibamba, XII.1931, leg. G. F. Overlaet, holotype MRAC. – Congo, Elisabethville, 7.XI.1923, leg. C. Seydel, 1 paratype TMSA.Angola, Huila Prov., 75 km N Kaconda, 1640 m, 10.XI.2011, leg. R. Müller, 1 ex. TMSA. – Angola, Huila Prov., 10 km S Kacula, 1560 m, 12.–13.XI.2011, leg. R. Müller & P. Schüle, 1 ex. TMSA, 1 ex. SMNS. – Angola, Huila Prov., 15–20 km S Caluquembe, 6.–7.XI.2011, leg. R. Müller & P. Schüle, 2 ex. TMSA, 2 ex. SMNS. – Angola, Huila Prov., 3.5 km SW Negola, 8.XII.2012, leg. P. Schüle, 2 ex. SMNS. – Zambia, 150 km S Kasemba, 11.XI.2002, leg. F. Wachtel, 11 ex. CRG. – Zambia, 40 km NE Livingstone, Mabula Camp, 14.XI.2002, leg. F. Wachtel, 1 ex. CRG. – Zambia, Lusaka, Kafue River, 1200 m, 22.XI.–2.XII.1987, leg. R. Mourglia, 4 ex. ZSM. – Zambia, Northern Prov., Chipona Falls, 30 km S Chinsali, 5.–6.XII.2002, leg. F. & L. Kantner, 1 ex. SMNS. – Zambia, Copperbelt Prov., NW Kapiri Mposhi, 8.XII.2002, leg. F. & L. Kantner, 1 ex. SMNS. – Zambia, Central Prov., 25 km NE Lilemone, 1250 m, 5.XII.2010, leg. F. Génier, 6 ex. CNC. – Zambia, Lusaka Prov., 9 km E Mulalika, 1100 m, 24.XI.2010, leg. F. Génier, 1 ex. CNC. – South Africa, Limpopo, Krüger NP, Shingwedzi, 19.–20.XI.1961, leg. Vári & Rorke, 1 ex. TMSA. – South Africa, Limpopo , Manyeleti Game Reserve, 18.XI.1987, leg. T. van Viegen, 1 ex. TMSA.“Lulua, Tshibamba”.Alphitobius leleupi and Alphitobius parallelipennis in TMSA are mislabelled, and that the actual depository of the holotype of Alphitobius parallelipennis seems unknown, at least it is not present in MRAC as published in the original description. De Meyer (VII.2013 in an email to the senior author) confirmed, that the holotype is lacking in MRAC with the remark “non renvoyé par Koch”. During the last visit of the senior author in TMSA, the mixture of locality and type labels could be confirmed, and also corrected. The holotype of Alphitobius parallelipennis could be recognised without any doubts among the type series of Alphitobius leleupi, although mislabelled, and was transferred with correct secondary labels from TMSA to MRAC.Congo ; Angola,Koch, 1953http://species-id.net/wiki/Alphitobius_rugosulusNone, not in TMSA.PageBreak19.XII.2006, leg. F. Kantner, 1 ex. SMNS. – Tanzania, Dodomo Prov., 70 km N Dodoma, 1350 m, 17.XII.2006, leg. F. Kantner, 1 ex. SMNS.Ethiopia, Oromia, 6.5 km SE Chichilla, 1550 m, 17.V.2012, leg. F. Wachtel, 1 ex. CRG. – Tanzania, Dodoma Prov., 15 km N Dodoma, 1550 m, “Campi Simba”.Kenya ; EthiopiMulsant & Godart, 1868http://species-id.net/wiki/Alphitobius_viatorUloma rufula Fairmaire, 1883, syn.PageBreak10.–13.XII.2008, leg. W. Schawaller, 2 ex. SMNS. – South Africa, Limpopo, Waterberg, Geelhoutbosch Farm, 15.XII.1997, leg. C. L. Bellamy, 1 ex. TMSA. – South Africa, Limpopo, 15 km NE Klaserie, Guernsey Farm, 18.–30.XII.1985, leg. S. & J. Peck, 1 ex. TMSA. – South Africa, Mpumalanga, Booysendal Farm, 25.X.2000, leg. TMSA staff, 1 ex. TMSA. – South Africa, Gauteng, Tswaing, 17.II.2003, leg. TMSA staff, 1 ex. TMSA. – South Africa, Free State, Bothaville, 15.II.1898, leg. H. Brauns, 5 ex. TMSA. – South Africa, KwaZulu-Natal, SW Magudu, 4.–5.I.2009, leg. P. Schüle, 3 ex. SMNS.Ethiopia, Ambo, 3.XI.1990, leg. L. Medvedev, 1 ex. SMNS. – Sudan, North Dafur, El Geneina, 10.VII.1978, leg. I. Abuzinid, 1 ex. ZSM (det. Kaszab). – Ivory Coast, Bingerville, 1962–1964, leg. J. Decelle, 6 ex. MRAC (det. Ardoin). – Ivory Coast, Kafolo/Comoé, 21.IV.1988, leg. F.-T. Krell, 1 ex. SMNS. – Burkina Faso (labelled as Haute Volta), Ouagadongou, no date, leg. Škulina, 1 ex. NMP. – Guinea, N’Zerekoré, 1.–15.XI.1951, leg. S. H. Olsen, 4 ex. NHMB. – Guinea Bissau, Bolama, VI.–XII.1899, leg. L. Fea, 1 ex. NHMB. – Congo, Tshiobo, N’Goy, 3.–4.VII.1926, leg. A. Collart, 2 ex. TMSA, 1 ex. SMNS. – Central African Republic, Uam, Bosum, IV.–VI.1914, leg. Tessmann, 11 ex. MNB. – Central African Republic, 40 km SW Bandoro, 530 m, 14.IV.2010, leg. Halada, 1 ex. SMNS. – Cameroon, Jaunde, X.1914, leg. Tessmann, 1 ex. MNB. – Kenya (labelled as Afr. or.), Ikutha, no further dates, 7 ex. MNB. – Kenya, Mt. Elgon, eastern slope, 2200 m, 23.–27.III.1988, leg. H. J. Bremer, 1 ex. ZSM. – Tanzania, 2 miles SE Mkomazi, 1500 ft. (460 m), 7.I.1970, leg. M. E. Irwin & E. S. Ross, 1 ex. CRA. – Malawi, Salima, 5.–6.I.2002, leg. J. Bezděk, 1 ex. SMNS. – Zambia (labelled as N Rhodesia), Lochinvar, near Monze, 13.–26.X.1962, leg. F. Zumpt, 3 ex. NHMB. – Zambia, Northern Prov., Chipona Falls, 30 km S Chinsali, 5.–6.XII.2002, leg. F. Kantner, 1 ex. SMNS. – Zambia, 150 km S Kasempa, 11.XI.2002, leg. F. Wachtel, 1 ex. CRG. – Zimbabwe, 60 km N Bulawayo, Maraposa Road, 3.XII.1998, leg. M. Snižek, 1 ex. ZSM. – Botswana, 10 km N Martin’s Drift, 7.I.2010, leg. P. Schüle, 1 ex. SMNS. – Angola, Sá da Bandeira, 2.X.1949, leg. B. Malkin, 1 ex. CRA. – Angola, Huambo Prov., 75 km N Caconda near Cuima, 10.–11.XI.2011, leg. P. Schüle, 1 ex. SMNS. – South Africa, Limpopo, Manyeleti Game Reserve, 17.–19.XI.1987, leg. T. van Viegen, 2 ex. TMSA. – South Africa, Limpopo, Naboomspruit, Torino Ranche, 15.I.1990, leg. S. Endrödy-Younga, 1 ex. TMSA. – South Africa, Limpopo, Kruger NP, Skukuza Research Camp, 1.–16.XII.2010, leg. J. Harrison, 1 ex. TMSA. – South Africa, Limpopo, Amatola, Scott Farm, 26.I.1998, leg. R. Müller, 1 ex. TMSA. – South Africa, Limpopo, Thabazimbi, 16.XI.2002, leg. F. Wachtel, 4 ex. CRG. – South Africa, Limpopo, N Makopane (Potgietersrus), Thabaphaspha Farm, 1400 m, 13.–14.XII.2003, leg. R. Müller & W. Schawaller, 1 ex. SMNS, 1 ex. TMSA. – South Africa, Limpopo, Mapungubwe, Little Muck NR, 500 m, 6.–9.XII.2008, leg. W. Schawaller, 1 ex. SMNS. – South Africa, Limpopo, 10 km N Modimolle (Nylstroom), Kuthaba Bush Lodge, 1300 m, viator), “Abyssinie” (rufula).“Marseille, importée” , or of hobohmi Koch, 1953 . In some localities (for example Modimolle and Magudu) Alphitobius viator was collected together with Alphitobius hobohmi.We could not clear, if Tropical and southern Africa, the holotype was imported to Marseille in southern France .Alphitobius grandis Fairmaire, 1897 and Alphitobius limbalis Fairmaire, 1901 from Madagascar could not be enlightened by the authors, thus both taxa are not included herein. Also not included is Alphitobius niger Ferrer, 1983, which was overlooked during the study and included herein only during the review process. Compare also Unfortunately, the taxonomic status of comb. n.http://species-id.net/wiki/Ulomoides_basilewskyiAlphitobius basilewskyi Ardoin, 1969Ivory Coast, Bingerville, VI.1962, leg. J. Decelle, male holotype MRAC. – Same locality and collector as holotype, but IV.1962–III.1963, 3 paratypes MRAC.“Bingerville”.PageBreakUlomoides basilewskyi  possesses distinctly crenulated outer margin of all tibiae , but not for Alphitobius. In the structure of tibiae, the shape of body, eyes, and antennae Ulomoides basilewskyi resembles Ulomoides dermestoides . The striking long apicale of the aedeagus comb. n.http://species-id.net/wiki/Peltoides_opacusAlphitobius opacus Gerstaecker, 1871Diaclina opaca  sensu Gebien (1940)Peltoides (Micropeltoides) crypticoides Pic, 1916, syn. n.opacus MNB. – Fort Crampel, no further data, holotype of crypticoides MNHN.Ugano, leg. v. d. Decken, no. 56752, no further data, holotype of Benin, Kokora, 52 km N Save, 21.VI.2001, leg. F. & L. Kantner, 1 ex. SMNS. – Uganda, Bwamba Forest, 2500 ft. (760 m), III.1948, leg. J. G. Williams, 1 ex. SMNS. – Angola, Huila Province, 20 km S Caluquembe, 6.–7.XI.2011, leg. P. Schüle, 1 ex. SMNS. – Guinea (labelled as French Guinea), Region Kindia, Ségueia, 10.V.1951, leg. J. Bechyné, 4 ex. NHMB, 1 ex. CRG. – SE Cameroon, Lolodorf, leg. L. Conradt, 1895, 1 ex. NHMB. – Tanzania (labelled as Deutsch Ostafrika), [residual label unreadable], 1 ex. NHMB. – Zambia, Ikengele, Nchila Reserve, 6.XI.2002, leg. F. Wachtel, 1 ex. CRG.opacus), “Fort Crampel (Kaga Bandora)” (crypticoides).“Ugano-Berge”  crypticoides Pic, 1916 fully coincide with opacus, and is thus a junior synonym.The examination of the type of opacus), Central African Republic ; Senegal (Peltoides (Micropeltoides) crypticoides); Benin, Cameroon, Guinea, Uganda, Angola, Zambia (new records).Tanzania (type locality  Senegal , Ivory C Senegal , Mali, T


Aberrant activation of β-catenin/TCF signaling is related to the invasiveness of pancreatic cancer. In the present study, we evaluated the effect of capsaicin on β-catenin/TCF signaling. In a concentration and time-dependent study, we observed that capsaicin treatment inhibits the activation of dishevelled (Dsh) protein DvI-1 in L3.6PL, PanC-1 and MiaPaCa-2 pancreatic cancer cells. Capsaicin treatment induced GSK-3β by inhibiting its phosphorylation and further activated APC and Axin multicomplex, leading to the proteasomal degradation of β-catenin. Expression of TCF-1 and β-catenin-responsive proteins, c-Myc and cyclin D1 also decreased in response to capsaicin treatment. Pre-treatment of cells with MG-132 blocked capsaicin-mediated proteasomal degradation of β-catenin. To establish the involvement of β-catenin in capsaicin-induced apoptosis, cells were treated with LiCl or SB415286, inhibitors of GSK-3β. Our results reveal that capsaicin treatment suppressed LiCl or SB415286-mediated activation of β-catenin signaling. Our results further showed that capsaicin blocked nuclear translocation of β-catenin, TCF-1 and p-STAT-3 (Tyr705). The immunoprecipitation results indicated that capsaicin treatment reduced the interaction of β-catenin and TCF-1 in the nucleus. Moreover, capsaicin treatment significantly decreased the phosphorylation of STAT-3 at Tyr705. Interestingly, STAT-3 over expression or STAT-3 activation by IL-6, significantly increased the levels of β-catenin and attenuated the effects of capsaicin in inhibiting β-catenin signaling. Finally, capsaicin mediated inhibition of orthotopic tumor growth was associated with inhibition of β-catenin/TCF-1 signaling. Taken together, our results suggest that capsaicin-induced apoptosis in pancreatic cancer cells was associated with inhibition of β-catenin signaling due to the dissociation of β-catenin/TCF-1 complex and the process was orchestrated by STAT-3. Today pancreatic cancer ranks as the fourth leading cause of cancer related deaths in the United States . In spitSignal transducer and activator of transcriptional 3 is a member of STAT family present in cytoplasm. Phosphorylated STAT-3s dimerize and translocate to the nucleus, where it regulates the transcription of genes and modulate various physiological functions, including cell cycle regulation, cell survival and angiogenesis , 19. IntCapsaicin is a major component of hot chili pepper. Several studies have shown significant chemopreventive and chemotherapeutic effects of capsaicin against certain mutagens and carcinogens –26. CapsIn order to determine the effect of capsaicin on β-catenin/TCF-1 signaling, PanC-1, L3.6PL and MiaPaCa-2 cells were treated with various concentrations of capsaicin for 24 h or with 75 μM capsaicin for various time points. Our results revealed that capsaicin treatment decreased the phosphorylation of GSK-3β at S9 and STAT-3 at Y705 and increased the phosphorylation of β-catenin at S33/37/T41 Figure . Capsaicβ-catenin upon translocation to the nucleus associates with transcription factor TCF and activates β-catenin/TCF-1 complex leading to the transcription of downstream gene such as c-Myc and cyclin D1, hence increase in cell proliferation. Since we observed decrease in c-Myc and cyclinD1 expression by capsaicin treatment, we next wanted to see β-catenin and TCF-1 protein levels in the nucleus. Our results show that β-catenin and TCF-1 levels decreased significantly in the nuclear fraction of capsaicin treated PanC-1 and L3.6PL cells Figure . STAT-3 We next wanted to see whether capsaicin disrupts the β-catenin/TCF-1 complex in the nucleus. In order to do that, PanC-1 cells were treated with 75 μM capsaicin for 24 h and nuclear fraction was isolated using nuclear extraction kit before being subjected to immunoprecipitation assay. β catenin and TCF-1 were immuno-precipitated and immuno-blotted with their respective antibodies. Our co-immunoprecipitation assay from nuclear fraction showed that capsaicin treatment inhibits β-catenin levels when immunoprecipated with β-catenin or TCF-1 antibodies. On the other hand, capsaicin treatment decreased TCF-1 levels when immunoprecipitated with TCF-1 or β-catenin antibodies, indicating capsaicin treatment disrupts association of β-catenin/TCF-1complex in the nucleus Figure .Inhibition of proteasome-mediated proteolysis stabilizes β-catenin leading to its accumulation in the nucleus. Thus, we wanted to see whether blocking proteosomal degradation of β-catenin with MG-132 could increase β-catenin levels. Pre-treatment of cells with MG-132 inhibited the proteosomal degradation of β-catenin, which in turn increased the expression of downstream molecules such as cyclin D1 and c-Myc in PanC-1 and MiaPaCa-2 cells Figure , suggestActivation of Wnt/β-catenin signaling inhibits APC/Axin/GSk-3β complex, which prevents phosphorylation of β-catenin leading to its translocation and accumulation in the nucleus. We therefore wanted to see whether GSK-3β inhibitor LiCl or SB415286 could block capsaicin mediated inhibition of β-catenin signaling. To do so, PanC-1 cells were pretreated with LiCl or SB415286 for 1 h, followed by 75 μM capsaicin treatment for 24 h. As expected, LiCl and SB415286 activated β-catenin/TCF-1 signaling by increasing β-catenin, p-GSK-3β and TCF-1 levels Figure . HoweverSTAT-3 plays a critical role in Wnt/β-catenin signaling. We wanted to see whether activation of STAT-3 by IL-6 or overexpression of STAT-3 by transient transfection increases β-catenin/TCF-1 signaling. Cells were pre-treated with IL-6 followed by 75 μM capsaicin treatment for 24 h. Our results revealed that activation of STAT-3 by IL-6 increased β-catenin and TCF-1 levels, but attenuated by capsaicin treatment Figure . To confin vivo. In order to further confirm that capsaicin mediated tumor growth suppression was associated with inhibition of β-catenin/TCF-1 signaling, tumors from control and capsaicin-treated mice were subjected to western blotting. Our results demonstrate that capsaicin treatment inhibited β-catenin, TCF-1, p-STAT-3, survivin and c-Myc levels whereas increased cleavage of caspase-3 in the tumors of capsaicin treated mice as compared to controls  is as a member of the STAT family which participates in normal cellular responses as transcription factor . SeveralCapsaicin (purity > 99%), anti-actin, LiCl and SB415286 were obtained from Sigma . The antibodies against Cl-caspase-3, Frizzled, DVI-1, p-β-Catenin (S33/37/T41), β-Catenin, p-GSK-3β (S9), GSK-3β, APC, Axin, p-STAT-3 (Tyr 705), STAT-3, TCF-1, c-Myc, Cyclin D1 were purchased from Cell Signaling . Lamin B was purchased from Santa Cruz Biotechnology, Inc (CA). Alexa fluor 488 (green), alexa fluor 594 (red) conjugated goat- anti-rabbit secondary antibody and DAPI were obtained from Invitrogen. MG-132 was procured from Calbiochem . AG-490, a specific inhibitor of STAT-3 was purchased from Sellack Chemicals LLC.2. Cell lines were verified by STR analysis at TTUHSC core facilities . All the three cell lines have mutant KRAS and TP53. Panc-1 has homozygous deletions in CDKN2A and p16 genes and wild type SMAD4. MiaPaCa-2 has homozygous deletion of p16 gene. L3.6PL exhibits homozygous deletion of SMAD4.Human pancreatic cancer cell lines PanC-1, L3.6PL and MiaPaCa-2 were obtained from ATCC . Monolayer cultures of PanC-1 and MiaPaca-2 cells were maintained in DMEM medium and L3.6PL was maintained in MEM medium, supplemented with 10% fetal bovine serum, PSN antibiotic mixture (10 ml/l), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate and 20% glucose. All the cultures were maintained at 37°C in a humidified chamber of 95% air and 5% CO6 PanC-1 cells were collected and suspended in 100 μl of nucleofection solution per sample and then combined with 2 μg STAT-3 DNA. Once the electroporator program was completed, 500 μl of pre-incubated respective medium was added to the cuvette and samples were transferred into the 6well plate for transfection. Transfected cells were treated with 75 μM capsaicin for indicated time periods.PanC-1 cells were transiently transfected with STAT-3 plasmid using Nucleofector transfection reagent  according to the manufacture instruction. Briefly 1 × 10In separate experiments, pancreatic cancer cells were treated with either 10 μM MG132, 20 μM AG-490, 40 mM LiCl or 50 μM SB415286 for 1 h or with 20 ng/ml IL-6 for 15min, followed by treatment with 75 μM capsaicin for 24 h. Cell lysate were prepared for western blotting as we described previously [To examine the effect of capsaicin on the nuclear interaction of β-catenin with TCF-1 protein, immunoprecipitaion assay was performed as described by us previously . BrieflyImmunofluorescence assay was performed as described by us previously . Briefly6 PanC-1 (PanC-1-luc) cells into the sub-capsular region on the pancreas. Skin incisions were then closed. On the very next day of surgery, animals were imaged for basal luminescence using IVIS Bio Luminescent System equipped with Living Image software  after injecting luciferin (3 mg/mouse). Twenty-one days after the surgical implantation of PanC-1-luc cells, mice were randomly divided into two groups with 5mice in each group. Group I served as controls and received 0.1 ml vehicle every day by oral gavage. Group II received 5 mg capsaicin/kg of body weight every day by oral gavage. Tumor luminescence and animal weight was measured thrice a week for seven weeks. At the end of the experiment (day 70), mice were sacrificed; tumors and pancreas were excised from each mouse, weighed and snap frozen.Pancreatic orthotopic implantation in nude mice was performed previously, and the tumors from control and capsaicin treated mice were used in this study . All aniCells were exposed to various concentrations of capsaicin for different time points and lysed on ice as described by us previously , 53. WhoP-values less than 0.05 were considered statistically significant.All statistical calculations were performed using Graph Pad Prizm 5.0. Analysis of variance (ANOVA) was used to test the statistical significance of difference between control and treated groups followed by Bonferroni's post-hoc analysis for multiple comparisons.


Embryonic stem cells (ESCs) are pluripotent stem cells and can differentiate into cardiomyocytes when cultured in appropriate conditions. The function of hypoxia-inducible factors (HIFs) has been identified in directing the formation of cardiac lineages. The purpose of this study was to investigate the ability of HIF2α to induce differentiation of ESCs into cardiomyocytes and to explore the potential underlying molecular mechanisms.HIF2α was then manipulated during cardiac differentiation with piggyBac transposon and the lentivirus system. The underlying mechanism was finally examined via administering selective inhibitors of the Wnt/β-catenin signaling pathway.Cardiac differentiation from mouse ESCs was analyzed using the “hanging drop” method, and success was determined by assaying the numbers of beating embryoid bodies and the expression level of cardiac markers. The expression of HIF2α can significantly drive mouse ESCs to form cardiomyocytes. Contrarily, knockdown of HIF2α inhibits the emergence of cardiac cells. In addition, the cardiomyogenesis-promoting effect of HIF2α occurred by increasing the protein level of β-catenin, an effector that contributes to cardiac differentiation at an early stage of ESC differentiation.Overexpressing HIF2α has a cardiomyogenesis-promoting effect in ESCs via enhancing the activation of the Wnt/β-catenin signaling pathway. Our results may be beneficial for generating and applying cardiomyocytes from ESCs safely and effectively in the future.The online version of this article (doi:10.1186/s12967-015-0447-7) contains supplementary material, which is available to authorized users. Cardiomyogenic development is a multistep process involving a cascade of signaling events and multiple transcription factors, which operate in a spatial and temporal fashion to specify the fate of cardiac cells . UnfortuHIF3α is normally expressed only in highly avascular tissues, such as the cornea. Additionally, they activate gene transcription while HIF3α inhibits the HIF1α- or HIF2α-mediated hypoxia responses 8] signalesponses . A previ factors . Similar factors , while oived EBs , 17. Notived EBs . HoweverIn this study, we investigated the role of HIF2α in cardiac differentiation using gain- and loss-of-function methods in mouse ESCs, and explored the possible intracellular signaling pathways by which HIF2α activates this process. Our study might provide expanded insight to create an effective strategy for promoting differentiation of ESCs cells into cardiomyocytes.2. The medium for routine maintenance was GMEM  supplemented with 10% FCS (HyClone), 1% MEM nonessential amino acids (Invitrogen), 2 mM GlutaMax (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen) and 100 units/ml LIF (Millipore). Cells were digested by 0.25% trypsin (Invitrogen) and passaged when confluence reached approximately 70%.46C ESCs, kindly provided by Dr. Smith A (University of Cambridge), were cultured on 0.1% gelatin-coated dishes at 37°C in 5% COESCs were differentiated into beating cardiomyocytes in vitro by the “hanging drop” method as described previously . BrieflyHIF2α were designed to target 21 base-pair gene specific regions and were then amplified into the pLKO.1-TRC (AgeI and EcoRI sites). The targeted sequences are as follows: HIF2α sh#1:GCTTCCTTCGGACACATAAGC; HIF2α sh#2: GGGACTTACTCAGGTAGAACT. pLKO.1-TRC-based lentiviral vectors were transfected into 293 T cells in combination with pMD2.G and psPAX2 plasmids. Virus-containing supernatant was collected after 48 hours and filtered through 0.45 μm filters (Millipore). ESCs were incubated in the virus supernatant for 48 hours. For gene overexpression, the coding region of HIF2α was cloned from mouse cDNA with Hot Start DNA Polymerase (Takara) and was inserted into the BglП and SalI sites of the PiggyBac transposon vectors. ESCs were transfected with 2 μg PiggyBac inserted with targets plus a 2 μg transposon vector using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The modified cells were screened by treatment with 2 μg/ml puromycin for about one week.For RNA interference in ESCs, short hairpin (shRNA) constructs for Total RNA was extracted with TRIzol (Invitrogen). cDNA was synthesized with 1 μg of total RNA using a PrimeScript 1st strand cDNA Synthesis Kit (Takara) according to the manufacturer’s instructions. QRT-PCR was performed with SYBR® Premix Ex Taq™ (Takara) in an ABI7500 Real-Time PCR machine (Applied Biosystems). Target gene expression was normalized to GAPDH expression. The primers that were used are listed in Additional file Cells were lysed in ice-cold RIPA cell buffer (Sigma) supplemented with protease inhibitors (Sigma). The proteins were separated with a 4–12% PAGE gel and electrotransferred onto a PVDF membrane. The membrane was probed with primary antibodies and subsequently detected by horseradish-peroxidase (HRP) conjugated antibodies (Santa Cruz). The primary antibodies were β-catenin (Santa Cruz), Flag antibody (Sigma), HIF1α (Santa Cruz) and HIF2α (Santa Cruz).TOP-Flash or FOP-Flash constructs (Addgene) were co-transfected with a Renilla luciferase plasmid (Promega) into mESCs overexpressing PB empty vectors or PB-HIF2α plasmids. Cells were harvested and the relative luciferase activity of the lysate was measured with the Dual-Luciferase reporter assay system (Promega).Cells were fixed in 4% paraformaldehyde for 20 minutes and blocked in blocking buffer (PBS containing 5% BSA and 0.2% Triton X-100). Cells were incubated in primary antibody solution at 4°C overnight, followed by Alexa Fluor 488  secondary antibodies for 1 h at 37°C. Nuclei were stained with Hoechst . The primary antibodies were Gata4 , Myosin  and Troponin T .Differentiated EBs were trypsinized into single cells, fixed in 2% PFA and permeabilized with 0.2% Triton × 100. Cells were suspended in antibody solution containing anti-cTnT and α-SMA antibodies . Cells were washed in PBS three times and then resuspended in FITC-conjugated secondary antibody solution . Cardiomyocytes were detected with a BECKMAN COULTER FACS.To induce a hypoxic condition, 100 μM CoCl2 (Sigma) were supplemented into ESCs or EBs. For analyzing the protein levels of HIF1α or HIF2α, 46C ESCs were treated with or without 100 μM CoCl2 for 10 h. For inducing cardiac differentiation, ESCs-derived EBs were exposed to 100 μM CoCl2 during the whole process of differentiation, and the medium was changed every two days.t test was used to determine the significance of differences in comparisons. Values of p < 0.05 were considered as statistically significant.All data are reported as the mean ± s.d. A Student’s HIF2α and analyzed by western blotting for expression of HIF2α  . To inveHIF2α during cardiac differentiation, we differentiated 46C ESCs into cardiomyocytes, which were immunopositive for cardiac markers Gata4, Myosin and Troponin T  was performed in EBs at day 15. Higher expression of α-MHC and cTnT was observed in HIF2α-EBs compared with empty vector-EBs  specific for HIF2α mRNA (shHIF2α). Stable knockdown of HIF2α transcript levels was observed following drug selection  Wnt reporter plasmid, and compared reporter activity in EBs at day 4. HIF2α significantly enhanced reporter activity  group, both inhibitors significantly reduced the number of beating EBs and the expression of cardiac transcription factors (Figures HIF2α-mediated cardiogenesis is β-catenin-dependent in ESCs.To verify the impact of Wnt/β-catenin signaling on HIF2α induced cardiac differentiation, two β-catenin specific inhibitors; i.e., XAV939 and IWR1 , were uss Figure C. Then,  Figures D and E. Wnt10b as well as upstream of the Wnt1 gene [HIF1α with potential subsequent effects on Wnt gene expression [HIF2α sh#2 cells unlike that of PB and PB-HIF2α groups (Figure HIF2α knockdown. These detailed mechanisms need to be clarified further.Indeed, the function of hypoxia is associated with Wnt/β-catenin signaling in many types of cells. For example, HIF1α promoted cellular adaptation to hypoxia by interacting with β-catenin . HIF2α ant1 gene . In cardpression . In our s Figure C, implyiThe present results provide valuable insight on the function of HIF2α in ESC-derived cardiomyocytes. The HIF2α-induced promoting effect on β-catenin signaling may be involved in this process. Our results will be particularly helpful in attempts to achieve effective cardiac differentiation from ESCs and will provide useful information for the clinical application of ESCs.