Fig 1: Islr stabilizes Dvl2 via antagonizing LC3-mediated autophagy. a Immunofluorescence analysis of GFP-LC3+ puncta in C2C12 cells transfected with GFP-LC3 and Dvl2-Flag plasmids, GFP-LC3 and Islr-HA plasmids, or GFP-LC3, Dvl2-Flag, and Islr-HA plasmids. N = 3 cell cultures in each group. Approximately, 20 cells total in each group. The numbers of GFP-LC3+ puncta per cell are shown on the right. b Western blot analysis of p62 and LC3II protein levels in shCtrl and shIslr C2C12 cells after 2 d in growth medium. c Immunofluorescence analysis of GFP-LC3+/Dvl2-Flag+ puncta in shCtrl and shIslr C2C12 cells transfected with GFP-LC3 and Dvl2-Flag plasmids. N = 3 cell cultures in each group. Approximately, 20 cells total in each group. The numbers of GFP-LC3+/Dvl2-Flag+ puncta per cell are shown on the right. d Immunofluorescence analysis of GFP-LC3+/Dvl2+ puncta in shCtrl and shIslr C2C12 cells treated with rapamycin (Rap) for 6 h. N = 3 cell cultures in each group. Approximately, 15 cells total in each group. The numbers of GFP-LC3+/Dvl2+ puncta per cell are shown on the right. e Immunofluorescence analysis of GFP-LC3+/Dvl2+ puncta in satellite cells from control, Islr cKO, Dact1 cKO, and Islr/Dact1 cKO mice treated with rapamycin (Rap) for 6 h. N = 3 cell cultures in each group. Approximately, 15 cells total in each group. The numbers of GFP-LC3+/Dvl2+ puncta per cell are shown on the right. Scale bars are all 25 µm. f Western blot analysis of Dvl2, Axin2, and Lef1 protein levels in primary myoblasts of control and Islr cKO mice incubated with BFA1 or DMSO. g Reciprocal co-immunoprecipitation analysis between GFP-tagged Islr and Flag-tagged Dvl2 in HEK293T cells. IB immunoblotting, IP immunoprecipitation. h Reciprocal co-immunoprecipitation analysis between Islr and Dvl2 in primary myoblasts cultured for 4 d in differentiation medium. i Western blot analysis of the ubiquitination level of Dvl2 in primary myoblasts of control and Islr cKO mice cultured for 4 d in differentiation medium. Error bars represent the means ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test
Fig 2: Long-term effects of neonatal Sev exposure on MT1 and Wnt signaling in adult hippocampus. (A, B) Western blotting and quantification of p-CREB, CREB, p-PKA, and PKA in hippocampus of control, Sev, and Sev-melatonin-treated mice at different time points. No changes of p-CREB, CREB, p-PKA, and PKA from 7 dps. (C) Heat map of RNA-seq results of top 20 differential genes in control, Sev-, and Sev-melatonin-treated mice. Notice the changes of SFRP1. (D, E) KEGG enrichment of signaling pathways in Sev-treated mice and Western blotting of SFRP1. (F) Western blotting and quantification of ß-catenin, p-ß-catenin (S33/S37/T41) in adult hippocampus of control and Sev-treated mice. (G) Western blotting and quantification of cytoplasmic and nuclear ß-catenin in adult hippocampus of control and Sev-treated mice. Notice the decrease in nuclear ß-catenin in Sev-treated mice. (H, I) qPCR and Western blotting and quantification of Axin2 in adult hippocampus of control and Sev-treated mice. (J) Double-immunohistochemistry and quantification of Axin2/NeuN- and Axin2/CC1-positive cells in normal hippocampus. N = 6 mice per group. Con, control. Sev, Sevoflurane. Cyt, cytoplasm. Nuc, nucleus. *p < .05. **p < .01. Student's t test. One-way ANOVA. Bars = 50 µm
Fig 3: WNT4 can promotes metastasis of colorectal cancer cells. a The mRNA level of WNT4 in colorectal cancer (CRC) cells transfected with WNT4-siRNA1, WNT4-siRNA2, and normal control. b The mRNA level of WNT4 in SW480 cells transfected with WNT4-vector and WNT4-HA. c CRC cells were transfected with WNT4-siRNA1, WNT4-siRNA2, then, WNT4, ß-catenin, AXIN2, E-cadherin, ZO-1, and nuclear ß-catenin and AXIN2 were analyzed by western blot. d Overexpression of WNT4 enhanced the TCF/LEF transcription activity. e and f Tumor cell metastasis assay was performed in vivo; the ratio of liver weight to body weight and the number of liver metastases in the Scramble group are much higher than the WNT4-shRNA group (n = 7). Scale bar, 50 µm. g and h A subcutaneous xenograft model in nude mice was used to identify if WNT4 promoted the proliferation of colon cancer cells (LoVo cells, n = 7; SW480 cells, n = 5). No significant difference was observed. g Scale bar, 20 µm. h Scale bar, 50 µm. Data are presented as the mean ± SD. Two-tailed Student’s t test was used for statistical analyses. *P < 0.05
Fig 4: BITC regulates the Wnt/ß-catenin pathway and the APC/ß-catenin complex. 4T1-Luc cells were treated with various concentrations of BITC as indicated for 24 and 48 h. Total protein lysates were immunoblotted for (A) APC, Axin2, GSK-3ß, ß-catenin, p-ß-catenin and Wnt2, and (B) cyclin D1 and c-Myc protein expression. ß-actin was used as the internal control. (C) Immunofluorescence assay was performed to evaluate the expression and subcellular location of APC and ß-catenin protein (magnification, ×1,000). (D) 4T1-Luc cells were treated with various concentrations of BITC as indicated for 24 and 48 h, and total RNA was isolated and reverse transcribed. The mRNA expression levels of APC and ß-catenin were detected via reverse transcription-quantitative PCR analysis. *P<0.05, **P<0.01 vs. C. (E) Protein lysates isolated from the tumors of control or BITC-treated mice were subjected to western blot analysis for APC and ß-catenin. 1–6 indicated different mice. (F) Tumors from control and BITC-treated mice were subjected to immunohistochemical analysis using APC and ß-catenin antibodies (magnification, ×400). APC, adenomatous polyposis coli; BITC, benzyl isothiocyanate; C, control; DAPI, 4',6-diamidino-2-phenylindole; GSK-3ß, glycogen synthase kinase-3ß.
Fig 5: Knockdown of Kindlin-2 inhibits Axin2 expression in HCC cells. a The microarray analysis indicated that Axin2 was significantly downregulated in LM3/LV-shK2 cells, with a fold change of 4.49. b Both quantitative real-time PCR and (c) Western blotting analyses revealed that Axin2 expression is decreased in Kindlin-2 knockout or knockdown cells but increased in Kindlin-2-overexpressing cells. ** represents P < 0.01
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