Fig. 6

The disruption of Islr downregulates the Dvl2-mediated canonical Wnt signaling pathway in myoblasts. a Immunofluorescence staining for MyHC in control shRNA (shCtrl) and Islr shRNA stable (shIslr) C2C12 cells. N = 3 cell cultures in each group. Scale bar = 50 μm. The percentage of MyHC⁺ cells is shown on the right. b Immunofluorescence analysis of cell morphologies in shCtrl and shIslr C2C12 cells by staining live cells with DiI. N = 3 cell cultures in each group. Scale bar = 25 μm. c Heatmap of the changes in selected gene expression levels in shCtrl and shIslr C2C12 cells at G2 and D3 by RNA-seq. d Expression analysis of Wnt target genes and myogenic genes in shCtrl and shIslr C2C12 cells at 3 d in differentiation medium using qRT-PCR. e Western blot analysis of active β-catenin protein levels in nuclear lysates extracted from shCtrl and shIslr C2C12 cells after 7 d in differentiation medium. f Luciferase activity of TOP/FOP in shCtrl and shIslr C2C12 cells after 3 d in differentiation medium. g Western blot analysis of Islr and Dvl2 protein levels in shCtrl and shIslr C2C12 cells after 2 d in growth medium. h Western blot analysis of Islr and Dvl2 protein levels in shCtrl and shIslr C2C12 cells after 7 d in differentiation medium. i Western blot analysis of Dvl2 protein levels in primary myoblasts of control and Islr cKO mice after 3 d in differentiation medium. Error bars represent the means ± s.d. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; Student’s t test