Fig 1: Islr is upregulated during satellite cell differentiation and C2C12 differentiation. a Immunohistochemistry analysis of Islr and Pax7 in uninjured TA muscles of wild-type (WT) mice. Scale bar = 10 μm. b Immunohistochemistry analysis of Islr and Pax7 in injured TA muscles of wild-type mice at 5 d postinjury using serial sections. Scale bar = 10 μm. c Immunohistochemistry analysis of Islr and MyoG in injured TA muscles of WT mice at 5 d postinjury using serial sections. Scale bar = 10 μm. d Flow cytometry analysis of CD31, CD45, Sca1, and α7-integrin expression in whole muscle-derived cells. e Western blot analysis of Islr, Pax7, MyoG, and MyHC in satellite cells from WT mice at 4 d in proliferation medium (G4) or 2 d in differentiation medium (D2). f Immunofluorescence staining for tubulin in C2C12 cells transfected with the Islr-GFP plasmid. Scale bar = 25 μm. g Immunofluorescence staining for tubulin and Flag in C2C12 cells transfected with the leader peptide-Flag-Islr (L-Flag-Islr) plasmid. Scale bar = 25 μm. h Immunohistochemistry analysis of Islr in C2C12 cells cultured in differentiation medium for 5 d. Scale bar = 50 μm. i Immunohistochemistry analysis of Islr in primary myoblasts derived from FACS-purified satellite cells cultured in differentiation medium for 2 d. Scale bar = 50 μm. j Expression analysis of Islr in C2C12 cells cultured in either growth medium for 1 or 2 d (G1 and G2) or in differentiation medium for 1, 3, 5, or 7 d (D1, D3, D5, and D7) using quantitative real-time PCR (qRT-PCR). k Western blot analysis of Islr in C2C12 cells cultured in either growth medium for 2 d (G2) or in differentiation medium for 4 or 7 d (D4 and D7). Error bars represent the means ± s.d. ***P < 0.001; Student’s t test
Fig 2: The differentiation ability of satellite cells is reduced in Islr KO mice in vitro. a Immunofluorescence analysis of MyoG+ cells in isolated EDL myofibers of control and Islr cKO mice after 3 d of culture. N = 3 in each group. Approximately, 30 clusters total in each group. Scale bar = 25 μm. The percentages of MyoG+ cells in the clusters are shown on the right. b Immunofluorescence staining for MyoG in FACS-purified satellite cells from control and Islr cKO mice cultured for 4 d in proliferation medium followed by 1 d in differentiation medium. N = 3 cell cultures in each group. Scale bar = 25 μm. The percentage of MyoG+ cells is shown on the right. c Western blot analysis of MyoG protein levels in isolated satellite cells of control and Islr cKO mice cultured for 4 d in proliferation medium followed by 1 d in differentiation medium. d Immunofluorescence staining for MyHC in FACS-purified satellite cells from control and Islr cKO mice cultured for 4 d in proliferation medium followed by 2 d in differentiation medium. N = 3 cell cultures in each group. Scale bar = 50 μm. The percentages of nuclei contained in the myotubes (a MyHC+ cell with at least two nuclei) are shown on the right. e The distribution of nuclei present in a MyHC+ cell differentiated from FACS-purified satellite cells of control and Islr cKO mice. N = 3 cell cultures in each group. f Western blot analysis of MyHC protein levels in isolated satellite cells of control and Islr cKO mice at 4 d in proliferation medium or 2 d in differentiation medium. Error bars represent the means ± s.d. NS: not significant, **P < 0.01, ***P < 0.001; Student’s t test
Fig 3: The deletion of Islr delays skeletal muscle regeneration. a A schematic illustration showing the location of loxP in the Islr gene. b Western blot analysis of Islr protein levels in isolated satellite cells of control and Islr cKO mice cultured for 4 d in proliferation medium. c Immunohistochemistry analysis of Islr in Pax7+ cells of control and Islr cKO mice at 5 d postinjury using serial sections. Scale bar = 10 μm. d Western blot analysis of Islr protein levels in injured TA muscles of control and Islr cKO mice at 5 d postinjury. e H&E staining of injured and contralateral TA muscles (CTL) in control and Islr cKO mice at 3.5 (N = 3), 5 (N = 4), and 14 (N = 3) d post injury. Scale bar = 100 μm. f Average cross-sectional area (CSA) of regenerating myofibers and g the number of myofibers containing two or more centrally located nuclei per field at 5 d postinjury. N = 3 in each group. h Distribution of myofiber CSAs at 5 d postinjury. N = 3 in each group. Error bars represent the means ± s.d. NS not significant, **P < 0.01, ***P < 0.001; Student’s t test. Control (Ctrl): Myf5-Cre+/−, Islrfl/fl; Islr cKO: Myf5-Cre+/−:Islrfl/fl
Fig 4: 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 5: 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
Supplier Page from MilliporeSigma for Anti-ISLR antibody produced in rabbit