Fig. 6.
Removal of p16Ink4a partially rescues regeneration and self-renewal of Slug-deficient SCs. a H&E staining of the double and triple BaCl2-injured TA muscles from Slug+/+p16+/+, Slug−/−p16+/+, and Slug−/−p16−/− mice (n = 3–6 mice per group). Left panel, the diagram for consecutive injury. Right panel, representative images of H&E staining of muscle tissue sections from injured mice. Scale bar, 100 μm. b Ratio of myofiber CSA in the intact and BaCl2-injured TA muscles between Slug+/+p16+/+, Slug−/−p16+/+, and Slug−/−p16−/− mice. *p < 0.05, ***p < 0.001 by one-way ANOVA; n.s. not significant. c Scheme of SC transplantation. SCs isolated from Slug−/−p16+/+ and Slug−/−p16−/− mice were infected with GFP-expressing retroviruses, and injected into either side of the BaCl2-pre-injured TA muscle of mdx recipient, respectively. Total mononucleated muscle cells were isolated separately from either TA muscle 4 weeks after transplantation, and subjected to flow cytometric analysis for the fraction of donor-derived SCs (GFP+) in MuSC (CD45−CD31−Sca1−Vcam I+) subpopulation of recipient mice. d Representative flow cytometry plots showing the frequency of donor-derived SCs (GFP+) within the total recipient MuSC (CD45−CD31−Sca1−Vcam I+) subpopulation in mononucleated TA muscle cells. e Percent of donor-derived cells (GFP+) in total MuSC (CD45−CD31−Sca1−Vcam I+) subpopulation of mononucleated TA muscle cells from mdx recipients (n = 4–6). ***p < 0.001 by student’s t-test. f Representative SA-β-Gal staining of cultured primary SC-derived myoblasts. 104 SCs of indicated genotype were plated in Matrigel-coated wells of 24-well plate, and passaged weekly. Bright-field imaging and SA-β-Gal staining were performed in cells on day 7 of culture at passage 3. Scale bar, 100 µm. g Percentage of the SA-β-Gal+ cells in serially passaged myoblasts on day 7 of culture at passage 3. ***p < 0.01; by student’s t-test. n.s. not significant. Data are shown as mean ± SEM of three independent replicates. Source data are provided as a Source Data file