Extended Data Fig. 6: Characterization of Cyclin D1 reduction and expression in MuSCs.
a, Gating strategy for FACS isolation of YFP+ MuSCs after tamoxifen administration to transgenic mice. Shown are MuSC yields in terms of the percentage of size- and doublet-gated cells that are YFP+DAPI-; NS, WT vs. HET and WT vs. KO (n values represent individual mice). Purity of isolated MuSCs is >94% or >98% as assessed by routine staining and quantification of YFP or Pax7, respectively, of cells fixed one hour after plating. b, TA muscles were isolated from twelve-month-old mice that had received tamoxifen injections at three months of age. Muscle sections were stained for Pax7 to identify MuSCs, YFP to identify recombined cells, and laminin to delimit muscle fibers and MuSCs from the interstitium. No MuSCs or YFP+ cells were identified in the interstitium, and no YFP+ cells were Pax7-. The MuSC pool was quantified by counting Pax7+ cells in sections (n=3 mice per group). c, FACS-isolated MuSCs were cultured continuously in the presence of EdU to assess S-phase progression (n=4 for HET(-Ex), 6 for HET(+Ex), 5 for KO(-Ex), 5 for KO(+Ex), and 6 for WT(-Ex) mice). d, To confirm maintenance of ex vivo quiescence by TubA, MuSCs were kept in culture for three days either in quiescence (with TubA) or during activation (with DMSO vehicle) in the continuous presence of EdU and then fixed for analysis. MuSCs were then released for two days in the presence of EdU by removing TubA. MuSCs were then fixed for analysis of exit from quiescence (n=3 mice per condition). e, MuSCs were infected as in Fig. 3j for three days and then harvested for Western blot. Each lane represents a pool of three to six mice split into the two infection conditions. f, MuSCs infected as in Fig. 3j were harvested for RT-qPCR analysis (n=3 mice per group). Scale bar in a, 50 μm, in b, 10 μm. Data are summarized with mean + s.e.m. NS, not significant; *P<0.05; two-tailed Welch’s t-test in a-c, one-tailed Welch’s t-test in d, one-tailed ratio-paired t-test in f.
