(A-C) Images (A) and quantification (B-C) of in situ assays for Rac/Cdc42 activity in T0 MuSCs ± QPs (B) or over 4 hours (C). Pak1GST binds to GTP-bound Rac/Cdc42. (D) Schematic showing NSC23766 (NSC) treatment during preparation of single myofibers. (E) Image of an NSC-treated T0 MuSC. (F-I) Quantification of control vs NSC-treated MuSCs showing: the frequency (F) and average length (G) of QPs, cMTOC formation at T0 (H), and the percentage of FOS+ MuSCs from T0-T2 (I). Control data are shared with Fig. 4 and Fig. S4. (J) Schematic showing mouse lines used for panels K-P. (K-L) Quantifications of the percentage of MYOD+ MuSCs (K) and Ki67+ cells/mm2 (L). (M) Image of a MYOD/Ki67+ MuSC in Rac1fl/+ muscle. (N-O) Quantification (N) and image (O) of interstitial cells/mm2. (P) Quantification of Pax7+ MuSCs/mm2. Data represent n=3-4 mice and show mean ± s.e.m. Control data (A-C, F-H) are shared with Fig. 4 and S4, see Methods. Comparisons by paired t-test (B), one-way ANOVA with Bonferroni’s multiple comparisons test (C,F-G,K-L,M,O), or two-way ANOVA with Šídák’s multiple comparisons test (I); *= p<0.05, **= p<0.01, ***=p<0.001, ****=p<0.0001. Scale bars: (A) 10μm; (E) 5μm; (M,O) 25μm. See also Figure S3.