Fig. 4

Loss of miR-31 drives Pax7-positive proliferating SCs into quiescence. a Representative photomicrographs of day 5 post injury TA muscle sections of WT and miR-31-KO mice after immunostaining for Pax7 (red) and Ki67 (green). Nuclei were labeled by staining with DAPI. Arrows point to Pax7⁺Ki67⁺ cells. Scale bar: 20 μm. b Quantification of the percentage of Pax7⁺ cells that did not co-express Ki67 among total Pax7⁺ cells. **P < 0.01. c Relative mRNA levels of Pax7 in day 5 post injury TA muscles of WT and miR-31-KO mice measured by qRT-PCR. ***P < 0.001. d The immunoblots presented here illustrate the protein levels of Pax7 and an unrelated GAPDH in TA muscles of WT and miR-31-KO mice at day 5 after BaCl₂ injection. e Representative individual and overlaid images of 1-day differentiated WT and miR-31-KO SCs after labeling Pax7, Ki67 and DAPI. Scale bars: 30 μm. f Quantification estimation of the percentage of Pax7⁺Ki67⁻ quiescent cells in WT and miR-31-KO cultures. ***P < 0.001. g Representative individual and overlaid images of WT and miR-31-KO proliferating SCs after labeling Pax7, Ki67 and DAPI. Scale bars: 30 μm. h Quantification estimation of the percentage of Pax7⁺Ki67⁻ quiescent cells in WT and miR-31-KO cultures. ***P < 0.001. i Single myofibers were isolated from the extensor digitorum longus (EDL) muscle of WT and miR-31-KO mice. After 5 days of culture, myofiber-derived SC clusters were fixed and stained for Pax7, MyoD, and DAPI. Representative merged photomicrographs of myofiber-derived SC clusters after immunostaining for Pax7 (red) and MyoD (green). Nuclei were identified by labeling with DAPI. Scale bar: 50 μm. j Percentage of self-renewing Pax7⁺MyoD⁻ SCs (normalized with DAPI) in WT and miR-31-KO myofiber-derived SC cultures. ***P < 0.001