Fig. 3

Inactivation of miR-31 promotes the myogenic lineage progression of SCs. a WT and miR-31-KO SCs were cultured in growth medium for 4 days and then switched to differentiation medium and cultured for 1 day. The cells were then fixed and labeled with anti-MyoD and anti-Ki67 antibodies. DAPI was used to identify nuclei. Representative individual and overlaid images of WT and miR-31-KO cultures after labeling with MyoD, Ki67, and DAPI. Scale bars: 30 μm. b Percentage of MyoD⁺Ki67⁻ cells (normalized to MyoD⁺ cells) in WT and miR-31-KO cell cultures. **P < 0.01. c The representative immunoblots presented here show MyoD and MyoG protein levels in WT and miR-31-KO cultures after undergoing differentiation for 1 day. d After 4 days in growth medium, the cells were induced to differentiate for 2 days. Myogenic differentiation was examined by staining for MyHC. Myogenic proliferation was determined by immunostaining against Ki67. Representative merged images of WT and miR-31-KO cultures after labeling with MyHC, Ki67, and DAPI. Scale bars: 30 μm. e The differentiation index was determined by the percentage of nuclei in MyHC-positive cells. **P < 0.01. f Percentage of Ki67⁺ cells (normalized to DAPI cells) in WT and miR-31-KO cell cultures. **P < 0.01. g SCs isolated from hindlimb muscles of WT and miR-31-KO mice were cultured in growth medium for 5 days. The cells were then fixed and measured by immunostaining against Pax7, MyoD, and DAPI. Representative individual and overlaid images of WT and miR-31-KO SC cultures after labeling with Pax7, MyoD, and DAPI. Scale bar: 30 μm. h Quantification of the percentages of self-renewing Pax7⁺MyoD⁻, proliferating Pax7⁺MyoD⁺ and differentiated Pax7^-MyoD⁺ cell populations in WT and miR-31-KO cultures. ***P < 0.001