Extended Data Fig. 6: PFKM depletion interferes with both muscle differentiation and fusion.
From: PFKM governs metabolic shifts throughout skeletal muscle differentiation
a, Immunoblotting (IB) of PFKM in undifferentiated cultured human muscle cells with siControl or siPFKM treatment. Right, protein levels normalized to loading control (Actin), assessed by densitometry of n = 3 biological replicates. Statistical significance was determined by unpaired two-tailed Student’s t-test and data are shown as mean ± s.e.m. b, IB of PFKM in C2C12 muscle cells after differentiation (3 days) with siControl or siPFKM treatment. Right, protein levels normalized to loading control (Actin), assessed by densitometry of n = 3 biological replicates. Statistical significance was determined by unpaired two-tailed Student’s t-test and data are shown as mean ± s.e.m. c, Representative immunofluorescence (IF) of myogenin (MyoG, pink) and DAPI (blue) in C2C12 muscle cells after differentiation with siControl or siPFKM treatment. Scale bar: 30 μm (n = 811 control nuclei and n = 1148 siPFKM nuclei from 4 regions of interest (ROIs)). Right, colocalization analysis of MyoG and DAPI normalized by total nuclei count (10x×10 magnification). Statistical significance was determined by unpaired two-tailed Student’s t-test and data are shown as mean ± s.e.m. d, Representative IF of myosin heavy chain (MyHC, red) and DAPI (blue) in C2C12 muscle cells after differentiation with siControl or siPFKM treatment. Scale bar: 30 μm (n = 3783 control nuclei and n = 3126 siPFKM nuclei from 4 fields of view). Right, quantification of mean fluorescence intensity (MFI) of MyHC per field of view (×10 magnification). Statistical significance was determined by unpaired two-tailed Student’s t-test and data are shown as mean ± s.e.m. e, Fusion index calculations were performed using the percentage of contacting nuclei over total nuclei in cultured muscle cells after differentiation with siControl or siPFKM treatment. Left, quantification of human muscle cells (n = 2586 control nuclei and n = 1868 siPFKM nuclei from 6 ROIs). Right, quantification of C2C12 muscle cells (n = 2203 control nuclei and n = 1458 siPFKM nuclei from 6 ROIs). Statistical significance was determined by unpaired two-tailed Student’s t-test and data are shown as mean ± s.e.m. f, Fusion index calculations were performed using the percentage of nuclei in MyHC+ multinucleated cells (myotubes) over total nuclei in cultured muscle cells after differentiation with siControl or siPFKM treatment. Left, quantification of human muscle cells (n = 2159 control nuclei and n = 1598 siPFKM nuclei from 5 fields of view). Right, quantification of C2C12 muscle cells (n = 1849 control nuclei and n = 1199 siPFKM nuclei from 5 ROIs). Statistical significance was determined by unpaired two-tailed Student’s t-test and data are shown as mean ± s.e.m. g, Total nuclei per ROI in cultured muscle cells after differentiation in siControl or siPFKM treatment. Left, quantification of human muscle cells (n = 1724 control nuclei and n = 1829 siPFKM nuclei from 5 ROIs). Right, quantification of C2C12 muscle cells (n = 1640 control nuclei and n = 1459 siPFKM nuclei from 5 ROIs). Statistical significance was determined by unpaired two-tailed Student’s t-test and data are shown as mean ± s.e.m. (ns P>0.05). h-i, IB of PFKM in cultured human muscle cells after differentiation with siControl or siPFKM treatment, with or without PFKM overexpression of wild-type (PFKM-WT) or methyl-mutant R774K (PFKM-RK). Right, protein levels normalized to loading control (Actin), assessed by densitometry of n = 3 biological replicates. Statistical significance was determined by two-way ANOVA with post-hoc Dunnett’s analysis and data are shown as mean ± s.e.m. (ns P>0.05). j, IB of PFKM in undifferentiated cultured human muscle cells in control or PFKM-WT conditions. Right, protein levels normalized to loading control (Actin), assessed by densitometry of n = 3 biological replicates. Statistical significance was determined by unpaired two-tailed Student’s t-test and data are shown as mean ± s.e.m. k, Cell proliferation assay in control or PFKM-WT conditions after 24 h in undifferentiated human muscle cells, (n = 3 biological replicates). Statistical significance was determined by unpaired two-tailed Student’s t-test and data are shown as mean ± s.e.m. Exact P values are shown in graphs.
