Fig. 4: Depletion of PFKM hinders muscle cell differentiation.
From: PFKM governs metabolic shifts throughout skeletal muscle differentiation
a, IB of PFKM, PFKP and PFKL in cultured human 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. (NS, P > 0.05). b, Quantitative PCR with reverse transcription (RT–qPCR) analysis of the relative transcript levels of myogenic factor 5 (MYF5) and myosin heavy chain 1 (MYH1) in cultured human muscle cells after differentiation (3 days) with siControl or siPFKM treatment (n = 6 biological replicates). Statistical significance was determined by multiple unpaired two-tailed Student’s t-test analyses with Šidák–Bonferroni correction (α = 0.05), and data are shown as mean ± s.e.m. c, IB of PFKM and MyHC in cultured human 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. d, Representative IF of myogenin (MyoG, pink) and DAPI (blue) in cultured human muscle cells after differentiation (3 days) with siControl or siPFKM treatment, with or without PFKM overexpression of PFKM-WT or PFKM-RK. Scale bars, 10 μm, n = 790 control nuclei, n = 1,050 siPFKM nuclei, n = 533 PFKM-WT nuclei, n = 595 siPFKM and PFKM-WT nuclei, n = 506 PFKM-RK nuclei and n = 562 siPFKM and PFKM-RK nuclei from three ROIs. e, Colocalization analysis of MyoG and DAPI normalized by total nuclei count (×10 magnification). 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). f, Representative IF of MyHC (red) and DAPI (blue) in cultured human muscle cells after differentiation (3 days) with siControl or siPFKM treatment, with or without PFKM overexpression of PFKM-WT or PFKM-RK. Scale bars, 20 μm, n = 1,665 control nuclei, n = 1,462 siPFKM nuclei, n = 1,239 PFKM-WT nuclei, n = 1,014 siPFKM and PFKM-WT nuclei, n = 886 PFKM-RK nuclei and n = 1,137 siPFKM and PFKM-RK nuclei from four fields of view. g, Quantification of mean fluorescence intensity (MFI) of MyHC per field of view (×10 magnification). 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). h, Representative IF of MyoG (pink) and DAPI (blue) in undifferentiated (day 0) cultured human muscle cells in control or PFKM-WT overexpression. Scale bars, 10 μm (n = 379 control cells and n = 298 PFKM-WT cells from five fields of view). Right, colocalization analysis of MyoG and DAPI normalized by total cell count (×20 magnification). Statistical significance was determined by unpaired two-tailed Student’s t-test, and data are shown as mean ± s.e.m. i, Representative IF of Myh1 (red) and DAPI (blue) in undifferentiated (day 0) cultured human muscle cells in control or PFKM-WT overexpression. Scale bars, 10 μm (n = 290 control cells and n = 233 PFKM-WT cells from four fields of view). Right, quantification of MFI of Myh1 per field of view (×20 magnification). Statistical significance was determined by unpaired two-tailed Student’s t-test, and data are shown as mean ± s.e.m. j, Schematic showing low PFKM leads to altered differentiation, and overexpression of both PFKM-WT and PFKM-RK rescues differentiation. Exact P values are shown in graphs.
