Fig. 3: PFKM is degraded in lysosomes in human muscle cells.
From: PFKM governs metabolic shifts throughout skeletal muscle differentiation
a, UMAP of single-cell (scRNA-seq) and single-nucleus (snRNA-seq) data from the human skeletal muscle atlas13, coloured by cell type. UMAP, uniform manifold approximation and projection. b, PFKM mRNA expression from the human skeletal muscle atlas13. PFKM expression is highest in differentiated myofibres, including type I slow-twitch myofibres and type II fast-twitch myofibres. PFKM expression is low in MuSCs. c, Track plots of differentiation and Wnt signalling genes in MuSC, myofibre type I and myofibre type II cell clusters across the human skeletal muscle atlas13. Within each track plot, individual cells are ranked from lowest expression (left) to highest expression (right), with bar height indicating relative expression per cell. Tracks are colour-coded as in a. Differentiation genes: paired box 7 (PAX7), myogenin (MYOG), myoblast determination protein (MYOD), myosin heavy chain 1 (MYH1). Wnt signalling genes: cMYC, glycogen synthase kinase-3β (GSK3B), beta catenin 1 (CTNNB1). d, PFKM mRNA expression from bulk RNA-seq37 in cultured human muscle cells during a 12 day differentiation time course (n = 2 biological replicates). TPM, transcripts per million. Statistical significance was determined by one-way ANOVA with post hoc Dunnett’s analysis, and data are shown as mean ± s.e.m. Associated data in Extended Data Fig. 4 include additional target gene markers of differentiation. e,f, IB of PFKM and MyHC in cultured human muscle cells during a 12 day differentiation time course (f). Protein levels normalized to loading control (Actin), assessed by densitometry of n = 3 biological replicates (e). Statistical significance was determined by one-way ANOVA with post hoc Dunnett’s analysis, and data are shown as mean ± s.e.m. (NS, P > 0.5). g, IB of PFKM in cultured human muscle cells after differentiation (3 days), treated with control buffer or a 20 min Wnt3a treatment following a bafilomycin pretreatment (4 h). Right, protein levels normalized to loading control (vinculin), 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). h, Track plots of lysosomal genes in MuSC, myofibre type I and myofibre type II cell clusters across the human skeletal muscle atlas13. Within each track plot, gene expression data are presented as described in c. Lysosomal genes: LAMP1, cathepsin D (CTSD), cathepsin B (CTSB), ATPase H+-transporting V1 subunit B2 (ATP6V1B2), mucolipin 1 (MCOLN1), solute carrier family 11 member 1 (SLC11A1), solute carrier family 11 member 2 (SLC11A2), transcription factor EB (TFEB), transcription factor binding to IGHM enhancer 3 (TFE3), charged multivesicular body protein 2A (CHMP2A), charged multivesicular body protein 4B (CHMP4B), vacuolar protein sorting 4 isoform a (VPS4A) and vacuolar protein sorting 4 isoform b (VPS4B). i,j, Representative IF of PFKM (green), LAMP1 (pink) and DAPI (blue) in mononuclear (i) and multinuclear (j) cultured human muscle cells after differentiation (3 days). Cells were treated with control buffer or 20 min Wnt3a treatment. Insets are shown at ×20, ×40 and ×100 magnification. Scale bars, 10 μm at ×20 and ×40; 5 μm at ×100 (n = 109 control mononuclear cells, n = 107 treated mononuclear cells, n = 215 control multinuclear cells and n = 119 treated multinuclear cells from three fields of view). Right, colocalization analysis of PFKM and LAMP1 normalized by total nuclei count (×20 magnification). 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). Exact P values are shown in graphs.
