Fig. 1: PFKM is degraded in lysosomes during Wnt signalling.
From: PFKM governs metabolic shifts throughout skeletal muscle differentiation
a, Schematic of metabolic pathways branching from glycolysis: G6P, F6P, FBP, pyruvate (Pyr), lactate (Lac), R5P, S7P, glutathione (GSH), glutathione disulfide (GSSG), glucosamine (GlcN), N-acetylglucosamine (GlcNAc) and N-acetylglucosamine-6-phosphate (GlcNAc-6P). b, Relative abundance of glucose-derived metabolites following treatment with control buffer or Wnt3a treatment (2 h) in HeLa cells by LC–MS (n = 3 biological replicates). Red, increase; blue, decrease. c, Representative immunofluorescence (IF) of GFP–PFKM (green), LAMP1 (pink) and DAPI (blue) in HeLa cells after a 20 min treatment with Wnt3a or control buffer. Scale bars, 5 μm; inset scale bar, 2.5 μm (n = 156 control cells and n = 137 treated cells from five fields of view). Right, colocalization analysis of PFKM and LAMP1 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. d, Immunoblotting (IB) of PFKM, PFKL and PFKP in HeLa cells after control buffer or Wnt3a treatment for 15 min and 5 h. Right, protein levels normalized to loading control (Actin), assessed by densitometry of n = 3 biological replicates. 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 > 0.05). FC, fold change. e, IB of PFKM, PFKL and PFKP in HEK293 cells treated with a Wnt3a treatment time course with 10 μM cycloheximide (CHX). Right, protein levels normalized to loading control (H3), assessed by densitometry of n = 3 biological replicates. 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 > 0.05). f, IB of PFKM in HEK293 cells after control buffer or Wnt3a treatment for 20 min following a bafilomycin pretreatment (12 h). 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). Exact P values are shown in graphs.
