Extended Data Fig. 2: mTORC1 signaling is enhanced during differentiation in XLCNM cellular models.
From: Lysosomal phosphoinositide turnover acts upstream of RagGTPase–mTORC1 and controls muscle growth
a–c, Effects of LY294002 (pan-PI3K inhibitor) and salubrinal (selective inhibitor of eIF2α dephosphorylation) on protein synthesis (HPG488 and AHA594 labeling) and differentiation capacity (myotube area and percentage of multinucleated myotubes) in Mtm1-KO cells. Scale bar, 200 µm. d, Inhibition of mTORC1 at early stages of differentiation does not restore protein synthesis rates or rescue the differentiation phenotype of Mtm1-KO myotubes. Quantification is shown as violin plots (HPG488 signal) and histograms (myotube area). e, Representative immunoblots showing mTORC1 activity (p-S6K/S6K) and AKT phosphorylation (p-AKT S473 and T308) in myotubes derived from two independent XLCNM patients (two biologically independent experiments). f, Representative immunoblots showing mTORC1 downstream targets (p-S6K and p-4E-BP1) in Mtm1 knockdown cells following re-expression of catalytically active (MTM1-FL) or inactive (MTM1-C375S) MTM1 (two biologically independent experiments). g, Representative myosin heavy chain (MHC) staining of CTRL and MTM1-KOCas9 myotubes, showing impaired differentiation in MTM1-KOCas9 cells. Scale bar, 100 µm. h, Representative hematoxylin and eosin (H&E) staining of tibialis anterior (TA) muscles from CTRL/WT and Mtm1-KO mice following notexin-induced regeneration or PBS injection (n = 4 mice per group). Scale bar, 200 µm. i, Morphometric analysis of nuclei number per fiber in transversal TA sections and isolated muscle fibers, showing reduced nuclear content in Mtm1-KO mice. Scale bar, 100 µm. All graphs show mean ± s.d. Individual data points represent individual myotubes, myotube areas, fibers, or mice, as indicated. For a–d, one-way ANOVA followed by Dunnett’s multiple-comparisons test was used (n = 57 myotubes for protein synthesis measurements in a, n = 42 myotubes for differentiation analyses in b,c, n = 59 myotube areas and n = 39 myotubes in d; three biologically independent experiments). For g, two-sided Mann–Whitney U tests were used (n = 44 myotube areas and n = 61 myotubes; three biologically independent experiments). For i, one-way ANOVA followed by Dunnett’s multiple-comparisons test was used (n = 4 mice per group; 20 fibers analysed per mouse). No statistical analysis was performed for representative immunoblots (e,f) or histological images (h). Exact P values are indicated in the graphs, except when P < 0.0001. Molecular weights in e and f are indicated in kDa.
