Fig. 4: The PI3K-Akt-mTORC1 axis is upregulated in the m.3243 A > G mutant cells.

a Analysis of RNA-seq data from the patient fibroblasts by QIAGEN ingenuity pathway analysis (IPA) showed cell signalling pathways, which are enriched in cells carrying the m.3243 A > G mutation. These include striking and significant increases in the expression of pathways involving PI3K-Akt, mTOR, EIF2, and NRF2. It is notable also that PTEN expression, a negative regulator of PI3K-Akt-mTOR signalling was downregulated (p < 0.05). b, c Immunoblotting of p-Akt (S473)/Akt, p-S6 (S235/236)/S6 and p-AMPK (T172)/AMPK in patient fibroblasts grown in the presence of 10 or 1% FBS media (b), showing a major increase in p-Akt/Akt (p < 0.0001) and p-S6/S6 (p < 0.0001) in the presence of 1% FBS media in the mutant cells; in contrast, there is no difference in p-AMPK/AMPK (c; n = 3 independent experiments). d, e Immunofluorescence staining for p-Akt (S473)/Akt (n = 66 and 78 muscle fibres for Control and Patient, respectively; p < 0.0001) and p-S6 (S235/236)/S6 (n = 30 and 23 muscle fibres for Control and Patient, respectively; p < 0.0001) in patient muscle biopsies (d) confirmed the increased phosphorylation of Akt and S6 in the patient muscle biopsies (e). Scale bar = 100 μm. Source data are provided as a Source Data file. All data, except Fig. 4a, are represented as mean ± SD and were analysed by one-way ANOVA with Tukey’s multiple comparisons test for fibroblasts and by unpaired t-test for biopsies (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).