Fig. 6: Reduction of the m.3243 A > G mutant load by inhibition of PI3K-Akt-mTORC1 requires neither selective cell death nor clonal expansion and is a cell-autonomous event.
From: Constitutive activation of the PI3K-Akt-mTORC1 pathway sustains the m.3243 A > G mtDNA mutation
a A flow chart describing the cell culture process used to assess cell growth/death over the 4- or 8-week drug pretreatments. Cells were pretreated with either vehicle, LY or RP for the time periods as specified in Fig. 6b (‘pretreatment’) and then seeded into 96-well plates for measuring cell growth/death with either vehicle, LY or RP (‘treatment’). Single-cell PCR for the m.3243 A > G mutant loads was performed at the end of pretreatments using TaqMan SNP genotyping. The chart was created with BioRender.com. b Cell growth (upper panel) and cell death (lower panel) over the 4- or 8-weeks drug pretreatments of A549 cybrid cells (left panel) and patient 1 fibroblasts (right panel) were measured using Incucyte (n = 6 culture wells; p < 0.0001). Although treatment with either LY or RP at 5 μM slowed cell proliferation in both patient 1 fibroblasts and cybrid cells, the cell death numbers were also lowered by the drug treatments. c, d A scatter plot showing the distribution of WT and mutant mtDNA measured in single A549 cybrid cells treated with LY or RP for 6 weeks showed a clear shift of mutant load distribution (c). d Mutant load distributions of single A549 cybrid cells (n > 850 cells) and patient 1 fibroblasts (n > 350 cells) treated with LY or RP for 6 and 12 weeks, respectively. Source data are provided as a Source Data file. All data represented as mean ± SD and were analysed by one/two-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
