Fig. 6: TORC1 and 2 inhibition reverse metabolic effects.

a Primary MpM cells Meso 7 T and Meso 8 T were treated with 100 nM Torin1 or left untreated for 18–20 h. The oxygen consumption rate (OCR) was measured in the SeaHorse extracellular flux assay. The vertical dashed lines indicate when oligomycin (a), FCCP (b) and rotenone + antimycin A (c) were injected. Error bars represent standard deviation, the centre of the error bar is the mean (n = 6 independent biological repeats). b The basal respiration from A was measured as the OCR value before oligomycin injection. The maximal respiration from A was measured as the highest OCR value after FCCP injection. Error bars represent standard deviation and the centre of the error bar is the mean. Significance was assessed using two-sided unpaired student’s t test adjusted for multiple comparisons, n = 6 independent biological repeats. P values are shown on the bar graph. c Cell extracts were prepared from Meso 8 T cells treated with vehicle or 100 nM AZD2014 for 24 h to inhibit mTORC1 and mTORC2, and analysed for metabolites representing multiple metabolic pathways. Volcano plot showing changes in intracellular metabolite abundance upon treatment with AZD2014. Red dots indicate metabolites for which intracellular abundance was altered significantly. Three biological experiments, each with three technical replicates absolute log2 fold change ≥0.58; two-sided paired t test was used with Benjamini–Hochberg correction to log2 transformed and centred data using R. d Intracellular abundance of TCA cycle metabolites: citrate, α-ketoglutarate, succinate, malate. Data represent the mean ± SD obtained from three biological experiments, each with three technical replicates. Significance was assessed using a two-sided Student’s t test with Benjamini–Hochberg correction, p values are shown on the graph. Blue bars untreated, red bars treated with 100 nM AZD2014. e Intracellular abundance of pyrimidine synthesis intermediates: carbamoyl aspartate, dihydroorotate, orotate, dTTP. Data represent the mean ± SD obtained from three biological experiments, each with three technical replicates. Significance was determined using two-sided Student’s t with Benjamini–Hochberg correction, p values are shown on the graph. Blue bars untreated, red bars treated with 100 nM AZD2014. f Intracellular abundance of dATP. Data represent the mean ± SD obtained from three biological experiments, each with three technical replicates. Significance was assessed using paired two-sided student’s t test with Benjamini–Hochberg correction, p values are shown on the graphs. g Electron micrographs of primary MpM cell lines (Meso 7 T and Meso 8 T) treated with 100 nM Torin 1 for 48 h. Scale bar 1 μm. h Enlarged and elongated mitochondria per field in G were calculated. In all, 19–23 different micrographs (for 369 mitochondrial particles) were analysed per sample. Error bars represent SEM and significance was assessed using a Mann–Whitney test, p values are shown. i Confocal images of primary MpM cells Meso 7 T and Meso 8 T treated with 100 nM Torin 1 for 24 h or left untreated, stained with DAPI (blue-nuclei) and ATPB (red-mitochondria). Scale bar 20 μm. Experiments were repeated on three independent occasions. j The mitochondrial networks in the samples from i were assessed in terms of the extent of elongated, mixed or enlarged/fragmented mitochondria and the data represented as pie charts, (black sections = elongated, light grey sections = mixed and dark grey = enlarged). The significance of changes in the elongated mitochondrial network was assessed using an unpaired two-sided student’s t test adjusted for multiple comparisons. Untreated vs Torin 1, p values are shown on the bar graphs.