Fig. 5: Aberrant signalling through mTOR correlates with increased protein synthesis in MpM.

a Schematic summary showing mTORC1 and mTORC2 signalling pathways upstream of protein synthesis regulation. b Western blot analysis of eight primary cell lines derived from patients with MpM and from untransformed mesothelium (NM), which were probed with antibodies against Akt and phosphorylated Akt, as a marker for mTORC2 activity, and 4EBP1 and phosphorylated 4EBP1, p70S6K and phosphorylated p70S6K, as a marker for mTORC1 activity. Actin was used as a sample integrity control. Source data are provided as a Source Data file. Experiments were repeated on three occasions and similar data were obtained in each case. c Western blot analysis of eight cell lines derived from patients with MpM and from a pool of untransformed mesothelium (NMS), which were probed with antibodies against eEF2 and phosphorylated eEF2. Beta-actin was used as a sample integrity control. Experiments were repeated on three occasions. d Primary MpM cells (Meso 7 T or Meso 8 T) were treated with 25 μM 4EGI-1 and the rate of cell growth was assessed over a 72 h time period with a crystal violet assay. Error bars represent standard deviation, the centre of the error bars is the mean (n = 4 where n = number of biological repeats of growth rate measurements as determined by change in absorbance). Blueline is untreated, red line treated with 25 μM 4EGI-1. e Primary MpM cells (Meso 7 T or Meso 8 T) were treated with 25 μM 4EGI-1. Puromycin incorporation from n = 3 independent experiments (representative in Supplementary Fig. 7 A). Error bars represent standard deviation and significance was assessed using unpaired Student’s t test, p values are shown on the bar graphs. f Primary MpM cells (Meso 7 T or Meso 8 T) were treated with 100 nM Hippuristanol and the rate of cell growth was assessed over a 72 h time period with a crystal violet assay. Error bars represent standard deviation and the centre of the error bars represents the mean (n = 4 where n = number of biological repeats of growth rate measurements). Blueline is untreated, red line treated with 100 nM Hippuristanol. g Primary MpM cells (Meso 7 T or Meso 8 T) were treated with 100 nM Hippuristanol. Puromycin incorporation from n = 3 independent experiments (representative in Supplementary Fig. 7 C). Error bars represent standard deviation and the centre of the error bars represents the mean. Significance was assessed using two-sided unpaired Student’s t test adjusted for multiple comparisons, p values are shown on the bar graphs. h Primary MpM cells (Meso 7 T or Meso 8 T) were treated with 100 nM Torin 1 and the rate of cell growth was assessed over a 72 h time period with a crystal violet assay. Error bars represent standard deviation, with the centre of the error bar the mean (n = 3, n = number of independent biological repeats of growth rate measurements). Blueline is untreated, red line treated with 100 nM Torin 1. i To assess protein synthesis rates, primary MpM-derived cell lines Meso 7 T and Meso 8 T were incubated with 100 nM Torin 1 for 30 m, followed by 33 μCi of 35 S methionine and the rate of incorporation over a 30 m period was assessed. Error bars represent standard deviation and the centre of the error bars represents the mean. (n = 3) and where n = number of independent biological repeats. Significance was assessed using a two-sided unpaired Student’s t test adjusted for multiple comparisons, p values are shown on the bar graphs. j Primary MpM cells (Meso 7 T or Meso 8 T) were treated with 100 nM AZD2014 and the rate of cell growth was assessed over a 72 h time period with a crystal violet assay. Error bars represent standard deviation, the centre of the error bars represents the mean (n = 8, n = number of independent biological repeats of growth rate measurements). Blueline is untreated, red line treated with 100 nM AZD2014. k Western blot analysis of primary MpM cell lines (Meso 7 T and Meso 8 T) treated with 100 nM Torin 1 for 24 h and probed with antibodies to assess changes in mTOR pathway, mitochondrial protein expression and mitochondrial dynamics. Actin was used as a sample integrity control. l m7GTP pull-down assay was performed on primary MpM cell lines (Meso 7 T and Meso 8 T), which were treated with 100 nM Torin 1 for 30 m. Input extracts and cap analogue pull-downs were probed with antibodies against eIF4G and 4EBP1. eIF4E was used as an input and pull-down control. Experiments were repeated on three occasions. m Correlation analysis between rpS6 phosphorylation (mTOR activity) and SDH expression in mesothelioma TMAs. Of the n = 812 individual TMA cores, 723 (89.04%) had a Phosho rpS6 H-score and 734 (90.39%) had an SDH H-score. SDH H-score was split into four equally sized groups and Spearman’s correlation analysis was performed for Phospho pS6 H-score against these groups. From n = 689 cores which had both a pS6 and SDH H-score, Spearman’s correlation coefficient was 0.172. The box plots show the box from the first quartile to the third quartile. The middle line represents the median. The whiskers go up to the maximum and minimum values.