Fig. 3: MCL1 controls cellular bioenergetics via mTORC1.

A–C Oxygen consumption rate (OCR) (A), basal (B) and maximal (C) respiration measured by Seahorse XF Mito Stress Test of CHL-1 cells transduced with the indicated shRNAs for 72 h. D, E Extracellular acidification rate (ECAR) of CHL1 cells measured as in (A). (n = 3 biologically independent samples). Mitochondria- (F) and glycolysis- derived (G) ATP production rate measured by Seahorse XF Real-Time ATP Rate Assay of CHL-1 cells transduced with the indicated shRNAs for 72 h. (n = 3 biologically independent samples). H ATP levels measured using LC-MS/MS in CHL-1 cells transduced with either scrambled shRNA or shRNA against MCL1. Lines connect values from biologically independent repeats (n = 3 biologically independent samples). I Immunoblotting analysis of lysates prepared from subcutaneous xenografts established in both flanks of mice with CHL-1 cells transduced with either scrambled shRNA or doxycycline-inducible shRNA against MCL1. The samples derived from the same experiment but different gels for pS6 and pS6K, another for S6K1 and S6 and another for MCL1 were processed in parallel. J Basal mitochondrial respiration of tumors derived from CHL-1 cells as in (I) (n = 3 mice per group). Tumors were isolated, immediately dissociated using GentleMACS tissue dissociator, plated in poly-D-lysine coated Seahorse plates and measured by Seahorse XF. K ATP levels in tumors derived from CHL-1 cells as in (I) measured using LC-MS/MS and. Lines connect values from the two tumors established on both flanks of the same mouse. n = 3 mice per group). L, M Qualification of gamma radiation count normalized to administrated ¹⁸FDG and dry weight [kBq/mg] of control (scr) and MCL1-depleted (shMCL1) tumors established as in (I) from CHL-1 (L) or SK-MEL30 (M) cells followed by ¹⁸FDG-PET assay and finally isolation of tumors and measurement of gamma radiation using automated gamma counter. Lines connect values from the two tumors established on both flanks of the same mouse. (n = 4 mice per group). N Representative image of coronal plane of mice bearing control or MCL1-depleted tumors on both flanks (circled) established as in (I) followed by ¹⁸FDG-PET assay. Mitochondria- (O) and glycolysis- derived (P) ATP production rate measured by Seahorse XF Real-Time ATP Rate Assay of CHL-1 cells transduced as indicated. (n = 3 biologically independent samples). Statistics are derived from 3 or more biological replicates. Data is presented as mean +/- SD and significance is determined by paired two-tailed t-test.