Fig. 2

CHCHD4-mediated tumour cell growth and mTORC1 signalling is coupled to CI activity. a Western blots show levels of CHCHD4, NDUFS3 (CI), SDHA (CII) UQCRC2 (CIII), COXIV (CIV) in whole cell lysates of control (Ctrl) U2OS cells, CHCHD4 (WT)-expressing cells (WT.cl1) or CHCHD4-(C66A/C68A)-expressing cells (C66A/C68A). α-Tubulin was used as a load control. b In-gel NTB assay of CI activity in mitochondrial fractions isolated from control U2OS cells, CHCHD4 (WT)-expressing cells (WT.cl1, WT.cl3) and CHCHD4 (C66A/C68A)-expressing cells (C66A/C68A). c Basal NADH fluorescence (as % of total NADH/NAD pool) in control U2OS cells or CHCHD4 (WT)-expressing cells (WT.cl1). ± SD. n = 3. d Chart shows GSEA of proteins upregulated in CHCHD4 (WT)-expressing cells (WT.cl1) relative to control U2OS cells, as assessed by SILAC [30]. n = 3. e Chart shows relative growth rates of cells described in (b) at 72 h relative to 0 h. ± SD. n = 3. f Western blots show levels of phosphorylated (P-) and total (T-) p70S6K, and puromycin labelled polypeptides in control U2OS cells and CHCHD4 (WT)-expressing cells (WT.cl1) either untreated (NT), or treated with rotenone (500 nM), BAY 87-2243 (50 nM) or sodium azide (100 μM) for 24 h. β-Actin was used as a load control. g Chart shows relative growth of control U2OS cells, and CHCHD4 (WT)-expressing cells (WT.cl1) stably expressing an empty vector (pWPI) or yeast NDI1, treated with BAY 87-2243 using a 10-fold dilution curve (starting dose 100 nM) for 72 h. ± SD. n = 3