Extended Data Figure 9: Effect of mitochondrial downregulation in human tumour spheres and metabolic stress mediated by inhibition of autophagy.

a, Effects of the combination of AZD8330 and BEZ235 (AZD+BEZ) on human tumour spheres. Some cells, usually doublets, are able to survive the treatment (×5). b, Immunoblots of human tumour spheres treated or not with AZD+BEZ probed with anti-phospho-p42/44 (pErk), total-Erk (Erk), phospho-Akt (pAkt), Akt and β-actin (Actin) antibodies; two independent tumours are reported. c, Annexin V staining of treated (AZD+BEZ) and control (Ctrl) cells after 4 days of treatment (n = 3). d, Mitochondrial transmembrane potential (Δψ_m) of untreated (Ctrl) and treated (AZD+BEZ) human spheres with AZD8330 and BEZ235 for 7 days (n = 3); representative flow-cytometry analysis of two tumours. e–h, TFAM and TUFM were downregulated using two inducible short hairpin RNAs (shRNAs) each (TFAM: #93, #95; TUFM: #63, #64) in human spheres expressing KRas (untreated) and cells surviving 1 week of treatment with AZD8330 and BEZ235 (AZD+BEZ); after 5 days of shRNA induction cells were replated for evaluating their spherogenic capacity. e, Immunoblots of tumour spheres after 72 h of shRNA induction (+Dox) probed with anti-TFAM, TUFM and HSP90 antibodies. f, Representative calcein staining after sphere replating. g, h, Effects of downregulation of TFAM and TUFM on spherogenic potential of untreated (g) and treated (h) cells; data represent the average of two independent human tumours. i, Immunoblot of KRas-expressing cells treated or not with oligomycin 200 nM (Oligo, +/−) probed with anti-Thr-172-phospho-AMPK and actin antibodies. j, k, Immunoblots of +KRas and −KRas cells treated with etomoxir (Eto, 100 μM for 6 h) (j) and bafilomycin (Baf, 50 nM for 24 h) (k) probed with anti-Thr-172-phospho-AMPK and vinculin antibodies. l, Annexin V staining of cells treated for 48 h with bafilomycin 50 nM (Baf) and etomoxir 100 μM (Eto) clearly shows a significant decrease in viability of SCs (−KRas). Control cells expressing KRas (+KRas) are not affected (n = 3); representative dot-plots are reported. Data are mean ± s.d.