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. Author manuscript; available in PMC: 2015 Apr 30.
Published in final edited form as: Nature. 2014 Aug 10;514(7524):628–632. doi: 10.1038/nature13611

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

Extended Data Figure 9

a, Effects of the combination of AZD8330 and BEZ235 (AZD+BEZ) on human tumor spheres. Some cells, usually doublets, are able to survive the treatment (5×). b, Immunoblots of human tumor spheres treated or not with the AZD+BEZ probed with anti-phospho-p42/44 (pErk), total-Erk (Erk), phospho- Akt (pAkt), Akt and (β-actin (Actin) antibodies, two independent tumors were reported. c, Annexin V staining of treated (AZD+BEZ) and control (Ctrl) cells after 4 days of treatment (n=3). d, Mitochondrial transmembrane potential (Δψm) of untreated (Ctrl) and treated (AZD+BEZ) human spheres with AZD8330/BEZ235 for 7 days (n=3), representative flow-cytometry analysis of two tumors. e-h, TFAM and TUFM were downregulated using two inducible shRNAs each (TFAM: #93, #95; TUFM: #63, #64) in human spheres expressing KRas (untreated) and cells surviving one week treatment with AZD8330 and BEZ235 (AZD+BEZ), after 5 days of shRNA induction cells were replated for evaluating their spherogenic capacity. e, Immunoblots of tumor spheres after 72hs of shRNA induction (+Dox) probed with anti-TFAM, TUFM and HSP90 antibodies, f, representative calcein staining after spheres replating. g-h, Effects of downregulation of TFAM and TUFM on spherogenic potential of untreated and treated cells respectively, data represent the average of two independent human tumors. i, Immunoblot of KRas expressing cells treated or not with oligomycin 200nM (Oligo, +/-) probed with anti-Thr172-phospho-AMPK and actin antibodies. Immunoblots of +KRas and -KRas cells treated with: j, etomoxir (Eto, 100 µM for 6hs) and k, bafilomycin (Baf, 50nM for 24hs) probed with anti-Thr172-phospho-AMPK and vinculin antibodies. l, AnnexinV staining of cells treated for 48hs with bafilomycin 50nM (Baf) and etomoxir 100µM (Eto) clearly shows a significant decrease in viability in surviving cells (-KRas). Controls cells expressing KRas (+KRas) are not affected (n=3); representative dot-plots are reported. Data are mean ± s.d.