Fig. 4

Platinum-induced increase in SIRT1 activity contributes to the enrichment of ALDH + cells. OVCAR5 (A), OVCAR3 (B) and OVSAHO (C) were untreated (U) or treated with respective IC50 dose of cisplatin (T) for 16 h (OVCAR5—12 μM, OVCAR3—15 μM and OVSAHO—4 μM). Cell lysates were collected and analyzed by western blot. A Graph displays mean ± SEM densitometric analysis of N = 3 biological replicates of H4K16ac relative to total H2A. Relative expression of SIRT1 in OVCAR5 (D), OVCAR3 (E) and OVSAHO (F) cells treated as in A, B and C, respectively. G OVCAR5 cells infected with empty vector (EV) or SIRT1 viral shRNA followed by treatment as in A. Lysates were collected and analyzed by western blot for the indicated proteins. H OVCAR5 cells infected EV or SIRT1 viral shRNA, untreated or treated as in A followed by ALDEFLUOR assay. Graph shows mean ± SEM percent ALDH + cells in N = 3 biological replicates. Percent ALDH + OVCAR5 (I) and OVSAHO (J) cells treated with cisplatin alone or in combination with DMSO or 3 µM Ex-527 for 16 h followed by ALDEFLUOR assay. Graph depicts mean ± SEM percent ALDH + cells in N = 3 biological replicates. (K) Correlation analysis of SIRT1 and TFAM expression using RNA-seq data from TCGA OC patient dataset. L Relative expression of SIRT1 and TFAM in OVCAR5 cells infected with EV or SIRT1 viral shRNA and treated as in A. Graph displays mean relative expression ± SEM to the untreated. Gene expression was normalized to the house keeping gene Actin B. For all comparisons, P—*< 0.05, **< 0.005, ***< 0.0005, ****< 0.0001