Fig. 6

CPI-613 causes mitochondrial collapse in chemoresistant ovarian xenografts. A & E Single cell suspensions were prepared from freshly isolated xenografts (n = 3), cells were pooled and cells in triplicates were labeled with epithelial cell marker Ep-CAM antibody and Mitoprobe TMRM (tetramethylrhodamine methyl ester) and mitochondria membrane potential was measured using flow cytometry. Representative flow cytometry pictures of Ep-CAM positive cells displaying TMRM fluorescence in (A) CaOV3 and (E) F2 xenografts. B, F Representative histograms showing the shift of polarization in cells from both xenografts. C, G Quantification of the Ep-CAM positive cells with TMRM measured by flow cytometry. ATP levels were measured as per manufacturer’s instructions in single cell suspensions from (D) CaOV3 and (H) F2 xenografts. Mitosox Red was used to measure the mitochondria superoxide in single cell suspensions from (I, J) CaOV3 and (L, M) F2 xenografts in Ep-CAM positive cells by flow cytometry. Bar graphs represent the quantification of the Mitosox Red signal. Lipid peroxidation was measured by measuring the malondialdehyde (MDA) in single cell suspensions from (K) CaOV3 and (N) F2 xenografts by ELISA. Caov3 cells (O) and (Q) F2 cells were plated on coverslips and treated with CPI-613 (100 µM) for 48 h and then stained with Mitosox Red and counterstained with DAPI. Representative fluorescence images are shown (400x magnification). P, R Bar graphs show quantification of the fluorescence intensity measured in 400x objective field from 3 slides per treatment. S Representative immunoblots showing expression of cytochrome C, APAF1 (apoptotic protease activating factor-1), cleaved caspase 3 (cleaved C3), cleaved caspase 9 (cleaved C9), cleaved PARP and beta-actin in proteins isolated from 3 individual mouse tumor tissue of CaOV3 and F2 xenografts. (T, U) Bar plots show normalized densitometry analysis from 2 individual blots from each xenograft. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 comparisons as indicated