Fig. 3

CPI-613 inhibits TCA cycle in ovarian cancer xenografts. Pooled tumor tissue (n = 4) was used to isolate mRNA from (A) OVCAR3, (B) CaOV3 and (C) F2 xenografts and subjected to quantitative polymerase chain reaction to estimate the expression levels of dihydrolipoamide dehydrogenase (DLD), oxoglutarate dehydrogenase (OGDH), dihydrolipoamide S-succinyltransferase (DLST), pyruvate dehydrogenase E1 subunit alpha 1 (PDH1a) and pyruvate dehydrogenase E1 subunit beta (PDHb). D Representative immunoblots showing expression of DLST, PDH1a and beta-actin in proteins isolated from 3 individual mouse tumor tissue of OVCAR3, CaOV3 and F2 xenografts. E, F, G Bar plots show normalized densitometry analysis from 2 individual blots from each cell line. H-J Single cell suspension was prepared from freshly isolated xenografts (n = 3), cells were pooled to plate 70,000 cells/well in triplicates and subjected to real-time XFe Seahorse analysis for bioenergetics profiling. Oxygen consumption rate (OCR), an indicator for mitochondrial respiration, was assessed at the basal and stressed conditions with port injections of (1) oligomycin, (2) FCCP (Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone), and a combination of (3) rotenone-antimycin in xenografts from (H) OVCAR3, (I) CaOV3 and (J) F2. The bar graph represents basal and stressed OCR. K-M Targeted analysis of the TCA cycle metabolites was performed to assess the levels of various metabolites in pooled xenografts (n = 3) in triplicates from (K) OVCAR3, (L) CaOV3 and (M) F2. The schematic figure represents a simplified version of the TCA cycle. NS = non-significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 untreated control (C) compared to CPI-613