**** mutant, 2594delC germline mutation in exon 11 and deletion of the wild type allele; RRID: CVCL_B079), UWB1.289 WT (RRID: CVCL_B078) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). of OvCa. mutated HGSOC lines: UWB1.289 WT, UWB1.289 MUT, PEO1, OVCAR4, and OVCAR3 (Figure S1A). The cells were treated with CPI-613 and CD133/CD117 levels, and cell viability were measured 7 days following the treatment by flow cytometry. Initially, we conducted dose-response curves with CPI-613 with all cell lines. Based on our results, we selected 75 M as our treatment concentration, given that it was near the IC50 (half maximal inhibitory concentration) for all cell lines. Carboplatin/paclitaxel treatment was used as a positive control since it has been demonstrated to increase the frequency of CSCs . Initially, we tested the effect of CPI-613 on its target enzyme pyruvate dehydrogenase (PDH) by assessing the expression of the phosphorylated form of PDH (pPDH), the inactivated form of the protein . To determine whether CPI-613 directly inhibited mitochondrial metabolism, we used the UWB1.289 MUT cell line. The cells were harvested following Dehydroepiandrosterone acute exposure (2 h) of CPI-613, and the lysates were subjected to a Western blot with an antibody that is specific for the phospho S293 residue of the PDH E1 subunit of the protein. Treatment with CPI-613 induced an increase in pPDH, demonstrating that a short time window was sufficient to observe the inhibitory effect of the drug (Figure 1A and Figure S3). To confirm that CPI-613 targets the TCA mitochondrial metabolic cycle by depleting cellular adenosine triphosphate (ATP), we assessed the phosphorylation status of 5 adenosine monophosphate-activated protein kinase (AMPK). AMPK is phosphorylated in the presence of a high adenosine monophosphate (AMP)/adenosine diphosphate (ADP) ratio as a consequence of ATP depletion in the cells . Acute treatment with CPI-613 resulted in an increase in AMPK phosphorylation (Figure 1A and Figure S3). These data confirmed that CPI-613 targets mitochondrial metabolism in our model. CPI-613 treatment alone was associated with a decrease in CD133+ and CD117+ cell frequency in all the cell lines compared to carboplatin/paclitaxel treatment which either Dehydroepiandrosterone had no (PEO1) or induced the expected CSC enrichment (Figure 1B). While carboplatin/paclitaxel alone was more efficient than CPI-613 in reducing overall tumor cell viability, its lack of negative effects on the CSC populations was clear (Figure 1B, left panel) again suggesting a preferential effect of CPI-613 on the CSCs. The combination of CPI-613 and carboplatin/paclitaxel had the capacity to offset either the resistance or enrichment of CD133+ and CD117+ cell frequency observed in response to the carboplatin/paclitaxel treatment alone (Figure 1B, < 0.0001). The error bars represent mean SEM. **** mutant, 2594delC germline mutation in exon Dehydroepiandrosterone 11 and deletion of the wild type allele; RRID: CVCL_B079), UWB1.289 WT (RRID: CVCL_B078) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Human OvCa cell line PEO1 (BRCA2 mutated, homozygous mutation 5193C>G; RRID: CVCL_2686) was purchased from SigmaCAldrich (St. Louis, Dehydroepiandrosterone MO, USA). OVCAR4 (RRID: CVCL_1672) and OVCAR3 (RRID: CVCL_0465) were provided by the National Cancer Institute C Developmental Therapeutics Program (NCI-DTP; Rockville, MD, USA). All cell lines were routinely tested for cultivated at 37 C in 5% CO2 humidity, and passaged until passage 12. UWB1.289 MUT and UWB1.289 WT Rabbit Polyclonal to BL-CAM (phospho-Tyr807) were maintained in 50% RPMI 1640 (GIBCO, Life Technologies; Carlsbad, CA, USA), 50% MEGM (Mammary Epithelial Growth Medium, MEGM Bullet Kit CC-3150; Lonza, Walkersville, MD, US), 10% FBS (GIBCO, Life Technologies; Carlsbad, CA, USA), and 1% Pen/Strep (Thermo Fisher Scientific; Waltham, MA, USA). UWB1.289 WT is a stable cell line derived from UWB1.289 MUT (ATCC CRL-2945), in which BRCA1 function was restored through transfection with a plasmid carrying the wild-type gene; selection was maintained by culturing the cells in 200 g/mL of Dehydroepiandrosterone G-418 (Life Technologies; Carlsbad, CA, USA). OVCAR3 cells were maintained in RPMI 1640 (GIBCO), 10% FBS (GIBCO), 1% Pen/Strep (Thermo Fisher Scientific), and 0.01 mg/mL of bovine insulin (SigmaCAldrich). OVCAR4 cells were maintained in RPMI 1640 (GIBCO), 10% fetal bovine serum (FBS) (GIBCO), and 1% Pen/Strep (Thermo Fisher Scientific). PEO1 cells were maintained in RPMI 1640 (GIBCO), 10% FBS (GIBCO), 1% Pen/Strep (Thermo Fisher Scientific), and 2 mM Sodium Pyruvate (GIBCO). 4.2. Drug Treatment (Carboplatin, Paclitaxel, Olaparib, and CPI-613)-MTT and Cell Counting Olaparib and CPI-613 were purchased from Selleckchem (Houston, TX, USA), and carboplatin and paclitaxel were obtained from Sigma (St. Louis, MO, USA). Olaparib and carboplatin concentrations were as previously described . IC50 value (the concentration required to kill and/or inhibit proliferation of cells by 50%.