Propium iodide

(S)-(+)-CPT (C9911), Evodiamine (E3531), sodium bicarbonate, Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propium iodide (PI), and Hochest 33342 were purchased from Sigma-Aldrich (St. Louis, MO, USA), Dulbecco’s modified Eagle’s medium (DMEM), trypsin, fetal bovine serum (FBS), L-glutamine, and sodium pyruvate were purchased from Gibco-BRL (Grand Island, NY, USA). An antibody against cyclin D1 was purchased from Ventana (Tucson, AZ, USA). γ-H2A.X, AMPK, phospho-AMPK, acetyl-CoA carboxylase (ACC), phospho-ACC, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling (Danvers, MA, USA). Ki-67 rabbit monoclonal primary antibody was purchased form DAKO (MIB-5, Denmark). Comet Assay kit was obtained from Trevigen (Gaithersburg, MD, USA). General Layer Medium (GLM) capacity chip was obtained from Bio-Rad (Hercules, CA, USA). The lentiviral vectors (LVs), luciferase (Luc) and green fluorescent protein (GFP) vectors, and beetle luciferin were obtained from Promega (Madison, WI, USA). iView Universal DAB Detection kit was obtained from Ventana (Tucson, AZ, USA). In Situ Cell Death Detection kit, POD, was obtained from Roche Applied Science (Penzberg, Germany).

Apoptosis assay using AnnexinV/PI staining was performed for HGSC and MOE cells. Cells were plated at 1000 cells/well in a 6 well plate. Cells were transfected for 72 hours as described above before collection. Cell media was centrifuged at 800 g to collect dead cells floating in the media. Live cells were collected using 0.25% trypsin, 1 mM EDTA (Invitrogen, Carlsbad, CA) to disrupt the cell monolayer. Total cell concentration of live and dead cells was approximately 1 × 10⁶ cells/ml. These cells were combined and resuspended in 300 ul 1X Annexin V Binding Buffer (140 mM NaCl, 4 mM KCl, 0.75 mM MgCl₂ and 10 mM HEPES in DDW). 4 samples were created for each cell line: No staining, Annexin only staining, PI only staining, Annexin/PI staining. Propium iodide was added to cells to a final concentration of 2 ug/ml (Sigma-Aldrich, St. Louis, MO). Annexin V was diluted 1:60 and 1 ul was added to cells (Thermo Fisher Scientific, Waltham, MA). Cells were incubated in the dark for 15 minutes before adding another 400 ul Annexin V Binding Buffer. Cells were transferred to a cell strainer (Corning Incorporated, Corning, NY) and sent to the flow cytometry core at the University of Illinois-Chicago.