Pan s6

The human ovarian cancer cell lines, HEY, SKOV3, OVCAR5, IGROV1 and OV433 were used. The SKOV3 and OVCAR5 cell lines were maintained in DMEM/F12 medium with 5% fetal bovine serum (FBS). The IGROV1 and OV433 cell lines were maintained in RPMI 1640 with 10% FBS. The HEY cell line was maintained in RPMI 1640 with 5% FBS. All medium was supplemented with 100 U/ml of penicillin and 100 ug/ml of streptomycin. The cells were cultured in a humidified 5% CO2 at 37°C. For glucose studies, the SKOV3 and OVCAR5 cell lines were cultured in DMEM medium without glucose (Cat # 11966–025, Gibco), containing 10% dialyzed FBS and supplied with specific concentrations of glucose (1 mM, 5 mM and 25 mM).
Glucose solution, MTT and DMSO were purchased from Sigma-Aldrich (St. Louis, MO). 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) was bought from Life Technologies (Grand Island, NY). Enhanced chemiluminescence (ECL) detection reagents were purchased from GE Health care (Piscataway, NJ). The non-fat milk, 20% bull serum albumin (BSA) and RNase A were purchased from Sigma (St. Louis, MO, USA), and all the primary antibodies for phosphorylated-S6, pan-S6, phosphorylated-AKT, pan-AKT, cyclin D1, CDK6, p21, Bcl-2, Mcl-1, Ki-67, VEGF, cleaved caspase 3, perk, bip, calnexin, MMP-9 and α-tubulin were obtained from Cell Signaling Technology (Danvers, MA, USA).

Three OC cell lines, SKOV3, Hey and IGROV-1, were used for the experiments. The SKOV3 cells were maintained in DMEM/F12 medium with 10% fetal bovine serum (FBS). The IGROV-1 cells were maintained in RPMI 1640 with 10% FBS. The Hey cell line was maintained in RPMI 1640 with 5% FBS. All medium was supplemented with 100 U/ml of penicillin and 100 ug/ml of streptomycin. The cells were cultured in a humidified 5% CO2 at 37°C. Phenformin was purchased from Sigma (St. Louis, MO). MTT (3-(,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), RNase A and RIPA buffer were purchased from Sigma (St. Louis, MO). Antibodies to phosphorylated-AMPK (Thr172), phosphorylated-S6 (Ser 235/236), β-actin, pan-AMPK and pan-S6 were obtained from Cell Signaling Technology (Beverly, MA). The Annexin V FITC kit was purchased from BioVision (Mountain View, CA). Enhanced chemiluminescence Western blotting detection reagents were purchased from Amersham (Arlington Heights, IL). All other chemicals were purchased from Sigma.

The ECC-1 and KLE cells treated with ONC201 for 24–36 h. Cell lysates were prepared in RIPA buffer. Protein concentration was measured by BCA assay. Equal amounts of protein were separated by 10–12% gel electrophoresis and transferred onto a PVDF membrane. The membrane was blocked with 5% nonfat dry milk and then incubated with different primary antibodies overnight at 4 °C. The primary antibodies from Cell Signaling (Beverly, MA) used in this study included as follows: MCL-1, BCL-2, Phos-S6, Pan-S6, Phos-AKT, Pan-AKT, PERK, Bip, Erol-1, IRE1-a, PD-I, CDK4, CyclinD1, E-cadherin, VEGF and Slug. DRD2 and DRD5 antibodies were from Santa Cruze Biotechnology Inc. (Dallas, TX).). β-Actin and α-Tubulin were from Sigma (St. Louis, MO). β-Actin and α-Tubulin were used at 1:5000 dilution, and the rest antibodies were diluted: 1:1500. The membrane was then washed and incubated with a secondary peroxidase-conjugated antibody for 1 h after washing. Antibody binding was detected using SuperSignal™ West Pico on the ChemiDoc™ Image System (Bio-Rad). After developing, the membrane was stripped and re-probed using antibodies against β-actin or α-tubulin to confirm equal loading. Intensity for each band was measured and normalized to β-actin or α-tubulin as an internal control.