2.1 Reagents
Thymoquinone (TQ) (#03416) was generously provided by Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). Primary antibodies against PI3K (#4292), ATG-7 (#8558), PARP (#9542), GAPDH (#2118), Caspase-3 (#9665), FASN (#3180), P62 (8025), Beclin-1 (#3495), Bax (#2772) and LC3B (#3868) were purchased from Cell Signaling Technology (Beverly, MA). Antibody against p53 (DO-I; #sc-126) was from Santa Cruz Biotechnology. Antibody against Lamp-1 (#611042) was from BD Transduction Laboratories. Secondary antibodies for immunoblotting were peroxidase goat anti-mouse IgG (#7076) and peroxidase goat anti-rabbit IgG (#7074) from Cell Signaling Technology. Secondary antibodies for immunofluorescence staining were Alexa Fluor 594 donkey anti-rabbit (A21207) and Alexa Fluor 488 donkey anti-mouse (A21202) from Thermo Fisher Scientific (Rockford, IL USA). Pefabloc SC was from Roche. Cisplatin injection (6 mL : 30 mg) was provided by Hansoh Pharma company.
2.2 Cell cultures
Human ovarian serous cystadenocarcinoma HO-8910 (Coriell Cell Repositories) and SKOV3 were obtained from Culture Collection of the Chinese Academy of Sciences (Shanghai, China) where they were characterized by mycoplasma detection, DNA-Fingerprinting, isozyme detection and cell vitality detection. Cells were both cultured in McCoy’s 5A Medium (#PYG0025) from Boster Biological Technology co. Ltd. (China), supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 100 unit/mL penicillin and 100 μg/mL streptomycin. For serum starvation, cells were washed once and incubated overnight in medium containing 0.1% FBS.
2.3 Proliferation assay
HO-8910 and SKOV3 cells were cultured as indicated. Confluent cells were detached with trypsin, seeded into 96-well plates (8000 cells/well) in 10% FBS-5A, and allowed to attach for 24 h. After starvation in 0.1% FBS-5A overnight, cells were treated with indicated doses of test compounds for 24 h, 48 h or 72 h. The number of viable cells in 96-well plates was determined by the instruction of CellTiter 96 AQueous One Solution Cell proliferation Assay kit (Promega, #G3580).
2.4 Cell cycle measurements
For the cell cycle study, 1 × 106 cells were seeded in 6-well plate and incubated at 37 °C in 5% CO2 overnight. The next day cells were starved for 12 h and then treated with test compounds for 24 h. Cell cycle was determined by flow cytometric analysis using cell cycle detection kit (#KGA511, KeyGENBioTECH, Nanjing, China) according to the manufacturer's instructions. Briefly, cells were harvested with trypsin and washed twice with PBS. After that, cells were fixed in cold 70% ethanol and stored at 4 °C. On the day of analysis, ethanol was removed by centrifugation and cells were washed twice with PBS, and then treated with RNase: PI mixture for 30 min at 37 °C. The staining samples were processed by a flow cytometer (NovoCyte, ACEA Biosciences, Inc. CA USA). The cell cycle analysis was performed with NovoExpress software (ACEA Biosciences).
2.5 Detection of cell apoptosis by FACS
Apoptosis of cells was determined by flow cytometric analysis using Annexin V-kFluor647/PI apoptosis detection kit (#KGA605, KeyGENBioTECH, Nanjing, China) according to the manufacturer's instructions. After trypsinization, cells were washed twice in cold PBS and suspended at 5×105/mL. Subsequently, 5 μL of Annexin V-kFluor647 reagent and 5 μL of PI reagent were added into cell suspension and stored for 15 min at room temperature in dark place. Apoptotic cells were examined and quantified by using flow cytometry (NovoCyte, ACEA Biosciences, Inc. CA USA).
2.6 Immunoblotting
Subconfluent cell cultures were starved and incubated with indicated doses of test compounds for 24 h. Cells were washed once in ice-cold phosphate-buffered saline (PBS) and lysed in RIPA buffer (0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 1% Triton X-100, 10% glycerol, 20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 150 mM NaCl), supplemented with 1 mM Pefabloc and 1 mM sodium orthovanadate. Total cell lysate (TCL) were then clarified by centrifugation at 14000 × g for 10 min at 4 °C. TCL samples were boiled for 5 min in SDS sample buffer containing 10 mM dithiothreitol (DTT) and the proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electro-transferred to PVDF membranes (Immobilon P), which were blocked in 5% bovine serum albumin (BSA) in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, containing 0.1% Tween 20. Primary antibodies were diluted according to the manufacturer's instructions, or used at 1 μg/ml for home-made antibodies, and incubated with the membranes overnight at 4°C. After washing, the membranes were incubated for 1 hour with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies (both from Invitrogen Life Technologies), and proteins were visualized using enhanced chemoluminescence (ECL) Western blotting detection system (SuperSignal West Dura Extended Duration Substrate, #34075) from Thermo Scientific on a cooled charge-coupled (CCD) device camera (Odyssey Fc).
2.7 Immunofluorescence staining
The immunofluorescence staining was performed as previously described with modification according to the primary antibody instructions (12). The cells were grown to confluency in 6-well plates onto glass cover slips. The cover slips were washed twice with PBS, prior to fixation with pre-cold methanol for 5 min at room temperature and permeabilized with 0.1% TritonX-100 (v/v) in PBS for 15 min. After washing three times with PBS, the cover slips were incubated with 2% BSA (w/v) in PBS for 1 h to block unspecific binding and incubated with primary antibodies (200 ng/mL) overnight at 4°C diluted in PBS containing 0.1% BSA. They were further washed in PBS and incubated with the secondary antibodies for 1h at room temperature. The cover slips were mounted on object slides with Vectashield H-1500 (Vector Laboratories). The cells were photographed under a fluorescence microscope (Axio Imager M2, Zeiss) with an AxioCam MRm digital camera, using a Plan-Apochromat 63 objective lens (Zeiss).
2.8 Statistical methods
Data are expressed as mean ± SD. Statistical significance was determined using the Student’s t test. P < 0.05 or P < 0.01 was considered statistically significant.