Cell line and cell culture Human pancreatic cancer cell line, PC-2, was purchased from the medical experimental animal center of the fourth military medical university. Cells were cultured in RPMI 1640 maximal medium containing 10% inactived fetal bovine serum (56°C, 30 min), 1 × 105 U/L penicillin and 100 mg/L streptomycin in a humidified atmosphere with 5% CO2 incubator at

37°C. MTT assay for the proliferation Nutlin-3 datasheet of PC-2 cells The proliferation of PC-2 cells was assessed using MTT dye reduction assay (Sigma, USA), which was conducted as described previously [9]. PC-2 cells were seeded in a 96-well plate at a density of 1 × 104 cells/well, cultured for 12 h under 37°C in 5% CO2, then treated with different concentration (50, 100, 150, 200 μmol/L) CoCl2 for 24-120 h. At the end of the treatment, MTT, 50 μg/10 μL, was added and the cells were incubated for another 4 hours. Dimethylsufloxide (DMSO; 200 μl) was added to each well after removal of the supernatant. After shaking the plate for 10 min, cell viability was assessed by measuring the absorbance at 490 nm using an Enzyme-labeling instrument (EX-800 type); all measurements were performed three times. Cell growth curve was completed using time as the abscissa and A value (mean

± SD) as the ordinate. Detection of morphological change by transmission electron microscope Uranyl acetate and lead citrate staining of cells were performed to detect morphological changes. Briefly, adherent PC-2 cells were treated MTMR9 with 200 μmol/L CoCl2 for 48 hours. After RG-7388 treatment, the treated cells were digested with pancreatin and fixed with 3% glutaraldehyde precooled in 4°C for 2 hours. To make ultra-thin sections of copper, cells were washed with phoisphate-buffered salein (PBS) once, fixed

with 1% osmic acid for 1 hour, dehydrated by acetone and embedded in see more epoxide resin. After staining with uranyl acetate and lead citrate, the sections were examined by a Hitachi-800 transmission electron microscope [10]. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) assay PC-2 cells were seeded in 6 cm culture capsules and treated with concentration gradient CoCl2 (0, 50, 100, 150, 200 μmol/L) separately for 8 h. In the group of 200 μmol/L, we selected cells at 0 h, 4 h, 8 h and 12 h point for further experiment. And then treated with 2.0 μmol/L YC-1 (0, 50, 100, 150,) for 2 h. As previously described [11], cells collected at specified time were used to extract total RNA using the Trizol reagent following the manufacturer’s instructions. 1 μgRNA synthetized cDNA through reverse transcriptase undergo listed below condition: 70°C 5 min, 42°C extended for 60 min, 95°C enzyme inactivated for 3 min and 4°C terminated reaction. Synthetical cDNA as template to carry out polymerase chain reaction.