These findings, for the first time, unified the current different observations about the effect of bortezomib on survivin expression, apoptosis induction and bortezomib resistance, and warranted further mechanistic studies and application of these findings in cancer therapeutics.

Methods Cell culture and reagents Colon cancer cell lines (HCT116p53+/+, HCT116p53-/-), lung cancer cell Bafilomycin A1 lines (EKVX and A549), prostate cancer cells (PC-3 and LNCaP) and multiple myeloma cell lines (KMS11 and RPMI8226) were maintained in RPMI 1640 medium. Breast cancer cells (MDA-MB-231 and MCF-7) were cultured in DMEM medium. All cell cultural mediums were supplied with 10% fetal bovine

serum (FBS, Atlanta Biologicals, Lawrenceville, GA) and penicillin (100 units/ml)/streptomycin (0.1 μg/ml) (Invitrogen, Grand Island, NY). Cells were routinely subcultured twice a week and maintained in a humidified incubator Selleckchem GSK872 with 5% CO2 at 37°C. Polyclonal anti-actin antibody and goat peroxidase-conjugated anti-rabbit IgG antibody were purchased from Sigma (St. Louis, MO). Survivin antibody (FL-142) was purchased from Santa Cruz (Santa Cruz, CA), MTT (tetrazolium salt, 3- [4,5-dimethylthiazol-2-yl]-2,5,-diphenyltetrazolium bromide) and leupeptin were purchased from Usb (Cleveland, OH). Cell treatment and siRNA/shRNA transfection/infection Cells grown in medium containing Thymidylate synthase 10% serum were treated with and without bortezomib in various concentrations (see text and results) for 24 – 72 hours were harvested and followed by various analyses. siRNA transfection [35] and shRNA infection [36] were performed as previously described MTT cell viability assay Effect of bortezomib on cell growth was determined by MTT assay. MTT was used as a colorimetric substrate for measuring cell viability. Non-viable cells, with altered cellular redox activity, are unable to reduce the MTT dye. After 72 hours with or

without bortezomib treatment, MTT was added (to a final concentration of 0.5 mg/ml). Cells in 96-well plates were incubated in a 5% CO2 incubator at 37°C for 4 hours, and then lysed thoroughly with lysis buffer (20% SDS, 50% N, N-dimethylformamide, pH 4.7, 100 μl/well). The absorbance in the relevant wells was measured at 570 nm using an Ultra Microplate Reader (GDC941 Bio-Tek Instruments). Flow cytometry analysis Cells at sub-confluence (~30%) were treated with bortezomib at 0, 5, 10 and 50 nM for 48 hours and then harvested by trypsinization and washed with PBS. Cells (~1 × 106) were resuspended in 5 ml 70% ethanol. After the initial fixation, cells were suspended in 0.5 ml PBS containing 25 μg/ml propidium iodide (PI), 0.2% Triton X-100 and 40 μg/ml RNase A and incubated for at least 30 minutes at 4°C.