Data from flow cytometry were analyzed using WinList software (Verity Software House Inc., Topsham, ME) and presented as DNA content profiles (X axle) over https://www.selleckchem.com/products/azd9291.html cell numbers (y axle). Triplicate assays were performed. Western blot analysis Cells with and without bortezomib treatment were washed with phosphate-buffered saline (PBS) and lysed on ice for 30 minutes in PBS FK866 manufacturer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 10 μg/ml phenylmethyl sulfonyl

fluoride, and 20 μM leupeptin. Cell lysates were then centrifuged at 15,000 g for 20 minutes at 4°C. Fifty μg total proteins from each sample were heated at 95°C for 5 minutes after mixing with equal volume of 2 × SDS loading buffer. Samples were separated on 12 – 15% SDS-polyacrylamide gel electrophoresis

(SDS-PAGE) gels and electrotransferred to Pure Nitrocellulose Membranes (Bio-Rad, Hercules, CA). The membrane was then blocked in 5% skim milk in TBS-T buffer (20 mM Tris/HCl (pH 7.5), 0.137 M NaCl, and 0.05% Tween 20) at room temperature for 2-3 hours; followed by incubation of the membrane with primary antibodies (against survivin or actin) in TBS-T containing 5% BSA overnight at 4°C in the range of dilutions from 1:1000 to 1:4000. After washing with TBS-T, the membrane was incubated in TBS-T buffer containing 5% skim milk containing the corresponding secondary antibody (1:5000) for 45-60 minutes at room temperature with shaking. Protein of interest was detected using ECL (Perkin Elmer, Waltham, MA) and visualized by autoradiography with various times (5-60 seconds) of exposure. Actin was detected as the internal control for normalization of total protein JPH203 cost Obatoclax Mesylate (GX15-070) loading in each lane. Cell death detection ELISA assay This assay is based on cell DNA fragmentation and the cell death/DNA fragmentation was detected using the Cell Death Detection ELISAPlus assay

kit (Roche) as described previously [37]. Briefly, transfected HCT116p53-/- cells were seeded in triplicates in 96-well plates and treated with and without bortezomib for 48 hours. After removing medium, cells were then lysed and 20 μl of lysate supernatant from each well were dispensed into streptavidin-coated well-removable 96-well plates followed by addition of 80 μl of immuno-reagents. After a 2-hour incubation at room temperature, unbound components were removed by washing with 1× incubation buffer for 3 times, followed by adding 100 μl of HRP substrate to each well, and the plate was placed on a shaker at 250 rpm for color development. Measurements were made at 405 nm against an ABTS solution as a blank control using a microplate reader. The absorbance value at 405 nm represents the quantities of DNA fragments/apoptosis induced by the treatment. Statistical analysis A t-test was performed for a pair-wise comparison of each experimental pair group with the control assuming equal variance. The significance (p-value marked with an asterisk “”*”") was set at equal to or less than 0.05.