Cells were treated with t BHP with or without exendin 4 for the indicated time, washed with PBS, and then stained with Hoechst 33342 and PI for 5 min at room temperature. One-hundred cells were counted under a fluorescence microscope and chosen at three independent times, and the rate of apoptosis was then determined. PI staining and annexin V FITC binding were done according to Hedgehog inhibitor Vismodegib the companies protocol and then analyzed by flow cytometry. Apoptotic cells were defined as the population that were PI bad and Annexin V FITC positive. 2The caspase 3 analysis was performed according to the manufacturers protocol. Quickly treated cells were washed once with ice-cold PBS and assayed for caspase 3 activity using a colorimetric assay. Cleavage of Ac DEVD pNA substrate by caspase 3 produces pNA, that has been quantified spectrophotometrically at 405nm utilizing an ELISA reader. The change in optical density is directly proportional to caspase 3 activity. The treated carcinoid syndrome cells were washed with ice cold PBS and then incubated with RIPA lysis buffer containing 50mM Tris HCl, 150mM NaCl, 1000 Triton X 100, 1mM EDTA, 1mM NaF, 1mM Na3VO4, salt deoxycholate, 1mM phenylmethanesulfonylfluoride, 10 ug/mL aprotinin, 1 ug/mL leupeptin, and 1 ug/mL pepstatin for 20 min. The cell lysates were then centrifuged at 12,000 g for 10min, and the protein concentrations were determined using the Bradford method. Whole cell protein was separated by 800-acre or 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and used in PVDF membranes. The membranes were incubated with these correct primary antibodies, P IRE1, IRE 1, JNK, p JNK, c Jun, p c Jun, caspase 3. Secondary horseradish peroxidase conjugated antibody detection LY2484595 was performed with enhanced chemiluminescence reagents. Quantification of the band density was performed by densitometric analysis. Data were analyzed by SigmaStat 3. 5 pc software and demonstrated by the mean standard deviation of no less than three separate experiments. Statistical differences between values were determined by Students test or ANOVA followed by Tukeys post hoc test. The importance level was set at P 0. 05. 3BThe treatment of T cells with 25 umol/L t BHP produced the maximal apoptotic reaction after 1 h as evidenced by results of the Hoechst/PI and Annexin V FITC/PI assays. B cells treated with 25 umol/L t BHP for 1 h clearly exhibited discoloration that has been indicative of apoptosis. Apparently, exendin 4 treatment markedly inhibited the apoptotic brilliant blue particle formation in MIN6 cells. An Annexin V FITC/PI quantification assay shown that exendin 4 protected MIN6 cells from t BHP induced apoptosis and that t BHP induced MIN6 cell death was mediated by apoptosis. The inhibitory influence of exendin 4 was 77. Six months, whereas JNK inhibitor produced a 72. 5% decrease in the level of apoptosis induced by t BHP, which suggested that JNK signaling is involved in this method. 3As shown in Figures 2 and 2, exposure of MIN6 cells to 25 umol/L t BHP for 1 h resulted in 2.