Fig. 5: Non-responders can be sensitized to IZI1551-induced cell death by Mcl-1 antagonism.

A Quantification of cell death kinetics, calculated as a percentage of PI-positive cell areas. Cells were co-treated with IZI1551 (1 nM) and marizomib (80 nM) simultaneously or pre-treated with marizomib for 24 h (MRZ −24 h) before the addition of IZI1551. Representative results from one out of 3 independent experiments are shown. Error bars represent the SD of 3 technical replicates. B Marizomib inhibited proteasome activities both in responder and non-responder cell lines. Cells were treated with 40 nM (low) or 80 nM of marizomib or bortezomib for 4 h and CT-L activities of the proteasomes were measured from total cell lysates by cleavage of Suc-LLVY-AMC. Bars indicate % of activity related to untreated controls. Data are shown as mean ± range of n = 2 measurements. C Surface expression of death receptors DR4 and DR5 in responsive and resistant cell lines. Cells were treated with marizomib (80 nM) and surface amounts were determined by flow cytometric measurements. Data show mean plus range. Similar results were obtained in independent repeat experiments. D Procaspase-8; FADD; FLIP and BID proteins are detected at different expression levels in responsive and resistant cells. Cells were treated with marizomib and the indicated proteins were detected in whole-cell lysates. Vinculin served as a loading control. Similar results were obtained in independent repeat experiments. E Anti-apoptotic Bcl-2 family proteins were detected in different cell lines. Cells were treated with marizomib (80 nM) and the indicated proteins were detected in whole-cell lysates. Vinculin or α-Tubulin served as loading controls. Similar results were obtained in independent repeat experiments. F Annexin V/PI-based flow cytometry of cells treated with 10 μM of S63845; 1 nM IZI1551 plus 80 nM MRZ or the combination thereof for 24 h. Data represent mean ± SEM of three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001; one-way ANOVA followed by Tukey post hoc test.