Fig. 3: Marizomib pre-treatment accelerates and enhances IZI1551-induced cell death.

A Caspase processing and caspase substrate cleavage in responder cell lines at early times. Cells were treated for 4 h with IZI1551 (1 nM) or marizomib (80 nM) or a combination of both in the presence or absence of Q-VD-Oph (50 µM). Whole-cell lysates were analyzed for the indicated proteins by western blotting. GAPDH, α-Tubulin, or β-Actin served as loading controls. Similar results were obtained in independent repeat experiments. c, cleaved. B Early cell death responses measured by Annexin V/PI-based flow cytometry. Cells were treated as in A. Data represent mean ± SEM from three independent experiments. *p ≤ 0.05; **p ≤ 0.01. ns = non-significant; one-way ANOVA followed by Tukey post hoc test. C 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. D Annexin V/PI-based flow cytometry of cells co-treated with reduced concentrations of IZI1551 (100 pM) and marizomib (40 nM). Data represent mean ± SEM from three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; ns = not significant; one-way ANOVA followed by Tukey post hoc test.