Figure 1.

Bortezomib and nutlin-3 synergistically induce the cell death accompanied by cytoplasmic vacuolation in various cancer cells. (a, j) Cells were treated with the indicated concentrations of bortezomib and/or nutlin-3 for 24 h and cellular viability was assessed using calcein-AM and EthD-1. The percentage of live cells was normalized to that of untreated control cells (100%). Data represent the means±s.d. One-way ANOVA and Bonferroni’s post hoc test. *P<0.005, **P<0.01 vs untreated cells. The experiment was repeated at least three times with similar results. (b) Isoboles for the combination of bortezomib and nutlin-3, which were isoeffective (IC₅₀) for inhibition of cell viability, are shown. (c) Two HCT116 isogenic cell lines were treated with bortezomib and/or nutlin-3 at the indicated concentration in the absence or presence of 20 μM pifithrin-α for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. Data represent the means±s.d. Kruskal–Wallis test was performed followed by Dunn’s test. *P<0.005 vs untreated cells, P<0.005, ^##P<0.01 vs cells treated with 20 μM nutlin-3, NS, not significant. (d, e, i) Cells were treated with the indicated concentration of bortezomib and/or nutlin-3 for 24 h and observed under the phase-contrast microscope. Scale bars, 10 μm. (f) MDA-MB 435S cells were treated with the indicated concentrations of carfilzomib and/or nutlin-3 or MG132 and/or nutlin-3 for 24 h and cellular viability was assessed using calcein-AM and EthD-1. Data represent the means±s.d. One-way ANOVA and Bonferroni’s post hoc test. *P<0.005, **P<0.01 vs untreated cells. (g) Isoboles for the combination of carfilzomib and nutlin-3 or isoboles for the combination of MG132 and nutlin-3, which were isoeffective (IC₅₀) for inhibition of cell viability, are shown. (h) MDA-MB 435S cells were treated with the indicated concentrations of carfilzomib and/or nutlin-3 or MG132 and/or nutlin-3 for 24 h and observed under the phase-contrast microscope. Scale bars, 10 μm.