Cell death induced by combined treatment with VPA/vorinostat and autophagy inhibitors depends on ROS and correlates with accumulation of ubiquitinated proteins. (A) Kasumi-1 cells were treated with VPA or vorinostat in the absence or presence of Baf or CQ for 16 hours. Cell viability was determined by fluorescence-activated cell sorter (FACS) analysis of PI-stained cells. (B) Kasumi-1 cells were treated with VPA for 16 hours in the absence or presence of Baf or CQ before fixation and PI staining for sub-G1 analysis. The figure shows representative histograms and presentation of cell viability as the fraction of non–sub-G1 cells. (C) Kasumi-1 cells transfected with siRNA oligoes against Atg7 and Ulk1 were treated with VPA for the last 16 hours. Cell viability was determined by FACS analysis of PI-stained cells. (D) Kasumi-1 cells were pretreated with NAC (5 or 10 mM) for 1 hour before addition of Baf or CQ. Then after 30 minutes, VPA was added, and the incubation was continued for 16 hours. Cell viability was determined by FACS analysis of PI-stained cells. (E) Cells were treated with the indicated concentrations of VPA for 16 hours in the absence or presence of Baf or CQ, and total cell lysates were prepared for immunoblotting. The blots were probed with antibodies against ubiquitin and GAPDH. Blots from 1 representative experiment are shown. (Right) Relative band intensities of ubiquitin at 1.5 mM VPA were normalized to GAPDH. All bars show mean values ± SEM quantified from ≥3 independent experiments. *P < .05; **P < .01; ***P < .001.