VPA or vorinostat activates autophagy in Kasumi-1 cells. (A) Kasumi-1 cells were treated with VPA or vorinostat for the indicated hours, and total cell lysates were prepared for immunoblotting. The blots were probed with the indicated antibodies. Blots from 1 representative experiment are shown. Similar results were obtained by probing for AML1-ETO using an AML1-reactive antibody (data not shown). AML1-ETO migrates more or less as a doublet.⁴⁷  (B) Kasumi-1 cells were treated with VPA or vorinostat (Vor) for 16 hours in the presence or absence of autolysosomal inhibitors, and autophagic activity was measured as degradation of long-lived proteins. Starvation in Hank’s balanced salt solution was used as a positive control. (C) Kasumi-1 cells were treated with VPA or vorinostat for 2 to 6 hours and then fixed and prepared for immunofluorescence staining of endogenous LC3 (green) and Hoechst staining of nuclei (blue). (Left) Representative images from the 4-hour time point are shown. Scale bar, 20 µm. (Right) Mean number of LC3 spots per cell and the mean intensity of LC3 spot pixels per cell were quantified from 1000 to 3000 cells per condition per experiment. The asterisks denote the statistical significances compared with the untreated control. (D) Autophagic flux was determined in Kasumi-1 cells by treatment with VPA or vorinostat for 4 hours in the presence or absence of Baf or CQ for the final 2 hours. Total cell lysates were prepared for immunoblotting, and the blots were probed with antibodies against LC3 and actin. Blots from 1 representative experiment are shown. The relative levels of LC3II were normalized to actin. All bars show mean values ± standard error of the mean (SEM) quantified from ≥3 independent experiments. *P < .05; **P < .01; ***P < .001.