VPA and vorinostat induce autophagy without reducing the AML1-ETO levels in SKNO-1 cells. (A) SKNO-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, and GAPDH was used as a loading control. Blots from 1 representative experiment are shown. (B) SKNO-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) SKNO-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). Representative images from the 4-hour time point are shown. Scale bar, 20 µm. (Right) The mean number of LC3 spots per cell and the mean intensity of LC3 spot pixels per cell were quantified from 1000 to 2000 cells per condition per experiment. The asterisks denote the statistical significances compared with the untreated control. (D-F) SKNO-1 cells were treated with the indicated concentrations of VPA and vorinostat for 16 hours in the absence or presence of autolysosomal inhibitors. Total cell lysates were prepared for immunoblotting with the indicated antibodies, and representative blots are shown. All bars show mean values ± SEM quantified from ≥3 independent experiments. *P < .05; **P < .01.