Role of autophagy in sorafenib/obatoclax-mediated lethality. (A) U937 cells were exposed to sorafenib (7.5μM) and obatoclax (1.5μM) for the designated intervals after which protein lysates were prepared and subjected to Western blot analysis. (B) U937 cells were stably transfected with LC3-EGFP, and EGFP-positive cells were sorted by FACS and exposed to sorafenib and obatoclax for 6 hours. Cells were then fixed and subjected to confocal microscopy. (C) Representative images with confocal microscopy of colocalized LC3-GFP (green) and LAMP1 (red) in U937 cells after 6-hour treatment with sorafenib (7.5μM) and obatoclax (1.5μM). Enlarged images of outlined areas are shown in supplemental Figure 5B. (D) U937 cells were pretreated with 3-MA (2.5 mM), chloroquine (CQ; 40μM), or bafilomycin A (BAF; 75nM) for 30 minutes, and exposed to sorafenib (7.5μM) and obatoclax (1.5μM) alone or in combination for an additional 16 hours after which the extent of cell death was monitored by the 7-AAD assay. *Significantly greater than values for cells not exposed to 3MA, CQ, or BAF; P < .01. (E) Alternatively, protein lysates were prepared from the indicated samples and subjected to Western blot analysis. (F) U937 cells in which VPS34 was stably knocked down with lentivirus-mediated shRNA and their control scrambled sequence counterparts (NC) were exposed to sorafenib (7.5μM) ± obatoclax (1.5μM) for 24 hours, after which the extent of cell death was determined using the 7-AAD assay. *Significantly different from values obtained for control cells (P < .05).