FIGURE 7.

PUMA contributes to Bax activation. A and B, Huh-7 (WT) and Huh-7 cells stably expressing short hairpin RNA complementary to PUMA (shPUMA) were incubated for 12 h with palmitic acid (PA) at 800 μm; vehicle (V)-treated cells were used as control. Cells were stained with Mito Tracker Red to visualize mitochondria. Next, cells were fixed, and Bax activation was assessed using conformation specific antisera (6A7), which only recognizes active Bax and using immunofluorescence microscopy. A, representative images of three independent experiments are depicted. B, 6A7-immunoreactive cells were quantified in 10 random 40× objective fields for each condition at the indicated time point with automated software. Data represent the mean and S.E. of three experiments; *, p < 0.01, palmitate-treated cells versus vehicle-treated cells; **, p < 0.01, palmitate-treated shPUMA versus palmitate-treated WT. C, effective down-regulation of PUMA protein levels in shPUMA Huh-7 cells compared with WT Huh-7 cells was verified by immunoblot analysis on whole cell lysates.