Figure 5.

Exogenously added HN peptide protects nonpolarized RPE cells from cell death. (A) Human nonpolarized RPE cells were treated with single dose of 150 μM tBH or 150 μM tBH plus varying doses of HN (5, 10, 25, 35 μg/mL) for 24 hours, and cell death was studied by TUNEL staining (red). Cell death was significantly higher in tBH-treated cells than in cells cotreated with HN and tBH. (B) Quantification of the TUNEL-positive cells. (C) Exogenously added HN inhibits mitochondrial ROS. The RPE cells were treated with 150 μM tBH or 150 μM tBH and varying doses of HN (10, 25, 35 μg/mL) for 24 hours. The tBH treatment induced mitochondrial ROS (red) formation in RPE cells, which was inhibited by HN cotreatment. (D) Colocalization of Bax and mitochondria in RPE cells treated with and without tBH and HN. Primary cultured human RPE cells were double-stained with Bax (green) and Mitotracker (red). Nuclei were stained with DAPI (blue). Colocalization of Bax and mitochondria is visualized by yellow staining in the merged images. In untreated control cells, Bax is mostly localized in the cytoplasm, while oxidative stress translocated Bax to the mitochondria. Humanin cotreatment inhibited Bax mitochondrial translocation as evidenced by cytoplasmic staining of Bax. (E) Western blot shows activation of caspase-3 by tBH and inhibition of caspase-3 activation with varying doses of HN cotreatment. *P < 0.01, **P < 0.001.