Fig. 3

AIF knockdown protects 661 W cells against light injury, but PARP-1 and mTOR function as upstream factors. a 661 W cells were transfected with specific shRNAs targeting the AIF gene, cells were lysed, and lysates were analyzed by Western blot. β-ACTIN was used as an internal control. Scramble: cells transfected with scrambled shRNA as a negative control; AIF KD: cells with AIF knockdown; Lt: 1500 lx light exposure for 72 h. b Cell mortality was quantified by PI/Hoechst staining. Scale bar = 100 μm. c Cells were cultured under light/dark conditions for 72 h, followed by cell lysis, and analysis of lysates with Western blot. β-ACTIN was used as an internal control. d-f Cells were cultured under light/dark conditions for 72 h, and the nuclear fractions were separated and analyzed by Western blot. LaminB1 and β-ACTIN were used as nuclear and cytosol fraction controls, respectively. The active form of 57 kDa AIF was quantitatively analyzed relative to LaminB1. mTOR KD: mTOR knockdown; PARP-1 KD: PARP-1 knockdown. All experiments were repeated in triplicate, and the results are shown as the means ± SEM (*: P < 0.05, **: P < 0.01, ***: P < 0.001, ns: no statistical significance). g 661 W cells expressing AIF-GFP were cultured under light/dark conditions for 72 h and images were captured with a confocal laser microscope. The nuclei were counter-stained with Hoechst staining. Scale bar = 20 μm