Fig. 6

FAF1 mediates JNK1-dependent necrosis in RGC5 cells upon ischemic stress. a Left panel: RGC5 cells were transfected with VC or GFP-FAF1 plasmid (4 μg). At 48 h after transfection, the cells were treated with oxygen glucose deprivation for 8 h. Cell death was measured by flow cytometry (n = 3). Right panel: representative immunoblots showing the expression levels of FAF1, GFP and β-actin (b) Left panel: RGC5 cells were transfected with scrambled-siRNA (scRNA) or FAF1-targeting siRNA (siFAF1) for 48 h and then exposed to oxygen glucose deprivation for 10 h. Cell death was measured using flow cytometry (n = 3). Right panel: representative immunoblots showing the expression levels of FAF1 and β-actin. c RGC5 cells were treated with oxygen glucose deprivation for the indicated times. Whole cell lysates were immunoprecipitated with anti-JNK1 antibody, followed by an immunoblot assay with the indicated antibodies. d Upper panel: RGC5 cells were transfected with VC or Flag-FAF1 plasmid (2 μg). At 48 h after transfection, the cells were treated with oxygen glucose deprivation for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. Lower panel: the graph shows the result of the quantitative analysis of the P-JNK level (n = 3). e Upper panel: RGC5 cells were transfected with scRNA or siFAF1. At 48 h post transfection, the cells were treated with oxygen glucose deprivation for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. Lower panel: the graph shows the results of the quantitative analysis of the P-JNK level (n = 3). Data (a, b, d, and e) are expressed as the mean ± S.E.M. of three independent experiments. Statistical comparisons were evaluated using ANOVA followed by Tukey’s HSD (a, b, d, and e) post hoc analysis. ***P < 0.001, and **P < 0.01