Fig. 2
FAF1 is essential for JNK1-dependent necrosis upon ischemic stress. a Faf1+/+ and Faf1gt/gt MEFs were treated with oxygen glucose deprivation for the indicated times, and then, cell lysates were immunoblotted using anti-FAF1 antibody. b Faf1+/+ and Faf1gt/gt MEFs were treated with oxygen glucose deprivation for the indicated times. Cell death was determined by measuring LDH release (n = 3). c Left panel: Faf1+/+ MEFs were transfected with the indicated concentrations of Flag-FAF1 plasmid. At 36 h after transfection, the cells were untreated or treated with oxygen glucose deprivation for 8 h. Cell death was determined by measuring PI uptake using a flow cytometer (n = 3). Right panel: representative immunoblots showing the Flag, FAF1 and β-actin expression levels. d Faf1+/+ MEFs were transfected with vector control (VC) or Flag-FAF1 plasmids (4 μg). At 36 h after transfection, the cells were treated with oxygen glucose deprivation for the indicated times. Cell death was determined by flow cytometry (n = 3). e Left panel: Faf1gt/gt MEFs were transfected with scrambled-siRNA (scRNA) or siRNA JNK1 (siJNK1) for 36 h, and subsequently transfected with VC or GFP-FAF1 plasmids (4 μg). At 24 h after transfection, the cells were untreated or treated with oxygen glucose deprivation for 6 h. Cell death was determined by flow cytometry (n = 3). Right panel: representative immunoblots showing the FAF1, GFP, JNK1 and β-actin expression levels. The data (b - e) are expressed as the mean ± S.E.M. of three independent experiments. Statistical comparisons were evaluated using ANOVA followed by Tukey’s HSD (b - e) post hoc analysis. ***P < 0.001, and **P < 0.01