Figure 4.

Knockdown of Asl decreases fumarate and desensitizes cells to erastin-induced ferroptosis. (A) MEF cells were transfected with siRNA-NC or siRNA-Asl (siAsl-1 and siAsl-2), the knockdown efficiency of Asl was determined by RT-qPCR. (B) Fumarate concentration was measured by HPLC in cells transfected with siRNA-NC or siRNA-Asl. (C) The GSH level was checked. (D) MEF cells were transfected with siRNA-NC or siRNA-Asl, then treated with erastin (2.5 µM) for 6 h in the absence or presence of Fer (10 µM) in the complete medium. Cell death was analyzed by PI staining followed by microscopy imaging and flow cytometry analysis. The representative images and the PI positive ratio are shown. Scale bar, 100 µm. (E) MEF cells were treated as in (D), and lipid peroxidation was measured through C11-BODIPY 581/591 staining followed by flow cytometry analysis. The representative histograms of flow cytometry and the relative mean fluorescence intensity are presented. (F) Lipid peroxidation was measured through C11-BODIPY 581/591 staining followed by confocal microscopy imaging and the representative images are shown. Scale bar, 10 µm. All data represent the mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. +Arg: complete medium, Era: erastin, Fer: Ferrostatin-1.