Fig. 4: NINJ1 overexpression enhanced ferroptosis by downregulating xCT level and activity.

A xCT expression was decreased following NINJ1 overexpression in HT-1080 cells, verified by Western blot. B–E Cell viability of control and NINJ1-overexpressing HT-1080 cells were determined by CellTiter-Glo assay following treatment with indicated concentrations of (B) erastin (23 h), (C) RSL3 (17 h), (D) FIN56 (17 h), and (E) FINO2 (23 h). F-G NINJ1 overexpression enhanced erastin-induced membrane rupture in HT-1080 cells. Membrane rupture of control and NINJ1-overexpressing HT-1080 cells were observed by CellTox Green under fluorescence microscopy (F) and quantified (G) following erastin treatment (1.25 μM, 21 h). Scale bar: 400 μm. H-I NINJ1 overexpression enhanced erastin-induced lipid peroxidation in HT-1080 cells. Lipid peroxidation of control and NINJ1-overexpressing HT-1080 cells were determined by C11-BODIPY staining (H) and quantified by % of lipid peroxidation positive cells (I) following erastin treatment (2.5 μM, 22 h). J-K Reduced xCT protein stability post-NINJ1 overexpression in HT-1080 cells was validated by cycloheximide treatment and subsequent Western blot analysis. L Glutamate release levels of control and NINJ1-overexpressing HT-1080 cells following erastin treatment (1.25 μM, 20 h) were determined by Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit. M Cell viability in HT-1080 cells with/without NINJ1 or xCT overexpression was determined by CellTiter-Glo assay following treatment with erastin (5 μM) and ferrostatin-1 (Fer-1, 10 μM) for 24 h. N Intracellular CoA levels in HT-1080 cells with/without NINJ1 or xCT overexpression were determined by Coenzyme A Assay Kit following erastin treatment (1.25 μM, 24 h). O The GSH/GSSG ratios in HT-1080 cells with/without NINJ1 or xCT overexpression were measured by the GSH/GSSG-Glo Assay following erastin treatment (1.25 μM, 24 h). Error bars in (B–E), (G), (I), and (K–O) represent SEM (n = 3+).