Figure 1.

Cystine and methionine deficiency induces ferroptosis of hepatocyte-derived cells. HepG2 and L-O2 cells were exposed to cystine (CST) and methionine (Met) deficiency (CST/Met (−)) for 1 (g—left), 12–24 (f—left), 24 (a,f—right, and g—middle and right), 0–30 (e), or 36 h (b–d). (a) Intracellular GSH and GSSG levels. (b) Morphological changes were observed under a light microscope after exposing HepG2 cells to CST/Met (−) (left). Effects of CST (200 μM) and Met (200 μM) supplementation on the viability of HepG2 (middle) and L-O2 cells (right) determined by MTT assay. (c) Viability of HepG2 cells exposed to CST/Met (−) in the presence of DFX (200 μM), Fer-1 (10 μM), Nec-1 (10 μM), and Z-VAD (20 μM). (d) Percentage of HepG2 cells showing high PI staining intensity was revealed by flow cytometer. (e) Necrotic cell death. Open circle and closed square indicate HepG2 cells incubated with control medium and medium without CST/Met, respectively. (f) Level of PTGS2 mRNA in both HepG2 (left) and L-O2 cells (right) determined by qPCR. (g) ROS (left) and lipid peroxidation (middle and right) were measured using DCFH-DA and C₁₁-BODIPY, respectively. Black, red, and blue lines indicate fluorescence intensities from HepG2 cells incubated with control, CST/Met (−), and CST/Met (−) + DFX, respectively (middle). ** p < 0.01, * p < 0.05, versus control; ## p < 0.01, # p < 0.05, versus CST/Met (−): Con, control; DFX, deferoxamine mesylate; Fer-1, ferrostatin-1; Nec-1, necrostatin-1; N.S., not significant; Z-VAD, Z-VAD-FMK.