Fig. 4

FRA affected HSFs behaviour by inducing ferroptosis. (A) The mRNA levels of GPX4, HO-1, SAT1 and SLC7A11 in HSFs treated with FRA (300 µM) for 24, 48 and 96 h were measured using qRT‒PCR. GAPDH was used as the internal reference gene. (B) WB results of the expression of GPX4 and HO-1 in HSFs incubated with or without FRA (300 µM) for 48 h. (C) Statistical analysis of the relative protein expression of GPX4 and HO-1 in HSFs. (D) MDA levels in different groups at 48 h were quantitatively evaluated by an MDA assay kit. (E) HSFs were treated with FRA (100 µM, 200 µM and 300 µM) for 48 h. Representative images of FerroOrange staining (scale bar = 50 μm) and C11-BODIPY staining (scale bar = 50 μm). (F) Representative images of MitoTracker staining (scale bar = 50 μm) and JC-1 staining (scale bar = 50 μm). Quantitative analysis of the relative fluorescence intensity of ferrous iron, lipid peroxide (G), MitoTracker Red area and the aggregate/monomer ratio (H). (I) TEM images of the mitochondrial ultrastructure in HSFs treated with or without FRA for 48 h (scale bar = 1 μm for the upper images; scale bar = 250 nm for the lower images). The results are expressed as the means ± SDs (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant