Fig. 4.

FTH1 and Mfrn1 regulate the sensitivity of hepatocyte to ferroptosis. The effects of the knockdown of FTH1 (shFTH1, A) and Mfrn1 (shMfrn1, B) on the protein expression of genes associated with iron metabolism and antioxidant defense by comparing to the scrambled control (shScrbl) in HepG2 cells. The relative changes are shown as mean ± SEM (n = 3). (C) Staining for baseline mtROS with MitoSOX in HepG2/shFTH1, HepG2/shMfrn1 and control shScrbl cells. (D) Staining of baseline mitochondrial Fe²⁺ content with Mito-FerroGreen (MFG) in HepG2/shFTH1 versus shScrbl cells. The fluorescence intensity of MitoSOX (C) and MFG (D) staining was quantified and is presented as mean ± SEM (n = 30 cells of 3 independent experiments). (E) HepG2 cells with the knockdown of FTH1, Mfrn1 or the scrambled control were treated with 100 μM FAC for 24 h, and ferroptotic cells was stained by PI. (F) HepG2/shFTH1 and control shScrbl cells were treated with 100 nM RSL3 for 4 h, followed by PI staining. The percentages of PI⁺ cells (E and F) were quantified, and the data shown are mean ± SEM (n = 9 fields of 3 independent experiments). (G) Lipid peroxidation was determined by staining with BODIPY C11 in RSL3 treated shFTH1 and control cells, with the green fluorescence denoting lipid oxidation. *, P < 0.01. Bars, 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)