Fig. 4.

Ferroptosis-related genes are regulated by HIC1 and HNF4A. (A) A promoter analysis revealed that HIC1 and HNF4A bind to the promoters of FUF and FDF. Software Homer was used to reveal TFs binding to the promoters, and the TCGA database was used to predict factors down-regulated or up-regulated in liver cancer. (B) Location of HIC1 and HNF4A motifs within the promoters of FUF and FDF. (C) Promoter activities of FUF and FDF with (WT) or without (Mut) HIC1 or HNF4A motifs in control cells, HepG2 and Bel-7402 cells treated with erastin (10 μM) in the presence or absence of ferrostatin-1 (1 μM) for 24 h, as measured by a dual-luciferase assay. The italic “P” indicates “Promoter”. (D) HIC1 and HNF4A respond to the treatment of erastin and ferrostatin-1. Enrichment of HIC1 in the FUF promoters and HNF4A in the FDF promoters were measured by ChIP in the control cells, HepG2 and Bel-7402 cells treated with erastin (10 μM) in the presence or absence of ferrostatin-1 (1 μM) for 24 h. The non-specific IgG antibodies were parallel used as the control. The italic “P” indicates “Promoter”. (E–F) mRNA (E) and protein (F) levels of HBA1 and STMN1 in control cells, HepG2 and Bel-7402 cells with HIC1/HNF4A knocked down in the presence or absence of HIC1/HNF4A overexpression. The cells were simultaneously treated with DMSO or erastin (10 μM), as measured by qPCR (E) or WB (F). The data are shown as the means + SD from three independent experiments. **, p < 0.01 indicate statistical significance. The data were analyzed by a one-way ANOVA test. Images of WB are representative ones of 3 independent experiments.