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. 2022 Feb 27;12(2):e747. doi: 10.1002/ctm2.747

FIGURE 5.

FIGURE 5

ELP3 is essential for RB1CC1‐mediated ferroptosis‐associated transcriptional reprogramming. (A) Venn diagram showing the common H4‐associated proteins following treating with DMSO, RSL3 (1 μM) and erastin (10 μM) for 4 h in HepG2 cells, as measured by IP using anti‐H4 or IgG antibodies followed by proteomics. (B) ChIP experiments showing ELP3 enrichment at ferroptosis‐associated peaks following treating with DMSO, RSL3 (1 μM) or erastin (10 μM) for 4 h in HepG2 cells. The IgG was parallel used as a negative control. (C) Re‐ChIP experiments showing co‐occupancy of RB1CC1 and ELP3 within the ferroptosis‐associated peaks in HepG2 cells treating with RSL3 (1 μM) or erastin (10 μM) for 4 h. (D) Heatmap showing changes of H4K12Ac within ferroptosis‐associated peaks in control and ELP3‐KO HepG2 cells treated with DMSO, RSL3 (1 μM) or erastin (10 μM) for 4 h. (E) CHCHD3 and SLC25A32 mRNA expression in control and ELP3‐KO HepG2 cells treated with DMSO, RSL3 (1 μM) or erastin (10 μM) for 4 h. (F) Chromosome conformation capture (3C) experiments demonstrating changes of promoter–enhancer associations in control and ELP3‐KO HepG2 cells treated with RSL3 (1 μM) or erastin (10 μM) for 4 h. (G) ChIP evaluating RB1CC1 enrichment within the Chr7‐p1 and Chr8‐p in control and ELP3‐KO HepG2 cells treated with DMSO, RSL3 (1 μM) or erastin (10 μM) for 4 h. (H) Interaction between RB1CC1 and ELP3 using proximity ligation assay (PLA) in HepG2 cells treated with DMSO, RSL3 (1 μM) or erastin (10 μM) for 4 h. (I) RB1CC1–ELP3 interaction relied on their C‐terminus, as measured by PLA in HepG2 cells expressing exogenous RB1CC1‐FLAG and ELP3‐Myc, as indicated, under the treatment of RSL3 (1 μM) or erastin (10 μM) for 4 h. (J) Cell death in control cells and RB1CC1‐KO and ELP3‐KO HepG2 cells reconstituted with exogenous RB1CC1△C‐FLAG and ELP3△C‐Myc following treating with DMSO, RSL3 (1 μM) or erastin (10 μM) for 12 h. Statistical analysis was performed using one‐way ANOVA (D, E, G, J). Data are presented as means ± SD from indicated samples. ****p < .0001, ***p < .001, **p < .01, indicates statistical significance and N.S. indicates non‐significance