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

FIGURE 2.

FIGURE 2

Nuclear translocation of RB1CC1 is critical for sensitising cells to ferroptosis. (A) RB1 mRNA in control, and HepG2 cells with RB1CC1 KO or OV, treating with DMSO, RSL3 (1 μM) or erastin (10 μM) for 4 h. (B) ChIP experiments for detecting RB1CC1 binding within the RB1 promoter in control and RB1CC1‐KO HepG2 cells treated with DMSO, RSL3 (1 μM) or erastin (10 μM) for 4 h. **p < .01 indicates significance between RSL3 and DMSO, or between erastin and DSMO. (C) Nuclear localisation of RB1CC1 in HepG2 cells following treating with RSL3 (1 μM) or erastin (10 μM) for indicated time. Scale bar, 10 μM. (D) Cellular fractionation experiments in HepG2 cells treated with RSL3 (1 μM) or erastin (10 μM) for indicated hours. (E) Subcellular localisation of truncated versions of RB1CC1 in HepG2 cells treated with DMSO, RSL3 (1 μM) or erastin (10 μM) for 4 h. Scale bar, 10 μm. (F) Phosphorylated and unphosphorylated RB1CC1 in HepG2 cells treated with DMSO, RSL3 (1 μM) or erastin (10 μM) for 4 h, as visualised by electrophoresis in gels containing Phostag™ reagent. (G) Schematic presentation of potential phosphorylation sites within the 499th–548th amino acid region of the RB1CC1 protein. (H) Phosphorylation of exogenous WT‐ or mutated‐RB1CC1 constructs, as indicated in HepG2 cells treated with DMSO, RSL3 (1 μM) or erastin (10 μM) for 4 h, as measured by gels containing Phostag™ reagent. Constructs expressing GFP was also co‐transfected as a loading control. (I) Subcellular localisation of WT‐, S537A‐ and S537E‐RB1CC1 constructs in HepG2 cells treated with DMSO, RSL3 (1 μM) or erastin (10 μM) for 4 h. Scale bar, 10 μm. (J) Cell death was measured in RB1CC1‐KO HepG2 cells reconstituted with WT‐, S537A‐, and S537E‐, RΔ5‐, RΔ6‐RB1CC1 constructs following treating with DMSO, RSL3 (1 μM), or erastin (10 μM) for 12 h. Statistical analysis was performed by Student's t‐test (B) or one‐way ANOVA (A and J). Data are presented as means ± SD from indicated samples. ***p < .001, **p < .01, *p < .05, indicates statistical significance and N.S. indicates non‐significance