FIGURE 3.

Sorafenib accelerates FSP1 ubiquitination degradation by promoting the interaction between TRIM54 and FSP1. (A) HCC cells were treated with vehicle or sorafenib (10 μΜ, 36 h), and the mRNA levels of FSP1 were assayed by RT‐qPCR. (B) Effect of sorafenib (10 μΜ, 36 h) on FSP1 stability in HCC cells treated with cycloheximide (30 μg/mL) at 0, 12, 24, 36 hours. (C) HCC cells were transient transfected with Ub plasmid, then treated with sorafenib (10 µM 36 h) and MG132 (100 μM 4 h). Lysates from HCC cells were immunoprecipitated with an anti-FSP1 antibody. The protein expression and ubiquitination degree were determined by western blotting. (D) The co-immunoprecipitation (Co-IP) assay was used to determine the interaction of FSP1 with TRIM54 in FSP1-overexpressing HCC cells. (E) The Co-IP assay was used to determine the interaction of FSP1 with TRIM54 in FSP1-overexpressing HCC cells treated with or without sorafenib (10 μM, 36 h). (F) HCC cells were cotransfected with Ub plasmid and si-NC or Ub plasmid and si-TRIM54, then treated with or without sorafenib (10 μM, 36 h). HCC cells were treated with MG132 (100 μM) for 4 hours before harvest. The levels of proteins and ubiquitination were assayed by western blot. (G–K) HCC cells were transfected with si-NC or si-TRIM54 for 24 hours, then treated with or without sorafenib (10 μM, 36 h); cell viability (G); MDA levels (H); GSH levels (I); C11-BODIPY flow cytometry analysis (J); and C11-BODIPY stain (×400 magnification) (K) were assayed. The data are the means ± SD of the 3 independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001. Abbreviations: FSP1, ferroptosis suppressor protein 1; GSH, glutathione; MDA, malondialdehyde; TRIM, tripartite motif.