Fig. 6: USP14 targets GPX4 for deubiquitination in a TRIM14-dependent manner.

A Co-IP and WB assays elucidated interactions among TRIM14, USP14, and GPX4. Huh7 cells overexpressing cGPX4-Myc with Flag-USP14 or HA-TRIM14 underwent 6 Gy IR, with samples collected 6 h post-IR. Non-irradiated groups served as controls. B Co-IP and WB assays evaluated the temporal dynamics of TRIM14/USP14/GPX4 complex assembly. Huh7 cells overexpressing cGPX4-Myc with Flag-USP14 and HA-TRIM14 were exposed to 6 Gy IR, with cellular lysates collected at specified intervals. C Co-IP and WB assays explored the interaction between USP14 and GPX4 in the absence of TRIM14. Huh7 cells with TRIM14-WT or TRIM14-KO, overexpressing cGPX4-Myc with Flag-USP14, were subjected to 6 Gy IR, with lysates collected at 0/2/6 h post-IR. D Co-IP and WB assays assessed the impact of TRIM14/USP14/GPX4 complex formation on GPX4 mutations or ML210 treatment. Huh7 cells overexpressing Flag-USP14 and HA-TRIM14 were transfected with either wild-type cGPX4-Myc or cGPX4-Myc with K48R/K118R mutations. Alternatively, cells treated with ML210 were exposed to 6 Gy IR; lysates were gathered 6 h later. E Lipid peroxidation levels were quantified via BODIPY-581/591-C11 staining in TRIM14-WT and TRIM14-KO Huh7 cells pre-treated with IU1-47 (5 μM) before IR. Samples were harvested 12 h post-IR, with comparative data provided below. F The GSH/GSSG ratio (Upper) and GPX4 activity (Below) were evaluated in TRIM14-WT and TRIM14-KO Huh7 cells pre-treated with IU1-47 (5 μM) followed by 6 Gy IR. Cells were harvested 4 h post-IR for analysis. G Co-IP and WB assays investigated the temporal formation of the TRIM14/USP14/GPX4 complex in IR-resistant Huh7 cells. Those overexpressing cGPX4-Myc with Flag-USP14 and HA-TRIM14 underwent 6 Gy IR, with lysates prepared at specified intervals. H Cell viability was assessed in both wild-type and TRIM14-KO parental Huh7 cells, as well as in IR-resistant variants. Cells were treated with IU1-47 prior to varying IR doses (0/4/8 Gy), using DMSO as a control. Viability data collected 72 h post-IR, with results based on three independent experiments. The IC₅₀ was calculated by normalizing cell viability to the drug-pretreated and IR control group or drug-pretreated control group (without IU1-47), which was set as 100% survival. Data are presented as mean ± SD. For (E) and (F): One-way ANOVA was employed for comparisons among the indicated groups.