Fig. 4: USP14 modulates defense mechanisms against radiation-induced ferroptosis via GPX4.

A Diagrammatic representation of the ferroptosis defense pathways and associated inhibitors explored in this study. B Evaluation of IU1-47 on inhibiting Huh7 cell proliferation in conjunction with Erastin (20 μM), ML210 (5 μM), DAHP (10 μM), or iFSP1 (5 μM) for 12 h, followed by 8 Gy IR. Viability was assessed 72 h post-IR. The IC₅₀ was calculated by normalizing cell viability to the drug-pretreated and IR control group (without IU1-47), which was set as 100% survival. C Lipid peroxidation levels were quantified by BODIPY-581/591-C11 staining in Huh7 cells pretreated with IU1-47 (5 μM) alongside Erastin (20 μM), ML210 (5 μM), DAHP (10 μM), or iFSP1 (5 μM) for 12 h prior to IR exposure. Lipid peroxidation levels in these groups are also shown. D Western Blot analysis for proteins SLC7A11, GPX4, FSP1, and GCH1 in Huh7 cells with/without USP14 expression 12 h after 6 Gy IR; and after treatment with IU1-47 (5 μM) for 2 h (DMSO as control) followed by 6 Gy IR 12 h later. Non-IR groups acted as controls. Actin was used as a loading control. E Detection of GPX4 via WB assay at the indicated time points in USP14-WT or USP14-KO Huh7 cells exposed to 6 Gy IR, or in parental Huh7 cells post IU1-47 treatment for 2 h (DMSO as control). Left: representative images. Right: normalized GPX4 grayscale intensity against GAPDH at specified times post IR. F, G GPX4 levels in PDX models. F USP14-WT/KO Huh7 xenografts and G PDX-1 model treated with IU1-47 combined with IR. Left: GPX4 IHC staining, Right: GPX4 staining score. Normal saline (NS) served as a control. Scale bars: 10 μm in (F) and 5 μm in (G). H Schematic illustration of GPX4’s function in this study. I GSH/GSSG ratio in USP14-WT/USP14-KO Huh7 cells at the indicated time points post IR (6 Gy) or cells treated with IU1-47 (5 μM) for 2 h before 6 Gy IR. DMSO was the control. J GPX4 activity detection in USP14-WT/USP14-KO Huh7 cells at the indicated time points post IR (6 Gy) or cells treated with IU1-47 (5 μM) for 2 h before 6 Gy IR. DMSO was the control. K Cell viability using the CCK-8 assay in GPX4-WT/GPX4-KO Huh7 cells following IU1-47 treatment and varying IR doses (0/4/8 Gy). DMSO served as vehicle control. Cells were collected 72 h post IR. 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. L Quantification of lipid peroxidation levels by BODIPY-581/591-C11 staining in GPX4-WT/GPX4-KO Huh7 cells pretreated with IU1-47 (5 μM) for 2 h prior to IR, collected 12 h post. Lipid peroxidation levels in these groups are shown. M GSH/GSSG ratio in GPX4-WT/GPX4-KO Huh7 cells pretreated with IU1-47 (5 μM) for 2 h before 6 Gy IR exposure. Cells were collected at the indicated time points post IR. Results are indicative of three independent experiments, expressed as mean ± SD. For (C, F, G)-Right and (I–M), one-way ANOVA was employed for comparisons among indicated groups. For (E)-Right, statistical significance was determined by two-way ANOVA.