Fig. 4: USP18-mediated NCOA4 deISGylation and degradation impairs sorafenib-induced ferritinophagy. | Cell Death & Disease

Fig. 4: USP18-mediated NCOA4 deISGylation and degradation impairs sorafenib-induced ferritinophagy.

From: Targeting USP18 overcomes acquired resistance in hepatocellular carcinoma by regulating NCOA4 deISGylation and ferroptosis

Fig. 4

A Heatmap showing the top 10 dysregulated proteins (red, upregulated proteins; purple, downregulated proteins) identified by proteomics assays. n = 3 replicates per group. B Western blot analysis of NCOA4 protein levels in HepG2 cells transfected with pcDNA, USP18, or USP18C64S plasmid for 48 h. The intensities of bands were analyzed by Image J and normalized to the corresponding pcDNA. C Representative IHC staining images of USP18 and NCOA4 in HepG2-Ctrl and HepG2-USP18-OE xenografts. Scale bars, 50 μm. D Protein expression of NCOA4 in HCC-P and HCC-SR cells. The intensities of bands were analyzed by Image J and normalized to the corresponding HCC-P cells. E Protein expression of NCOA4 in the HepG2 xenografts from the indicated groups. The intensities of bands were analyzed by Image J and normalized to the mean of the corresponding NT group. F The Kaplan–Meier curves demonstrate the association between NCOA4 expression and overall survival among HCC patients in the TCGA cohort. G The HepG2 cell lysates were immunoprecipitated with anti-USP18 antibodies and blotted with anti-NCOA4 antibodies. H Expression of NCOA4 ubiquitination and ISGylation in anti-NCOA4 immunoprecipitation and whole-cell lysates (input) derived from HepG2 cells transfected with pcDNA, USP18, or USP18C64S plasmid for 48 h and treated with MG132 (10 μM) for 4 h. *, heavy chain. I The effect of USP18 overexpression on NCOA4-mediated ferritinophagy. Scale bars, 5 μm. J FerroOrange staining. The impact of USP18 knockdown on the sorafenib-induced elevation of Fe2+ levels in HepG2-SR cells, with or without NCOA4 siRNA transfection. Scale bars, 5 μm. K The tumor growth curve in mice from the indicated groups. Tumor volume was measured every 3 days beginning from the first treatment (means ± SEM, *p < 0.05, unpaired Student’s t-test). n = 8 mice per group. L Representative H&E staining images and IHC images of Ki67 in excised xenografts. Scale bars, 100 μm.

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