Fig. 1: DUB siRNA library screening identifies USP18 as a key driver of sorafenib resistance.

A The schematic diagram illustrates the workflow of the DUB siRNA library screening strategy used in this study. B Volcano map characterization screening results. The rose red triangles indicate resistance-associated DUBs (p < 0.05 and cell viability <75%). Blue-gray squares indicate sensitive associated DUBs (p < 0.05 and cell viability >125%). The cell viability was determined by comparing it to the control group, which consisted of cells treated with sorafenib alone. C Protein expression of USP18 in parental (P) and sorafenib-resistant (SR) cells. The intensities of bands were analyzed by Image J and normalized to the corresponding parental (P) cells. D Representative immunoblot images of USP18 in HepG2 and HCCLM3 cells treated with sorafenib (0, 2.5, 5, and 10 μM) for 24 h. The band intensities were quantified using Image J and normalized to the control cells treated with DMSO. E The USP18 levels in HCC tissue samples of non-responders (NR) and responders (R) to sorafenib treatment were assessed using the GSE109211 dataset (means ± SEM, ****p < 0.0001, unpaired Student’s t-tests). F The Kaplan–Meier curves demonstrate the association between USP18 expression and overall survival among HCC patients in the TCGA cohort. G Representative IHC images of USP18 at different staining intensity levels (levels 1–4 represent progressive low to high staining) in resected HCC samples from patients who went on to receive sorafenib treatment (left). Kaplan–Meier survival analysis comparing the cumulative survival rate of patients with different USP18 expression levels (n = 80 cases) (right). Patients were distinguished by the median expression level of USP18, using the Log-rank (Mantel-Cox) test. Scale bars, 100 μm. H Schematic overview of an HCC xenograft model that acquired resistance to sorafenib. I Representative H&E staining images and IHC images of USP18 and Ki67 in excised xenografts. Scale bars, 100 μm.