Fig. 1: Identification of USP14 as a radioresistant DUB in HCC using a DUB-based CRISPR screen.

A Schematic illustration of the experimental framework for analyzing deubiquitination activity in PDX models and HCC cells. Assessment of relative deubiquitinase (DUB) activity through ubiquitin-AMC hydrolysis assay in (B) PDX models, C Huh7 and MHCC97H cells, and D both parental and IR-resistant Huh7 and MHCC97H cells. DUB activity was adjusted to the respective control group. Non-IR indicates non-radiation; IR denotes ionizing radiation. Experiments were conducted in triplicate, with data presented as mean ± SD. Statistical significance was determined using a two-tailed Student’s t-test to compare the specified groups. Colony formation assays in (E) parental and (F) IR-resistant Huh7 cells treated with N-ethylmaleimide (NEM) for 12 h, followed by a single dose of 0, 2, 4, or 6 Gy IR. Representative images and survival curves are displayed. DMSO served as a vehicle control. Experiments were performed in triplicate, data presented as mean ± SD. Statistical significance was determined via two-way ANOVA. G–I MHCC97H xenografts were subjected to alternating treatments with NEM (5 mg/kg/day for 10 sessions) and IR (2 Gy/day for 5 sessions). G Representative tumor images. Quantitative assessment of (H) tumor volume and (I) weight. Experiments were triplicated, data shown as mean ± SD. Statistical significance determined by two-way ANOVA. J Treatment outline for DUB sgRNA library screening. Huh7 cells received continuous radiation at 2 Gy/dose × 5, while MHCC97H xenografts received interval radiation at 1 Gy/dose × 5. Relative DUB abundance from the CRISPR-Cas9 screen in (K) Huh7 cells and (L) nude mice with subcutaneous MHCC97H xenografts. DUBs with log₂ (fold change) < −2 and a false discovery rate (FDR) < 0.01 were identified as radioresistance-related genes. Colony formation assays in (M) Huh7, (N) MHCC97H, and (O) SUN449 cells (including USP14-WT, knockout-KO, and KO + OE) groups with single doses of 0, 2, 4, 6, or 8 Gy IR. Representative images and survival curves are provided. Experiments run in triplicate, data as mean ± SD. Statistical significance assessed by two-way ANOVA. USP14-WT/KO MHCC97H xenografts exposed to IR (2 Gy/day for 5 sessions every alternate day). P Representative tumor images. Q Quantitative tumor volume analysis. Experiments duplicated in triplicate, data as mean ± SD. Statistical significance via two-way ANOVA. Kaplan–Meier analyses of (R) overall survival and (S) progression-free survival in HCC patients from SYSUFAH, stratified by USP14 immunoreactive scores (IRS). Results showcase three independent experiment representations.