Fig 2: Verification of UBA1 levels in HCC cells. (A, B) UBA1 expression in some HCC cell lines was detected by qRT–PCR and Western blotting. (C, D) Effects of UBA1 interference within Huh7 cells at the mRNA or protein level. (E) Cell proliferation ability detected by CCK-8 viability following UBA1 expression interference. (*p < 0.05; **p < 0.01; ##p < 0.01).

Fig 3: The Nrf2 signal transduction pathway. (A) Coexpression correlation analysis in the TCGA HCC RNA-Seq cohort. (B) Following UBA1 silencing, Nrf2 expression, along with the downstream members (HO-1, NQO1, and FTH1) (C), was downregulated, as evidenced by Western blotting. (D, E) Best oltipraz dose screening. *p < 0.01, #p < 0.05.

Fig 4: Effects of changed UBA1 expression on malignant phenotypes and ferroptosis in HCC cells. (A) Wound healing assay. Migratory cells to the wound were determined following different treatments and imaged. (B, C) Transwell migration and invasion assays. Huh7 cells treated with different drug doses were analyzed for their invasion and migration capacities. (D) Ferrous iron and the MDA content (E) in Huh7 cells subjected to si-UBA1 transfection (* si-UBA1 versus si-UBA1+oltipraz, **p < 0.01, *p < 0.05; # si-NC versus si-UBA1, ##p < 0.01, #p < 0.05).

Fig 5: HIV-1 ubiquitinates and degrades C99, but not C83.a Transfection of HIV-1 JR-CSF reduces Flag-C99, but not Flag-C83 or GAPDH-HA in HEK293A cells. b Densitometry analysis of effects in (a) from 3 independent replicates. c Ubiquitination inhibitor TAK-243 stabilizes exogenous Flag-C99 but not C83-V5 in HEK293A cells transfected with either empty vector control (pcDNA3.1) or JR-CSF. d Densitometry analysis of effects in c from 3 independent replicates. e MG132 and TAK-243, but not DMSO control, stabilizes exogenous Flag-C99 in HEK293A cells transfected with JR-CSF. f Densitometry analysis of effects in (e) from 3 independent replicates. g Ubiquitin-activating enzyme E1 (UBE1) specific siRNA, but not negative control siRNA (NC2), suppresses downregulation of exogenous Flag-C99 in JR-CSF transfected HEK293A cells. h Densitometry analysis of effects in (g) from 3 independent replicates. i Schematic of N-terminally Flag-tagged WT or mutant C99 constructs used in (j). K: Lysine; A. Alanine. Each mutation site at the corresponding amino acid is represented by X. WB (j) and densitometry analysis (k) of effects of single or combined lysine-alanine (K-A) mutations on Flag-C99 stability in control pcDNA3.1 or JR-CSF transfected HEK293A cells. Complete stabilization mirroring TAK-243 treatment occurs in the C99-7KA mutant. l Representative (n = 3) anti-Flag IP showing ubiquitination of Flag-C99 but not Flag-C99-7KA mutant in lysates from HEK293A transfected with JR-CSF. Red arrows highlight ubiquitinated bands. Quantification of the protein levels from 3 independent replicates is presented in b, d, f, h, k. Data is presented as mean, SEM. b one-way ANOVA with Tukey’s multiple comparisons test; d unpaired two-tailed t test with Welch’s correction; f unpaired two-tailed t test with Welch’s correction; h and k: two-way ANOVA with Sidak’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant. Source data are provided as a Source Data file.