Fig 2: RNF2 is required for K63-linked ubiquitination of TRAF2 A Correlation analysis between RNF2 and TRAF2 expression using TIMER 2.0. RNF2 expression positively correlates with TRAF2 levels in LIHC samples. B HEK-293 cells were transfected with HA-tagged RNF2 and Flag-tagged TRAF2. Lysates were prepared and immunoprecipitated using anti-HA or anti-Flag antibodies. Immunoblot analysis was performed as indicated. Immunoprecipitation assay further confirmed the interaction between endogenous RNF2 and TRAF2 in Hepa1-6 cells. C Immunofluorescence demonstrating co-localization of RNF2 and TRAF2 in Sk-Hep1 cells. D HEK-293 cells were transiently transfected with HA-tagged RNF2 and Flag-tagged TRAF2. Rabbit anti-HA and mouse anti-Flag antibody were used for the proximity ligation assay. Red dots present the interaction of RNF2 with TRAF2. Scale bars, 20 μm. E HEK-293 cells were transfected with HA-RNF2 and Flag-TRAF2 as indicated. Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-ubiquitin, anti-TRAF2, or anti-HA as indicated. (F and G) RAF2 ubiquitination increased upon overexpression of wild-type RNF2 (RNF2-WT), but not the ΔR mutant. 293 T cells were transfected with HA-RNF2-WT or ΔR mutant. Lysates were subjected to immunoprecipitation with anti-Flag antibodies F or Ni–NTA pull-down under denaturing conditions G, followed by immunoblotting with the indicated antibodies. H TRAF2 ubiquitination decreased after RNF2 depletion. 293 T cells were co-transfected with the indicated plasmids or shRNAs. Immunoprecipitation of Flag-TRAF2 followed by immunoblot analysis was performed. I Sk-Hep1 cells transfected with control or RNF2 shRNA. Endogenous TRAF2 was immunoprecipitated and analyzed for ubiquitination. G RNF2 mediates K63-linked ubiquitination of TRAF2. Lysates from E were immunoprecipitated with anti-Flag antibodies and immunoblotted with antibodies against K63- and K48-linked ubiquitin

Fig 3: RNF2 inhibition sensitizes HCC to anti-PD-1 therapy by recruiting MDSCs into the tumor microenvironment A Representative dot plots comparing matched pre- and post-treatment melanoma patient RNF2 mRNA levels in melanoma patients. Statistics calculated using two-sided Wilcoxon matched pair rank test with significance at p < 0.05. Kaplan–Meier curves predicting survival of melanoma patients receiving anti-PD-1 therapy based on net changes in RNF2 mRNA levels in the melanoma-GSE91061-anti-PD-1 datasets. (B-C) C57BL/6 J mice were subcutaneously injected with Hepa1-6-Ctrl or Hepa1-6-RNF2 KO cells and treated with either anti-PD-1 or isotype control. Tumor growth was monitored until the experimental endpoints, with data presented as mean ± SEM. Tumor growth curves are shown. D Representative images of IHC for CD8, Gr1, and S100A8 + S100A9 in indicated mouse tumors and IHC quantification. The scale bars represent 50 μm. E Representative H&E staining (upper), immunostaining and quantification of Ki-67- and TUNEL-positive cells (lower). The scale bars represent 50 μm. F A schematic diagram illustrates the RNF2-TRAF2-CXCL1 axis driving MDSC accumulation, leading to suppression of T cell function in HCC. High RNF2 expression is positively correlated with MDSC infiltration in HCC patients. RNF2 enhances NF-κB activation by regulating K63-linked ubiquitination of TRAF2, resulting in elevated CXCL1 expression, a chemoattractant for MDSCs via the CXCL1-CXCR2 axis. RNF2-recruited MDSCs inhibit the activity of effector CD8 + T cells within the tumor immune microenvironment, thereby promoting immunosuppression and tumor progression in HCC. Statistics calculated using one-way ANOVA post hoc Tukey test for multi-group or two-tailed Student’s t-test for two-group comparisons. **p < 0.01

Fig 4: RNF2 activates NF-κB signaling A HEK-293 cells were co-transfected with the indicated plasmids along with pNF-κB-luc plasmids or the control-luciferase plasmid and subjected to a reporter assay. Luciferase assay indicated that RNF2 but not the ΔR mutant induces the activation of NF-κB signaling. B Western blot analysis of p-IκBα, IκBα, p-IKKα/β, IKKα, IKKβ, and β-actin in RNF2 KO Hepa1-6 cells or shRNF2 Sk-Hep1 cells. C Western blotting analysis of IκBα expression in the indicated cells treated with TNF-α (10 ng/ml) in RNF2 KO Hepa1-6 cells or shRNF2 Sk-Hep1 cells. β-Actin is used as a loading control. D Assay of NF-κB luciferase reporter gene activity in RNF2-overexpressing Hepa1-6 or Sk-Hep1 cells transfected with vector or the IκBα dominant-negative mutant (IκBα-mu). Statistics calculated using one-way ANOVA post hoc Tukey test for multi-group or two-tailed Student’s t-test for two-group comparisons. **p < 0.01

Fig 5: RNF2 positively correlates with progression and poor prognosis of HCC by recruiting MDSCs into the tumor microenvironment. A Comparison of RNF2 expression between tumor and normal tissues from TCGA database analyzed by TIMER 2.0. B K-M analysis of the OS of HCC patients in the TCGA liver hepatocellular carcinoma (LIHC)cohort based on the expression level of RNF2. Data were analyzed by log-rank test. C Representative images of IHC staining of RNF2. Scale bars: 100 μm (black). Patients were divided into two groups based on RNF2 expression levels. The low expression group consisted of patients exhibiting negative or weak RNF2 expression, while the high expression group included those with moderate to strong expression. Overall survival curves were generated using the log-rank test to assess differences in survival rates between the two groups. (D-E) The expression of RNF2 was positively correlated with the infiltration levels of MDSCs in LIHC analyzed by TIMER 2.0. Correlation analysis indicated that RNF2 expression was positively correlated with CD33 expression. F Representative mIHC staining of CD33 and RNF2 in tissue from human HCC tumors. Multiplexed immunofluorescence staining images showing the expression of RNF2, CD8, CD33 and DAPI in HCC. G High infiltration levels of MDSCs were associated with poor survival. Cox regression analysis of TCGA data demonstrated a significant correlation between increased MDSC infiltration and worse prognosis in HCC patients. Furthermore, elevated RNF2 expression and a high proportion of MDSCs significantly correlated to poorer OS compared to their counterparts, strongly suggesting that RNF2 influenced patient prognosis through an immune-related mechanism