Fig 2: DUBs regulate type I IFN signaling by altering ubiquitination in both catalytic activity–dependent and –independent manners.(A) Heat map summary of regulatory roles of DUBs on total ubiquitination of key molecules in the type I IFN signaling pathway. (B) Heat map summary of regulatory roles of DUBs on ubiquitination of key molecules with different linkages in the type I IFN signaling pathway. Data are obtained by band intensity analysis (IP-HA/IP-Flag) and are normalized to the control sample. (C) Luciferase activity in 293T cells transfected with an IFN-β luciferase reporter, together with vectors for RIG-I (2CARD), along with an empty vector or with expression vectors for the indicated WT DUB or catalytically inactive mutants. (D) Coimmunoprecipitation and immunoassay of extracts of 293T cells transfected with Flag-TRAF3, Myc-BRCC3, and Myc-BRCC3-H122Q with the indicated antibodies. (E) Coimmunoprecipitation and immunoassay of extracts of 293T cells transfected with Flag-RIG-I and HA-K11-Ub together with Myc-USP5 or Myc-USP5-C335A followed by SeV infection with the indicated antibodies. (F) Coimmunoprecipitation and immunoassay of extracts of 293T cells transfected with Flag-STING and HA-K27-Ub together with Myc-USP22 or Myc-USP22-C185A followed by HSV-1 infection with the indicated antibodies. (G) Coimmunoprecipitation and immunoassay of extracts of 293T cells transfected with Flag-RIG-I and HA-K48-Ub together with Myc-USP5 or Myc-USP5-C335A with the indicated antibodies. Data are representative of three independent experiments [mean and SEM in (C)]. NS, nonsignificant (P > 0.05); ***P < 0.001.

Fig 3: TRIM14 recruits USP14 and BRCC3 to deubiquitinate KDM4D. (A) Co-IP and immunoblot analysis demonstrate decreased ubiquitination of KDM4D in Flag-TRIM14 inducible HeLa cells treated with Doxy (200 ng/mL) in the presence of 3MA (5 mM). (B) WT or Trim14−/− BMDCs were treated with 0.3 μM BafA1 for 6 h, and then cell extracts were collected for co-IP and immunoblot analysis of K63-linked ubiquitination of KDM4D. (C) Co-IP and immunoblot analysis of the interaction of KDM4D and TRIM14, USP14, or BRCC3 in BMDCs with or without LPS (100 ng/mL) treatment. (D) HeLa cells were transfected with siTRIM14 (#1 + 4) or scrambled siRNA (Scr), and treated with BafA1 (0.3 μM, 6 h). Then, cell extracts were collected for co-IP and immunoblot analysis of the interaction of KDM4D and USP14 or BRCC3. Quantifications of IP: USP14/KDM4D and IP: BRCC3/KDM4D were performed in ImageJ software (NIH). **P < 0.01, ***P < 0.001. (E and F) Immunoblot analysis of KDM4D levels in WT or USP14/BRCC3 DKO HEK293T cells transfected with increasing doses of HA-TRIM14 (E) or treated with TNF-α (50 ng/mL) for the indicated times (F). (G) Co-IP and immunoblot analysis of K63-linked ubiquitination of KDM4D in WT or DKO HEK293T cells transfected with HA-TRIM14 and treated with BafA1 (0.3 μM, 6 h). In A–G, all of the experiments were repeated three times with similar results.

Fig 4: The depletion of BRCC3 inhibits bladder cancer tumorigenesis in vivo. (A) Xenograft models (n = 6) were established by subcutaneously inoculating wild-type or BRCC3-KO cells and allowing the cells to grow for 5 weeks. Then the mice were sacrificed and the tumors were removed and weighed. (B–D) The tumor volume and weight measurements are shown. (E) Representative H&E staining and immunohistochemical staining of the xenograft tumors from the tumor-bearing mice in the wild-type and BRCC3-KO groups, showed the deficiency of BRCC3 (the fifth panel) and the upregulation of IκBα expression (the second panel), the downregulation of Ki67 expression (the top panel), TNFα expression (the third panel) and p-P65 expression (the fourth panel) in the BRCC3-KO groups. Statistical analysis of the tumor volume and weight were performed using one-way ANOVA. The means ± standard error of three independent experiments is shown. *P < 0.05, **P < 0.001, and ***P < 0.001 compared with controls.

Fig 5: BRCC36 stabilizes the HMGCR protein through deubiquitination. The overexpression or depletion of BRCC36 cells was treated with MG132 A) or CHX B). The expression levels of BRCC36 and HMGCR was detected by using western blot assay. C) Liver cancer cells overexpressing or depleting BRCC36 were treated with MG132 for 12 h, and the ubiquitinated HMGCR was detected by using co‐IP assay. 293T cells were transfected with plasmids containing various polyubiquitin chains D), and transfected with BRCC36 wildtype or mutant plasmids E) for 48 h, and the ubiquitinated HMGCR was detected using co‐IP assay.