Fig 2: TRIM37 Promotes Hepatic Fibrosis In Vivo(A–D) AAV8-HBV 1.2 vector was injected in mice through the tail vein along with control shRNA (ad-shNC) or Trim37 shRNA (ad-shTRIM37), and the effect on (A) collagen secretion was measured by Masson’s trichrome staining in liver tissue, and HBV infection was verified by HBsAg and hepatitis B core antigen (HBcAg) staining; (B) the level of Hyp secretion was measured by an A030 Hyp assay kit; (C) the amounts of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were quantified by commercial kits; and (D) protein levels of TRIM37, SMAD7, collagen I/III, α-SMA, and MMP-2 were measured by western blotting using the indicated antibodies. Scale bar, 100 μm. ∗∗∗p < 0.01 compared with control; ##p < 0.01, ###p < 0.001 compared with AAV8-HBV 1.2 + ad-shNC.

Fig 3: HBV-Mediated NF-κB Activation Transcriptionally Regulates the Expression of TRIM37(A–D) LX-2 cells were transfected with HBV replicon or control vector, cells were treated with NAC (10 mM) for 12 h, and (A) ROS production was measured by dichlorofluorescin diacetate (DCF-DA) staining followed by fluorescence-activated cell sorting (FACS), and the fluorescence intensity was quantified; (B–D) expression levels of TRIM37 and NF-κB p65 were measured by RT-PCR and/or western blotting. (E and F) LX-2 cells were transduced with either control vector or HBV replicon, treated with PDTC (10 μM) or vehicle for 24 h, and (E) luciferease reporter activity of TRIM37 promoter was measured, and (F) mRNA and protein levels of TRIM37 were measured by RT-PCR and western blotting, respectively. (G) The NF-κB binding site in the the TRIM37 promoter was predicted by the JASPAR algorithm. (Left) The predicted sequence, binding site (BS), and schematic diagram of TRIM37 gene, promoter region, and 3′ UTR are shown. (Right) Chromatin from LX-2 cells was immunoprecipitated using control IgG or NF-κB p65 antibody, and binding of the antibody in the BS region of the promoter or negative control 3′ UTR region of TRIM37 (NC) was measured by qPCR. ∗∗∗p < 0.001 compared with control or IgG; ###p < 0.001 compared with HBV.

Fig 4: TRIM37 Regulates SMAD7 Ubiquitination(A) TRIM37-associated proteins in LX-2 cells were identified by mass spectrometric analysis and are listed in (B). (C) LX-2 cell lysates were immunoprecipitated with either control IgG or TRIM37 antibody (top panel) and control IgG or SMAD7 antibody (bottom panel), and immunoblotted with indicated antibodies. (D) LX-2 cells were transduced with TRIM37 overexpression (oeTRIM37) or empty vector, and TRIM37 expression was measured by RT-PCR and western blot. (E) LX-2 cells transduced with oeTRIM37) or empty vector were treated with vehicle or MG132, and western blot was performed with the indicated antibodies. (F) Cell lysates from oeTRIM37 or empty vector-transfected LX-2 cells were immunoprecipitated with anti-SMAD7 or IgG and then immunoblotted with anti-ubiquitin. ∗∗∗p < 0.01 compared with vector.

Fig 5: TRIM37 Promotes HBV-Induced Hepatic Fibrosis(A and B) LX-2 cells were transfected with HBV replicon or control vector, and (A) the concentrations of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) in cell culture supernatant were assessed by ELISA, and (B) the HBV DNA copy number was determined using qPCR. (C) LX-2 cells were transfected with shTRIM37 (−1, −2, and −3), and mRNA and protein levels of TRIM37 were measured. (D–H) LX-2 cells were transfected with control vector or HBV along with control shRNA or shTRIM37 (shTRIM37-2 and shTRIM37-3), and the effect on (D) cell viability was measured by a CCK-8 kit, (E) LX-2 morphology was observed, (F) Hyp secretion was measured by an A030 Hyp assay kit, (G) collagen I/III in cell culture supernatant was measured by ELISA, and (H) protein levels of TRIM37, α-SMA, and MMP-2 were assessed by western blot. Scale bar, 50 μm. ∗∗∗p < 0.01 compared with control or shNC; ##p < 0.01, ###p < 0.001 compared with HBV + shNC.