Journal: Scientific Reports

Article Title: TRIM39 aggravates hepatocellular carcinoma growth through targeting the p62-KEAP1-NRF2 axis

doi: 10.1038/s41598-025-32976-x

Figure Lengend Snippet: p62 is a binding partner of TRIM39. ( A ) Cell lysates from HepG2 cells transfected with empty vector or Flag-TRIM39 were immunoprecipitated with anti-Flag antibody. p62 was identified via mass spectrometry. ( B ) HepG2 cells were co-transfected with Flag-TRIM39 and Myc-p62 for 48 h. Total cell lysates were immunoprecipitated with anti-Flag or anti-Myc antibodies. Myc-p62 and Flag-TRIM39 were detected by western blot. ( C-D ) Immunofluorescence assay of Flag-TRIM39 and Myc-p62 in HepG2 cells. Representative confocal microscopy images were shown. Scale bars, 5 μm. The colocalization of TRIM39 and p62 in HepG2 cells was analyzed. ( E ) The analysis of p62 mRNA levels in TRIM39 stably overexpression or knockdown HCC cells. ( F - G ) The analysis of p62 protein levels in TRIM39 stably overexpression HepG2 and Huh7 cells. *, P < 0.05. ( H - I ) The analysis of p62 protein levels in TRIM39 stably knockdown MHCC97 and HCCLM3 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ( J ) HepG2 cells transfected with Flag-TRIM39 were treated with cycloheximide (CHX, 10µM) for indicated time points and subjected to western blot analysis. The quantitative result of p62 protein level was analyzed. ( K ) TRIM39 stably knockdown MHCC97 cells were treated with cycloheximide (CHX, 10µM) for indicated time points and subjected to western blot analysis. The quantitative result of p62 protein level was analyzed. ( L ) TRIM39 stably knockdown MHCC97 cells were treated with cycloheximide (CHX, 10µM) and MG132 (10µM) for indicated time and subjected to western blot analysis. The quantitative result of p62 protein level was analyzed. ( M ) TRIM39 stably overexpression and control cell lysates were immunoprecipitated with anti-p62 antibody. Immunoprecipitation complex was detected by anti-p62 and anti-Ub antibodies. ( N ) TRIM39 stably knockdown and shNC cell lysates were immunoprecipitated with anti-p62 antibody. Immunoprecipitation complex was detected by anti-p62 and anti-Ub antibodies. ( O ) TRIM39 facilitated p62 Ub conjugates by in vitro ubiquitination assay. ( P ) TRIM39 stably overexpression cells were co-transfected with indicated HA-Ub (K-R) mutants for 48 h. Total lysates were immunoprecipitated with anti-p62 antibody. The immunoprecipitation complex was analyzed by western blot with anti-HA and anti-p62 antibodies. ( Q ) TRIM39 stably overexpression cells were transfected with Flag-p62 and its mutants (K420R, K435R). The expression of Flag-p62 was analyzed by western blot.

Article Snippet: Briefly, Purified recombinant His-p62 and GST-TRIM39 proteins were incubated in a mixture of ubiquitin ligation reaction containing 5 mM MgCl2, 50 mM Tris-HCl (pH 7.5), 2 mM DTT, 4 mM ATP, 1 μg ubiquitin (Yeasen, 20431ES08), 300 ng E1-UBA1 (Targetmol, TMPY-02840), and 500 ng E2-UBE2L3 (Targetmol, TMPY-02842) at 30 °C for 3 h. The reaction was terminated by boiling for 10 min in 2× SDS sample buffer, and the proteins were resolved by SDS-PAGE followed by western blotting with the indicated antibodies.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Mass Spectrometry, Western Blot, Immunofluorescence, Confocal, Microscopy, Analysis, Stable Transfection, Over Expression, Knockdown, Control, In Vitro, Ubiquitin Proteomics, Expressing, Co-Immunoprecipitation Assay