Fig 1: Regional association plots for the ABCG8 and TRAF3 signals in the GWAS discovery stage. Locus zoom for ABCG8 (a) and TRAF3 (b) signals in chromosome 2 and 14, respectively. To the left is the P value related to GSD in a log10 scale. SNPs with the highest association to GSD are colored purple. The insert panel denotes the imputed quality score (r2) and the appropriate SNPs are colored in the plot.
Fig 2: Effect size for ABCG8 and TRAF3 association signals in admixed Chilean populations. Forest plot calculated for GSD and GBC samples. The effect is shown in OR values and their 95% confidence intervals. Sample size for GSD case-control populations and for GBC cases are shown in parenthesis.
Fig 3: TRAF3 expression in human gallbladder and duodenal tissues. (a) Immunohistochemical analysis of TRAF3 expression in normal human gallbladder. Left panel: Negative control without primary antibody; Middle panel: Positive staining was observed for TRAF3 in the cell epithelium (1), muscle fibers (2), arterioles (3), and veins (4); Right panel: Zoom-in (60x) showing TRAF3 localization in the mucosal epithelium. (b) Western blot for TRAF3 in biopsies of duodenal mucosa tissue from GSD cases and control individuals. (c) Determination of TRAF3 protein levels in the duodenal mucosa (as is observed in b). (d) Differential expression analyses for TRAF3 mRNA in duodenal (left panel) and gallbladder mucosa tissues (right panel) in GSD cases and control samples.
Fig 4: USP11 stabilized TRAF3 by deubiquitination. a, b) Western blot assay for the determination of USP11 and TRAF3 protein levels in the myocardial tissues of rat and H9C2 cells. ***p < 0.001 and **p < 0.01 compared with the sham + Ad-shNC group. ###p < 0.001 and ##p < 0.01 compared with the /IR + Ad-shNC group, c) Co-IP revealing USP11 and TRAF3 interaction, d) TRAF3 ubiquitination level shown by immunoprecipitation after USP11 overexpression, e) TRAF3 stability after getting treated with CHX in the presence of USP11. ***p < 0.001 and **p < 0.01 compared with the NC group. USP11, ubiquitin-specific protease 11.
Fig 5: TRAF3 overexpression abrogated the protection of USP11 knockdown on H9C2 cells. a) Protein expressions of USP11 and TRAF3 in cells were detected by Western blot, b) The MTT assay was used to assess cell viability, c) The level of LDH was quantified by ELISA, d) Flow cytometry analysis of cell pyroptosis, e) ELISA detection of the levels of IL-6, IL-18, IL-1ß, and TNF-a in the supernatant of cells. ***p < 0.001 compared with the control group. #p < 0.05 and ###p < 0.001 compared with the H/R+Ad-shNC + NC group. &p < 0.05, &&p < 0.001, and &&&p < 0.001 compared with the H/R + Ad-shUSP11 + NC group.LDH, lactate dehydrogenase; IL, interleukin; TNF-a, tumor necrosis factor a; USP11, ubiquitin-specific protease 11.
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