Fig. 2

STAT3 directly regulates TRIM6 expression in hepatocellular carcinoma. (A) Identification of STAT3 as a potential upstream regulator of TRIM6 using the TF_Target_Finder tool, which integrates data from KnockTF, CHEA, GTRD, ChIP_Atlas, and cor_TCGA databases. (B) Scatter plot demonstrating the correlation between TRIM6 and STAT3 expression levels across different cancer types, highlighting the significant positive correlation in LIHC. (C) Correlation analysis of TRIM6 and STAT3 expression in LIHC, indicating a positive correlation with a linear regression line. (D) Schematic representation of the TRIM6 promoter region, showing the two predicted STAT3 binding sites. Red bars represent the positions of the putative binding sites. (E) Diagram of the wild-type (WT) and mutated (Mut1, Mut2, Mut3) TRIM6 promoter constructs used in luciferase reporter assays. The WT construct includes the full promoter region with both intact STAT3 binding sites, while the mutant constructs have specific mutations that disrupt one (Mut1, Mut2) or both (Mut3) STAT3 binding sites. (F) Luciferase reporter assay results showing TRIM6 promoter activity in STAT3-overexpressing (STAT3-OE) cells compared to control cells (Con-OE). Overexpression of STAT3 significantly increases luciferase activity in the WT construct, indicating enhanced TRIM6 promoter activity. In contrast, mutations in either of the STAT3 binding sites (Mut1, Mut2) lead to a partial reduction in luciferase activity, while mutation of both sites (Mut3) results in a substantial decrease in promoter activity, demonstrating that both STAT3 binding sites are crucial for full activation of the TRIM6 promoter. Statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (G) Chromatin immunoprecipitation (ChIP) assay confirming STAT3 binding to the TRIM6 promoter in HCC cells. *p < 0.05. (H) Relative TRIM6 mRNA levels in HCC cells treated with DMSO or the STAT3 inhibitor Stattic, showing that inhibition of STAT3 reduces TRIM6 expression. **p < 0.01. (I) Relative mRNA expression levels of SOCS3, TRIM6, and STAT3 in STAT3-overexpressing (STAT3-OE) and control (Con-OE) cells, as determined by qRT-PCR. Overexpression of STAT3 significantly increases the expression of TRIM6 and SOCS3. ****p < 0.0001. (J) Relative mRNA expression levels of SOCS3, TRIM6, and STAT3 in STAT3-knockdown (STAT3-shRNA) and control (Con-shRNA) cells. Knockdown of STAT3 leads to a significant reduction in TRIM6 and SOCS3 mRNA levels. ****p < 0.0001. (K) Western blot analysis of STAT3 and TRIM6 protein levels in HCC cells treated with DMSO (control) or the STAT3-targeted degrader SD-36. Overexpression of STAT3 increases TRIM6 protein levels, while STAT3 knockdown or targeted degradation by SD-36 significantly reduces TRIM6 protein levels. β-Actin serves as the loading control.