Fig. 6
TRIM6 Promotes Ubiquitination and Degradation of DDX58 in Hepatocellular Carcinoma Cells. (A, B) Western blot analysis of DDX58 protein levels following TRIM6 overexpression in HCCLM3 (A) and Huh-7 (B) cells. TRIM6 overexpression significantly reduces DDX58 protein levels. β-Actin was used as a loading control. (C, D) Quantification of DDX58 protein and mRNA levels in HCCLM3 (C) and Huh-7 (D) cells after TRIM6 overexpression. The reduction in DDX58 protein levels is not accompanied by changes in DDX58 mRNA levels, suggesting post-transcriptional regulation. **p < 0.01, ***p < 0.001, ns = not significant. (E, F) Western blot analysis of DDX58 protein levels following TRIM6 knockdown in HCCLM3 (E) and Huh-7 (F) cells. TRIM6 knockdown increases DDX58 protein levels. β-Actin was used as a loading control. (G, H) Quantification of DDX58 protein and mRNA levels in HCCLM3 (G) and Huh-7 (H) cells after TRIM6 knockdown. The increase in DDX58 protein levels is not reflected in DDX58 mRNA levels. **p < 0.01, ***p < 0.001, ns = not significant. (I, J) Cycloheximide (CHX) chase assay showing the degradation rate of DDX58 in HCCLM3 cells. (I) TRIM6 overexpression accelerates DDX58 degradation, while (J) TRIM6 knockdown slows down DDX58 degradation. ***p < 0.001. (K, L) TUBE2 pulldown assay detecting ubiquitinated DDX58 in HCCLM3 cells. (K) TRIM6 overexpression increases DDX58 ubiquitination, whereas (L) TRIM6 knockdown reduces DDX58 ubiquitination. (M, N) Quantitative RT-PCR analysis of DDX58 mRNA levels in HCCLM3 (M) and Huh-7 (N) cells treated with the STAT3 inhibitor Stattic. ns = not significant. (O, P) Western blot analysis (O) and quantification (P) of DDX58 protein levels in HCCLM3 cells following Stattic treatment. Pharmacological inhibition of STAT3 with Stattic markedly elevates DDX58 protein abundance. **p < 0.01.
