Fig. 4.

Effects of USP inhibition on SMAD3 phosphorylation and collagen gene expression in TGF-β-stimulated fibroblasts

(A, B) Total and phospho-SMAD3 western blots in unstimulated or TGF-β-stimulated synovial fibroblasts, cultured in multiplicate wells in the presence or the absence of NSC632839, a pan-USP inhibitor. (A) Representative experiment. (B) β-actin-normalized SMAD3 and phospho-SMAD3 quantification in four different experiments. P-values were calculated using paired Student’s t-tests. *P < 0.01; ***P < 0.0001. (C) Quantitative PCR evaluation of COL1A1, COL3A1 and fibronectin (FN1) gene expression in unstimulated or TGF-β-stimulated synovial fibroblasts, cultured in duplicate wells in the presence or the absence of NSC632839. Results are displayed as mean (± s.e.m.) fold-changes in β2-microglobulin-normalized gene expression compared with unstimulated cells, from four different experiments. P-values were calculated using paired Student’s t-tests. **P < 0.005; ***P < 0.0001. USP: ubiquitin-specific peptidase.