Figure 3.

CSE exposure affected protein expression levels of canonical TGF-β signaling mediators and their nuclear translocation. SCP-1 cells (N  =  3, n  =  3) were exposed to CSE (5%) twice a week. After 14 days, the cells were treated with rhTGF-β1 (10 ng/mL) for 1 h. Protein expression of phospho-Smad2 (a), phospho-Smad3 (b), and Smad4 (c) was measured in cell lysates by Western blot and normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (d) A representative Western blot of the measured proteins is shown. SCP-1 cells (N  =  3, n = 3) were treated overnight with or without CSE (5–10%) or CH (0.5–1 mM). Next, cultures were incubated with rhTGF-β1 (10 ng/mL) for 24 h and then stained for Smad3. (e) Representative immunostaining images of nuclear localization of Smad3. The immunofluorescence signal was pseudocolored for better visualization using the fire tool in ImageJ. (f) Quantification of Smad3 nuclear translocation was performed with ImageJ. The results are expressed as mean ± SEM. Statistical significance was determined by the Kruskal–Wallis H, test followed by Dunn’s post-test. Significance was established as ** p < 0.01 or *** p < 0.001 compared to TGF-β and ° p < 0.05 or °°° p < 0.001 compared to untreated cells.