Fig. 2

Specific activin A inhibition attenuates TGFβ1-induced Smad3 activation and profibrotic responses in MC. a ELISA demonstrates that TGFβ1 (24 h) increases actA and actB secretion (n = 3) to 19.5 ng/ml and 2.5 ng/ml, representing an 8.9- and 1.06-fold induction respectively. b TGFβ1 increases actA in whole cell lysate by 1.8-fold (n = 3). c ActA (20 ng/ml) upregulates FN (n = 3–4), CTGF (n = 4) and αSMA (n = 6) at 48 h. d TGFβ1 and actA both increase Smad3 transcriptional activity; no synergistic effect is seen (n = 6–12). e An actA neutralizing antibody attenuates TGFβ1-induced FN (n = 5–6), αSMA (n = 5), CTGF (n = 5–6), and Smad3 activation (n = 10–12). f ActA neutralization decreases TGFβ1-induced Smad3 transcriptional activity at 24 h (n = 9–15), but this is not decreased by actB neutralization (n = 6) (g). h MC were stimulated with TGFβ1 or actA for 1 h, then treated with their type I receptor inhibitor SB431542 (50 µM). Restimulation with the same ligand shows that cells become refractory to TGFβ1, but not actA (n = 4). *, **, ***, ****P < 0.05, 0.01, 0.001, 0.0001; one-way ANOVA with Tukey’s multiple comparisons post hoc test