<p>(A) The effect of CX-4945 on TGF-β1-induced activation of Smad and expression of Snail and Twist was evaluated using Western blot analysis. Briefly, after 24 h serum starvation, A549 cells were treated with TGF-β1 (5 ng/ml) alone or in combination with CX-4945 in media containing 0.1% FBS for 48 h. Cytosolic and Nuclear fractions were obtained as described in ‘Materials and Methods’. Actin and histone H3 were used as internal control of cytosolic and nuclear fraction, respectively. The relative, normalized ratio between p-Smad2 and actin was presented. (B) Nuclear translocation of Smad2/3 and its inhibition by CX-4945 were confirmed by immunocytochemistry. Nuclei were counterstained with Hoechst 33342. All scale bars represent 50 µm.</p
