Figure 1.

Azithromycin suppresses TGFB-induced NOX4 and myofibroblast differentiation in LF. (A) Western blotting (WB) using anti-EDA-FN1, anti-COL1A1/2 (type I collagen), anti-ACTA2 (actin, α 2, smooth muscle, aorta), anti-NOX4, and anti-ACTB of cell lysates from control (lane 1, 2) and azithromycin (AZM; 10 μg/ml)-treated (lane 3, 4) LF. AZM treatment was started 1 h before TGFB1 (2 ng/ml) stimulation and protein samples were collected after 24 h treatment with TGFB1. In the lower panels are the average ( ± SEM) taken from 7 independent experiments shown as relative expressions. *p < 0.05. (B) WB of cell lysates from control siRNA (lane 1, 2) and NOX4 siRNA- (lane 3, 4) transfected LF. AZM (10 μg/ml) treatment was started 48 h post transfection and 1 h before TGFB1 (2 ng/ml) stimulation. Protein samples were collected after 24-h treatment with TGFB1. The lower panels show the average ( ± SEM) of relative expressions, which were taken from 5 independent experiments. *p < 0.05 and **p < 0.001. (C) WB using anti-phospho-SMAD2, anti-SMAD2, anti-phospho-SMAD3, anti-SMAD3, and anti-ACTB of cell lysates from control (lane 1, 2) and AZM- (10 μg/ml) treated (lane 3, 4) LF. In the lower panels are the average ( ± SEM) taken from 5 independent experiments shown as relative expressions. *p < 0.05. (D) LF were treated with TGFB1 in the presence or absence of AZM (10 μg/ml) and mRNA samples were collected after 24 h treatment with TGFB1 (n = 4). Real time-PCR was performed using primers to NOX4 or ACTB, as a control. NOX4 mRNA expression was normalized to ACTB. Shown is the fold increase ( ± SEM) relative to control-treated cells. *p < 0.05.