Fig. 3

ALLN attenuates the fibrogenic phenotype of cultured human lung fibroblasts stimulated with TGF-β1. Normal human lung fibroblasts were pretreated with DMSO as control or various concentrations of DMSO-diluted ALLN for 1 h, followed by stimulation with 10 ng/ml recombinant human TGF-β1 for an additional 24h. A After harvest, mRNA and protein expression of the indicated molecules was evaluated using real-time reverse transcription-polymerase chain reaction (RT-PCR). Values were normalized to GAPDH and are shown as relative levels (mean ± SEM). All values represent mean ± SEM; n = 5 each group; *, p ≤ 0.05; **, p ≤ 0.01, ***, p ≤ 0.001. Fibroblasts were also immunostained for αSMA, COL1A2, or FN1 (green, B) and phospho-Smad2/3, SNAIL2, or ZEB1 (green, C). Nuclear counterstaining (blue) was performed with DAPI. Representative images of three experiments are shown (400-fold magnification)