Figure 6.

FK506-binding protein 10 (FKBP10) is up-regulated by transforming growth factor (TGF)-β₁ in primary human lung fibroblasts (phLF), and knockdown of FKBP10 attenuates synthesis of collagen I, collagen V, and α-smooth muscle actin (α-SMA). (A) Western blot analysis of phLF treated with increasing concentrations of TGF-β₁ (0.1, 0.2, 1.0, 2.0, and 5.0 ng/ml) shows up-regulation of FKBP10 at 48 hours in the presence of 1.0 ng/ml and higher TGF-β₁ concentrations. Efficacy of TGF-β₁ treatment was confirmed by monitoring phosphorylation of mothers against decapentaplegic homolog 3 (SMAD3, depicted as pSMAD3) in comparison with total SMAD3 levels (SMAD3). (B) 2.0 ng/ml TGF-β₁ significantly increased FKBP10 in phLF after 48 h, as quantified by densitometric analysis from Western blot analysis (cf. Figure 7C for representative Western blot, lanes with scrambled [−] small interfering RNA [siRNA] only). The effect of the knockdown was always significant (P < 0.01 or P < 0.001) but is not specified in the interest of clarity. (C) FKBP10 knockdown effects in phLF in combination with 24 and 48 hours of TGF-β₁ treatment (2.0 ng/ml). A representative Western blot analysis for detection of FKBP10, collagen I, collagen V, fibronectin, and α-SMA is shown. (D) Densitometric quantification of collagen I, collagen V, fibronectin, and α-SMA protein levels. β-actin (ACTB) was used as loading control. Scrambled siRNA was used as control. Data are based on eight completely independent experiments and are given as mean ± SEM. Statistical analysis for comparison of FKBP10 siRNA vs. scrambled siRNA control was performed by paired two-tailed t test. *P < 0.05, **P < 0.01. The well-known effect of TGF-β₁ on these proteins was mostly significant but is not specified in the interest of clarity. ctrl = control.