Figure 2. NOX4 is expressed in lungs of human subjects with idiopathic pulmonary fibrosis (IPF) and mediates H₂O₂ production, myofibroblast differentiation, and serum-stimulated proliferation of IPF-derived mesenchymal cells.

(a) Immunohistochemical staining demonstrating expression of NOX4 in myofibroblastic foci in lungs of a representative human subject with IPF. Length bar = 100 μm. (b–i) Mesenchymal cells isolated from IPF lung tissues (IPF-MCs) by explant tissue culture and analyzed at passage 2–5. (b) IPF-MCs were transfected with nontargeting (control) siRNA or NOX4 siRNA and treated with/without TGF-β1 (2 ng/ml) for 16 h and analyzed for NOX4 protein (inset) and extracellular H₂O₂ production. (c–f) The effect of siRNA knockdown of NOX4 in IPF-MCs with/without TGF-β1 (2 ng/ml) on the expression of α-SMA mRNA (c) and protein (f); fibronectin mRNA (d) and protein (f); and NOX4 mRNA (e) and protein (f), as determined by real-time PCR (at 24 h) and Western immunoblotting (at 48 h). (g) Control (nontargeting) and NOX4 siRNA transfected IPF-MCs were treated with/without TGF-β1 (2 ng/ml) for 48 h and conditioned culture media was collected and analyzed for acid-soluble collagen using the Sircol assay. (h,i) The effect of siRNA knockdown of NOX4 on proliferation of IPF-MCs treated with/without serum was determined at 24 h by BrdU incorporation assay (h) and at 48 h by assessment of cell numbers using a coulter counter (i). Values represent mean ± S.E.M.; n = 3–5. ^*P < 0.001 compared to control (without TGF-β1 or serum) and nontargeting siRNA.