Figure 1. Identification of NOX4 as the enzymatic source of extracellular H₂O₂ production by myofibroblasts and its role in mediating myofibroblast differentiation and contractility.

(a) RNA was isolated from human fetal lung mesenchymal cells (hFLMCs) treated with/without TGF-β1 (2 ng/ml) for 18 h and analyzed by Affymetrix (U133A) microarray for members of the NOX/DUOX gene family. Values represent mean ± S.D., n = 3 per group. ^*P < 0.001 compared to control. ND indicates “not detected” (below threshold). (b) hFLMCs were treated with/without TGF-β1 (2 ng/ml) for the times indicated and cell lysates subjected to SDS-PAGE and Western immunoblotting for NOX4 and GAPDH. (c) Effect of NOX4 siRNA (duplex 4) on extracellular release of H₂O₂ by hFLMCs treated with/without TGF-β1 (2 ng/ml for 16 h). (d) hFLMCs were pretreated with pharmacologic inhibitors against ALK5 receptor kinase (SB431542; 1 μM), MEK (PD98059; 20 μM), p38 MAPK (SB203580; 6 μM), JNK (SP600125; 100 nM), and then stimulated with TGF-β1 (2 ng/ml × 16 h) prior to measurement of extracellular H₂O₂ release. (e) Effect of SMAD3 siRNA knockdown on TGF-β1-induced NOX4 expression in hFLMCs, as determined by Western immunoblotting. (f) Effect of siRNA-mediated knockdown of SMAD3 on extracellular H₂O₂ production stimulated by TGF-β1 (2 ng/ml × 16 h) in hFLMCs. (g) hFLMCs in 3-D collagen matrix were stimulated with/without TGF- β1 (2 ng/ml × 16 h) in the presence/absence of catalase (750 U/ml) and effects on α-smooth muscle actin (α-SMA), fibronectin, and β-actin were determined by Western immunoblotting. (h) Effect of siRNA-mediated silencing of NOX4 in 3D-collagen matrix-embedded hFLMCs on cellular expression of α-SMA, fibronectin, and procollagen-1 treated with/without TGF-β1 (2.5 ng/ml × 72 h), as determined by Western immunoblotting. (i,j) Effect of exogenous catalase (750 U/ml) (i), and siRNA-mediated NOX4 silencing (j) on TGF-β1-induced contractility in 3D-collagen matrices. Values represent mean ± S.E.M.; n = 4. ^*P < 0.001 compared to controls.