Figure 2. Brd4 inhibition blocks TGF-β1–induced Nox4 gene upregulation in human lung fibroblasts.

(A–C) Normal human lung fibroblasts (IMR90) were transfected with siRNA Brd4 or NT and then treated with vehicle or TGF-β1 (2 ng/mL) for 48 hours. (A) The whole cell lysate were collected to examine Brd4 expression by Western blots. (B) Densitometry of Brd4-associated signals detected (ratio to β-actin) in A. *P < 0.05, Brd4 siRNA–transfected cells compared with NT control of the same cell line, by 2-tailed t test. (C) Treated as in A, cells were analyzed for Nox4 mRNA by real-time PCR (mean ± SD; n = 3 in each group). *P < 0.05, each group compared with vehicle only; ^#P < 0.05, TGF-β1–treated siRNA Brd4 vs. NT cells, by 2-tailed t test. (D) IMR90 fibroblasts were incubated overnight with 1% fetal bovine serum at 70% confluence and then treated with vehicle or various Brd4 inhibitors with the same concentration as in Figure 1 for 2 hours before stimulation with TGF-β1 (2 ng/mL) for 48 hours. Cells were analyzed for Nox4 mRNA by real-time PCR (mean ± SD; n = 3 in each group). *P < 0.05, each group compared with TGF-β1 with vehicle only (Vehl/TGF-β1), by 2-tailed t test. (E) IMR90 fibroblasts were pretreated with or without OTX015 for 2 hours and then with or without TGF-β1 for 48 hours. Cells were collected and subjected to SDS-PAGE and Western blot analysis for Nox4 and β-actin (loading control). (F) The densitometry of Nox4-associated signals detected (ratio to β-actin) in E. *P < 0.05, OTX015 pretreated cells with TGF-β1 compared with TGF-β1, by 2-tailed t test. (G) IMR90 fibroblasts stimulated with/without TGF-β1 (2 ng/mL for 24 hours) in the presence/absence of OTX015 (0.5 μM) were analyzed for extracellular H₂O₂ production, as a marker of Nox4 activity (mean ± SD; n = 6 in each group). *P < 0.05, compared with TGF-β1/OTX015, by 2-tailed t test.