Figure 5

PI3K and Rac modulate TGFβ1-mediated NOX4 expression. (A,B) NHLF were pre-treated (30 minutes) in the presence or absence of a PI3K inhibitor, LY294002 (50 µM) followed by treatment with TGFβ1 (2 ng/mL; 48 h). (A) qPCR analysis of NOX4 transcript expression expressed as ΔΔct. Data are means + SEM from three experiments. (B) ROS (H₂O₂ generation) was measured using Amplex Red assay. After treatment, cells were washed twice in KRPG and incubated for 30 minutes with Amplex Red reagent (50 µM) and 0.1 U/mL HRP in KRPG. Fluorescence is measured expressed as relative fluorescence units (RFU) compared to control. Data are means + SEM from three independent experiments. (C–E) NHLF were stimulated with TGFβ1 (2 ng/mL; 48 h) in the presence or absence of indicated concentrations of a rac inhibitor EHT, and (C) NOX4 transcript was measured using qPCR and expressed as ΔΔct. (D) SDS-PAGE immunoblotting was performed on cell lysates using Abs specific for α-SMA and FN proteins after which, the blots were stripped and re-probed for GAPDH. Representative blots are from a single experiment of three performed. (E) Represents quantitative densitometric analysis of indicated proteins from (D) using AlphaView software and expressed as a percentage of control cells. Results are means + SEM from three independent experiments. *P < 0.05; **P < 0.01.