Fig. 4

NOX4-mediated ROS is involved in the mechanisms for SMAD phospholylation and myofibroblast differentiation in LF. a Fluorescence intensity of CM-H2DCFDA staining for intracellular ROS production. After 24 h treatment with TGF-β, incubation with CM-H2DCFDA (10 μM) was performed for 30 min, fluorescence of DCF was measured by a fluorescence microplate reader. The fluorescence level in the control treated cells in the absence of metformin was designated as 1.0. Shown panels are the average (±SEM) taken from three independent experiments. *p < 0.05. b Fluorescence intensity of CM-H2DCFDA staining for intracellular ROS production. Metformin treatment was started 48 h post-siRNA transfection and 1 h before TGF-β (2 ng/ml) stimulation. After 30 min incubation with CM-H2DCFDA, fluorescence of DCF was measured by a fluorescence microplate reader. The fluorescence level in the control siRNA transfected cells without TGF-β and metformin treatment was designated as 1.0. Shown panels are the average (±SEM) taken from six independent experiments. *p < 0.05. c WB using anti-phospho-SMAD2, anti-SMAD2, anti-phospho-SMAD3, anti-SMAD3, anti-type I collagen, anti-αSMA, and anti-β-actin of cell lysates from control (lane 1, 2), NAC (1 mM) (lane 3, 4), and NAC (10 mM) (lane 5, 6) treated LF. NAC treatment was started 1 h before TGF-β (2 ng/ml) stimulation and protein samples were collected after 24 h treatment for type I collagen and anti-αSMA WB, but 30 min for SMAD WB. Shown panels are the average (±SEM) taken from three independent experiments shown as relative expression. Open bar is control and filled bar is TGF-β treated. *p < 0.05 and **p < 0.001