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Fig. 2 | Respiratory Research

Fig. 2

From: Metformin attenuates lung fibrosis development via NOX4 suppression

Fig. 2

Metformin-mediated AMPK activation is involved in suppression of TGF-β-induced NOX4 expression in LF. a WB using anti-NOX4, and anti-β-actin of cell lysates from control (lane 1, 2) and metformin (lane 3, 4) treated LF. Metformin treatment was started 1 h before TGF-β (2 ng/ml) stimulation and protein samples were collected after 24 h treatment with TGF-β. Lower panel is the average (±SEM) taken from three independent experiments shown as relative expression. Open bar is control and filled bar is metformin treated. *p < 0.05. b Left panel: LF were treated with TGF-β and mRNA samples were collected at indicated time points (n = 9). *p < 0.05. Right panel: LF were treated with TGF-β in the presence or absence of metformin (10 mM) and mRNA samples were collected after 12 h treatment with TGF-β (n = 6). Open bar is control and filled bar is metformin treated. Real time-PCR was performed using primers to NOX4 or β-actin, as a control. NOX4 expression was normalized to β-actin. Shown is the fold increase (±SEM) relative to control treated cells. *p < 0.05. c WB using anti-NOX4, anti-type I collagen, anti-α-smooth muscle actin (SMA) and anti-β-actin of cell lysates from control siRNA (lane 1, 2) and NOX4 siRNA (lane 3, 4) transfected LF. TGF-β (2 ng/ml) treatment was started 48 h post transfection. Protein samples were collected after 24 h treatment with TGF-β. In the right panels are the average (±SEM) taken from four independent experiments shown as relative expression. Open bar is control and filled bar is TGF-β treated. *p < 0.05. d WB using anti-phospho-AMPK, anti-NOX4, anti-type I collagen, anti-αSMA, and anti-β-actin of cell lysates from control siRNA (lane 1, 2, 3, 4) and AMPK siRNA (lane 5, 6) transfected LF. Metformin treatment was started 48 h post transfection and 1 h before TGF-β (2 ng/ml) stimulation, and protein samples were collected after 24 h treatment with TGF-β. In the right panels are the average (±SEM) taken from five independent experiments shown as relative expression. Open bar is control and filled bar is TGF-β treated. *p < 0.05

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