Figure 3

Carbachol induces EMT-related changes in A549 cells. Cells were stimulated with TGF-β1 (5 ng/mL) or carbachol (0.1–10 μM) for 72 h (A), carbachol (1 μM) for the indicated times (B), or with the addition of pirenzepine (10 μM), methoctramine (10 μM), or 4-DAMP (1 μM) administered 1 h before carbachol (1 μM) for 72 h (C). Cell lysates were assayed for EMT markers and GAPDH. Representative western blots of E-cadherin, vimentin, α-SMA and GAPDH are shown. (D) Cells were treated with 4-DAMP (1 μM) 1 h before stimulation with carbachol (1 μM) for 72 h. Cells were fixed and stained with E-cadherin, vimentin and α-SMA antibodies, and then incubated with Alexa Fluor-conjugated secondary antibodies and DAPI for nuclei labeling. E-cadherin (green), vimentin (purple), α-SMA (red) and nuclei (blue) were visualized using confocal fluorescence microscopy. Data are expressed as mean ± SEM of 3–5 independent experiments after densitometric analysis. ^# p < 0.05, ^## p < 0.01 vs. control; *p < 0.05, **p < 0.01 vs. carbachol.