Determination this explanation of ACh by LC MS/MS ACh levels in the supernatants of A549 cells were deter mined by LC MS/MS as previously described. Immunofluorescence Cells were grown on chamber slides and treated as designed. After intermediate washes with cold phosphate buffered saline, the cells were fixed with 4. 0% para formaldehyde in PBS for 15 min at RT. The cells were rinsed in cold PBS and blocked in 5% bovine serum albumin for 1 h at RT. The cells were then incubated with primary antibodies overnight at 4 C, washed with cold PBS, incubated with Alexa Fluor conjugated secondary antibodies at RT for 1 h, washed with PBS again, and then stained with 1 ug/mL 4,6 diamidino 2 phenylindole for 5 min at RT. After washing, images were col lected using an Axioscope microscope system at 40�� magnification.

The following antibodies were used E cadherin, SMA, and vimentin. Measurement of TGF B1 The amount of TGF B1 in the supernatants of A549 cells was determined using enzyme linked immunosor bent assay kits ac cording to the manufacturers instructions. Statistics analysis All data are expressed as mean SEM. Data were analyzed by one way ANOVA or the Mann Whitney test where appropriate and statistical significance was accepted at a p value of 0. 05. Statis tical analyses were performed using Prism version 5. 0. Results TGF B1 induced EMT is attenuated by mAChR antagonist EMT is defined by changes in gene expression in which epithelial markers are decreased while mesenchymal markers are increased. We examined whether TGF B1 induced EMT events could be modulated by mAChRs in lung epithelial cells.

As expected, A549 cells exposed to TGF B1 for 72 h resulted in a decrease in the epithelial marker E cadherin, as well as an increase in the mesenchymal markers vimentin and SMA. Interestingly, TGF B1 induced EMT events were significantly arrested by the non selective mAChR antagonist atropine in a dose dependent manner. This result sug gested a modulatory effect of mAChRs and prompted us to surmise a potential effect of endogenous ACh in EMT induction. TGF B1 induced EMT is modulated by non neuronal cholinergic system To further assess the potential effect of endogenous ACh in EMT events in A549, the acetylcholinesterase inhibitor physostigmine was used to increase the amount of ACh by blocking ACh degradation.

Interestingly, how ever, a significant, additive effect was observed by the combined administration of physostigmine and TGF B1 at 72 h post stimulation. The change in the expression of TGF B1 induced E cadherin, vimentin, and SMA was significantly enhanced by physostigmine compared with TGF B1 alone. In addition, the EMT event also occurred in the presence of physostigmine alone compared with GSK-3 controls. Having observed the effect of AChE inhibitor on EMT process, we went on to measure ACh levels in the su pernatants of cultured A549 cells. This was evaluated by western blot analysis.