Mecamylamine hydrochloride mec

The human histocytic lymphoma cell line U937 was obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and cultured according to the protocol described for mouse PBMCs. Cells in the log-phase of growth were transferred to 24-well plates (1 × 10⁶ cells/ml and per well) and primed with 1 μg/ml LPS from Escherichia coli (L2654; Sigma-Aldrich) for 5 h. Thereafter, BzATP (100 μM) was applied for 30 min, in the presence or absence of ACh (10 μM), Nic (100 μM), PC (100 μM), L-α-G-PC (Sigma-Aldrich; 0.1 μM to 1000 μM) or 1-palmitoyl-sn-glycero-3-phosphocholine LPC (C16:0), Sigma-Aldrich; 0.1 μM to 100 μM). To test the involvement of different subunits of nAChR, the following antagonists were applied: mecamylamine hydrochloride (Mec, 100 μM, Sigma-Aldrich), α-bungarotoxin (α-Bun, 1 μM, Tocris Bioscience, Bristol, UK), strychnine hydrochloride (Stry, 10 μM, Sigma-Aldrich) or conotoxins ArIB [V11L, V16D] (500 nM) or RgIA4 (200 nM; Ramarao and Cohen, 1998 (link); Whiteaker et al., 2007 (link); Innocent et al., 2008 (link); Richter et al., 2016 (link); Romero et al., 2017 (link)).

Human Het-1A squamous esophageal cells were obtained from American Type Culture Collection (ATCC, CRL-2692). Cells were cultured at 37°C and 5% CO₂ in BEBM media (Lonza/Clonetics Corporation) according to ATCC recommendations. Culture flasks and plates were precoated with a mixture of 0.01 mg/mL fibronectin, 0.03 mg/mL bovine collagen type I and 0.01 mg/mL bovine serum albumin dissolved in culture medium. Cells were seeded in 96-well plates (1×10⁴ cells per well), and 24 h later the analyzed compounds were added to cells. Next, cells were incubated for 48 h, examined under microscope, and characterized using WST-1 assay and fluorescent Hoechst/propidium iodide assay as described elsewhere [19 (link),25 (link)]. Hoechst/propidium iodide assay allows evaluation of cell viability by staining all cell nuclei with Hoechst 33342 and dead cell nuclei with propidium iodide. For inhibition of nAChRs and muscarinic acetylcholine receptors (mAChRs), 1 h preincubation of keratinocytes with 1 μM α-bungarotoxin (α-Bgtx, Tocris), 10 μM mecamylamine hydrochloride (Mec, Sigma), 1 μM atropine (Sigma), or 1 μg per 50 μl anti-α7-nAChR antibody (#ab10096, Abcam, Cambridge, UK) was performed before rSLURP-1 application. Antibodies at the same concentration were additionally added to the cells after 24 and 40 hours of incubation with rSLURP-1.