A, H1838 cells were cultured with Dihydro-β-erythroidine (DhbetaE, 1 μM) or TC2559 (0.1 μM) in the presence or absence of nicotine for up to 5 days. Afterwards, the luminescence of viable cells was detected using Cell Titer-Glo Luminescent Cell Viability Assay Kit according to the protocol of the manufacturer (Promega). All data are depicted as mean ± SD. * indicates significant difference as compared to the untreated cells group. ** indicates significant difference of combination treatment as compared the DhbetaE or TC2559 alone (P<0.05). Con, indicates untreated control cells. B, H1838 cells were transfected with control or α4 nAChR siRNA (100 nM each) for 40 hr before exposure of cells to nicotine for up to 5 days. Afterwards, the luminescence of viable cells was detected using Cell Titer-Glo Luminescent Cell Viability Assay Kit according to the protocol of the manufacturer. All data are depicted as mean ± SD. * indicates significant difference as compared to the untreated cells group. ** indicates significant difference of combination treatment as compared the nicotine alone (P<0.05). The insert on the top showed the Western blot result for α4 nAChR protein production. Con, indicates untreated control cells. C, H1838 cells were transfected with control or α4 nAChR siRNA (100 nM each) for 40 h before exposure of cells to α-bungarotoxin (α-BT, 1 μM) and nicotine for up to 5 days. Afterwards, the luminescence of viable cells was detected using Cell Titer-Glo Luminescent Cell Viability Assay Kit according to the protocol of the manufacturer. All data are depicted as mean ± SD. * indicates significant difference as compared to the untreated cells group. ** indicates significant difference of combination treatment as compared the nicotine alone (P<0.05). *** indicates significant difference of combination α4 nAChR siRNA and α-bungarotoxin (α-BT) plus nicotine treatment as compared to the α4 nAChR siRNA plus nicotine (P<0.05). Con, indicates untreated control cells.