Fig. 2.

CSE inhibit CFTR mRNA expression and activity. A: CFTR mRNA levels in Calu-3 cells treated with 100 μg/ml CSE (Cigarette Smoke Extract) or DMSO (vehicle) for 24 h, measured by quantitative real-time RT-PCR (qRT-PCR). The results were expressed as arbitrary units (A.U.). Measurements correspond to four independent experiments (n = 4), each done in duplicate. B: CFTR channel halide transport activity was measured in Calu-3 cells treated with 100 μg/ml CSE or DMSO by using the SPQ Cl^- sensitive probe. Arrows indicate the times of buffers addition; I: NaI buffer, II: NaNO₃ buffer, III: NaNO₃ buffer + cAMP cocktail, IV: KSCN 150 mM + 5 μM valinomycin. Fluorescence values were calculated as F= (F-Fq)/(Fi-Fq) −1; Fi, are the initial fluorescence values in NaI buffer. Fq corresponds to background values (fluorescence quenching in the presence of KSCN+valinomycin). The graph is the mean of four independent experiments (n = 4). C: Halide efflux slopes, corresponding to SPQ fluorescence changes of Calu-3 cells incubated with CSE or DMSO. The slopes were calculated from the linear regressions of the first 6 points after CFTR stimulation and were plotted as percentage (%) relative to controls. All data were expressed as mean ± SEM. * * indicates p < 0.01 and * p < 0.05, as compared to control cells. Statistical analyses were performed by ANOVA and Tukey's test.