Fig. 5. Activation of adenosine receptors regulates connexin expression in Calu-3 cells. (A) qRT-PCR analysis showed an increase in the mRNA level of Cx26, Cx30 and a decrease in the mRNA level of Cx43 induced by NECA (10 µM, 24 h). The PKA inhibitor Rp-cAMPs (Rp-c, 200 µM) as well as the Sp1 inhibitor withaferin A (witA, 1 µM) blocked the NECA-induced increase in Cx26 mRNA. (B) Western blot experiments showed an increase in Cx26 protein level and a decrease in Cx43 protein level induced by NECA. The results are given as average ± SEM from three cell culture passages. (C) The mRNA levels of Cx26 and Cx43 were significantly reduced after 48 h treatment with specific siRNAs. The Cx26 mRNA level was increased after Cx43-siRNA treatment, the Cx30 mRNA level was not significantly affected by both Cx26- and Cx43-siRNAs. The results are given as average ± SEM from at least three experiments. (D) The dye uptake rate of Calu-3 cells was significantly reduced after knockdown of Cx26 with specific siRNA, and NECA was not able to affect it. The results are given as average ± SEM from at least six experiments with three cell culture passages. The data were statistically compared to the control (contr, neg siRNA) using student's t test (P<0.05 *, P<0.01 **, P<0.001 ***).