Figure 5.

siRNA knockdown of the α9 subunit reduces the nicotine‐induced proliferation and activation of p‐ERK and p‐Akt in A549 cells. (A) q‐PCR analysis of α9 subunit mRNA level in A549 cells transfected for 48 h with the indicated α9 siRNAs. Data are shown as relative expression ±SD of three independent experiments performed in triplicate and are expressed with respect to the level of α9 subunit mRNA level in the cells transfected with scrambled siRNA set as 1, according to the 2^−ΔΔCt method. *P < 0.05, statistically significant with respect to control (ctrl) cells (one‐way ANOVA, post‐Tukey's test). ns: not significant. (B) Effect of knocking down the α9 subunit on nicotine‐induced cell proliferation. A549 cells transfected with siRNA II or siRNA IV were treated for 48 h with 100 nM nicotine and then counted. (C) Western blot analysis of ERK and Akt pathway activation in A549 cells in the presence or absence of 100 nM nicotine, with or without α9 siRNA II. (D) Western blot analysis of ERK and Akt pathway activation in A549 cells in the presence or absence of 100 nM nicotine, with or without α9 siRNA IV. The Western blot results are expressed as the p‐Akt/Akt and p‐ERK/ERK ratios, taking the ratio of cells treated with scrambled siRNA as 100. The statistical analysis of nicotine‐induced proliferation and ERK and Akt pathway activation in the presence of scrambled siRNA or α9 siRNAs was made in seven independent experiments performed in triplicate using one‐way ANOVA followed by Bonferroni's test (*P < 0.05 vs. untreated cells) or Dunn's test.