Figure 4. Effect of the CHRNα3 gene knockdown on nAChR signalling and apoptotic proficiency of lung cancer cells.

A) H1650 and H1299 cells were stably transfected with an inducible shRNA expression vector targeting CHRNα3 gene, and the efficiency of shRNA-mediated silencing was revealed by Western blots using anti-nAChRα3 antibody. To silence the CHRNα3 gene, expression of shRNA was induced with 1μM doxicycline (+Dox) for 48 hours. B) Analysis of nicotine-induced Ca²⁺ influx in H1299 cells expressing different levels of nAChRα3. H1299 cells were grown in the presence (+Dox) or absence (-Dox) of doxycycline, and following addition of 10 mM nictone (time point, 0 sec), the imaging of intracellular Ca²⁺ was performed using the Ca²⁺ indicator fluo-4 (10μM) and confocal laser microscopy (upper panel). RGFP expression is under the control of the same doxycyclin-inducible promoter as the α3-shRNA and thus is used as a reporter to shRNA expression. Plot comparing the quantified fluorescent intensity changes in CHRNα3-depleted (+Dox) and CHRNα3-expressing (-Dox) cells (lower panel). C) Knockdown of CHRNα3 expression triggers activation of the Akt-signalling pathway. H1299 cells were treated with nicotine (10 mM) for 48 hours following prior incubation in the presence (+Dox) or absence (-Dox) of doxycycline for 48 hours. Phosphorylation of Akt (PO4-Akt) and p42/44 (PO4-p42/44), and native Akt and p42/44 protein levels in total protein lysates were monitored by Western blot analysis. Equal loading was verified by anti-actin antibody. D) Knockdown of CHRNα3 gene impairs cell death response. H1650 and H1299 cells stably transfected with inducible CHRNα3 shRNA were incubated in the presence or absence of doxycycline for 24 hours and treated with etoposide, cyclohexamide or dexamethasone (100μM) for a further 24 hours. Cells were then stained with Annexin V/propidium iodide, and apoptotic fraction was analysed by FACS. Shown are mean values (+/-SD) from three different experiments. Statistical significance was calculated using Student’s unpaired ‘t’ test (P < 0.05; two tailed) and significant values are indicated by an asterisk.