Figure 8.

α5SNP enhanced in vitro basal cell proliferation and migration in a prostate cancer cell model. Cells were transfected with either the wild-type α5 construct (α5-WT) or with its mutated version α5D398N (α5-MUT), and functional consequences were studied. (A) Depiction of the expression of the α5 subunit in non-transfected DU145 cells (NT; native expression) and in cells transfected with α5-WT (native plus foreign expression) or α5-MUT (native plus foreign expression); mRNA levels were determined via qPCR, and data show the mean ± SEM of three independent cell culture experiments. (B) Immunofluorescence images showing the successful overexpression of α5-WT (left) and α5-MUT (right) constructs in DU145 cells; the V5-tagged subunit was detected using incubation with the anti-V5 primary antibody followed by the appropriate Alexa Fluor 488 secondary antibody. Objective 100×, scale bar 40 µm. (C,D) Cell proliferation index and migration index (respectively) of DU145 cells transfected with α5-WT or α5-MUT-containing plasmids; cells were treated with 1 μM nicotine. The bar diagram shows data (mean ± SEM). (E) Cell proliferation index of DU145 cells transfected with α5-WT or α5-MUT-containing plasmids. In the cells transfected with the latter, proliferation was analyzed in the presence or absence of SQ22536 (non-selective inhibitor of ACs, SQ, 10 µM) or PTX (G-protein inhibitor, pertussis toxin, 0.1 µM). The bar diagram shows the data (mean ± SEM). (F) Cell proliferation index of non-transfected DU145 cells treated with 1 μM nicotine in the presence or absence of the inhibitors SQ22536 (SQ, 10 µM) or PTX (0.1 µM). Measurements were made using 3 independent experiments performed in triplicate. Statistical significance was analyzed using the Kruskal–Wallis test for non-parametric data with the GraphPad Prism (GraphPad 9.1.1 Software) software. A p-value of <0.05 was considered significant. * p ≤ 0.05, ** p ≤ 0.001, *** p ≤ 0.0001.