Fig. 5.

The DU-145 and LNCaP cell lines were transiently transfected with the pcDNA3.1-AP-2alpha expression vector and pcDNA3.1 empty vector. Twenty-four hours after transfection, cells were subjected to fluorescence-activated cell sorting. After another 24 h of incubation in cell medium, cells were harvested and RT–qPCR assay (A), western blot assay (B) were performed. (C) In order to asses whether transfection of DU-145 and LNCaP cells with the pcDNA-AP-2alpha expression vector has an effect on the regulation of intracellular zinc uptake, transfected DU-145 and LNCaP cells were incubated with 2 μg/ml of zinc for 1 h, washed twice in phosphate-buffered saline and incubated with the cell-permeable fluorescent zinc indicator FluoZin-3 (5 μM) for additional 15 min. Fluorescence was examined by flow cytometry. Data is representative of three independent experiments. *P < 0.01; **P < 0.05 compared with pcDNA3.1-AP-2alpha transfected cells. (D) The effect of AP-2alpha expression on cell growth was examined using the CellTiter-Blue Assay. DU-145 and LNCaP prostate cancer cells were transfected as described above, plated in a 96-well plate (5 × 10³ to 10 × 10³ cells per well) in triplicates and incubated for 48 h in complete cell growth medium. After the 48 h incubation period ended, the CellTiter-Blue Assay was performed. Error bars represent ±SD of triplicate fluorescence means.