Enhanced cell proliferation of gastric cancer cells overexpressing t-DARPP. (A) AGS cells stably transfected with t-DARPP (clones #1 and #2) demonstrate an increased rate of cell proliferation (48% and 45%) as compared to pcDNA empty vector control cells (26%, p < 0.01). ClickiT® EdU Assay (Invitrogen) was utilized to measure cell proliferation in t-DARPP expressing cells. EdU (5-ethynyl-2'-deoxyuridine) is incorporated as a thymidine analog during active DNA synthesis. EdU labeling in cells is detected by the binding of azide group of Alexa Fluor 488 dye (depicted as green fluorescence) to alkyne group of EdU. Ratio of EdU positive cells to total number of cells (represented by blue nuclei stain, DAPI) is a direct index of the number of proliferating cells. (B) t-DARPP overexpression in AGS stable clones is demonstrated by Western blot from total proteins. (C) Quantification data represents results obtained from two independent t-DARPP stable clones (#1 and #2). 500 cells were counted for each experiment and the percentage of EdU positive cells for each clone was averaged from at least four independent microscopic fields. Results are representative of at least three independent experiments and shown as mean ± SD. Significance of difference was calculated using one-way ANOVA.