Fig. 2

Increased expression of AT2R in BCa cells: effects on cell proliferation and apoptosis. a AT2R mRNA expression in EJ cells following transduction with gradient doses of Ad-G-AT2R-eGFP (10 ifu, 20ifu, 50 ifu, 100 ifu, 200 ifu/cell). Columns, mean (n = 3); *P < 0.05 and ** P < 0.01 vs cells transduced with 10 ifu of Ad-G-AT2R-eGFP. b EJ cells were transduced with either Ad-CMV-eGFP or Ad-G-AT2R-eGFP (100 ifu/cell) for 24 h as described in Materials and Methods. Incubations were followed by detection of eGFP fluorescence and AT2R immunoreactivity using an anti-AT2R antibody. Representative fluorescence micrographs from Ad-CMV-eGFP–transduced and Ad-G-AT2R-eGFP–transduced cells, showing eGFP fluorescence, AT2R immunostaining and DAPI in each treatment condition. Scale bars, 50 μm. c BCa cells underwent transduction with either Ad-CMV-eGFP or Ad-G-AT2R-eGFP (100 ifu/cell) for 24 h. Following this, WST-1 reagent was added to the culture medium (1:10 dilution), and absorbance was measured at 450 nm. Columns, mean A450 nm from three experiments; bars, SD. * P < 0.01 vs Ad-CMV-eGFP–transduced cells. d BCa cells were transduced with either Ad-CMV-eGFP or Ad-G-AT2R-eGFP (100 ifu/cell) for 2 d as described in the Methods. Incubations were followed by detection of eGFP fluorescence and counting of cells that display apoptotic-like morphology from 10 random fields per well. Columns, mean from three separate experiments; bars, SD. * P < 0.01 vs corresponding eGFP-transduced cells. e EJ cells were transduced with either Ad-CMV-eGFP or Ad-G-AT2R-eGFP (100 ifu/cell) for 2 d, and apoptotic cells were detected using the DeadEnd Colorimetric TUNEL System kit. A representative phase-contrast micrograph was shown from each treatment condition. The brown-colored nuclei represent TUNEL-positive (apoptotic) cells. f quantification of the TUNEL-positive cells as a percent of the total number of cells in the dish. Columns, mean of three experiments; bars, SE. * P <0.001 vs eGFP group