<p><b>A</b>. pBI-G expression vector plasmid or HA-ATF3 expression plasmid (pBIG-HA-ATF3) were co-transfected with the tTA (tet-off) expression plasmid into HEK-293T cells in the presence (+) or absence (-) of doxycycline (Dox, 10µg/ml). Nuclear cell lysate was separated on 12.5% SDS-PAGE followed by Western blot analysis with anti-HA, anti-ATF3, and anti-α tubulin (loading control). <b>B</b>. Schematic representation of the mating cages. Gender matched heterozygotes αMHC-tTA driver mice were mated with ATF3-tg responder mice line. Mating in the presence of doxycycline represents ATF3-Off expression during embryonic development therefore, mice are designated adult-ATF3 expressing. Mating in the absence of doxycycline represents ATF3-On expression through embryonic development therefore, mice are designated embryonic-ATF3 expressing. <b>C</b>. RT-qPCR analysis for cDNA derived from atria and ventricles of either wild-type or ATF3 transgenic mice treated with doxycycline as indicated. The expression level of ATF3 was examined by either mouse- (black) or human-specific primers (gray). The results represent the mean expression relative to GAPDH of the indicated number of animals (n). Asterisks (*/**) represent P value <0.05 or <0.01 of a one-tailed t-test compared to wild-type mice. <b>D</b>. Representative Western blot analysis (Top panel) of cell lysates derived from ventricles of wild type mice (Wt.), adult-ATF3 expressing and wild type mice following 2 h PE injection. The membrane was probed with anti-ATF3 and GAPDH for loading control. The asterisks (*) represent non-specific cross reactive proteins (non-specific). Densitometry analysis (bottom panel) of ATF3 expression was normalized with the GAPDH level. The results represent the mean and SEM from six independent animals. <b>E</b>. Immunohistochemistry of left ventricle sections stained with αATF3 (1:200). Representative sections derived from mice positive for HA-ATF3 responder and αMHC driver (ATF3 Tg, right panel) and Wild-type mouse (left panel). The magnification shown is X20. The black arrow indicates an ATF3-stained nucleus.</p
