Figure 4

The effect of doxorubicin on B7-H1 and AKT expression and induction of apoptosis. A) Column chart of the FACS data showing the effect of siRNA-B7-H1 inhibition on the percentage of apoptosis induced after 48-hours treatment of MDA-MB-231 cells with doxorubicin (n = 6, **indicated P < 0.001) (Top panel), representative FACS data of one of the experiments (middle panel) and image of the cells right before harvesting (bottom panel). B) Column chart of the FACS data showing the effect of specific AKT-inhibitor (LY294002) on B7-H1 expression and induction of apoptosis in MDA-MB-231 cells after 48 hours doxorubicin treatment (n = 5, **indicates P = 0.016) (Top panel), representative FACS data of one of the experiments (middle panel) Diamond, total percentage of apoptotic cells, Spade, numbers in brackets are specific doxorubicin induced apoptosis (SDA) calculated by subtracting the percent of apoptotic cells in non-treated control. Image of the cells right before harvesting is indicated in the bottom panel. (The columns are means and error bars are SEMs in the top panel of A and B). C) Western blot for nuclear proteins from the same cells as in B stained with B7-H1 or GAPDH. D) Representative immunofluorescence images of the same cells as in B stained with a specific antibody for phospho-AKT (green). E) Cytoplasmic and nucleus AKT were quantified manually from the presented image shown in D. Nuclei were counterstained with DAPI (Red).