Figure 6

Overexpression of PAX2 enhances estradiol-induced decrease of cell invasion in luminal breast cancer cells. MCF-7 cells were cultivated in a steroid- and serum-depleted growth medium, transfected with PAX2 cDNA or empty vector and then treated with 10 nM estradiol. A) Following 30 min-treatment with estradiol, PAX2 phosphorylation on ser393 residue and total levels of PAX2 protein were determined (se: short exposition time, to ensure comparable levels of exogenous PAX2 protein between PAX2-transfected cells treated with estradiol or not; le: long exposure time, to detect endogenous PAX2 protein in empty vector-transfected cells), whereas following 24 h-treatment with estradiol, total levels of ERBB2 protein were determined, using western blot. GAPDH was used as a control to normalize for protein content. Results are representative of three independent experiments. B) Following treatment with estradiol for the indicated times, cell proliferation was determined, using MTT assay. Results are mean +/- SD of four independent experiments. *statistically significant difference. C) Following treatment with estradiol for the indicated times, the presence of cleaved fragments of PARP was assessed using western blot. Positive control for PARP cleavage was MCF-7 cells treated with 10 μM cisplatin for 24 h. D) Following 24 h-treatment with estradiol, cell nuclei were stained with Hoechst dye and number of apoptotic cells was determined under a fluorescent microscope. E) Following 24 h-treatment with estradiol, cells were subjected to Matrigel invasion assay. In C-E, results are representative or mean value +/-SD of three independent experiments. *statistically significant difference.