Figure 5

Knockdown of PAX2 abrogates 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 shRNAs, control shRNA (Ctl: scrambled sequence from PAX2 shRNA #600) or transfection reagent only (None) for 24 h and then treated with 10 nM estradiol. A) PAX2 phosphorylation on ser393 residue and total levels of PAX2 protein were determined using western blot after 30 min of treatment with estradiol. B) Total levels of ERBB2 was determined following 24 h-treatment with estradiol. GAPDH was used as a control to normalize for protein content. C-D) Following treatment with estradiol for the indicated times, cell proliferation was determined, using MTT assay (C), and the presence of cleaved fragments of PARP was assessed using western blot (D). Positive control for PARP cleavage was MCF-7 cells treated with 10 μM cisplatin for 24 h. E-F) 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), and cells were subjected to Matrigel invasion assay (F). All results are representative or mean value +/-SD of three independent experiments; different subscript letters indicate a statistically significant difference.