Figure 2

PAX2 is activated by estradiol in breast cancer cells of the luminal subtype selectively. Breast cancer cells were cultivated in a steroid- and serum-depleted growth medium, before they were treated with estradiol. A) MCF-7 cells were treated for the indicated time periods with 10 nM estradiol and total levels of PAX2 protein, PAX2 phosphorylation on ser393 residue, and ERBB2 protein levels were determined using western blot. GAPDH was used as a control to normalize for protein content. B) MCF-7 cells were treated for the indicated time periods with 10 nM estradiol and the impact on PAX2 gene expression were determined using RT-PCR. GAPDH was used as a control to normalize for total mRNA content. C-D) MCF-7 cells were treated with 10 nM estradiol for 1 h and subcellular localization of PAX2 was determined using immunofluorescence (C) and subcellular fractionation (D). In C), Hoescht dye was used to visualize cell nuclei; magnification: 400×. In D), ERα was used as a positive control for estradiol-induced nuclear import, PARP was used as a marker of nuclear fraction (N) purity, and GAPDH was used as a marker of cytosolic fraction (C) purity. E) Non-luminal breast cancer cell lines MDA-MB-231 (231) and HS578T were treated with the indicated doses of estradiol. Following 30 min-treatment with estradiol, PAX2 phosphorylation on ser393 residue and total levels of PAX2 protein were determined, 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. All results are representative of three independent experiments.