Estradiol negatively regulates PAX2 activity and invasion in an ERα-dependent manner in MCF-7 cells. A) MCF-7 cells were cultivated in a steroid- and serum-depleted growth medium and treated with 10 nM estradiol for 24 h before they were subjected to Matrigel invasion assay. Numbers above bars indicate total number of cells recovered in the lower chamber at the end of the assays. Pictures show the density of invasive cells that reached the porous filter. B-D) MCF-7 cells were cultivated in a steroid- and serum-depleted growth medium and pretreated with ERα antagonist ICI 182780 (1 μM) for 1 h before they were treated with 10 nM estradiol. (B) Following 24 h-treatment with estradiol, total protein content of ERBB2, and of ERα as a marker of ICI 182780 efficiency, were determined using western blot. GAPDH was used as a control to normalize for protein content. (C) Following 24 h-treatment with estradiol, cell invasion was determined using Matrigel invasion assay. (D) Following 30 min-treatment with estradiol, the extent of phosphorylation of PAX2 on ser393 residue and total levels of PAX2 protein were determined, using western blot. GAPDH was used as a control to normalize for protein content. All results are representative or mean value +/-SD of three independent experiments. Different subscript letters and * indicate a statistically significant difference.