Nuclei were counterstained with 4��, 6-diamidino-2-phenylindole (DAPI), and specimens were visualized using an Axioplan2 fluorescence microscope (Carl Zeiss MicroImaging, Thornwood, NY). In addition, adult C57BL/6 mice were euthanized according to IACUC-approved protocols and urinary bladders were isolated, fixed in neutral-buffered selleck formalin for 2 h, dehydrated in graded alcohols, paraffin-embedded and cut into 5 ��m thick sections. Sections were analyzed for urothelial and epithelial markers as described above. Flow cytometry Following 14 d of culture, RA-stimulated and spontaneously differentiating UP2-GFP ESCs were dissociated into a single cell suspension and quantified for GFP fluorescence on a FACScaliber flow cytometer (BD Biosciences, Franklin Lakes, NJ).

Untransfected ESCs either maintained as controls or differentiated similarly were analyzed in parallel in order to control for background fluorescence. CellQuest 3.0 software (BD Biosciences) was used to acquire and analyze FACScan data. In some experiments, GFP+ populations were sorted and collected from RA-treated UP2-GFP ESCs utilizing a MoFlo high-speed sorter (Dako-Cytomation, Carpinteria, CA) and subjected to further analysis detailed below. Protein Analysis Whole cell lysates were retrieved in 20 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1% Triton X-100 supplemented with protease and phosphatase inhibitors. Nuclear extracts were obtained according to methods reported in the literature [37]. Immunoblot analysis was performed as described previously [38].

Primary antibodies included: anti-p63 and GATA4/6 as described above. Electromobility shift analysis Electromobility shift analyses (EMSAs) for GATA-4 and GATA-6 binding to murine uroplakins 1B and 2 promoter sequences were carried out as using methods previously described [39]. Briefly, 3�C5 ��g of nuclear extract and 0.5 ng of 32P-labeled oligonucleotide probe were combined in a reaction mix (20 ��l reaction volume) containing 25 mM Tris-HCl, pH 8, 50 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 8% glycerol, 2 ��g of BSA, and 0.5 ��g of poly(dI-dC). The reaction was incubated for 20 min at room temperature, and the DNA-protein complexes were resolved on 5% polyacrylamide gels in 0.5X Tris-borate-EDTA (TBE) buffer at 4��C. The gels were dried, and the complexes were visualized by autoradiography using a Typhoon Trio variable mode phosphorimager (GE Healthcare Biosciences, Piscataway, NJ).

For competition experiments, a 25-fold molar excess of the cold competitor oligonucleotide was added simultaneously with the probe. For supershift experiments, a GATA4 or GATA6 antibody (as described above) was preincubated on ice for 1 h with nuclear extract followed by addition of the other components for 20 min at Cilengitide room temperature. Species-matched IgG antibodies were used in parallel as additional controls.