Fig. 6. GATA5 and NF-ATc collaborate in endocardial differentiation. (A) Blocking NF-ATc activation using cyclosporine A (CsA) inhibits TC13 differentiation and reduces GATA5 induction in response to retinoic acid (RA). Immunocytochemistry using the GATA5 antibody was carried out as described in Materials and Methods. Note how cells treated with CsA do not elongate or align (arrowheads) and show reduced but persistant staining for GATA5 (red nuclei) suggesting that GATA5 induction precedes NF-ATc activation in endocardial differentiation. (B) GATA5 and NF-ATc synergistically activate the endothelin 1 (ET-1) promoter. (Top) A schematic representation of the ET-1 promoter showing the close proximity of the NF-ATc and GATA binding sites. (Bottom) A 1.4 kbp endothelial-specific ET-1 promoter is transcriptionally activated by GATA5 and NF-ATc as evidenced in cotransfection with increasing amounts (0.25, 50, 100, 250, 500 ng) of either GATA5 or NF-ATc expression vectors in TC13 cells (left panel). At limiting amount of expression vectors (25 ng) GATA5 and NF-ATc synergistically activate the ET-1 promoter (right panel). The empty pCDNA3 and pCD-SR backbone vectors of GATA5 and NF-ATc, respectively, are used as controls. The results are expressed as fold activation and represent the mean of three independent experiments, each done in duplicate.