Fig. 5. Inhibition of endocardial differentiation in GATA5 antisense transfectants. (A) Schematic representation of the GATA5 antisense construct. The cDNA fragment spanned from 850 bp to 1200 bp where 1 is the A of the initiating methionine. (B) RT-PCR analysis was conducted on RNA isolated from two different TC13 clones expressing antisense GATA5 (AS5) as well as TC13 cells transfected with the control pCDNA3 vector. The GATA5 primers used detect only the endogenous transcripts. Note how the antisense inhibits accumulation of GATA5 transcripts following RA treatment. (C) Western blot analysis confirms that antisense clones do not express GATA5 protein. GATA4 levels are only slightly decreased in the GATA5 antisense but are further decreased in response to RA, as in control cells. 20 µg of nuclear extracts were resolved on a 15% SDS-PAGE and blotted on a nylon membrane with the respective antibodies. (D) GATA5-deficient TC13 cells do not elongate and remain negative for Von Willebrand factor staining even after 5 days of treatment with RA. No arrest in cell proliferation was observed in these cells compared to control cells. The data shown are from two independent clones, AS5(1) and AS5(2). (E) RT-PCR analyses show that terminal endocardial markers (EPAS-1, ET-1, Tn-X, ErbB3) are not induced in the antisense GATA5 transfectants after 4 days of RA treatment. (F) Gel shift analysis using extracts from RA-treated GATA5 antisense transfectants (AS) or RA-treated control (Ctl) in presence of cyclosporine A (CsA). NF-AT binding is decreased in AS- and in CsA-treated cells. Note that in both cases, the decrease is greater for NF-ATc than NF-ATp. Binding was competed by 100x self probe (S) but not a mutant probe (M).