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. 2009 May 28;4(5):e5698. doi: 10.1371/journal.pone.0005698

Figure 2. Down-regulation of NKX2-5 alters expression of cardiac-associated genes.

Figure 2

(A) Cardiac myocytes and fibroblasts isolated from human fetus at 20-22 weeks of age and cultured for 5 days were immunostained with anti-MyHC antibody (MF-20) to determine the percentage of myocytes in the preparations. The isolated myocyte culture contained >90% MyHC+ cells while the fibroblasts revealed <5% MyHC+ cells. (B) The graph summarizes the western blot analysis after treatment with AdGFP and AdXKN (antisense Nkx2-5 RNA) at 8 MOI, and immunoblotting with NKX2-5, GATA4, β-catenin, MEF2C, PCNA, and GAPDH antibodies. The level of Nkx2.5 was reduced to less than 5% in antisense RNA-treated cells (n = 3, P = 0.002). Similarly, GATA4 and MEF2C were downregulated significantly while no difference was detected in the level of PCNA. β-catenin was upregulated in the myocytes treated with AdXKN (n = 3, P = 0.016). (C) Western blot analysis of 20-wk old cardiac myocytes treated with increasing concentration of AdXKN showed changes in GATA4 and β-catenin that corresponded with the level of NKX2-5. Lane 1 represents cells treated with AdGFP control at 8 MOI. (D) Immunostaining of cardiomyocytes with MF20 (myosin heavy chain) revealed a reduction in the amount of myosin heavy chain (indicated by arrows) and with a punctate pattern of staining in cells exposed to antisense NKX2-5 for 48 hrs (AdXKN) compared to control cells (AdGFP).