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. 2013 Mar 12;22(15):2211–2220. doi: 10.1089/scd.2012.0611

FIG. 3.

FIG. 3.

Hoxa10 inhibits cardiogenesis. (A) Percentage of EB day 4, Flk-1-PDGFRα double-positive cardiac progenitor cells are significantly decreased following Hoxa10 overexpression for 48 h (day 2–4) of EB formation; 15%±9% versus 43.6%±7.9%, *P<0.05, n=8. (B) Reduction of beating in EBs overexpressing Hoxa10 following induction with doxycycline (Dox) on days 3–6 versus control EBs plus doycycline; 12.5±10.3 versus 78.4±21.7, *P<0.05, n=8. (C) Western analysis reveals absence of cTroponin I in day 12 EBs overexpressing Hoxa10 following the induction with doxycycline (Dox) on days 3–6 versus day 12 control EBs treated with doxycycline (Dox) on days 3–6). Western analysis reveals absence of cTroponin I (D) and cTroponin T (E) in day 12 Hoxa10 doxycycline-inducible EBs following the induction with doxycycline (Dox) on days 3–6 versus no doxycycline. (F) Protein expression of Connexin 43 is preserved in day 12 EBs following Hoxa10 induction. (G) The Nkx2-5-GFP reporter ES cell line was infected with Hoxa10 or empty lentivirus. Overexpression of Hoxa10 for 24 h (day 3–4 of EB formation) in cardiac crescent equivalent progenitors results in a reduction of total Flk-1/PDGFRα double-positive cardiac progenitor cells; 35.5%±7% versus 48.6%±7%; *P<0.05, n=5. (H) Percentage of Nkx2-5 expressing GFP-positive cells double-positive for Flk-1 and PDGFRα is also significantly decreased with Hoxa10 overexpression; 49%±10% versus 71%±5%; *P<0.05, n=5. (I) qRT-PCR analyses revealed an increase in Brachyury expression and reduction in the cardiac transcription factors Nkx2-5, Gata-4, Tbx-5, and Isl-1 as well as a small decrease in Aldh1a2 in GFP-positive cells collected from day 4 Nkx2-5-GFP EBs following the induction of Hoxa10 with doxycycline (Dox) for 24 h (day 3–4); *P<0.05, n=3. Hes1 expression, however, was not significantly altered. (J) The Nppa promoter was fused to the luciferase (luc) reporter gene and was transfected into Cos7 cells with and without addition of the Nkx2-5, Gata4, or Hoxa10 expression plasmids. Empty pGL3 and Nppa promoter-driven luciferase expression is shown. Nppa promoter revealed a 10.65±0.44-fold activation with Nkx2-5, which was reduced to 7.41±0.50 with addition of Hoxa10 and a 20.55±0.49-fold activation with Nkx2-5 and Gata4, which was reduced to 12.62±0.17 with the addition of Hoxa10; *P<0.05, n=3.