DEV02305 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Mutation of the tyrosine-rich domain (YRD) does not affect the ability of Nkx2-5 to activate the Nppa promoter in C3H 10T¹/₂ cell in vitro. Nkx2-5Y-A was unable to activate/repress the Nppa-Luc reporter gene ANF700-Luc (Stennard et al., 2003) to the same degree as wild type in conjunction with cardiac transcription factors: (A) Gata4 and/or Tbx20b short isoform lacking the potent transrepression domain (Stennard et al., 2003); (B) Tbx5 and/or Tbx2; (C) myocardin and/or serum response factor (SRF) transfected SRF represses the activity of myocardin (Wang et al., 2001); and (D) SRF alone.

  Additional reference

  Wang, D., Chang, P. S., Wang, Z., Sutherland, L., Richardson, J. A., Small, E., Krieg, P. A. and Olson, E.N. (2001). Activation of cardiac gene expression by myocardin, a transcriptional cofactor for serum response factor. Cell 105, 851-862.

• Supplemental Figure 2 -

  Fig. S2. Validation of GFP, lacZ and Nkx2-5 expression from targeted Nkx2-5 alleles in ES cell-derived embryoid bodies (EBs). (A-D) EBs of indicated genotypes after plating on collagen-coated slides showing GFP fluorescence (green) and nuclear fluorescence (H33342; blue) (A,B) or staining for lacZ (C,D). GFP fluorescence and lacZ staining were coincident with each other and with areas that underwent spontaneous beating (data not shown). (E-J) EBs grown for 10 days in rolling culture bottles were fixed and adjacent sections were immunostained for Nkx2-5 and cardiac muscle-specific a-actin antibodies. Nuclear staining for Nkx2-5 was detected in regions expressing a-actin in Nkx2-5^GFP/+ and Nkx2-5^GFP/Y-A EBs. No staining was detected in regions over actin-positive regions in Nkx2-5^GFP/lacZ (null) EBs demonstrating the specificity of the antibody. Although not quantitative, these data show stable expression of Nkx2-5Y-A in EB-derived cardiomyocytes, supporting previous experiments in transfected COS cells and C3H 10T¹/₂ fibroblasts (Figs 1F, 3B).

• Supplemental Figure 3 -

  Fig. S3. Amino acid comparison showing a YRD-like element in the Drosophila bagpipe gene family. Tyrosines and phenylalanines are highlighted in red; amino acids represented at least four times in this grouping are shaded in green. Alignment generated by the Clustal algorithm in the MacVector software suite.