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Additional Supporting Information may be found in the online version of this article.

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STEM_723_sm_SuppFig1.tif1626KSupplementary Fig. 1. TAp63 is expressed during ES cardiogenesis (A) Real-time RTPCR analysis for TAp63 andδNp63 expression at the indicated time points of ES cell differentiation. The value for each gene was normalized to undifferentiated wt ES cultures at day 0 and the graph represents the average of three independent experiments ± sd. (B) Validation of siRNAs on epithelial cells. Hela cells were transiently transfected with a TAp63 orδNp63 expressing construct along with a control siRNA (si ctrl) or siRNAs specific for total p63,δNp63 or TAp63 (two different siRNAs tested) and analyzed 48 h later by Western blot with anti-p63 and anti-tubulin (as loading control) antibodies. The TAp63 siRNA did not modify the expression ofδNp63 and vice versa (not shown). (C) Real-time RT-PCR analysis for Islet1, Nkx2.5 and Tbx5 cardiac-relateds genes, and MLC2v and α-actinin and TnT2 sarcomeric genes expressed at differentiation days 5 or 12 of ES cells treated with si ctrl, siTAp63 or sip63 specific siRNA. The value for each gene was normalized to si ctrl transfected ES cultures at the indicated time points and the graph represents the average of three independent experiments ± sd.
STEM_723_sm_SuppFig2.tif2952KSupplementary Fig. 2. (A) Left: co-immunofluorescence detection at day 11 of Nkx2.5 with p63 during differentiation of wt ES cells. No single cell displays both proteins. Scale bar = 50 μm. Right: staining of E12.5 wt embryo with p63 antibody. The embryonic heart is devoted of p63 nuclear staining (the cytoplasmic staining in the atrium is non specific; yellow arrow) while the developing epidermis (insert; blue arrow) is strongly positive for p63 in the nucleus. Scale bars = 200 μm. (B) Detection of p63 trancripts by in situ hybridization with pan-p63 antisens probe on E8.5 wt embryos (specific staining highlighted by white arrowheads). No signal was detected withδNp63-specific probe.
STEM_723_sm_SuppFig3.tif2220KSupplementary Fig. 3. (A) Detection of p63 and sox-17 proteins by immunofluorescence staining in the endodermal END2 cell line. Dapi is shown in blue. (B) Detection of TAp63 gamma expression in END2 endodermal cells by Real-time RT-PCR (normal thymic cells and undifferentiated ES cells as positive and negative controls, respectively) and (C) by Western blot analysis with anti-p63 and anti-tubulin (as loading control) antibodies. Extracts from Hela cells transfected with the different p63 isoforms were loaded as size controls. (D) Real-time RT-PCR analysis for Sox17 at days 3 and 9 of sh-ctrl and sh-p63 ES cell differentiation, in the presence of BMP-2. The values were normalized to undifferentiated sh-ctrl ES cultures and represents the average of three independent experiments ± sd. *: p<0.02, **: p<0.1.
STEM_723_sm_SuppFig4.tif719KSupplementary Figure 4. Real-time RT-PCR analysis for TAp63,δNp63 and total p63 gene expression at day 6 of differentiation of wt and sh-p63 ES cells treated or not with 5ng/ml of Activin A at day 1 before differentiation. The value for each gene was normalized to undifferentiated wt ES cultures and represents the average of three independent experiments ± sd.
STEM_723_sm_SuppTable1.pdf12KSupplementary Table 1.

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