LEGENDS TO SUPPLEMENTARY DATA
Supplementary Data S1: Effects of anti-p53 siRNAs or homozygote gene knock-out
of p53 on ESC cardiomyogenesis and endothelial differentiation
Undifferentiated CGR8 ESCs were transfected overnight with control siRNAs (si Ct) or
anti-p53 siRNAs (si p53). The day after, day0, these cells and p53-/- ESCs (p53-/-) and their
wild-type counterparts (p53+/+) were induced to differentiate, cultivated until day7 and
replated on Petri dishes until day12 for analysis of cardiomyogenesis (A) or endothelial
differentiation (B, C). mRNAs were extracted and analyzed by real time RT-PCR for Nkx2.5
(A), Tie2 (B) and VEGF Receptor 1 (Flk1) (C) gene expression. Results are expressed in
arbitrary units, with the values of untransfected CGR8 control cells or wild type control ESCs
taken as 1, and are the means ± S.E.M. of at least 3 independent experiments. Significance to
Ct values is given as: *p < 0.05, **p < 0.01, ***p < 0.001.
Supplementary Data S2: Oct4 and TBX1 gene regulation in p53-/- ESCs
p53-/- ESCs (p53-/-) and their wild-type counterparts (p53+/+) were induced to
differentiate and mRNAs were extracted at various time from day0 to day12, as indicated, and
analyzed by real time RT-PCR for the expression of TBX1 (A) or Oct4 (B) gene expression.
Results are expressed in arbitrary units, with the values of wild type control ESCs at day0
taken as 1, and are the means ± S.E.M. of at least 3 independent experiments. Significance to
wild type control ESC values is given.
Supplementary Data S3: Effects of anti-Nanog transfections in p53-/- ESCs
Undifferentiated p53-/- ESCs were transfected overnight with control siRNAs (si Ct) or
anti-Nanog siRNAs (si Nanog) and induced to differentiate until either day7 (A and B) or
day12 (C). mRNAs were extracted at various time, as indicated and analyzed by real time RTPCR for Pax6 (A), Bcl2 (B), and Nkx2.5 (C). Results are expressed in arbitrary units, with
1
the values of control cells taken as 1, and are the means ± S.E.M. of at least 3 independent
experiments.
2
Gene
Supplementary data Table 1: sequences of the primers used in RT-qPCR
Forward
Reverse
Housekeeping gene
36B4
TCCAGGCTTTGGGCATCA
CTTTATCAGCTGCACATCACTCAGA
Endothelial markers
FLK-1
TCTGTGGTTCTGCGTGGAGA
GTATCATTTCCAACCACCCT
Tie2
ATGTGGAAGTCGAGAGGCGAT
CGAATAGCCATCCACTATTGTCC
Smooth muscle markers
α-SMA
GAGAAGCCCAGCCAGTCG
CTCTTGCTCTGGGCTTCA
Cardiomyocytes markers
NKX2.5
ACCTTTCTCCGATCCATCCCACTT
GCGTTAGCGCACTCACTTTAATGG
Troponin T2
GCGGAAGAGTGGGAAGAGACAGAC
GCACGGGGCAAGGACACAAG
GCATCAACCTGCTGCAATCC
GAGAAGTATTCACAAGCCCTGCTT
Neural markers
MAP2
Myogenesis markers
Myh1
GGTCGAAGTTGCATCCCTAA
CACAAACACCGATGACTTGG
Adipogenic markers
aP2
TTCGATGAAATCACCGCAGA
GGTCGACTTTCCATCCCACT
PPARγ
CTGTTTTATGCTGTTATGGGTGAAA
GCACCATGCTCTGGGTCAA
Nanog
AACGATATGGTGGCTACTCTC
TCGGTTCATCATGGTACAGTC
Oct-4
TGTGGACCTCAGGTTGGACT
CTTCTGCAGGGCTTTCATGT
ESCs and iPSCs
Mesoderm comittment
Brachyury
GTGACTGCCTACCAGAATGA
ATTGTCCGCATAGGTTGGAG
Tbox-1
CTGTGGGACGAGTTCAATCAG
TTGTCATCTACGGGCACAAAG
Mesp-1
TGTACGCAGAAACAGCATCC
TTGTCCCCTCCACTCTTCAG
Ectoderm comittment
Pax-6
TGCCCTTCCATCTTTGCTTG
TCTGCCCGTTCAACATCCTTAG
Mouse p53 and p53 targets
3
p53
AGAGACCGCCGTACAGAAGA
GCATGGGCATCCTTTAACTC
P21
CCTGGTGATGTCCGACCTG
CCATGAGCGCATCGCAATC
BCL2
ACAGTGGCAAGTGTCTTAGAAAG
CTCCTCACCGGGTTTGGAAC
4