Supplementary Data. Supplementary Table S1.

Transcription

1 Supplementary Data Supplementary Table S1. Primer Sequences for Real-Time Reverse Transcription-Polymerase Chain Reaction Gene Forward 5?3 Reverse 3?5 Housekeeping gene 36B4 TCCAGGCTTTGGGCATCA CTTTATCAGCTGCACATCACTCAGA Endothelial cells Flk-1 TCTGTGGTTCTGCGTGGAGA GTATCATTTCCAACCACCCT PECAM GTCATGGCCATGGTCGAGTA CTCCTCGGCGATCTTGCTGAA Tie-2 ATGTGGAAGTCGAGAGGCGAT CGAATAGCCATCCACTATTGTCC vwf AACCTGCCAGAGCCTGTCTA GTTGCAGTCCTGAGAGAGGG Smooth muscle cells Alpha-SMA GAGAAGCCCAGCCAGTCG CTCTTGCTCTGGGCTTCA Cardiomyocytes Nkx2.5 ACCTTTCTCCGATCCATCCCACTT GCGTTAGCGCACTCACTTTAATGG GATA-4 TTCCTTGTCCTCATCACCCACAGA GACAATGTTAACGGGTTGTGGAGG Neurons MAP2 GCATCAACCTGCTGCAATCC GAGAAGTATTCACAAGCCCTGCTT Skeletal muscle cells MyH1 GGTCGAAGTTGCATCCCTAA CACAAACACCGATGACTTGG Myogenin TAGTCTCTTCCTGAAGCCAGTTGC GGTATCATCAGCACAGGAGACCTT Early mesoderm Brachyury GTGACTGCCTACCAGAATGA ATTGTCCGCATAGGTTGGAG ES cells Nanog AACGATATGGTGGCTACTCTC TCGGTTCATCATGGTACAGTC Oct-4 TGTGGACCTCAGGTTGGACT CTTCTGCAGGGCTTTCATGT Transgene expression pcag=p38 GACGGTATCGATAAGCTTGC GTAACGCTCGGGCACCTC SMA, smooth muscle actin; ES, embryonic stem.

2 A Percentage Cardiomyocyte Phenotype D Relative Ratio/ (+/+) d CGR8 +/+ 1 -/- 15 -/- PECAM * B Relative Ratio/ (+/+) d Nkx2.5 d d12 d d12 d d12 E Relative Ratio/ (+/+) d CGR8 +/+ 1 -/- 15 -/- vwf * C Relative Ratio/ (+/+) d GATA-4 d d12 d d12 d d12 * F Percentage Myotube Phenotype G Relative Ratio/ (+/+) d Myogenin d d26 d d26 d d26 SUPPLEMENTARY FIG. S1. p38 = ES cells do not differentiate into cardiac, endothelial cells, or skeletal myotubes. For cardiomyogenesis, EBs from wild-type CGR8 and 1 = and 15 = ES cells were plated on gelatin at day 7 of differentiation and analyzed at day 12. (A) Cardiomyocyte phenotype was expressed as the percentage of EBs with beating cell areas at day 12. Values are expressed as means of at least 3 independent experiments SEM. mrnas at days and 12 were analyzed by real-time RT-PCR for expression of the specific cardiac markers Nkx2.5 (B) or GATA-4 (C). For endothelial cells, EBs from wild-type CGR8 and 1 = and 15 = ES cells were plated on gelatin at day 7 of differentiation. mrnas were extracted at days 3, 5, and 7 and analyzed by real-time RT-PCR for the expression of the endothelial markers PECAM (D) and vwf (E). For skeletal muscle differentiation, EBs from wild-type CGR8 and 1 = and 15 = ES cells were plated on gelatin at day 7, and the ratio of EBs with spontaneous contractile myotubes was evaluated by day 26 for each cell line; at least different EBs per cell line were counted (F). Histogram shows the means SEM of 3 independent experiments. In these experiments, mrnas were extracted at day 26 and analyzed by real-time RT-PCR for expression of the specific skeletal muscle marker Myogenin (G). InB E and G, results are expressed in arbitrary units, with the values of wild-type CGR8 at day taken as 1, and are the means SEM of at least 3 independent experiments. Significance between wild-type and KO cells is given as *P <.5, P <.1, and P <.1. ES, embryonic stem; EB, embryoid body; KO, knock-out; RT-PCR, reverse transcription-polymerase chain reaction; SEM, standard error of the mean.

3 A Percentage Neuronal Phenotype B MAP2 2 C RA RA Neuronal Phenotype D 2 MAP2 Percentage 25 Relative Ratio/ (+/+) d 1 +/+ 1 -/- R /- R15-1 R15-2 d d12 d d12 d d12 d d12 d d12 R15-1 R15-2 SUPPLEMENTARY FIG. S2. Neuronal phenotype of p38mapk KO and reexpressing ES cells. EBs from wild-type CGR8, 1 =, R1-1, 15 =, R15-1, and 2 ES cells were treated (RA) or not with retinoic acid between days 3 and 5, plated on gelatin at day 7 of differentiation, and analyzed at day 12. (A, C) Neuronal phenotype, at day 12, was expressed as the percentage of EBs with neurons in outgrowths (means SEM of at least 3 independent experiments). (B, D) mrnas were extracted at days and 12 and analyzed by real-time RT-PCR for expression of the specific neuronal marker MAP2. Results are expressed in arbitrary units, with the values of wild-type CGR8 at day taken as 1, and are the means SEM of at least 3 independent experiments. Significance between KO and reexpressing cells is given.

4 SUPPLEMENTARY FIG. S3. mrnas from wild-type CGR8, 1 =, R1-1, 15 =, R15-1, and 2 ES cells were extracted at day and analyzed by real-time RT-PCR for expression of the specific markers of pluripotency Nanog (A) and Oct-4 (B). (C) mrnas were extracted at various times, as indicated, during differentiation and analyzed by real-time RT-PCR for the p38mapk transgene expression. In A and B, results are expressed in arbitrary units, with the values of wild-type CGR8 at day taken as 1, and are the means SEM of at least 3 independent experiments. Significance between wild-type and KO cells is given as *P <.5. In C, transgene expression was set as 1 at day for the R15-1 cell line. In D, p38a protein expression at day 2 of differentiation of various ES cell lines was analyzed by western blotting with a-ha, a-phosphomapkap-k2 (a- MK2-p), and a-mapkap-k2 total (a-mk2) antibodies. Membranes were reprobed with a-erk2 antibodies as loading control. a-ha, a-hemaglutinin antigen; a-mapkap-k2, MAPK, mitogen activated protein kinase-activated protein kinase-2; a-erk2, a-extracellular signal-regulated kinase 2.

5 SUPPLEMENTARY FIG. S4. Reexpression of p38 protein in KO cell lines partially rescues their cardiac and endothelial cell defects. EBs from wild-type CGR8, 1 =, R1-1, 15 =, R15-1, and 2 ES cells were plated on gelatin at differentiation day 3 for endothelial differentiation or at differentiation day 7 for cardiomyogenesis and analyzed at day 7 or 12. (A) mrnas at day 12 were analyzed by real-time RT-PCR for expression of the specific cardiac marker Nkx2.5. (B) mrnas at day 7 were analyzed by real-time RT-PCR for expression of the specific Flk-1 endothelial marker. Results are expressed in arbitrary units, with the values of wild-type CGR8 at day (B) or day 12 (A) taken as 1, and are the means SEM of at least 3 independent experiments. Significance between KO and reexpressing cells is given as *P <.5 and P <.1. (C) Differentiated cells at day 12 were analyzed by flow cytometry analysis of Troponin T. A histogram representative of 3 independent experiments is shown. For each experiment, control value (irrelevant) was determined as no more than 5% of positivity. (D) Differentiated cells at day 12 were analyzed by immunofluorescence using anti-troponin T antibodies (magnification: ). Nuclei were stained with 4,6-diamidino-2-phenylindole.

6 SUPPLEMENTARY FIG. S5. Reexpression of p38 protein in KO cell lines partially rescues their skeletal muscle cell defects. EBs from wild-type CGR8, 1 =, R1-1, 15 =, R15-1, and R15-2 ES cells were plated on gelatin at day 7 and analyzed at day 26. mrnas were analyzed by real-time RT-PCR for expression of the specific skeletal muscle marker Myogenin (A). Results are expressed in arbitrary units, with the values of wild-type CGR8 at day 26 taken as 1, and are the means SEM of at least 3 independent experiments. Significance between KO and reexpressing cells is given as P <.1. (B) Differentiated cells at day 12 were analyzed by flow cytometry analysis of Myogenin. A histogram representative of 3 independent experiments is shown. For each experiment, control value (irrelevant) was determined as no more than 5% of positivity. (C) Differentiated cells at day 12 were analyzed by immunofluorescence using anti-mhc antibodies (magnification: ). Nuclei were stained with 4,6-diamidino-2-phenylindole. wt, wild-type.

7 A Relative Ratio/ Control d12 Relative Ratio/ Control d12 B Nkx2.5 Control d7-d12 GATA-4 Control d7-d12 C %/ Control d7 D %/ Control d7 2 2 Control d7-d Control d7-d12 Flk-1 Alpha-SMA SUPPLEMENTARY FIG. S6. EBs from wild-type CGR8 ES cells were differentiated in the presence or absence of PD for different periods as indicated. For cardiomyogenesis, EBs were plated at day 7 and analyzed at day 12 of differentiation. mrnas were extracted at day 12 and analyzed by real-time RT-PCR for expression of the specific cardiac markers Nkx2.5 (A) and GATA-4 (B). Results are expressed in arbitrary units, with the values of control cells at day 12 taken as 1, and are the means SEM of at least 3 independent experiments. For endothelial and smooth muscle cell formation, EBs were plated at day 3 and mrnas were analyzed at days, 3, 5, and 7 of differentiation by real-time RT-PCR for expression of the specific endothelial marker Flk-1 (C) and the smooth muscle marker a-sma (D). Results are expressed as the percentage of the expression level in control cells at day 7 and are the means SEM of at least 3 independent experiments. Significance between control and treated cells is given. a-sma, a-smooth muscle actin.

8 A B Relative Ratio/ Control d Nkx2.5 Control d-d3 d3-d5 d-d12 Flk-1 C %/ Control d7 Relative Ratio/ Control d26 Control d-d3 d3-d5 d-d Myogenin Control d-d3 d3-d5 d-d26 SUPPLEMENTARY FIG. S7. EBs from wild-type CGR8 ES cells were differentiated in the presence or absence of PD for different periods as indicated schematically in Fig. 6A. For cardiomyogenesis, EBs were plated at day 7 and analyzed at day 12 of differentiation. mrnas were extracted at day 12 and analyzed by real-time RT-PCR for expression of the specific cardiac marker Nkx2.5 (A). Results are expressed in arbitrary units, with the values of control cells at day 12 taken as 1, and are the means SEM of at least 3 independent experiments. Significance between control and treated cells is given. For endothelial cell formation, EBs were plated at day 3 and mrnas were analyzed at days, 3, 5, and 7 of differentiation by real-time RT-PCR for expression of the specific endothelial marker Flk-1 (B). Results are expressed as the percentage of the expression level in control cells at day 7 and are the means SEM of at least 3 independent experiments. Significance between control and treated cells is given. For skeletal myogenesis, EBs were plated at day 7 and analyzed at day 26 of differentiation. mrnas were extracted at day 26 and analyzed by real-time RT-PCR for expression of the specific skeletal muscle marker Myogenin (C). Results are expressed in arbitrary units, with the values of wild-type CGR8 at day 26 taken as 1, and are the means SEM of at least 3 independent experiments. Significance between control and treated cells is given as P <.1, and P <.1.