JCS028282 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Schematic of experimental procedures. Undifferentiated mouse ESCs were cultured for 3 days on collagen IV-coated dishes with 10% FCS to induce VEGFR2⁺ cells, which were purified by cell sorting using anti-VEGFR2 antibody. After an additional 3 days of culture of VEGFR2⁺ cells with 10% FCS and VEGF, we obtained PECAM1⁺ CD34⁺ sheets of endothelial cells and SMA⁺ mural cells. MESECs were obtained by cell sorting using anti-CD34 antibody, and were used to study the effects of TGFβ signals and Snail.

• Supplemental Figure S2 -

  Fig. S2. Effects of TGFβ isoforms on MESECs. MESECs were cultured in the presence of 10% FCS and VEGF (A) in combination with TGFβ1 (B), TGFβ2 (C) or TGFβ3 (D), followed by PECAM1 (red) and SMA (green) immunofluorescence staining. Scale bars: 100 µm.

• Supplemental Figure S3 -

  Fig. S3. Effect of tetracycline (Tc)-regulated Snail expression on undifferentiated ESCs. (A) Undifferentiated ESCs carrying a Tc-regulated transgene encoding FLAG-epitope-tagged mouse Snail (Tc-Snail) or control transgene (Tc-Empty) were cultured with leukemia inhibitory factor (LIF) in the presence (+) or absence (−) of Tc for 4 days, followed by observation of their morphology. (B) Protein levels of E-cadherin (top) and α-tubulin (bottom) were examined by immunoblotting of total lysates of the ESCs described above.

• Supplemental Figure S4 -

  Fig. S4. Effect of TβR-I inhibitor on the EndMT induced by tetracycline (Tc)-regulated Snail expression in MESECs. (A) MESECs derived from ESCs carrying a Tc-regulated transgene encoding Snail (Tc-Snail) were cultured in the presence (+) or absence (−) of Tc in combination with LY364947 (TβR-I inhibitor), and subjected to immunofluorescence staining for PECAM1 (red) and SMA (green). (B) Quantitative analysis of the effects of Snail on colony formation from single MESECs, performed as described in Fig. 1F. Briefly, MESECs derived from Tc-Empty or Tc-Snail ESCs were cultured sparsely with 10% FCS in the absence (−) or presence (+) of Tc in combination with TβR-I inhibitor for 4 days, followed by staining of colonies for PECAM1 and SMA. E, pure endothelial colony; M, mural-containing colony.

• Supplemental Figure S5 -

  Fig. S5. Effect of tetracycline (Tc)-regulated Snail expression on MESECs in the absence of Smad4-dependent signals. MESECs were sorted from the vascular cells derived from ESCs carrying a Tc-regulated transgene encoding FLAG-epitope-tagged mouse Snail (Tc-Snail), transfected with siRNA for Smad4 or with scrambled sequence as a negative control (NTC), and cultured in the presence (+) or absence (−) of Tc. (A) MESECs described above were subjected to immunofluorescence staining for PECAM1 (red) and SMA (green). (B,C) Levels of expression of claudin 5 (B) and SMA (C) in MESECs described above were analyzed by quantitative real-time RT-PCR. Error bars indicate s.d.

• Supplemental Figure S6 -

  Fig. S6. Binding of Smad3 and FLAG-Snail to the endogenous SMA and claudin 5 promoters. Tc-Snail ESC-derived endothelial cells were cultured in the presence of Tc, and treated with or without TGFβ2 for 3 hours (A), or cultured in the absence of Tc (B). Chromatin samples prepared from these cells were subjected to ChIP analysis. PCR was carried out to detect the SMA promoter. Input lanes show amplification of total input DNA. Control (C) lanes show PCR amplification of samples precipitated with control IgG antibodies. Smad3 and M2 lanes show amplification of target sequences within the immunoprecipitates using antibodies for Smad3 and FLAG (M2), respectively.

• Supplemental Figure S7 -

  Fig. S7. Effect of BMP7 on the TGFβ-mediated alteration of endothelial and mural marker expression in MESECs. Levels of mRNA expression of claudin 5 (A) and SMA (B) in MESECs cultured in the absence (−) or presence (Tβ) of TGFβ2 in combination with BMP7 were analyzed by quantitative real-time RT-PCR. Error bars indicate s.d.

• Supplemental Table S1 - Adobe PDF