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First published online 16 September 2008
doi: 10.1242/jcs.028282

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Snail is required for TGFβ-induced endothelial-mesenchymal transition of embryonic stem cell-derived endothelial cells

Department of Molecular Pathology, Graduate School of Medicine and the Global Center of Excellence Program for `Integrative Life Science Based on the Study of Biosignaling Mechanisms', The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

View larger version (86K): [in this window] [in a new window]   Fig. 1. Effects of TGFβ family members on mouse ESC-derived endothelial cells (MESECs). (A-E) MESECs were obtained by CD34-sorting of vascular (endothelial and mural) cells derived from mouse ESCs, and cultured in the presence of 10% fetal calf serum (FCS) and VEGF (A), followed by immunofluorescence staining for PECAM1 (red) and SMA (green). MESECs were also treated with BMP4 (B), TGFβ2 (C), activin (D) and TβR-I inhibitor (E). Scale bars: 100 µm. (F) Quantitative analysis of the effects of TGFβ signals on colony formation from single MESECs. MESECs were cultured at low density with 10% FCS and VEGF in the absence (–) or presence of TGFβ2 (Tβ) or TβR-I inhibitor (inhib) for 4 days, and then stained for PECAM1 and SMA. The number of colonies per well was counted to assess the effect of TGFβ signals on colony formation of MESECs. Two colony types were observed: those consisting of pure endothelial cells (E) and those also containing mural cells (M). Experiments were repeated at least three times with essentially the same results.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Effect of TGFβ signals on expression of endothelial and mural markers in MESECs. (A-D) Levels of mRNA expression for claudin 5 (A), SMA (B), SM22 (C) and calponin (D) in MESECs cultured in the absence (–) or presence of TGFβ2 (Tβ) or TβR-I inhibitor (inhib) as analyzed by quantitative real-time RT-PCR. Error bars indicate s.d. (E) Protein levels of claudin 5 (top), SMA (middle) and -tubulin (bottom) were examined by immunoblotting of total lysates of the MESECs described in A-D.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Effect of TGFβ signals on Snail expression in MESECs. (A) Levels of expression of Snail in MESECs cultured in the absence (–) or presence of TGFβ2 (Tβ) or TβR-I inhibitor (inhib) as analyzed by quantitative real-time RT-PCR. Error bars indicate s.d. (B) Protein levels of Snail (top) and -tubulin (bottom) were examined by immunoblotting of total lysates of the HUVECs cultured in the absence or presence of TGFβ2 or TβR-I inhibitor.

View larger version (83K): [in this window] [in a new window]   Fig. 4. Effect of tetracycline (Tc)-regulated Snail expression on MESECs. (A) MESECs were sorted from the vascular cells derived from ESCs carrying a Tc-regulated transgene encoding FLAG-epitope-tagged mouse Snail (Tc-Snail) or control transgene (Tc-Empty), and cultured in the absence (–) or presence (+) of Tc. Expression of FLAG-Snail (top and bottom rows, green) was examined, in addition to nuclear staining (bottom row, red). (B) MESECs in A were subject to immunofluorescence staining for PECAM1 (red) and SMA (green). (C) 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 at low density with 10% FCS in the absence (–) or presence (+) of Tc for 4 days, followed by staining of colonies for PECAM1 and SMA. E, pure endothelial colony; M, mural-containing colony. Scale bars: 100 µm.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Effect of Snail on expression of endothelial and mural markers in MESECs. (A-D) Levels of expression of claudin 5 (A), SMA (B), SM22 (C) and calponin (D) in MESECs derived from Tc-Empty or Tc-Snail ESCs cultured in the absence (–) or presence (+) of Tc were analyzed by quantitative real-time RT-PCR. Error bars indicate s.d. (E) Protein levels of claudin 5 (top), SMA (middle) and -tubulin (bottom) were examined by immunoblotting of total lysates of the MESECs described in A-D.

View larger version (62K): [in this window] [in a new window]   Fig. 6. Effect of Snail knockdown on MESECs. MESECs were sorted from the vascular cells derived from ESCs, transfected with Snail siRNA or with scrambled sequence as a negative control (NTC), and cultured in the absence (–) or presence of TGFβ2 (Tβ). (A) The levels of endogenous expression of Snail in the MESECs were analyzed by quantitative real-time RT-PCR. Error bars indicate s.d. Black and gray bars, represent +TGFβ2 and –TGFβ2, respectively. (B) The MESECs were subjected to immunofluorescence staining for PECAM1 (red) and SMA (green). (C) Quantitative analysis of the effects of Snail on colony formation from single MESECs, performed as described in Fig. 1F. Briefly, MESECs transfected with Snail siRNA or scrambled sequence were cultured at low density with 10% FCS in the absence (–) or presence of TGFβ2 (Tβ) for 4 days, followed by staining of colonies for PECAM1 and SMA. E, pure endothelial colony; M, mural-containing colony. Scale bars: 100 µm.

View larger version (23K): [in this window] [in a new window]   Fig. 7. Effect of Snail on expression of endothelial and mural markers in MESECs. (A-D) Levels of expression of claudin 5 (A), SMA (B), SM22 (C) and calponin (D) in MESECs transfected with Snail siRNA or scrambled sequence as a negative control (NTC), and cultured in the absence (–) or presence of TGFβ2 (Tβ) were analyzed by quantitative real-time RT-PCR. Error bars indicate s.d. (E) Protein levels of claudin 5 (top), SMA (middle) and -tubulin (bottom) were examined by immunoblotting of total lysates of the MESECs described in A-D.

View larger version (58K): [in this window] [in a new window]   Fig. 8. Effect of Smad4 knockdown on TGFβ-induced EndMT of MESECs. MESECs were sorted from the vascular cells derived from ESCs, transfected with Smad4 siRNA or with scrambled sequence as a negative control (NTC), and cultured in the absence (–) or presence of TGFβ2 (Tβ). (A) Levels of endogenous expression of Smad4 in the MESECs were analyzed by immunoblotting. (B,C,E,F) Levels of expression of PAI1 (B), Snail (C), claudin 5 (E) and SMA (F) in the MESECs were analyzed by quantitative real-time RT-PCR. Error bars indicate s.d. (D) The MESECs were subject to immunofluorescence staining for PECAM1 (red) and SMA (green).

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