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.