An EMT event is required for Bra::GFP expression. (A) Expression of Bra::GFP in mESCs subject to (a) Act, (b) Chi and (c) Act/Chi, in the presence of CsA (3 μm; grey) or vehicle (DMSO; white) analysed by FACS. Error bars show standard deviation, n = 3. (B,B',B") E14-Tg2A mESCs subject to Chi in the presence of CsA (3 μm) or DMSO, stained for Hoechst, E-cadherin, β-catenin and Bra. Scale bars denote 50 μm. In the presence of CsA, E-cadherin is not effectively cleared from the membrane, β-catenin does not enter the nucleus and there is no effective expression of Bra. (C) Stills from live imaging of E14-Tg2A mESCs in Chi with DMSO or CsA. Notice that in CsA, cells stretch out filopodia but do not undergo an EMT. Scale bars denote 50 μm. (D) Cell velocities (μm/h) measured from multiple films as in (C). Tracking for Act + DMSO ceased after 48 h due to loss of focus. Refer to Figure 4 for comparison. Analysis of the average instant velocities shows that CsA reduces the movement of the cells. (E) Distribution of instant velocities. AC, activin A + chiron; Act, activin A; β-cat, β-catenin; Bra, brachyury; Chi, chiron CHIR99021; CsA, cyclosporine A; DMSO, dimethyl sulfoxide; E-Cad, E-cadherin; EMT, epithelial-to-mesenchymal transition; GFP, green fluorescent protein; Vel. velocity.