Figure 8.

5T4−/− ES cells exhibit altered phenotype after treatment with DECMA-1 antibody and decreased motility after differentiation. (a) E14 and 5T4−/− ES cells were cultured on gelatin-treated plates in the presence of LIF and an excess (23.2 μg/ml IgG component) of either (i) control antibody (cAb) or (ii) E-cadherin neutralizing antibody DECMA-1 (nAb) and colony phenotype assessed after 24 h using phase contrast microscopy. (iii) Enlarged image of the marked areas in panel ii. Note that the cellular phenotype of 5T4−/− ES cells is altered compared with wild-type ES cells. (b) 5T4−/− ES cells cultured for 24 h in FCS+LIF and neutralizing antibody DECMA-1 (nAb) and assessed for DAPI, OCT-4, and E-cadherin protein expression by immunofluorescent microscopy. Bar, 5 μm. (c) Analysis of EMT-associated transcripts was determined by RT-PCR in 5T4−/− ES cells treated with either (i) control (cAb) or (ii) DECMA-1 (nAb) antibody for 72 h (C, control; β-tub, β-tubulin). (d) Cellular motility of undifferentiated and differentiating (3 d in the absence of LIF) wild-type and 5T4−/− ES cells was assessed using Costar Transwell 5-μm pore size plates. Data represents the fold change in motility compared with undifferentiated cells. Note that 5T4−/− cells cultured in the absence of LIF exhibit decreased motility compared with control cells. (e) (i) Phase-contrast microscopy images showing wild-type (wt) and 5T4−/− ES cell colony morphology 24 h after induction of differentiation. (ii) Enlarged images of the marked areas shown in panel i. (f) Cellular motility of undifferentiated and differentiating (3 d in the absence of LIF) wild-type (D3) and N-cadherin−/− ES cells was assessed using Costar Transwell 5-μm pore size plates. Data represents the fold change in motility compared with undifferentiated cells. Note that Ncad−/− cells cultured in the absence of LIF exhibit decreased motility compared with control cells.