Figure 5.

E-Cadherin Expression Partially Blocks ESC Differentiation

(A) Schematic of ZHBTc4-derived inducible E-cadherin-GFP (ECG) rescue ESC line. Randomly integrated FKBP-E-cadherin-GFP was constitutively expressed from the CAG promoter.

(B) Induction of E-cadherin-GFP with Shield-1. ECG ESCs were cultured under self-renewing conditions with or without Shield and imaged for GFP expression. The scale bar represents 50 μm.

(C–D) Induction of E-cadherin in ECG cells supports low-level expression of Nanog (C), but not Essrb (D). Cells were cultured in the presence/absence of Shield and/or tetracycline and then fixed for immunofluorescence. The scale bar represents 50 μm.

(E) E-cadherin supported limited AP-positive clonal growth of ESCs in the absence of Oct4. ECG ESCs were plated at clonal density in the presence/absence of Shield and/or tetracycline and colonies stained for tissue-nonspecific alkaline phosphatase (AP; red) after 7 days of growth.

(F) Quantitation of clonal growth in ESCs supported by E-cadherin in place of Oct4. Colonies were scored as undifferentiated (dark green), mixed (light green), and differentiated (gray).

(G) E-cadherin and POUV proteins interact. Postnuclear membrane fractions were purified from FLAG-tagged POUV-protein-rescued cell lines (see Figure 6) grown in self-renewing conditions. The figure shows immunoblot of coimmunoprecipitations, precipitating with either E-cadherin or the FLAG tag antibody. F-O4, FLAG-Oct4; F-X25, FLAG-Xlpou25; F-X91, FLAG-Xlpou91; Ig, IgG control.

See also Figure S5.