Fig. 3. Klf5 constitutive expression prevents ESC differentiation. (A) ESCs that were mock-transfected (right panel) or transfected with Klf5 expression vector (left panel) were plated at low density (80 cells/cm²) and grown for 6 days without LIF. Scale bars: 1 mm. (B,C) Mock- or Klf5-transfected ESCs were grown for 5 days in differentiation conditions and stained with anti-Nanog and -Oct3/4 (B) or -Klf5 and -Nanog (C) antibodies. Scale bars: 100 µm. (D) Western blot analyses of Oct3/4 and Nanog in undifferentiated and differentiated ESCs. Protein extracts are from four clones stably expressing Klf5-overexpressed (Klf5 ov.) and one from mock-transfected cells.