Fig. (1) Generation of GATA4- and eGFP-expressing D3 mESC cell lines. (A) Dual expression cassettes in nucleofected GATA4 (top) and eGFP (bottom) plasmids. CAGp, CAG promoter; EF1p, EF1α promoter; PuroR, puromycin-resistance gene. (B) eGFP expression in undifferentiated cells from a D3-eGFP clone at low (upper panels) and high (lower panels) magnification. Cells are shown from left to right in brightfield, fluorescence, and overlay. (C) Western blot for GATA4 detection in three D3G4 clonal lines. (D) Cloned D3-eGFP cells (4 panels at left) and D3G4 clone B cells (4 panels at right). Upper row, brightfield; lower row, the corresponding fields stained with anti-OCT4 and fluorescent secondary antibody (αOCT4) or secondary antibody alone (-). (E) Brightfield image of undifferentiated D3G4-B cells at high magnification as in (B).