Figure 2.

Loss of E-cadherin expression shows its necessity during reprogramming. (A) Ecad protein expression in OCT4, SOX2, KLF4 and c-MYC (OSKM)-transduced non-Cre-treated (−) and His-tat-NLS (HTN)-Cre-treated (+) Ecad^flox/flox MEFs and equally treated wild-type MEFs, respectively. (B) Determination of colony number by counting of mouse embryonic stem cell-like colonies in pools of non-Cre-treated, transduced Ecad^flox/flox MEFs or wild-type MEFs (black) or Cre-treated Ecad^flox/flox MEFs or wild-type MEFs (grey), respectively. Colony numbers were as follows: MEFs Ecad^flox/flox without HTNCre (Ctl), 319 colonies (set to 100%); with HTNCre (+HTNCre), 88 colonies (28%); wild-type MEFs Ctl, 72 colonies (set to 100%); and +HTNCre, 93 colonies (129%). The difference in absolute colony numbers between Ecadv^flox/flox and wild-type MEFs is due to the different viability of cells isolated from different animals at different times. The experiment was repeated four times and the values from one representative experiment are shown. (C) Relative mRNA expression level of Ecad, NANOG, DPPA5 and NR5A2 of a pool of OSKM-induced Cre-treated and non-treated Ecad^flox/flox MEFs 10 days after infection. Median values from biological duplicates are presented as bars; one representative experiment is shown of four independent experiments. (D) Immunofluorescence analysis of cell colonies from OSKM-induced Ecad^flox/flox MEFs for Ecad and NANOG either untreated (Ctl) or Cre-treated (HTNCre) showing no recombination (non-recombined first and second column) or recombination (recombined). DAPI was used for nuclear staining. Magnification × 400; scale bars, 50 μm. AU, arbitrary units; Ctl, control; DAPI, 4,6-diamidino-2-phenylindole; MEF, mouse embryonic fibroblast; mRNA, messenger RNA; wt, wild type.