Fig. 4. Intact SMAD2/3 signaling is not required for the maintenance of the undifferentiated state of mESCs, but is required for the maintenance of the stem cell compartment of blastocyst outgrowths. (A) Western blot analysis of 129/SVJ mESCs. mESCs were cultured for 3 days in mESC medium with (+) and without (-) LIF, in mESC medium with LIF plus 10 µM SB431542, in mESC medium with 25 ng/ml activin A, in mESC medium with 2 µM BIO, in mESC medium with 2 µM BIO plus 10 µM SB431542, in CM, and in CM with 10 µM SB431542. Membranes were probed with antibodies specific for phosphorylated SMAD2, SMAD2/3, OCT3/4 and -tubulin (as a control for protein loading). (B) Whole-mount immunofluorescent confocal microscopy of pre-implantation stage mouse embryos. Mouse embryos were extracted and fixed at two-cell, four-cell, eight-cell, compacted morula and blastocyst stages. Embryos were decorated with an antibody specific for phosphorylated SMAD2 and SytoxGreen nuclear counterstain. Scale bars: 20 µm. (C) Confocal immunofluorescent microscopy of blastocyst outgrowths. Mouse blastocyst stage embryos were extracted and cultured in the presence of mESC medium supplemented with DMSO or 20 µM SB431542 for 4 days. Outgrowths were decorated with an antibody specific for OCT3/4 and SytoxGreen nuclear counterstain. Table gives percentage of embryos exhibiting OCT3/4-positive cells. Scale bars: 100 µm.