The properties of DKO ESCs can be reverted to those of WT ESCs upon stable expression of WT, but not kinase-dead, GSK-3. (A) Immunoblot analyses of cytosolic lysates prepared from WT and DKO ESCs as well as DKO ESC cell lines stably expressing V5-tagged WT- or K148A-GSK-3α. (B) Semi-quantitative RT-PCR analysis of transcripts expressed in the same cell lines examined in panel A. (C–N) Immunofluorescence images of EBs maintained for 14 days in LIF-free medium. EBs derived from (C, F) V5-WT-GSK-3α-expressing DKO ESCs; (D, G) V5-K148A-GSK-3α expressing DKO ESCs; and (E, H) DKO ESCs double-stained for Oct-4 and GFAP, respectively. EBs derived from (I, L) WT ESCs; (J, M) V5-WT-GSK-3α-expressing DKO ESCs and (K, N) V5-K148A-GSK-3α-expressing DKO ESCs stained for Nestin and Nestin/DAPI (nuclear stain) merge, respectively. Bar = 100 μm in all micrographs.