(A) Retention of the pluripotency markers Oct4, Nanog, and Sox2 as well as expression of the neuronal marker β-III-tubulin was determined by immunofluorescent staining of the indicated EBs, maintained under differentiating conditions for 14 days.
(B) H&E staining of 3-week teratomas derived from WT (E14K), DKO-GSK3β, DKO-GSK3β-K85A, and DKO-TCF4DN mESCs.
(C) Immunohistochemical staining of teratomas revealed retention of the pluripotency marker Nanog and absence of neural tissue, as determined by staining for β-III-tubulin and GFAP, in teratomas derived from DKO-TCF4DN mESCs.
(D) β-catenin is maintained at low levels in teratomas derived from WT mESCs, whereas some regions of high β-catenin levels are observed in DKO-GSK3β teratomas, resulting from the loss of GSK3β transgene expression and reversion to DKO phenotype. In contrast, β-catenin levels remain high in DKO-GSK3β-K85A and DKO-TCF4DN teratomas.
Scale bars represent 20 μm (A) or 200 μm (B–D). See also Figure S2