Knockdown of β-Catenin in DKO mESCS Expressing TCF4DN Partially Rescues Differentiation into the Neural Lineage
(A) Stable expression of β-catenin-specific short hairpin RNAs (shRNAs) reduces cytosolic β-catenin protein levels to approximately that of DKO-GSK3β mESCs. Two negative controls were assayed, including empty vector (β-sh cont. #1) and β-catenin scrambled shRNA (β-sh cont. #2) alongside two distinct β-catenin-specific shRNAs (β-sh #1 and β-sh #2).
(B) Validation of β-catenin knockdown by qRT-PCR. Bars represent n = 3 ± SEM.
(C) Morphology of DKO-TCF4DN β-sh ctrl #1 and DKO-TCF4DN β-sh #2 mESCs grown on gelatin-coated culture dishes. Inset highlights the flatter morphology of β-catenin knockdown DKO-TCF4DN mESCs. Scale bar represents 200 μm.
(D) Immunofluorescent staining of β-catenin knockdown DKO-TCF4DN mESCs reveals a significant reduction in the cytosolic and nuclear β-catenin pools as a result of shRNA expression.
(E) H&E staining of DKO-TCF4DN-β-sh ctrl #1, DKO-TCF4DN-β-sh #1, and DKO-TCF4DN-β-sh #2 teratomas. Scale bar represents 200 μm.
(F) Immunohistochemical analyses of teratomas derived from β-catenin knockdown DKO-TCF4DN mESCs reveals that suppression of β-catenin alleviates the differentiation blockade to the neural lineage. Scale bar represents 100 μm.