Figure 2
Bioactive lipids S1P and LPA enhance β-catenin nuclear accumulation and activate Wnt signaling during early cardiac differentiation from hiPSCs. (A) Immunofluorescence for β-catenin (green), pluripotency marker Nanog (red), and DAPI (DNA) (blue) following 2-hour treatment of hiPSCs with DMSO, small molecule GSK3β inhibitor/Wnt activator CHIR99021 (CHIR), bioactive lipids S1P + LPA, or CHIR + bioactive lipids. Arrows indicate cells exhibiting characteristic β-catenin nuclear accumulation. (B) Quantification of β-catenin staining represented as nuclear intensity over cytoplasmic intensity for the treatment groups normalized to DMSO control. (C) Luciferase luminescence intensity after transfection of hiPSCs with TOPFlash Wnt pathway activity reporter and 2-hour treatment with CHIR, S1P/LPA, or both, represented as fold increase over DMSO control. (D) Model illustrating the signaling cascade linking bioactive lipids and the Wnt/β-catenin signaling pathway in the context of hiPSCs. Treatment with S1P/LPA on hiPSCs dissociates β-catenin from adherens junctions and E-cadherin, thus increasing the overall β-catenin pool that can be utilized for downstream signaling and gene transcription. Treatment with GSK3β inhibitor CHIR frees β-catenin and increases the overall intracellular β-catenin pool for downstream signaling and gene transcription. (E) Microarray analysis illustrating key alterations in gene expression following 48-hour treatment of hiPSCs with small molecule GSK3β inhibitor/Wnt activator CHIR with or without bioactive lipids S1P/LPA. A list of up- (red) and down- (blue) regulated genes after treatment with bioactive lipid is shown. Experiments were performed in 3–4 biological replicates.
