DMSO promoted a low dose Activin-mediated decrease in Oct4 expression and apoptosis and increased the G1 population and SMAD2 phosphorylation.
The molecular mechanism underlying DMSO potentiation of low dose (6.25 ng/ml) Activin-induced DE differentiation was analysed. (A,B) DMSO decreased the proportion of OCT4-expressing cells induced by Activin at low dose, as analysed by immunocytochemistry. (C) DMSO arrested the cell cycle and decreased cell death, as revealed by flow cytometry to measure DNA quantities using DyeCycle. (D) Flow cytometric analysis of cleaved caspase3 revealed that DMSO inhibited apoptosis, triggered by a low dose of Activin. (E,F) Flow cytometric analysis of phosphorylated SMAD2. DMSO promoted SMAD2 phosphorylation, induced by a low dose Activin. (G) Western blot analysis. DMSO increased pSMAD2 in cells induced by Activin at a low dose. However, Activated β-catenin was unchanged. (H) Suppression of Activin signalling by SB231542 completely inhibited DE differentiation by DMSO, as shown by immunocytochemistry. (I) Wnt and BMP signalling were not required for DE differentiation by DMSO, as revealed by immuncytochemical analysis of SOX17. Scale bar; 50 μm.