Figure 5.

β-catenin knockdown blocked or reversed TCM-induced decidualization and MET in ESCs. A. Supernatant of plasmid lentiviruses, sh-scramble, sh1-β-catenin, sh2-β-catenin, were used to transfect ESCs. Western blot analysis showed decreased expression of β-catenin. B. CTNNB1 mRNA levels were significantly reduced in experimental conditions (sh1-β-catenin, sh2-β-catenin), compared with negative control (sh-scramble) by RT-PCR analysis. Error bars indicate SD from two independent experiments where *P < 0.05, **P < 0.01, or ***P < 0.001 by one-way ANOVA with Tukey post hoc test. β-catenin knockdown showed morphological changes and ESC transition using phase-contrast microscopy. C. Round or elliptical shapes were representative features of negative control decidual cells treated with sh-scramble. D, E. Stromal cells, with spindle-shapes, clear outlines, and sparse intercellular linkages were observed in β-catenin-shRNA treated ESCs (sh1-β-catenin, sh2-β-catenin). Scale bar = 50 μm. F-J. mRNA expression was analyzed by RT-PCR in four groups of ESCs (TCM, sh-scream, sh1-β-catenin, sh2-β-catenin). Relative mRNA expression was determined by normalizing to GAPDH levels. Error bars show SD from two independent experiments, where ns = non-significant, *P < 0.05, **P < 0.01, or ***P < 0.001 by ANOVA with Tukey post hoc test. K. Western blot analysis showed that β-catenin downregulation via shRNA1 and shRNA2 treatment in ESCs decreased expression cyclin-D3, desmin, E-cadherin, and FOXO1. Increased expression of N-cadherin was also observed compared to two controls. β-tubulin levels remained unchanged and used as the loading control.