Fig. 5.

Loss of TEAD4 impairs human TSC self-renewal. (A) TEAD4 and YAP1 protein expressions were tested in human TSCs without (control) and with TEAD4 depletion by RNAi [shTEAD4 (1) and shTEAD4(2)]. (B) Plot showing reduced cell proliferation in TEAD4-KD human TSCs (mean ± SE; n = 3, *P ≤ 0.005). (C) Equal numbers of control and TEAD4-KD human TSCs were plated and cultured in the stem state culture condition. Micrographs confirm reduced cell proliferation and loss of stem state colony morphology (white circle) in TEAD4-depleted TSCs. (D) Confocal images of organoids generated with human TSCs. Immunostaining shows expressions of E-cadherin (CDH1, green) in the outer cell layer and human CG-β subunit (HCG-β, ρεδ) in the inner cell TSC organoids, confirming that the organoids develop an inside-out pattern of CTB vs. STB populations with respect to the first-trimester placental villi. (E) Micrographs show inefficient organoid formation by TEAD4-KD human TSCs. (F) Venn diagrams showing the number of genes that are direct targets of TEAD4 and also showing significant changes in their mRNA expression in Tead4-KD human TSCs. (G) The table shows alteration of mRNA expression of cell cycle regulators and STB and EVT markers in TEAD4-KD human TSCs. (H) Western blot analyses showing loss of protein expressions of cell cycle regulators in TEAD4-KD human TSCs. (I) Venn diagrams showing the number of unique and common TEAD4-regulated genes in mouse and human TSCs. (J) Ingenuity pathway analysis showing major biofunctions that are associated with genes regulated by TEAD4 in both mouse and human TSCs.