OPN silencing limits trophoblast outgrowth and invasion <i>in vitro</i>.

<p>(A) Real-time PCR of <i>Opn</i> mRNA levels in OPN-knockdown mESC by OPN siRNA transfection (NC: negative control siRNA, si-OPN: OPN siRNA). (B) A western blot confirmed the knockdown effect of OPN-targeted siRNA on OPN protein expression in mESCs or on secreted OPN proteins in the culture medium. (C) The adhesion rate of mouse blastocysts in control and OPN-knockdown mESCs. (D) OPN-targeted, siRNA-pretreated mESCs significantly decreased the trophoblast-spreading areas in the mESCs compared to the control group. The data for calculating the mean spreading area included at least ten embryos for each group, and all of the experiments were repeated at least three times. *, <i>p</i><0.05; <i>error bars</i>, S.E. (E). Immunofluorescence was performed to detect the trophoblast outgrowth and invasion in OPN-targeted siRNA pretreated mESCs (Control, negative control siRNA pretreated mESC; si-OPN, OPN-targeted siRNA). Mouse embryos were marked by E-cadherin (Green signal), and nuclei were marked by DAPI (Blue). All of the experiments were repeated three times.