Fig 2: In vitro decidualization of mouse primary ESCs.(A) qRT-PCR analysis of DTPRP mRNA expression in ESCs cultured for up to 72 h. (B) qRT-PCR analysis of NDRG4 mRNA expression in cultured ESCs. (C) Western blot analysis of NDRG4 protein expression in ESCs cultured for up to 48 h. Densitometric analyses of NDRG4 compared with 0 h is shown. The values represent the mean ± SEM, as determined from three separate experiments. *, significantly different (P < 0.05).

Fig 3: Uterine NDRG4 expression during early pregnancy in mice.(A) Quantitative PCR analysis of NDRG4 mRNA expression in the uterus during early pregnancy (n = 3, *P < 0.05). (B) Western blot and densitometric analyses of uterine NDRG4 protein levels during early pregnancy (n = 3). All experiments were repeated three times. The data are shown as the mean ± SEM. *P < 0.05. (C) Immunohistochemical analysis of uterine NDRG4 protein expression on days 1, 3, 4, 5, and 8 of pregnancy. IS, implantation sites; NI, non-implantation sites. *, indicates the location of the embryo. le, luminal epithelium; ge, glandular epithelium; st, stroma; pdz, primary decidual zone; sdz, secondary decidual zone; D, day of pregnancy; gc, giant cell; Scale bar represents 100 μm.

Fig 4: Down-regulation of NDRG4 expression in ESCs inhibits in vitro decidualization.Quantitative PCR analyses of DTPRP mRNA expression (A) and NDRG4 mRNA expression (B) in ESCs transfected with NDRG4-targeting siRNAs or non-targeting siRNAs at 24 h and 48 h. Western blot and densitometric analyses of NDRG4 protein levels in ESCs at 72 h after transfection (C). The relative fold induction of NDRG4 protein expression compared with its expression in non-siRNA-treated group is shown. The values represent the mean ± SEM, as determined from three separate experiments. *, significantly different (P < 0.05).

Fig 5: Uterine NDRG4 expression following artificial decidualization and activation of delayed implantation.Immunohistochemical analysis of uterine NDRG4 protein expression following artificial decidualization (B) and in its contralateral uninjected uterine horn (A), under delayed implantation (D) and activation (E). Quantitative PCR analysis of NDRG4 mRNA expression in the uterus following artificial decidualization (C) and activation of delayed implantation (F) (n = 3). The thick arrow indicates the luminal epithelium. The small arrow shows the glandular epithelium. De, decidua; *, significantly different from control (P < 0.05).