siRNA-mediated downregulation of caveolin-1 expression in BeWo cells.

<p><i>A</i>, BeWo cells were transfected with the indicated concentrations of caveolin-1(Cav-1) siRNA or a non-silencing control siRNA. After 48 h cells were lysed and levels of caveolin-1 and β-actin were assessed by immunoblotting. <i>B</i>, densitometric analysis of immunoblots assessed for caveolin-1 expression and normalised to β-actin expression, in cells transfected for 48 h with 50 nM Cav-1 siRNA or non-silencing control siRNA. Results are presented as mean ± SEM for three separate experiments, *p<0.001 compared with control transfected cells (determined by ANOVA).</p

Labour study.

<p>Labour study.</p

sEndoglin levels in maternal plasma during labour (A, B) and Caesarean section (C,D).

<p>Median and ranges for sEndoglin ng/ml. In normal pregnancy labour (A) the levels declined by 24 hr (a***p<0.001, pre-labour vs 24 hr, b**p<0.01-full dilatation vs 24 hr, and placental delivery vs 24 hr). In pre-eclampsia labour (B) a similar decline by 24 h was noted (a*p<0.05, pre-labour vs 24 hr, full dilatation vs 24 hr, and placental delivery vs 24 hr). At Caesarean section (C,D) a significant decline in levels of sEndoglin by 24 hr was noted with placental delivery in both normal pregnancy and pre-eclampsia (a**p<0.01).</p

Inhibin A levels (n = 10) in maternal plasma during labour and Caesarean section.

<p>Median and ranges for inhibin A levels pg/ml in labour (A, B). In normal pregnancy labour (A) there was a significant decline in inhibin A levels by 24 h (a**p<0.01-prelabour vs 24 hr, full dilatation vs 24 hr, and b* p<0.05-placental delivery vs 24 hr. A similar decline was present in PE labour (B) (a*p<0.05, a-pre-labour vs 24 hr, b **p<0.01-placental delivery vs 24 hr), but the increase noted between pre-labour and full dilatation in pre-eclamptic women was not significant. At Caesarean section (C,D) a significant decline in levels of inhibin A by 24 hr was noted with placental delivery. (NP and PE a**p<0.01 placental delivery vs 24 hr).</p

Activin A levels (median (ranges) ng/ml) in maternal plasma during labour (A,B) and Caesarean section (C,D) in normal pregnancy (n = 10) and women with pre-eclampsia (n = 10).

<p>In normal pregnancy labour (A) there was a significant decline in activin A levels by 24 hr (a**p<0.01, pre-labour vs 24 hr, b ***p<0.001-full dilatation vs 24 hr, and c***p<0.001-placental delivery vs 24 hr. In pre-eclampsia labour (B) there was a significant increase in levels of activin A, (a*p<0.05 pre-labour vs full dilatation) and a significant decline postpartum (a*p<0.05 pre-labour vs 24 hr, b***p<0.001-full dilatation vs 24 hr, and c**p<0.01-placental delivery vs 24 hr). At Caesarean section a significant decline in levels of activin A by 24 hr was noted with placental delivery in normal pregnancy and pre-eclampsia (C,D). (NP a*p<0.05 and PE, a ***p<0.001 placental delivery vs 24 hr).</p

Elective Caesarean Section study.

<p>Elective Caesarean Section study.</p

RhoE is expressed in primary human villous cytotrophoblasts.

<p>Lysates were prepared from BeWo cells or freshly isolated primary human villous cytotrophoblasts (three separate preparations) and assessed for RhoE and β-actin expression by immunoblotting.</p

Effect of RhoE knockdown on BeWo cell fusion and differentiation.

<p>BeWo cells were transfected with 50nM RhoE siRNA or a non-silencing control then treated with 1mM dbcAMP and studied at the time points indicated. Cell lysates were made and expression of RhoE and β-actin was assessed by immunoblotting (A). Cells were also fixed and immunostained for desmosomal protein and cell fusion quantified (B) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030453#s2" target="_blank">Materials & Methods</a>. Cell lysates were also studied for expression of PLAP, β-hCG and β-actin by immunoblotting (C) with densitometric analysis normalised to β-actin expression (D). Results are presented as mean ± SEM for four separate experiments. *p<0.05 compared with non-silencing control (determined by ANOVA).</p

Effect of hypoxia on cAMP-induced RhoE expression.

<p>BeWo cells were cultured at 20% O₂ (normoxia) or 1% O₂ (hypoxia) for 24h, then treated with 1mM dbcAMP at 20% or 1% O₂ for a further 24h. Cell lysates were made and RhoE and β-actin expression was assessed by immunoblotting (A) with densitometric analysis of RhoE expression normalised to β-actin expression (B). A representative blot from three separate experiments is shown.</p

Effect of cyclic AMP on RhoE expression and fusion in BeWo cells.

<p>BeWo cells were treated with or without 1mM dbcAMP and studied at the indicated times. Cell lysates were made and expression of RhoE and β-actin was assessed by immunoblotting (A) and densitometric analysis of blots (B). Cells were fixed, immunostained for desmosomal protein (green) and counterstained with Hoechst 33258 (blue) (C) and cell fusion was quantified (D) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030453#s2" target="_blank">Materials & Methods</a>. Results are presented as mean ± SEM for three separate experiments. *p<0.05, **p<0.01 compared with control (determined by ANOVA).</p