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

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 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

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 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

Attenuation of RhoE expression in response to cyclic AMP in JEG-3 cells.

<p>BeWo or JEG-3 cells were treated with 1mM dbcAMP. At the indicated times cell lysates were made and expression of RhoE and β-actin was assessed by immunoblotting (A) with densitometric analysis of RhoE expression normalised to β-actin expression (B). Cells were fixed, immunostained for desmosomal protein (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 BeWo cells (determined by ANOVA).</p