Figure 1.
Effect of forskolin treatment on GRP78 expression in trophoblastic cells.
Primary first trimester or term CTBs and BeWo cells were treated or not with 100 µM forskolin for 96 h or 48 h respectively. A- Total RNA was then extracted, reverse transcribe andGRP78 cDNA was quantified by qPCR and normalized to cyclophilin A. B- Proteins (40 ug) were analysed by western blot with anti-GRP78 and anti-GAPDH antibodies. C- Membrane GRP78 expression was quantified by cell-ELISA in BeWo cells. Results are presented as mean ± SEM. n = 3 * p<0.05.
Figure 2.
Downregulation of GRP78 in Bewo cells.
A- Cells were untreated (CTRL) or treated with 20 µM VST or CB106 for 48 h. B- Cells were transfected with GRP78 or control siRNA for 48 h. A–B Upper panel: Immunoblots of GRP78 and GAPDH. Lower panel: The intensity of the GRP78 bands from three independent experiments was quantified and normalized to GAPDH.
Figure 3.
Role of GRP78 on fusion properties of BeWo cells.
A- Cells were untreated (CTRL) or treated with anti-syncytin (AbSyn) or anti-GRP78 (AbC20) antibodies, 20 µM VST or CB106 for 48 h in the absence or presence of 100 µM forskolin. B- Cells were transfected with GRP78 or control siRNA for 48 h in presence or not of 100 µM forskolin. Fusion index was calculated for 3 independent experiments. * p<0.05.
Figure 4.
Role of GRP78 on phosphatidylserine flip of BeWo cells.
A- BeWo cells were seeded on gelatin layer and treated or not with anti-syncytin (AbSyn) or anti-GRP78 (AbC20) antibodies, 20 µM VST or CB106 in the absence or presence of 100 µM forskolin for 48 h. B- BeWo cells were transfected with GRP78 or control siRNA and seeded on gelatin layer. The following day, cells were treated or not with forskolin for 48 h. Phosphatidylserine flip was evaluated by a colorimetric assay (APOPercentage). n = 3, * p<0.05.
Figure 5.
Effect of GRP78 down regulation on hCG secretion.
BeWo cells were transfected with GRP78 or control siRNA and seeded on gelatin layer and then treated or not with 100 µM forskolin for 48 h. hCG level in culture supernatant was determined by ELISA and normalized to protein extract concentration. n = 3, * p<0.05.
Table 1.
Demographic and clinical characteristics of the study groups.
Figure 6.
GRP78 expression in trophoblastic cells.
A- GRP78 mRNA from control (CTRL) and preeclamptic (PE) trophoblastic cells was quantified by qPCR and normalized to cyclophlin A. B- GRP78 expression from control (CTRL) and preeclamptic (PE) trophoblastic cells was quantified by Cell-ELISA. C- Relative proportion of membrane GRP78 in trophoblastic cells purified from control (CTRL) and preeclamptic (PE) trophoblastic cells was evaluated by Cell-ELISA. Results are presented as mean ± SEM. n = 3 * p<0.05; AU: arbitrary unit.