Activated α2-macroglobulin binding to cell surface GRP78 induces trophoblastic cell fusion

The villous cytotrophoblastic cells have the ability to fuse and differentiate, forming the syncytiotrophoblast (STB). The syncytialisation process is essential for placentation. Nevertheless, the mechanisms involved in cell fusion and differentiation are yet to be fully elucidated. It has been suggested that cell surface glucose-regulated protein 78 (GRP78) was involved in this process. In multiple cancer cells, cell membrane-located GRP78 has been reported to act as a receptor binding to the active form of α2-macroglobulin (α2M*), activating thus several cellular signalling pathways implicated in cell growth and survival. We hypothesised that GRP78 interaction with α2M* may also activate signalling pathways in trophoblastic cells, which, in turn, may promote cell fusion. Here, we observed that α2M mRNA is highly expressed in trophoblastic cells, whereas it is not expressed in the choriocarcinoma cell line BeWo. We thus took advantage of forskolin-induced syncytialisation of BeWo cells to study the effect of exogenous α2M* on syncytialisation. We first demonstrated that α2M* induced trophoblastic cell fusion. This effect is dependent on α2M*-GRP78 interaction, ERK1/2 and CREB phosphorylation, and unfolded protein response (UPR) activation. Overall, these data provide novel insights into the signalling molecules and mechanisms regulating trophoblastic cell fusion.


Results
High α 2 M expression in vCTB prevents the use of these cells in studying the effects of α 2 M on syncytialisation. In order to determine the best model to study the effects of α 2 M* in trophoblastic cell fusion, we first evaluated the mRNA expression of α 2 M in placental vCTB and in BeWo cells, which are commonly used as a model to mimic the syncytialisation of placental villous cytotrophoblast. We cultured vCTB cells purified from early first-trimester placenta, late first-trimester placenta and term placenta for 24 h, 48 h, 72 h and 96 h to obtain a time course of syncytialisation (as previously described by Bastida-Ruiz et al. 16 ), allowing us to analyse basal α 2 M expression and determine if cell fusion increases the expression of α 2 M in trophoblastic cells. With a similar objective, BeWo cells were cultured for 48 h with or without Forskolin (FSK), a cell fusion inducer. mRNA expression of α 2 M was detected in primary trophoblastic cells observing that the α 2 M expression was independent of cell fusion rate, though it increased with gestational age (Fig. 2). On the contrary, no α 2 M expression was detected in treated or untreated BeWo cells (Fig. 2). The technical difficulties we encountered while trying to silence α 2 M expression in primary vCTB cells due to a low transfection efficiency led us to use BeWo cells to study the effects of exogenous α 2 M* on trophoblastic cell fusion and differentiation. α 2 M* increases cell fusion through its interaction with cell membrane-located GRP78. We hypothesised that α 2 M* binding to cell membrane-located GRP78 would activate signalling pathways that, in turn, may increase syncytialisation in trophoblastic cells. Firstly, we measured the fusion index (FI), a calculation that indicates the fusion rate in BeWo cells after α 2 M* treatment. We observed an increased cell fusion rate upon α 2 M* addition, which was independent of FSK treatment (Fig. 3A). Nevertheless, α 2 M* treatment exhibited no effect on hCG expression (Fig. 3B), demonstrating that α2M* induces trophoblastic cell fusion but not cell differentiation. We then decided to determine if the increased cell fusion that we observed after α 2 M* treatment was caused by its interaction with cell membrane GRP78. The blockage of GRP78 with anti-GRP78 antibodies prior to treatment of BeWo cells in vitro with α 2 M* caused a decrease in cell fusion, reaching the levels of α 2 M*-untreated BeWo cells (Fig. 3C). These results collectively demonstrated that cell fusion events are favoured by the interaction of α 2 M* and cell membrane GRP78 in BeWo cells. α 2 M*-GRP78 interaction increases ERK1/2, JNK and CREB phosphorylation as well as GRP78 and CHOP expression. The interaction of α 2 M* and cell membrane-located GRP78 activates several signalling pathways implicated in cell survival, growth and proliferation in prostate cancer, which have been reported to promote syncytialisation in trophoblastic cells (Fig. 1). The activation of these pathways was thus investigated in BeWo cells. Firstly, the protein levels of the active phosphorylated forms of several proteins -the expression of which was incremented in prostate cancer, concretely CREB, ERK1/2 and JNK -, were measured in BeWo cells (Fig. 4A) treated or not with α 2 M*. We observed increased expression of the phosphorylated forms of CREB, ERK1/2 and JNK upon α 2 M* treatment, suggesting that these proteins and their signalling pathways play a role in α 2 M*-cell fusion promotion. Subsequently, we decided to measure the expression of the phosphorylated form of Akt, the involvement of which in trophoblastic cell fusion is controversial. We detected no modulation of Akt phosphorylation after α 2 M* treatment in BeWo cells (Fig. 4B). Additionally, we tested the possible implications of NF-kB signalling in trophoblastic cell fusion by transfecting BeWo cells with a plasmid expressing the luciferase protein under NF-kB control. The luciferase glowing signal was not statistically different in cells treated or not with α 2 M* (Fig. 4C). These results suggest that Akt phosphorylation and NF-kB activity are not induced by α 2 M* in BeWo cells. Finally, we evaluated the expression of GRP78 and CHOP, the main markers of UPR activation (Fig. 4D), which is known to occur due to GRP78-α 2 M* interaction in prostate cancer cells. We observed an increased expression of CHOP and GRP78 upon α 2 M* treatment, suggesting the activation of UPR and its possible involvement in α 2 M*-dependent cell fusion promotion. α2M*-GRP78 interaction triggers several signalling pathways that are implicated in cell fusion. In order to elucidate the role of the different activated signalling pathways in trophoblastic cell fusion, selective inhibitors of the different activated proteins such as KT5720 (which inhibits PKA and thus affects CREB Interaction between GRP78 and α 2 M* promotes the activation of ERK1/2, JNK, PI3K, Akt, NF-kB and UPR in prostate cancer cells. The activation of ERK1/2 and JNK is known to enhance cell survival and proliferation through their derived signalling pathways. Additionally, the activation of Akt, NF-kB and UPR is known to promote anti-apoptotic signals, which, in turn, promote cell survival and proliferation. Overall, the proteins activated by α 2 M-GRP78 interaction have been reported to enhance the survival and proliferation of prostate cancer cells 22,29 . (B) Effects of ERK1/2, PKA, CREB, JNK, Akt signaling pathways and UPR activation on syncytialisation. The activated forms of ERK1/2 and PKA are known to activate CREB, a protein that has been reported to increase the expression of hCG and syncytins, promoting trophoblastic cell fusion and differentiation 30 . Furthermore, active JNK has been shown as well to increase the expression of syncytins 31 . In a more controversial manner, Akt activation was described as a negative modulator of syncytialisation in trophoblastic cells 32 . However, some studies confer no effects on cell fusion and differentiation to this protein 30 . Finally, UPR was recently reported to activate autophagy during trophoblastic syncytialisation, promoting cell survival and favouring cell fusion and differentiation 16 . Summing up, some of the proteins activated in prostate cancer cells by GRP78-α 2 M* interaction are capable of inducing syncytialisation in trophoblastic cells. activation), UO126 (ERK1/2) and SP600125 (JNK) were used in BeWo cells. The expression of the different targeted proteins was measured by Western blot, observing a diminution in the expression of the phosphorylated active forms of CREB, ERK1/2 and JNK after treatment with the corresponding inhibitors (Fig. 5A). The phosphorylation reduction achieved by the inhibitors, in BeWo cells treated or untreated with α 2 M*, demonstrated the correct inhibition of the signalling pathways. Afterwards, the cell fusion rate was measured and compared with the corresponding controls before and after α 2 M* treatment (Fig. 5B). We then verified that these treatments did not alter cell viability that could affect the fusion index results (Fig. 1SA). CREB, ERK1/2 and JNK activate signalling pathways that are known to increase hCG secretion and the expression of syncytins in trophoblastic cells. We have already demonstrated that α 2 M* treatment does not affect hCG secretion (Fig. 3B). Therefore, we next investigated syncytin-1 and -2 expression in BeWo cells treated or not with α 2 M*. We observed that α 2 M* treatment did not affect their expression at the mRNA level (Fig. 5C). This result was also confirmed at the protein level for syncytin-1 (Fig. 5D), suggesting α 2 M*-GRP78 interaction played a role in cell fusion independent of hCG secretion and syncytin expression.
In order to elucidate the role of the different activated signalling pathways in trophoblastic cell fusion, selective inhibitors of the different activated proteins such as KT5720 (which inhibits PKA and thus affects CREB activation), UO126 (ERK1/2) and SP600125 (JNK) were used in BeWo cells. The expression of the different targeted proteins was measured by Western blot, observing a diminution in the expression of the phosphorylated active forms of CREB, ERK1/2 and JNK after treatment with the corresponding inhibitors (Fig. 5A). The phosphorylation reduction achieved by the inhibitors, in BeWo cells treated or untreated with α 2 M*, demonstrated the correct inhibition of the signalling pathways. Afterwards, the cell fusion rate was measured and compared with the corresponding controls before and after α 2 M* treatment (Fig. 5B). We then verified that these treatments did not alter cell viability that could affect the fusion index results (Fig. 1SA). CREB, ERK1/2 and JNK activate signalling pathways that are known to increase hCG secretion and the expression of syncytins in trophoblastic cells. We have already demonstrated that α 2 M* treatment does not affect hCG secretion (Fig. 3B). Therefore, we next investigated syncytin-1 and -2 expression in BeWo cells treated or not with α 2 M*. We observed that α 2 M* treatment did not affect their expression at the mRNA level (Fig. 5C). This result was also confirmed at the protein level for syncytin-1 (Fig. 5D), suggesting α 2 M*-GRP78 interaction played a role in cell fusion independent of hCG secretion and syncytin expression.
In order to elucidate the role of the different activated signalling pathways in trophoblastic cell fusion, selective inhibitors of the different activated proteins such as KT5720 (which inhibits PKA and thus affects CREB activation), UO126 (ERK1/2) and SP600125 (JNK) were used in BeWo cells. The expression of the different targeted proteins was measured by Western blot, observing a diminution in the expression of the phosphorylated active forms of CREB, ERK1/2 and JNK after treatment with the corresponding inhibitors (Fig. 5A). The phosphorylation reduction achieved by the inhibitors, in BeWo cells treated or untreated with α 2 M*, demonstrated the correct inhibition of the signalling pathways. Afterwards, the cell fusion rate was measured and compared with www.nature.com/scientificreports www.nature.com/scientificreports/ the corresponding controls before and after α 2 M* treatment (Fig. 5B). We then verified that these treatments did not alter cell viability that could affect the fusion index results (Fig. 1SA). CREB, ERK1/2 and JNK activate signalling pathways that are known to increase hCG secretion and the expression of syncytins in trophoblastic cells. We have already demonstrated that α 2 M* treatment does not affect hCG secretion (Fig. 3B). Therefore, we next investigated syncytin-1 and -2 expression in BeWo cells treated or not with α 2 M*. We observed that α 2 M* treatment did not affect their expression at the mRNA level (Fig. 5C). This result was also confirmed at the protein level for syncytin-1 (Fig. 5D), suggesting α 2 M*-GRP78 interaction played a role in cell fusion independent of hCG secretion and syncytin expression. CREB and ERK1/2 are involved in the control of UPR, which is implicated in cell fusion. We decided to explore the possible modulation of UPR by CREB activation in trophoblastic cells since previous studies conferred this function to CREB 33 . Additionally, ERK1/2 is known to be an activator of CREB 34 , and we speculated that it could be involved in α 2 M-induced CREB phosphorylation and, consequently, UPR modulation. Firstly, we treated BeWo cells with KT5720, a potent PKA inhibitor preventing CREB phosphorylation (Fig. 6A), or U0216, a potent inhibitor of MKK1/2 preventing both ERK1/2 ( Fig. 6A) and CREB phosphorylation (Fig. 2S). We then analysed their impact on the expression of UPR-related proteins, which we have already demonstrated to be involved in trophoblastic cell fusion promotion 16 . As we previously showed in Fig. 4A, we observed that the addition of α 2 M* to the BeWo cells without blocking CREB and ERK1/2 activation led to increased expression of GRP78 and CHOP (Fig. 6A). On the contrary, BeWo cells that were pre-treated with KT5720 or UO126 did not show increased expression of GRP78 or CHOP after the addition of α 2 M*. These results suggest that α 2 M*-induced CREB and ERK1/2 activation may affect BeWo cell fusion through UPR activation. Finally, we blocked UPR activation by pre-treating BeWo cells before α 2 M* addition with inhibitors of the different UPR branches (4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), STF-083010 (STF) and GSK2656157 (GSK)). α 2 M*-induced cell fusion is inhibited by inhibitors of UPR (Fig. 6B), while not affecting cell viability (Fig. 1SB), suggesting that UPR mediates the effect of α 2 M* on BeWo cell fusion. www.nature.com/scientificreports www.nature.com/scientificreports/ Discussion PE and other pregnancy disorders are characterised by incorrect STB formation [review 35 ] and, therefore, abnormal placentation, together with reduced expression of cell membrane-located GRP78 in trophoblastic PE cells 19 . Syncytialisation seems to be essential for correct placental development, and understanding the mechanisms controlling this process is fundamental to medical treatment. α 2 M* bound to cell surface-located GRP78 has been found to activate some signalling pathways in cancer cells, leading to cell survival, growth and proliferation 22,29 . Some of the proteins implicated in these signalling pathways have been reported to induce syncytin expression and hCG secretion, inducing cell fusion 30 . We hypothesised that some of these signalling pathways could also be activated by α 2 M*-GRP78 interaction in trophoblastic cells and, therefore, be implicated in cell fusion.
In this study, we have taken advantage of BeWo cells, a cell line that is used as a trophoblastic cell fusion model, which lacks α 2 M expression. The characteristics of this cell line allowed us to study the effect of exogenous α 2 M in trophoblastic cells and the involvement of GRP78 during the different triggered events. Firstly, we demonstrated that α 2 M* treatment led to an increase in cell fusion through GRP78 interaction, phosphorylation of ERK1/2, CREB and JNK, and UPR activation. The ways these proteins promote cell fusion have not been completely elucidated; we evaluated syncytin-1 and -2 expression and hCG secretion since they were previously demonstrated to be increased by ERK1/2, CREB and/or JNK activation 30,31 . However, we obtained negative results, showing α 2 M*-GRP78 interaction affects trophoblastic cell fusion independent of hCG secretion and syncytin expression. This unexpected result led us to hypothesise that CREB and ERK1/2 could be implicated in trophoblastic cell fusion by different mechanisms. It was reported that CREB phosphorylation had an impact on UPR activation in breast cancer cells 33 . Furthermore, we recently demonstrated that UPR activation in trophoblastic cells is involved in syncytialisation 16 . In this paper, we have demonstrated that α 2 M* treatment of BeWo cells induced UPR activation, and, thus, cell fusion but not differentiation. We have also shown that inhibition of CREB phosphorylation (using PKA or ERK1/2 inhibitors) eliminated the effect of α2M* on UPR activation in BeWo cells, suggesting that α 2 M* could activate the CREB pathway and, thus, UPR in BeWo cells (Fig. 7).
However, the impact of α 2 M*-induced CREB and ERK1/2 activation on UPR activation and, consequently, on trophoblastic syncytialisation was surprising since we previously demonstrated that UPR was implicated in both cell fusion and differentiation (hCG secretion) 16 , while, in this study, we have only observed effects on cell fusion. BeWo cells were seeded for 24 h prior to treatment with or without 100 pM of α 2 M* for 48 h. Western blotting was performed. GRP78 and CHOP levels were quantified using the ImageJ software, and data are expressed as the fold change relative to the control. n = 3. The images of bands for the target protein and GAPDH were taken from the same gel, and each image was cropped, as delineated by black dividing lines, as well as adjusted for image intensity for optimal visualisation. (2020) 10:9666 | https://doi.org/10.1038/s41598-020-66554-0 www.nature.com/scientificreports www.nature.com/scientificreports/ Conveniently, it was observed that CREB modulates the IRE1α and PERK branches of the UPR 33 , while no effect was described in the ATF6 branch. We have seen that UPR-induced hCG secretion is controlled primarily by ATF6 (data not shown) in trophoblastic cells. This observation may explain why α 2 M*-induced UPR activation does not lead to increased hCG secretion.
Interestingly, we also observed an increased trophoblastic expression of α 2 M with gestational age, displaying strong α 2 M expression at term, which suggests that this glycoprotein plays a role in pregnancy. Some previous observations already highlighted the implication of α 2 M in pregnancy by using murine models 36,37 . It was demonstrated that α 2 M participates in the spiral artery remodelling, an essential step necessary for the successful pouring of blood into the placenta 36 . Additionally, it was proven that murine α 2 M and other members of the α 2 M family, namely the murinoglobulins, were implicated in trophoblastic invasion 37 . Moreover, functional deficiency of α 2 M has not been described, suggesting that α 2 M is essential for gestational success 37 . Nevertheless, the role of human α 2 M in spiral artery remodelling was never studied. Here, for the first time, we suggested that α 2 M* could also play an essential role in trophoblastic cell fusion. Unfortunately, we were unable to abolish or significantly decrease α 2 M expression in primary cells to confirm the role of endogenous α 2 M in the fusion of trophoblastic cells, and we had to pursue our investigation by using BeWo cells. Nevertheless, we managed to unravel a new mechanism by which α 2 M is implicated in pregnancy, demonstrating the importance of this protein to gestation.
The results obtained in this study regarding the impact of α 2 M* on trophoblastic cell fusion lead us to hypothesise that members of the same family, such as the pregnancy zone protein (PZP), could have similar effects. PZP is a macroglobulin protein, normally detected as a trace plasma protein (<0.01 mg/ml), the expression of which is highly modulated by reproductive hormones, detectable by week 5 of pregnancy and peaking at term 38 . Blood concentrations at term have been reported to be 100-200-fold above pre-conception levels and matches, or even exceeds, that of α 2 M 39 . This pregnancy protein shares a 71% homology with α 2 M 40 , and it has been identified in syncytiotrophoblast, immune cells, placental villous mesenchyme and endothelium 41 . The PZP functions have been hypothesised to be similar to those attributed to α 2 M 42,43 . Additionally, it was reported that PZP possesses a receptor-binding domain with high homology to the one observed in α 2 M 28, 43 . We speculate that due to its Western blotting was performed. p-CREB, CREB, p-ERK1/2, ERK1/2, p-JNK and JNK levels were quantified using the ImageJ software, and data are expressed as the fold change relative to the control. n = 3. The images of bands for the target proteins were taken from the same gel, and each image was cropped, as delineated by black dividing lines, as well as adjusted for image intensity for optimal visualisation. (B) Nuclei and syncytia were counted, and a fusion index was calculated. n = 3. Data represented as mean±SEM. ns (not significant), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005; ANOVA comparison test. (C,D) BeWo cells were seeded for 24 h prior to treatment with or without 100 pM of α 2 M*. (C) RNA was retrotranscribed, and 10 ng of cDNA was used to perform qPCR using syncytin-1 and syncytin-2 primers. n = 3. Data represented as mean±SEM. **P ≤ 0.01; t-test comparison test. (D) BeWo cells were seeded for 24 h prior to treatment with or without 100 pM of α 2 M* for 48 h. Western blotting was performed. Syncytin-1 and GAPDH levels were quantified using the ImageJ software, and data are expressed as the fold change relative to the control. n = 3. The images of bands for the target proteins were taken from the same gel, and each image was cropped, as delineated by black dividing lines, as well as adjusted for image intensity for optimal visualisation. Scientific RepoRtS | (2020) 10:9666 | https://doi.org/10.1038/s41598-020-66554-0 www.nature.com/scientificreports www.nature.com/scientificreports/ elevated expression during pregnancy and its high homology to α 2 M 38 , PZP may bind GRP78 and also play a role in trophoblastic cell fusion by activating similar signalling pathways to those activated by α 2 M*. Unfortunately, this possibility has not been investigated yet, and it could be essential to understanding the potential mechanisms triggered by PZP during pregnancy.
Furthermore, looking from a structural perspective, it is evident that macroglobulin proteins are oligomeric complexes formed by several identical subunits. α 2 M is a homo-tetrameric protein, and, therefore, it contains four different receptor-binding domains, one per monomer; while PZP is a homo-dimer, containing two receptor-binding domains 28,38 . This spatial conformation could provide α 2 M and PZP with the ability to bind to several GRP78 proteins from different cells, conferring to these proteins a bridge function, bringing cells together in close proximity and favouring cell fusion. It may be interesting to investigate whether the monomeric versions of α 2 M could induce trophoblastic cell fusion or if the bridge function derived from the structural conformation of this protein favours cellular interactions and is needed for the attainment of total fusion competence.
We could conclude that the cell surface-located GRP78 is implicated in trophoblastic cell fusion through the interaction of α 2 M* and the subsequent activation of ERK1/2 and CREB, which, in turn, modulates UPR activation in BeWo cells. These results reinforce the critical role of GRP78 and UPR in trophoblastic cell fusion 16,19 and encourage further investigation into the roles of α2M family proteins during pregnancy.

Materials and Methods
ethics statement. This research was approved by the Geneva Hospital Ethics Committee (#10-001 and 02-088). Informed written consent was obtained from all patients before inclusion in the study. All methods were carried out in accordance with relevant guidelines and regulations.
Purification of vCTB. vCTB were isolated from first-trimester trophoblast (n = 3 early first trimester, n = 3 late first trimester) and normotensive term placentae (n = 3). Purification took place according to the protocol previously detailed by Bischof et al. 44 Briefly, isolation of small placental tissue pieces was followed by enzymatic tissue digestion with a Difco Trypsin solution (BD, Le Pont de Claix, France). Next, cell separation was performed in a Percoll gradient (GE Healthcare, Uppsala, Sweden), and immunopurification of the vCTB was performed using monoclonal mouse anti-human CD45 immobilised antibodies (Dako, Glostrup, Denmark).  Briefly, insoluble material from human plasma was pelleted, and the supernatant plasma solution was dialyzed prior to performing metal chelate chromatography in a zinc-sepharose-4B column. Bound protein was pulsed from the column, and peak protein fractions were pooled and concentrated prior to gel filtration. The high molecular weight peak containing pure α2M was pooled and concentrated for storage.
To evaluate the role of membrane GRP78 in α 2 M*-induced cell fusion, a pre-treatment with rabbit anti-GRP78 antibodies (GL-19, 3 µg/ml from Sigma, Darmstadt, Germany) or normal rabbit IgG antibodies (sc-2027, 3 µg/mL from SantaCruz Biotechnology, Labforce, Switzerland) was performed in a 96-well tissue culture plate (  The control vector was used to determine the NF-kB pathway-specific effects and background luciferase activity (ratio of firefly luminescence from the NF-kB reporter to firefly luminescence from the control firefly luciferase vector). polymerase chain reaction (pcR). The extraction of total RNA was performed in BeWo cells (from three independent experiments) or vCTB (n = 3 for each gestational stage) using the PureLinkRNA Mini Kit (AMBION, Austin, TX, USA). Reverse transcription with 1 µg of total RNA was completed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies). A PCR using 50 ng of cDNA was performed using the REDTaq ReadyMix PCR Reaction Mix (Sigma, Steinheim, Germany) and the following primers for α 2 M detection: α 2 M forward 5′-GAAGTGTTTGGGACCAGATG-3′ and α 2 M reverse 5′-AGTCGGAAGCGTCACTATAC-3′; and GAPDH detection: GAPDH forward 5′-CGTATTGGGCGCCTGGTCACC-3′ and GAPDH reverse 5′-GGGATGATGTTCTGGAGAGCCC-3′. A 1% agarose gel was prepared using Basic agarose premier (MP Biomedicals, Illkirch, France) and SYBR Safe DNA gel stain (Cartshad, CA, USA), as indicated by the manufacturer. Acquisition was accomplished using the Geneflash machine (Syngene Bioimaging).
Quantitative polymerase chain reaction (qpcR). PureLinkRNA Mini Kit (AMBION, Austin, TX, USA) was used to extract total RNA from BeWo (from three independent experiments). Reverse transcription was performed with 1 µg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies). Detection of the real-time qPCR product was performed using the KAPA SYBR FAST qPCR Kit Master Mix (Kapa Biosystems, Axon Lab, Baden, Switzerland) on an Eco Real-Time PCR System (Labgene Scientific, Châtel-St-Denis, Switzerland). The relative expression of syncytin-1 and syncytin-2 genes was normalised to the two housekeeping genes GAPDH and Cyclophilin A. The primers used for quantification of these genes' expression are described in Table 1.
Western blot. As previously described by Bastida-Ruiz et al. 16 , 40 µg of proteins from whole BeWo (from three independent experiments) was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. The antibodies used for immunoblotting are described in Table 2. A specific signal was detected using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK) or Immobilon Western Chemiluminescent HPR Substrate (Billerica, MA, USA). fusion index (fi). A frequently used marker of trophoblastic cell fusion is the FI 46 . It describes the number of nuclei inside syncytia as a percentage of the total number of nuclei.
Trophoblastic cell FI was determined by immunocytochemistry, as previously described by Bastida-Ruiz et al. 16 . Briefly, cells were washed in PBS, fixed in 3% paraformaldehyde and immunostained using mouse anti-desmoplakin antibodies (sc-390975, 1:500 dilution from Santa Cruz Biotechnology, Heidelberg, Germany). Revelation was performed with diaminobenzidine (Dako, Carpinteria, CA, USA) after incubation with secondary antibodies anti-mouse IgG-HRP (sc-2005, Santa Cruz Biotechnology, Heidelberg, Germany). Nuclei were stained with haematoxylin. Image acquisition was completed using a Ceti Inverso TC-100 inverted biological microscope (Medline scientific, UK). FI was expressed in percentages and calculated as follows: [(N − S)/T], where N equals the number of nuclei in syncytia, S equals the number of syncytia, and T equals the total number of nuclei counted 19 . The syncytia are recognised as cells containing several nuclei inside the same cell. Three different fields Luciferase expression. BeWo cells were lysed, without discarding the supernatant, with the Dual-Glo Luciferase assay system (Promega, Madison, WI, USA) following the manufacturer's instructions 24 hours after treatment. The resulting lysate was used to analyse the luminescence with the GloMax 96-well Luminometer (EG501, Promega Biosystems Sunnyvale, Inc., Sunnyvale, CA, USA) and the Glomax 1.9.3 software. Luciferase expression measurement was done for three independent experiments, run in triplicate.
Proliferation/viability assay. To determine the effect of the different treatments on BeWo cell viability, we performed an MTT assay. After the corresponding treatment, the medium was replaced with a medium containing 20% MTT (Sigma-Aldrich Corporation, USA) solution (5 mg/mL in medium) for 2 h. Acidic isopropanol solution (150 µL) was added, and then each well was vigorously mixed to dissolve the precipitated formazan. UV-visible absorption was measured at 540 and 690 nm. A proliferation/viability assay was performed for three independent experiments, run in triplicate.
Statistics. Data were represented as means ± standard error of the mean (SEM) for at least 3 different samples. Statistical differences between samples were assessed by the Student's t test or ANOVA test, followed by Tukey's multiple comparison test, as specified in each experiment, and the p-value<0.05 was considered significant. GraphPrism software was used to perform the different statistical analyses.