Journal: Frontiers in Cell and Developmental Biology

Article Title: Reduced Wiskott-Aldrich syndrome protein expression in preeclampsia placenta impairs trophoblast syncytialization by modulating syncytin-2 via FAK/β-catenin pathway

doi: 10.3389/fcell.2025.1678878

Figure Lengend Snippet: Reduced Expression of N-WASP and Syncytin-2 and Analysis of WNT/β-Catenin Pathway and WASL Gene Expression in Preeclamptic Placentas. (A) The violin plot depicts WASP like actin nucleation promoting factor (WASL) gene expression in the GSE75010 dataset, comparing control (n = 77) and preeclampsia (n = 80) placentas. A significant difference is observed ( P = 0.0072). (B) Positive correlation between WNT/β-catenin pathway activity and WASL gene expression in GSE75010 (r = 0.36, P = 0.03). Histograms show expression distributions. (C) WASL expression positively correlated with WNT/β-catenin signaling (Pearson r = 0.36, P = 0.03), with moderate Bayesian support ( log BF = -0.83) in GSE204835 . (D) GSEA for WNT/β-catenin pathway in GSE204835 reveals significant enrichment in preeclampsia ( P = 0.02938, adj. P = 0.03938). (E) Hematoxylin staining and representative immunostaining of N-WASP, syncytin-2, GCM1, and CK7 protein in the control group and preeclamptic placenta. Scale bar, 50 μm. (F) Representative N-WASP, syncytin-2 and the GCM1 immunoblot of preeclamptic and placenta, normalized by β-actin. (G) WASL (N-WASP) and syncytin-2 mRNA expression levels were quantified using quantitative polymerase chain reaction (qPCR) in placental samples from the preeclampsia group (n = 30) and control group (n = 35). The analysis revealed a significant reduction in both WASL and syncytin-2 mRNA expression levels in the preeclampsia group compared with the control group. (H) WASL (N-WASP) mRNA expression is positively correlated with syncytin-2 mRNA expression. SYN-2, syncytin-2; ACTB, β-actin; CK7, Cytokeratin 7; PE, preeclampsia; ***, P < 0.001.

Article Snippet: The primary antibodies were diluted in phosphate-buffered saline (PBS): WASL Polyclonal antibody, 1:100; Rabbit Anti- ERVFRD-1 antibody, 1:100; GCM1 Polyclonal antibody, 1:50; Cytokeratin 7 Polyclonal antibody (1:100, Proteintech, China); E-cadherin Polyclonal antibody, 1:200.

Techniques: Expressing, Gene Expression, Control, Activity Assay, Staining, Immunostaining, Western Blot, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reduced Wiskott-Aldrich syndrome protein expression in preeclampsia placenta impairs trophoblast syncytialization by modulating syncytin-2 via FAK/β-catenin pathway

doi: 10.3389/fcell.2025.1678878

Figure Lengend Snippet: N-WASP protein expression during trophoblast cell syncytialization. (A) Western blot showed the expression of E-cadherin (ECAD), neural Wiskott‐Aldrich syndrome protein (N-WASP), β-catenin, LEF1, GCM1, and syncytin-2 (SYN2) protein in BeWo cells stimulated by forskolin (FSK) gradient concentration (0 μm, 10 μm, 25 μm, and 50 μm). Data were normalized to GADPH. (B) Quantitative real-time polymerase chain reaction (RT-qPCR) result showed the expression of β-hCG mRNA in BeWo cells stimulated by FSK gradient concentration. Data were normalized to GADPH. (C) Immunofluorescence staining of E-cadherin (red) and nuclei (DAPI, blue) showing changes in E-cadherin localization and expression in cells treated with gradient concentration of FSK. Dashed lines highlight cellular boundaries. Scale bar = 50 μm. (D) Immunofluorescence images of β-catenin (green) and nuclei (DAPI, blue) in 0μm (Control) and 25 μm forskolin-treated Bewo cells. FSK treatment results in increased nuclear β-catenin. Scale bar = 100 μm. (E) Western blot analysis of β-catenin in cytoplasmic (CE) and nuclear (NE) extracts from control and 25 μm FSK-treated cells, with Lamin B1 and GAPDH as nuclear and cytoplasmic markers, respectively. (F) Fusion index of cells treated with varying concentrations of FSK. **, P <0.01, ***, P < 0.001, NS = not significant.

Article Snippet: The primary antibodies were diluted in phosphate-buffered saline (PBS): WASL Polyclonal antibody, 1:100; Rabbit Anti- ERVFRD-1 antibody, 1:100; GCM1 Polyclonal antibody, 1:50; Cytokeratin 7 Polyclonal antibody (1:100, Proteintech, China); E-cadherin Polyclonal antibody, 1:200.

Techniques: Expressing, Western Blot, Concentration Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Staining, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reduced Wiskott-Aldrich syndrome protein expression in preeclampsia placenta impairs trophoblast syncytialization by modulating syncytin-2 via FAK/β-catenin pathway

doi: 10.3389/fcell.2025.1678878

Figure Lengend Snippet: Role of N-WASP in the forskolin-stimulated BeWo cells. (A) During the process of forskolin (FSK)-induced BeWo cell fusion, western blot showed the expression of neural Wiskott-Aldrich syndrome protein (N-WASP) was upregulated, and the expression levels of β-catenin and LEF1 proteins also increased, indicating the activation of the Wnt/β-catenin signaling pathway. Additionally, the expression levels of syncytin-2 and glial cells missing 1 (GCM1) were significantly elevated. (B) Quantitative polymerase chain reaction (qPCR) analysis revealed that β-HCG mRNA expression, normalized to GAPDH, across the same treatment groups, highlighting the impact of N-WASP knockdown on β-HCG transcription during FSK-induced BeWo cell fusion. (C) Immunofluorescence images of β-catenin (green) and nuclei (DAPI, blue) across treatments. FSK treatment alone increases the nuclear localization of β-catenin, which is reduced when N-WASP is knocked down (FSK + sh-N-WASP). Scale bar = 100 μm. (D) Immunofluorescence staining of E-cadherin (red) and nuclei (DAPI, blue) illustrating changes in E-cadherin expression and cellular boundary morphology across treatments. FSK enhances E-cadherin localization along cell-cell contacts, while sh-N-WASP alters this pattern. Scale bar = 50 μm. (E) Western blot of β-catenin in cytoplasmic (CE) and nuclear (NE) extracts under the same treatment conditions, with Lamin B1 and GAPDH as nuclear and cytoplasmic markers, respectively. FSK promotes β-catenin nuclear translocation, which is diminished by N-WASP knockdown. (F) Fusion index quantifying cell fusion percentage across treatments. FSK significantly increases cell fusion, an effect partially reduced by N-WASP knockdown. ECAD, E-cadherin; SYN-2, syncytin-2; ACTB, β-actin. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Article Snippet: The primary antibodies were diluted in phosphate-buffered saline (PBS): WASL Polyclonal antibody, 1:100; Rabbit Anti- ERVFRD-1 antibody, 1:100; GCM1 Polyclonal antibody, 1:50; Cytokeratin 7 Polyclonal antibody (1:100, Proteintech, China); E-cadherin Polyclonal antibody, 1:200.

Techniques: Western Blot, Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Knockdown, Immunofluorescence, Staining, Translocation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reduced Wiskott-Aldrich syndrome protein expression in preeclampsia placenta impairs trophoblast syncytialization by modulating syncytin-2 via FAK/β-catenin pathway

doi: 10.3389/fcell.2025.1678878

Figure Lengend Snippet: Illustration of how N-WASP promotes syncytin-2 expression to drive trophoblast syncytialization. N-WASP, through its interaction with MYO1B, activates the FAK signaling pathway, stabilizing β-catenin and promoting its nuclear translocation. In the nucleus, β-catenin enhances GCM1 expression, which drives syncytin-2 transcription. Syncytin-2 facilitates trophoblast fusion, forming the syncytial layer essential for syncytialization.

Article Snippet: The primary antibodies were diluted in phosphate-buffered saline (PBS): WASL Polyclonal antibody, 1:100; Rabbit Anti- ERVFRD-1 antibody, 1:100; GCM1 Polyclonal antibody, 1:50; Cytokeratin 7 Polyclonal antibody (1:100, Proteintech, China); E-cadherin Polyclonal antibody, 1:200.

Techniques: Expressing, Translocation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reduced Wiskott-Aldrich syndrome protein expression in preeclampsia placenta impairs trophoblast syncytialization by modulating syncytin-2 via FAK/β-catenin pathway

doi: 10.3389/fcell.2025.1678878

Figure Lengend Snippet: Establishment of preeclampsia rat model and expression changes of N-WASP, GCM1, and syncytin-2 in placental tissue. (A) Mean arterial pressure (MAP) is significantly elevated in the preeclampsia (PE) group from day 7 of pregnancy, confirming the development of hypertension characteristic of PE. (B) Urinary protein levels increase substantially in the PE group from day 11 of pregnancy. (C) Elevated serum creatinine levels in the PE group suggest renal dysfunction. (D) Histological and immunohistochemical analysis reveals disrupted placental architecture in the PE group, with reduced labyrinth zone (LZ) and decreased expression of neural Wiskott-Aldrich syndrome protein (N-WASP), syncytin-2 (SYN-2), and elevated expression of E-cadherin (ECAD). (E) Western blot results show significantly reduced N-WASP expression in the PE group. HE staining scale bar=100 μm, immunohistochemistry scale bar=50 μm. (F) Decreased syncytin-2 levels in the PE group. (G) GCM1 expression is significantly lower in the PE group.

Article Snippet: The primary antibodies were diluted in phosphate-buffered saline (PBS): WASL Polyclonal antibody, 1:100; Rabbit Anti- ERVFRD-1 antibody, 1:100; GCM1 Polyclonal antibody, 1:50; Cytokeratin 7 Polyclonal antibody (1:100, Proteintech, China); E-cadherin Polyclonal antibody, 1:200.

Techniques: Expressing, Immunohistochemical staining, Western Blot, Staining, Immunohistochemistry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reduced Wiskott-Aldrich syndrome protein expression in preeclampsia placenta impairs trophoblast syncytialization by modulating syncytin-2 via FAK/β-catenin pathway

doi: 10.3389/fcell.2025.1678878

Figure Lengend Snippet: Effects of N-WASP expression changes in a rat model of preeclampsia. (A) Western blot analysis of neural Wiskott-Aldrich syndrome protein (N-WASP), glial cells missing 1 (GCM1), and syncytin-2 (SYN-2) protein expression in BeWo cells treated with different concentrations of hydroxychloroquine (HCQ) (0, 2.5, 5, and 10 μM). The results indicate that HCQ upregulates N-WASP expression in a dose-dependent manner, with a concomitant significant increase in GCM1 and SYN-2 expression. **, P < 0.01, ***, P < 0.001, compared with the 0 μM group. (B) In a forskolin (FSK)-induced BeWo cell model, Western blot results show that N-WASP-shRNA significantly reduced N-WASP expression. However, HCQ treatment reversed this downregulation and concurrently upregulated GCM1 and SYN-2 expression. *, P < 0.05, **, P < 0.01, ***, P < 0.001. (C) Immunohistochemical analysis of N-WASP, SYN-2, and E-cadherin (E-CAD) expression in placental tissues from the control, control+HCQ, preeclampsia (PE), and PE+HCQ groups. Results reveal decreased N-WASP and SYN-2 expression and elevated E-CAD expression in the PE group. HCQ treatment restored N-WASP and SYN-2 levels while reducing E-CAD expression. (D) Mean arterial pressure (MAP) changes during pregnancy in each group. MAP was significantly elevated in the PE group, whereas HCQ treatment markedly reduced MAP levels in PE rats (# P < 0.05, ## P < 0.01 compared with the PE group)., compared with day 1 of pregnancy, **, P < 0.001; #, compared with the Control group at the same gestational day, #, P < 0.05, ##, P < 0.01, ###, P < 0.001; △, compared with the Control+HCQ group at the same gestational day; □, compared with the PE group at the same gestational day. (E) Changes in urinary protein concentration during pregnancy across groups. Urinary protein levels were significantly increased in the PE group, while HCQ treatment markedly reduced urinary protein levels (△ P < 0.05, △△ P < 0.01 compared with the PE group). , compared with day 1 of pregnancy, *, P < 0.01, ***, P < 0.001; #, compared with the Control group at the same gestational day, ##, P < 0.01; △, compared with the Control+HCQ group at the same gestational day, △, P < 0.05, △△△, P < 0.001; □, compared with the PE group at the same gestational day, □□, P < 0.01. (F) Comparison of serum creatinine levels among groups. Serum creatinine levels were significantly elevated in the PE group but were substantially decreased following HCQ treatment. *, P < 0.05, **, P < 0.01, ***, P < 0.001. (G) Western blot analysis shows downregulation of N-WASP, GCM1, and SYN-2 expression in the PE group. HCQ treatment effectively restored N-WASP expression and simultaneously upregulated GCM1 and SYN-2 levels. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Article Snippet: The primary antibodies were diluted in phosphate-buffered saline (PBS): WASL Polyclonal antibody, 1:100; Rabbit Anti- ERVFRD-1 antibody, 1:100; GCM1 Polyclonal antibody, 1:50; Cytokeratin 7 Polyclonal antibody (1:100, Proteintech, China); E-cadherin Polyclonal antibody, 1:200.

Techniques: Expressing, Western Blot, shRNA, Immunohistochemical staining, Control, Protein Concentration, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reduced Wiskott-Aldrich syndrome protein expression in preeclampsia placenta impairs trophoblast syncytialization by modulating syncytin-2 via FAK/β-catenin pathway

doi: 10.3389/fcell.2025.1678878

Figure Lengend Snippet: Reduced Expression of N-WASP and Syncytin-2 and Analysis of WNT/β-Catenin Pathway and WASL Gene Expression in Preeclamptic Placentas. (A) The violin plot depicts WASP like actin nucleation promoting factor (WASL) gene expression in the GSE75010 dataset, comparing control (n = 77) and preeclampsia (n = 80) placentas. A significant difference is observed ( P = 0.0072). (B) Positive correlation between WNT/β-catenin pathway activity and WASL gene expression in GSE75010 (r = 0.36, P = 0.03). Histograms show expression distributions. (C) WASL expression positively correlated with WNT/β-catenin signaling (Pearson r = 0.36, P = 0.03), with moderate Bayesian support ( log BF = -0.83) in GSE204835 . (D) GSEA for WNT/β-catenin pathway in GSE204835 reveals significant enrichment in preeclampsia ( P = 0.02938, adj. P = 0.03938). (E) Hematoxylin staining and representative immunostaining of N-WASP, syncytin-2, GCM1, and CK7 protein in the control group and preeclamptic placenta. Scale bar, 50 μm. (F) Representative N-WASP, syncytin-2 and the GCM1 immunoblot of preeclamptic and placenta, normalized by β-actin. (G) WASL (N-WASP) and syncytin-2 mRNA expression levels were quantified using quantitative polymerase chain reaction (qPCR) in placental samples from the preeclampsia group (n = 30) and control group (n = 35). The analysis revealed a significant reduction in both WASL and syncytin-2 mRNA expression levels in the preeclampsia group compared with the control group. (H) WASL (N-WASP) mRNA expression is positively correlated with syncytin-2 mRNA expression. SYN-2, syncytin-2; ACTB, β-actin; CK7, Cytokeratin 7; PE, preeclampsia; ***, P < 0.001.

Article Snippet: The concentrations of antibodies were WASL Polyclonal antibody (1:2000, 14306-1-AP, Proteintech); Rabbit Anti- ERVFRD-1 antibody (1:2000, bs-15466R, Bioss), GAPDH Monoclonal antibody (1:3000, 60004-1-Ig, Proteintech); GCM1 Polyclonal antibody, (1:1000, 21724-1-AP, Proteintech); E-cadherin Polyclonal antibody (1:1000, 20874-1-AP, Proteintech); Beta Catenin Polyclonal antibody (1:1000, 51067-2-AP, Proteintech); GSK3β Polyclonal Antibody (1:1000, YT2082, Immunoway); GSK3β(phospho Ser9) Polyclonal Antibody (1:1000, YP0124, Immunoway); Lymphoid Enhancer-Binding Factor (LEF1) Antibody (N-term) (1:1000, AP12048A, Abcepta); β-actin (1:20000, 66009-1-Ig, Proteintech).

Techniques: Expressing, Gene Expression, Control, Activity Assay, Staining, Immunostaining, Western Blot, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reduced Wiskott-Aldrich syndrome protein expression in preeclampsia placenta impairs trophoblast syncytialization by modulating syncytin-2 via FAK/β-catenin pathway

doi: 10.3389/fcell.2025.1678878

Figure Lengend Snippet: Role of N-WASP in the forskolin-stimulated BeWo cells. (A) During the process of forskolin (FSK)-induced BeWo cell fusion, western blot showed the expression of neural Wiskott-Aldrich syndrome protein (N-WASP) was upregulated, and the expression levels of β-catenin and LEF1 proteins also increased, indicating the activation of the Wnt/β-catenin signaling pathway. Additionally, the expression levels of syncytin-2 and glial cells missing 1 (GCM1) were significantly elevated. (B) Quantitative polymerase chain reaction (qPCR) analysis revealed that β-HCG mRNA expression, normalized to GAPDH, across the same treatment groups, highlighting the impact of N-WASP knockdown on β-HCG transcription during FSK-induced BeWo cell fusion. (C) Immunofluorescence images of β-catenin (green) and nuclei (DAPI, blue) across treatments. FSK treatment alone increases the nuclear localization of β-catenin, which is reduced when N-WASP is knocked down (FSK + sh-N-WASP). Scale bar = 100 μm. (D) Immunofluorescence staining of E-cadherin (red) and nuclei (DAPI, blue) illustrating changes in E-cadherin expression and cellular boundary morphology across treatments. FSK enhances E-cadherin localization along cell-cell contacts, while sh-N-WASP alters this pattern. Scale bar = 50 μm. (E) Western blot of β-catenin in cytoplasmic (CE) and nuclear (NE) extracts under the same treatment conditions, with Lamin B1 and GAPDH as nuclear and cytoplasmic markers, respectively. FSK promotes β-catenin nuclear translocation, which is diminished by N-WASP knockdown. (F) Fusion index quantifying cell fusion percentage across treatments. FSK significantly increases cell fusion, an effect partially reduced by N-WASP knockdown. ECAD, E-cadherin; SYN-2, syncytin-2; ACTB, β-actin. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Article Snippet: The concentrations of antibodies were WASL Polyclonal antibody (1:2000, 14306-1-AP, Proteintech); Rabbit Anti- ERVFRD-1 antibody (1:2000, bs-15466R, Bioss), GAPDH Monoclonal antibody (1:3000, 60004-1-Ig, Proteintech); GCM1 Polyclonal antibody, (1:1000, 21724-1-AP, Proteintech); E-cadherin Polyclonal antibody (1:1000, 20874-1-AP, Proteintech); Beta Catenin Polyclonal antibody (1:1000, 51067-2-AP, Proteintech); GSK3β Polyclonal Antibody (1:1000, YT2082, Immunoway); GSK3β(phospho Ser9) Polyclonal Antibody (1:1000, YP0124, Immunoway); Lymphoid Enhancer-Binding Factor (LEF1) Antibody (N-term) (1:1000, AP12048A, Abcepta); β-actin (1:20000, 66009-1-Ig, Proteintech).

Techniques: Western Blot, Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Knockdown, Immunofluorescence, Staining, Translocation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reduced Wiskott-Aldrich syndrome protein expression in preeclampsia placenta impairs trophoblast syncytialization by modulating syncytin-2 via FAK/β-catenin pathway

doi: 10.3389/fcell.2025.1678878

Figure Lengend Snippet: The fusion-promoting effect of N-WASP Overexpression in BeWo Cells is significantly attenuated by the Wnt/β-catenin pathway inhibitor XAV-939. (A) Western blot analysis of protein levels in BeWo cells under different treatment conditions. neural Wiskott-Aldrich syndrome protein (N-WASP) overexpression significantly increased the expression of fusion-related markers, while XAV-939 treatment counteracted this effect. (B) N-WASP overexpression led to a significant increase in β- human chorionic gonadotropin (HCG) mRNA levels, which was reduced upon co-treatment with XAV-939. (C) Quantification of fusion index (%) is shown in the adjacent bar graph. Forskolin-induced fusion was enhanced by N-WASP overexpression; however, XAV-939 treatment partially reversed this effect. (D) Immunofluorescence staining for E-cadherin (E-cad; red) and DAPI nuclear stain (blue) in BeWo cells across different conditions. Merged images indicate cell-cell adhesion structures and cellular fusion events. In the Control and N-WASP overexpression (OE) groups, E-cadherin staining reveals diminished continuity of E-cadherin staining along cell borders, with prominent fusion in the N-WASP OE group. XAV-939 treatment (both alone and with N-WASP OE) leads to reduced cell fusion, as evidenced by distinct cell-cell borders of E-cadherin staining along cell borders. Scale bars represent 50 μm. **, P <0.01, ***, P <0.001.

Article Snippet: The concentrations of antibodies were WASL Polyclonal antibody (1:2000, 14306-1-AP, Proteintech); Rabbit Anti- ERVFRD-1 antibody (1:2000, bs-15466R, Bioss), GAPDH Monoclonal antibody (1:3000, 60004-1-Ig, Proteintech); GCM1 Polyclonal antibody, (1:1000, 21724-1-AP, Proteintech); E-cadherin Polyclonal antibody (1:1000, 20874-1-AP, Proteintech); Beta Catenin Polyclonal antibody (1:1000, 51067-2-AP, Proteintech); GSK3β Polyclonal Antibody (1:1000, YT2082, Immunoway); GSK3β(phospho Ser9) Polyclonal Antibody (1:1000, YP0124, Immunoway); Lymphoid Enhancer-Binding Factor (LEF1) Antibody (N-term) (1:1000, AP12048A, Abcepta); β-actin (1:20000, 66009-1-Ig, Proteintech).

Techniques: Over Expression, Western Blot, Expressing, Immunofluorescence, Staining, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reduced Wiskott-Aldrich syndrome protein expression in preeclampsia placenta impairs trophoblast syncytialization by modulating syncytin-2 via FAK/β-catenin pathway

doi: 10.3389/fcell.2025.1678878

Figure Lengend Snippet: Interaction between N-WASP and MYO1B activates the FAK/β-catenin signaling pathway. (A) Identification and analysis of neural Wiskott-Aldrich syndrome protein (N-WASP) interacting proteins in N-WASP overexpressing BeWo cell models using co-immunoprecipitation coupled with mass spectrometry. Gene ontology (GO) enrichment analysis results are presented as a bar chart, showing that the enriched functions are primarily associated with protein binding, cytoskeletal organization, and cellular component biogenesis. (B) Functional enrichment bubble plot of myosin 1B (MYO1B), displaying various biological interaction networks associated with cell fusion. Each bubble represents a significantly enriched GO term, with the bubble size and color indicating the level of enrichment. (C) Molecular docking analysis of MYO1B and N-WASP binding using the HDOCK server, with the binding interface visualized in surface representation. MYO1B is shown in yellow, N-WASP in purple, and the protein-protein contact area in red. Interface interactions analyzed by Protein-Ligand Interaction Profiler (PLIP) are as follows: (A) A salt bridge is formed between ARG-746 of MYO1B and GLU-271 of N-WASP (yellow dashed line), along with hydrogen bonds between ARG-746 and N-WASP residues ARG-274 and ASN-270 (blue solid lines). Additional hydrogen bonds are observed between GLN-749 and LYS-745 of MYO1B and GLU-266 of N-WASP, as well as between TYR-738 of MYO1B and ASN-270 of N-WASP. (B) A hydrogen bond is formed between SER-752 of MYO1B and GLN-210 of N-WASP. (C) Hydrogen bonds are observed between ALA-111 of N-WASP and HIS-775 of MYO1B, ARG-778 of MYO1B and TYR-95 of N-WASP, ARG-711 of MYO1B with ASN-97 and LEU-138 of N-WASP, and ARG-767 of MYO1B with GLN-142 and GLU-146 of N-WASP, forming additional hydrogen bonds and salt bridges. Gray dashed lines indicate regions of multiple hydrophobic interactions. (D) Immunofluorescence staining shows colocalization of N-WASP (green) and MYO1B (red) within cells. DAPI staining marks the cell nuclei (blue). The merged image indicates colocalization of N-WASP and MYO1B, suggesting a possible interaction between them. Scale bars represent 50 μm. (E) Co-immunoprecipitation experiment verifies the interaction between N-WASP and MYO1B in BeWo cells. The left panel shows the detection of N-WASP after MYO1B immunoprecipitation (IP), and the right panel shows the detection of MYO1B after N-WASP immunoprecipitation, confirming a physical interaction between the two proteins in cells. (F) Western blot analysis of the effects of N-WASP overexpression and MYO1B knockdown on the FAK/β-catenin signaling pathway. Results indicate significant changes in the phosphorylation levels of FAK (p-FAK/FAK), GSK-3β (p-GSK-3β/GSK-3β), and β-catenin expression following MYO1B knockdown. **, P <0.01, ***, P <0.001.

Article Snippet: The concentrations of antibodies were WASL Polyclonal antibody (1:2000, 14306-1-AP, Proteintech); Rabbit Anti- ERVFRD-1 antibody (1:2000, bs-15466R, Bioss), GAPDH Monoclonal antibody (1:3000, 60004-1-Ig, Proteintech); GCM1 Polyclonal antibody, (1:1000, 21724-1-AP, Proteintech); E-cadherin Polyclonal antibody (1:1000, 20874-1-AP, Proteintech); Beta Catenin Polyclonal antibody (1:1000, 51067-2-AP, Proteintech); GSK3β Polyclonal Antibody (1:1000, YT2082, Immunoway); GSK3β(phospho Ser9) Polyclonal Antibody (1:1000, YP0124, Immunoway); Lymphoid Enhancer-Binding Factor (LEF1) Antibody (N-term) (1:1000, AP12048A, Abcepta); β-actin (1:20000, 66009-1-Ig, Proteintech).

Techniques: Immunoprecipitation, Mass Spectrometry, Protein Binding, Functional Assay, Binding Assay, Immunofluorescence, Staining, Western Blot, Over Expression, Knockdown, Phospho-proteomics, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reduced Wiskott-Aldrich syndrome protein expression in preeclampsia placenta impairs trophoblast syncytialization by modulating syncytin-2 via FAK/β-catenin pathway

doi: 10.3389/fcell.2025.1678878

Figure Lengend Snippet: Illustration of how N-WASP promotes syncytin-2 expression to drive trophoblast syncytialization. N-WASP, through its interaction with MYO1B, activates the FAK signaling pathway, stabilizing β-catenin and promoting its nuclear translocation. In the nucleus, β-catenin enhances GCM1 expression, which drives syncytin-2 transcription. Syncytin-2 facilitates trophoblast fusion, forming the syncytial layer essential for syncytialization.

Article Snippet: The concentrations of antibodies were WASL Polyclonal antibody (1:2000, 14306-1-AP, Proteintech); Rabbit Anti- ERVFRD-1 antibody (1:2000, bs-15466R, Bioss), GAPDH Monoclonal antibody (1:3000, 60004-1-Ig, Proteintech); GCM1 Polyclonal antibody, (1:1000, 21724-1-AP, Proteintech); E-cadherin Polyclonal antibody (1:1000, 20874-1-AP, Proteintech); Beta Catenin Polyclonal antibody (1:1000, 51067-2-AP, Proteintech); GSK3β Polyclonal Antibody (1:1000, YT2082, Immunoway); GSK3β(phospho Ser9) Polyclonal Antibody (1:1000, YP0124, Immunoway); Lymphoid Enhancer-Binding Factor (LEF1) Antibody (N-term) (1:1000, AP12048A, Abcepta); β-actin (1:20000, 66009-1-Ig, Proteintech).

Techniques: Expressing, Translocation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reduced Wiskott-Aldrich syndrome protein expression in preeclampsia placenta impairs trophoblast syncytialization by modulating syncytin-2 via FAK/β-catenin pathway

doi: 10.3389/fcell.2025.1678878

Figure Lengend Snippet: Establishment of preeclampsia rat model and expression changes of N-WASP, GCM1, and syncytin-2 in placental tissue. (A) Mean arterial pressure (MAP) is significantly elevated in the preeclampsia (PE) group from day 7 of pregnancy, confirming the development of hypertension characteristic of PE. (B) Urinary protein levels increase substantially in the PE group from day 11 of pregnancy. (C) Elevated serum creatinine levels in the PE group suggest renal dysfunction. (D) Histological and immunohistochemical analysis reveals disrupted placental architecture in the PE group, with reduced labyrinth zone (LZ) and decreased expression of neural Wiskott-Aldrich syndrome protein (N-WASP), syncytin-2 (SYN-2), and elevated expression of E-cadherin (ECAD). (E) Western blot results show significantly reduced N-WASP expression in the PE group. HE staining scale bar=100 μm, immunohistochemistry scale bar=50 μm. (F) Decreased syncytin-2 levels in the PE group. (G) GCM1 expression is significantly lower in the PE group.

Article Snippet: The concentrations of antibodies were WASL Polyclonal antibody (1:2000, 14306-1-AP, Proteintech); Rabbit Anti- ERVFRD-1 antibody (1:2000, bs-15466R, Bioss), GAPDH Monoclonal antibody (1:3000, 60004-1-Ig, Proteintech); GCM1 Polyclonal antibody, (1:1000, 21724-1-AP, Proteintech); E-cadherin Polyclonal antibody (1:1000, 20874-1-AP, Proteintech); Beta Catenin Polyclonal antibody (1:1000, 51067-2-AP, Proteintech); GSK3β Polyclonal Antibody (1:1000, YT2082, Immunoway); GSK3β(phospho Ser9) Polyclonal Antibody (1:1000, YP0124, Immunoway); Lymphoid Enhancer-Binding Factor (LEF1) Antibody (N-term) (1:1000, AP12048A, Abcepta); β-actin (1:20000, 66009-1-Ig, Proteintech).

Techniques: Expressing, Immunohistochemical staining, Western Blot, Staining, Immunohistochemistry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reduced Wiskott-Aldrich syndrome protein expression in preeclampsia placenta impairs trophoblast syncytialization by modulating syncytin-2 via FAK/β-catenin pathway

doi: 10.3389/fcell.2025.1678878

Figure Lengend Snippet: Effects of N-WASP expression changes in a rat model of preeclampsia. (A) Western blot analysis of neural Wiskott-Aldrich syndrome protein (N-WASP), glial cells missing 1 (GCM1), and syncytin-2 (SYN-2) protein expression in BeWo cells treated with different concentrations of hydroxychloroquine (HCQ) (0, 2.5, 5, and 10 μM). The results indicate that HCQ upregulates N-WASP expression in a dose-dependent manner, with a concomitant significant increase in GCM1 and SYN-2 expression. **, P < 0.01, ***, P < 0.001, compared with the 0 μM group. (B) In a forskolin (FSK)-induced BeWo cell model, Western blot results show that N-WASP-shRNA significantly reduced N-WASP expression. However, HCQ treatment reversed this downregulation and concurrently upregulated GCM1 and SYN-2 expression. *, P < 0.05, **, P < 0.01, ***, P < 0.001. (C) Immunohistochemical analysis of N-WASP, SYN-2, and E-cadherin (E-CAD) expression in placental tissues from the control, control+HCQ, preeclampsia (PE), and PE+HCQ groups. Results reveal decreased N-WASP and SYN-2 expression and elevated E-CAD expression in the PE group. HCQ treatment restored N-WASP and SYN-2 levels while reducing E-CAD expression. (D) Mean arterial pressure (MAP) changes during pregnancy in each group. MAP was significantly elevated in the PE group, whereas HCQ treatment markedly reduced MAP levels in PE rats (# P < 0.05, ## P < 0.01 compared with the PE group)., compared with day 1 of pregnancy, **, P < 0.001; #, compared with the Control group at the same gestational day, #, P < 0.05, ##, P < 0.01, ###, P < 0.001; △, compared with the Control+HCQ group at the same gestational day; □, compared with the PE group at the same gestational day. (E) Changes in urinary protein concentration during pregnancy across groups. Urinary protein levels were significantly increased in the PE group, while HCQ treatment markedly reduced urinary protein levels (△ P < 0.05, △△ P < 0.01 compared with the PE group). , compared with day 1 of pregnancy, *, P < 0.01, ***, P < 0.001; #, compared with the Control group at the same gestational day, ##, P < 0.01; △, compared with the Control+HCQ group at the same gestational day, △, P < 0.05, △△△, P < 0.001; □, compared with the PE group at the same gestational day, □□, P < 0.01. (F) Comparison of serum creatinine levels among groups. Serum creatinine levels were significantly elevated in the PE group but were substantially decreased following HCQ treatment. *, P < 0.05, **, P < 0.01, ***, P < 0.001. (G) Western blot analysis shows downregulation of N-WASP, GCM1, and SYN-2 expression in the PE group. HCQ treatment effectively restored N-WASP expression and simultaneously upregulated GCM1 and SYN-2 levels. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Article Snippet: The concentrations of antibodies were WASL Polyclonal antibody (1:2000, 14306-1-AP, Proteintech); Rabbit Anti- ERVFRD-1 antibody (1:2000, bs-15466R, Bioss), GAPDH Monoclonal antibody (1:3000, 60004-1-Ig, Proteintech); GCM1 Polyclonal antibody, (1:1000, 21724-1-AP, Proteintech); E-cadherin Polyclonal antibody (1:1000, 20874-1-AP, Proteintech); Beta Catenin Polyclonal antibody (1:1000, 51067-2-AP, Proteintech); GSK3β Polyclonal Antibody (1:1000, YT2082, Immunoway); GSK3β(phospho Ser9) Polyclonal Antibody (1:1000, YP0124, Immunoway); Lymphoid Enhancer-Binding Factor (LEF1) Antibody (N-term) (1:1000, AP12048A, Abcepta); β-actin (1:20000, 66009-1-Ig, Proteintech).

Techniques: Expressing, Western Blot, shRNA, Immunohistochemical staining, Control, Protein Concentration, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reduced Wiskott-Aldrich syndrome protein expression in preeclampsia placenta impairs trophoblast syncytialization by modulating syncytin-2 via FAK/β-catenin pathway

doi: 10.3389/fcell.2025.1678878

Figure Lengend Snippet: N-WASP protein expression during trophoblast cell syncytialization. (A) Western blot showed the expression of E-cadherin (ECAD), neural Wiskott‐Aldrich syndrome protein (N-WASP), β-catenin, LEF1, GCM1, and syncytin-2 (SYN2) protein in BeWo cells stimulated by forskolin (FSK) gradient concentration (0 μm, 10 μm, 25 μm, and 50 μm). Data were normalized to GADPH. (B) Quantitative real-time polymerase chain reaction (RT-qPCR) result showed the expression of β-hCG mRNA in BeWo cells stimulated by FSK gradient concentration. Data were normalized to GADPH. (C) Immunofluorescence staining of E-cadherin (red) and nuclei (DAPI, blue) showing changes in E-cadherin localization and expression in cells treated with gradient concentration of FSK. Dashed lines highlight cellular boundaries. Scale bar = 50 μm. (D) Immunofluorescence images of β-catenin (green) and nuclei (DAPI, blue) in 0μm (Control) and 25 μm forskolin-treated Bewo cells. FSK treatment results in increased nuclear β-catenin. Scale bar = 100 μm. (E) Western blot analysis of β-catenin in cytoplasmic (CE) and nuclear (NE) extracts from control and 25 μm FSK-treated cells, with Lamin B1 and GAPDH as nuclear and cytoplasmic markers, respectively. (F) Fusion index of cells treated with varying concentrations of FSK. **, P <0.01, ***, P < 0.001, NS = not significant.

Article Snippet: The concentrations of antibodies were WASL Polyclonal antibody (1:2000, 14306-1-AP, Proteintech); Rabbit Anti- ERVFRD-1 antibody (1:2000, bs-15466R, Bioss), GAPDH Monoclonal antibody (1:3000, 60004-1-Ig, Proteintech); GCM1 Polyclonal antibody, (1:1000, 21724-1-AP, Proteintech); E-cadherin Polyclonal antibody (1:1000, 20874-1-AP, Proteintech); Beta Catenin Polyclonal antibody (1:1000, 51067-2-AP, Proteintech); GSK3β Polyclonal Antibody (1:1000, YT2082, Immunoway); GSK3β(phospho Ser9) Polyclonal Antibody (1:1000, YP0124, Immunoway); Lymphoid Enhancer-Binding Factor (LEF1) Antibody (N-term) (1:1000, AP12048A, Abcepta); β-actin (1:20000, 66009-1-Ig, Proteintech).

Techniques: Expressing, Western Blot, Concentration Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Staining, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Reduced Wiskott-Aldrich syndrome protein expression in preeclampsia placenta impairs trophoblast syncytialization by modulating syncytin-2 via FAK/β-catenin pathway

doi: 10.3389/fcell.2025.1678878

Figure Lengend Snippet: Role of N-WASP in the forskolin-stimulated BeWo cells. (A) During the process of forskolin (FSK)-induced BeWo cell fusion, western blot showed the expression of neural Wiskott-Aldrich syndrome protein (N-WASP) was upregulated, and the expression levels of β-catenin and LEF1 proteins also increased, indicating the activation of the Wnt/β-catenin signaling pathway. Additionally, the expression levels of syncytin-2 and glial cells missing 1 (GCM1) were significantly elevated. (B) Quantitative polymerase chain reaction (qPCR) analysis revealed that β-HCG mRNA expression, normalized to GAPDH, across the same treatment groups, highlighting the impact of N-WASP knockdown on β-HCG transcription during FSK-induced BeWo cell fusion. (C) Immunofluorescence images of β-catenin (green) and nuclei (DAPI, blue) across treatments. FSK treatment alone increases the nuclear localization of β-catenin, which is reduced when N-WASP is knocked down (FSK + sh-N-WASP). Scale bar = 100 μm. (D) Immunofluorescence staining of E-cadherin (red) and nuclei (DAPI, blue) illustrating changes in E-cadherin expression and cellular boundary morphology across treatments. FSK enhances E-cadherin localization along cell-cell contacts, while sh-N-WASP alters this pattern. Scale bar = 50 μm. (E) Western blot of β-catenin in cytoplasmic (CE) and nuclear (NE) extracts under the same treatment conditions, with Lamin B1 and GAPDH as nuclear and cytoplasmic markers, respectively. FSK promotes β-catenin nuclear translocation, which is diminished by N-WASP knockdown. (F) Fusion index quantifying cell fusion percentage across treatments. FSK significantly increases cell fusion, an effect partially reduced by N-WASP knockdown. ECAD, E-cadherin; SYN-2, syncytin-2; ACTB, β-actin. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Article Snippet: The concentrations of antibodies were WASL Polyclonal antibody (1:2000, 14306-1-AP, Proteintech); Rabbit Anti- ERVFRD-1 antibody (1:2000, bs-15466R, Bioss), GAPDH Monoclonal antibody (1:3000, 60004-1-Ig, Proteintech); GCM1 Polyclonal antibody, (1:1000, 21724-1-AP, Proteintech); E-cadherin Polyclonal antibody (1:1000, 20874-1-AP, Proteintech); Beta Catenin Polyclonal antibody (1:1000, 51067-2-AP, Proteintech); GSK3β Polyclonal Antibody (1:1000, YT2082, Immunoway); GSK3β(phospho Ser9) Polyclonal Antibody (1:1000, YP0124, Immunoway); Lymphoid Enhancer-Binding Factor (LEF1) Antibody (N-term) (1:1000, AP12048A, Abcepta); β-actin (1:20000, 66009-1-Ig, Proteintech).

Techniques: Western Blot, Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Knockdown, Immunofluorescence, Staining, Translocation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: OVO-like 1 regulates progenitor cell fate in human trophoblast development

doi: 10.1073/pnas.1507397112

Figure Lengend Snippet: OVOL1 represses genes that promote the trophoblast progenitor state. (A and B) qRT-PCR (A) and Western blot (B) analysis of MYC, TP63, ID1, and ASCL2 transcript and protein levels, respectively, in BeWo trophoblast cells cultured under standard conditions (Undiff, 0 h) or following 6-, 24-, or 48-h exposure to differentiating (Diff) conditions. TFIID was used as a loading control. (C) Phospho-histone H3 immunofluorescence in BeWo trophoblast cells cultured in undifferentiated or differentiating conditions. DAPI was used to identify nuclei. The percentage of phospho-histone H3-positive nuclei is shown on the right. (D) Protein expression of MYC, TP63, ID1, and ASCL2 in BeWo trophoblast cells expressing shCTRL, shOVOL1-a, or shOVOL1-b maintained in undifferentiated or differentiating conditions. (E) Immunofluorescence of MYC, ID1, TP63, and ASCL2 in 8-wk human placenta. Antibodies that detect PCNA and CGB were used to identify proliferating cytotrophoblast cells and syncytiotrophoblast, respectively. (Scale bars, 50 μm.) (F) ChIP analysis using antibodies for OVOL1 or IgG (negative control) in lysates of BeWo trophoblast cells maintained in undifferentiated or differentiating conditions. *P < 0.05, **P < 0.05, n ≥ 3.

Article Snippet: Membranes were subsequently probed using antibodies for OVOL1 (1 μg/mL; clone {"type":"entrez-protein","attrs":{"text":"ARP38500","term_id":"1190169411","term_text":"ARP38500"}} ARP38500 ; Aviva Systems Biology), ERVFRD-1 (1 μg/mL; ARP56008_050; Aviva Systems Biology), MYC (1:1,000; clone D84C12; Cell Signaling Technology), TP63α (1:1,000; 4892, Cell Signaling Technology), ID1 (1:1,000; clone EPR7098; Abcam), ASCL2 (1 μg/mL; AF6539; R&D Systems), GCM1 (0.1 μg/mL; HPA011343; Sigma-Aldrich), transcription factor II D (TFIID) (0.2 μg/mL; sc-56794; Santa Cruz Biotechnology), and α-tubulin (0.05 μg/mL; clone CP06; EMD-Millipore Corp.).

Techniques: Quantitative RT-PCR, Western Blot, Cell Culture, Control, Immunofluorescence, Expressing, Negative Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: OVO-like 1 regulates progenitor cell fate in human trophoblast development

doi: 10.1073/pnas.1507397112

Figure Lengend Snippet: Effect of OVOL1 knockdown in primary cytotrophoblast cells on genes linked with the cytotrophoblast progenitor state. Transcript expression of MYC, ID1, TP63, and ASCL2 in primary cytotrophoblasts transduced with shRNAs targeting OVOL1 (shOVOL1). *P < 0.05; n = 6.

Article Snippet: Membranes were subsequently probed using antibodies for OVOL1 (1 μg/mL; clone {"type":"entrez-protein","attrs":{"text":"ARP38500","term_id":"1190169411","term_text":"ARP38500"}} ARP38500 ; Aviva Systems Biology), ERVFRD-1 (1 μg/mL; ARP56008_050; Aviva Systems Biology), MYC (1:1,000; clone D84C12; Cell Signaling Technology), TP63α (1:1,000; 4892, Cell Signaling Technology), ID1 (1:1,000; clone EPR7098; Abcam), ASCL2 (1 μg/mL; AF6539; R&D Systems), GCM1 (0.1 μg/mL; HPA011343; Sigma-Aldrich), transcription factor II D (TFIID) (0.2 μg/mL; sc-56794; Santa Cruz Biotechnology), and α-tubulin (0.05 μg/mL; clone CP06; EMD-Millipore Corp.).

Techniques: Knockdown, Expressing, Transduction