Journal: Genes & Diseases
Article Title: Unraveling the role of the WIPF1/ACTN4 complex in podosome formation of human placental EVTs: Insights into recurrent spontaneous abortion
doi: 10.1016/j.gendis.2025.101665
Figure Lengend Snippet: WIPF1-ACTN4 interaction and its role in podosome function during extravillous trophoblast (EVT) invasion. (A) Venn analysis of the WIPF1 interactome identified in the WIPF1-OE HTR-8/SVneo cells. The light red and grey represent anti-WIPF1 and anti-IgG immunoprecipitates, respectively. The dark red represents the non-specific interacting protein of the WIPF1 immunoprecipitates. (B) Protein–protein interaction network via STRING. Nodes, circles. Edges, lines. (C) WIPF1 and ACTN4 immunoprecipitates stained with ACTN4 and WIPF1 in WIPF1-OE HTR8/SVneo cells. (D – F) Immunofluorescence images of the expression of ACTN4 (green) and WIPF1 (red) in HTR-8/SVneo cells. The blue colors represent nuclei stained with Hoechst. Scale bars, 10 μm. Colocalization analysis was performed, and Pearson's correlation coefficient was calculated to assess their interaction. A gray value analysis plot of fluorescence intensity along the crossed-out distance is shown. (G) mRNA levels of ACTN4 in human trophoblast stem cells (hTSCs) and EVTs, relative to the mRNA levels of GADPH. ∗∗ p < 0.01. (H) Protein levels of ACTN4 in hTSCs and EVTs. (I) Immunofluorescence images of the expression of ACTN4 in hTSC and EVT differentiation. Scale bars, 25 μm. (J) Protein levels of WIPF1 and ACTN4 after single and double knockdown of WIPF1 and ACTN4 in HTR-8/SVneo cells. (K) Protein levels of invasion markers after single and double knockdown of WIPF1 and ACTN4 in HTR-8/SVneo cells. (L) Actin polymerization assay was conducted using HTR-8/SVneo cells stably transfected with lentiviral vectors containing shRNA constructs targeting WIPF1 (WIPF1 knockdown), ACTN4 (ACTN4 knockdown), WIPF1 and ACTN4 (WIPF1/ACTN4 double knockdown), and vector control groups. The assay monitored the kinetics of pyrene actin polymerization in 60 min. The experiment was performed in triplicate. (M) Immunofluorescence images for F-actin (red), Oregon Green® 488 conjugate gelatin (green), cortactin (white), and nuclei (blue) in HTR-8/SVneo cells with WIPF1 and/or ACTN4 knockdown following incubation for 12 h on the matrix. The black areas mark areas of podosome-mediated degradation, which appear beneath the cells. Scale bars, 10 μm. (N) Quantitative analysis of the relative area of gelatin degradation zones associated with podosomes for different treatment groups. n = 30 fields; ∗∗ p < 0.01 and ∗∗∗ p < 0.001. (O) Quantitative evaluation of the proportion of cells containing podosome-associated gelatin degradation area for different treatment groups. n = 30 fields; ∗ p < 0.05 and ∗∗ p < 0.01. ACTN4, alpha-actinin 4; WIPF1, WAS/WASL interacting protein family member 1.
Article Snippet: The actin polymerization assay was performed following the manufacturer's instructions (BK003, Cytoskeleton, CO, USA) , , to assess the impact of specific protein silencing on actin polymerization in HTR-8/SVneo cells.
Techniques: Staining, Immunofluorescence, Expressing, Fluorescence, Knockdown, Polymerization Assay, Stable Transfection, Transfection, shRNA, Construct, Plasmid Preparation, Control, Incubation
Cytoskeleton Inc is a verified supplier 
Cytoskeleton Inc manufactures this product 
