Fig. 4

Activin A increases human primary EVT migration and human villous explant outgrowth by upregulating fibronectin expression through ALK4-SMAD4 signaling. A, Cellular morphology of primary EVT outgrowth (left panel, white arrow indicates the fragment of villous tissue) and purity of the primary human EVTs tested by IF staining for HLA-G (right panel). B, IF staining for fibronectin in human primary EVTs after transfection for 48 h with 20 nM si-Ctrl or si-FN1. The knockdown efficiency of fibronectin was evaluated by quantification of fibronectin IF integrated density (IntDen) normalized to Hoechst IF IntDen using ImageJ software. C and D, Human primary EVTs were transfected for 48 h with 20 nM si-Ctrl or si-FN1 prior to treatment with Ctrl or 50 ng/mL activin A for an additional 24 h. The expression levels of FN1 mRNA (C) and fibronectin protein (D) were examined by RT–qPCR and Western blot. E, Human primary EVT migration was examined by wound healing assay. Representative photographs of wound healing assays were taken at 0, 12 and 24 h. F, An ex vivo extravillous explant culture model was employed to further explore the role of activin A and fibronectin in the function of the human first trimester placenta. The placental villous explants were transfected with si-Ctrl or si-FN1 after 24 h of culture, followed by Ctrl or 50 ng/mL activin A for an additional 24 h. Representative images were taken at 24 and 48 h of culture, and the right panel shows summarized quantitative results from image analysis of the explant outgrowth area. G, Human primary EVTs were transfected with 20 nM si-Ctrl, 20 nM si-ALK4, or 20 nM si-SMAD4 and then treated with a vehicle control or 50 ng/mL activin A for an additional 24 h. Protein levels of SMAD4 and fibronectin were measured by Western blot. The results are presented as the mean ± SEM of three independent experiments. The P value in (B) was calculated by two-tailed Student’s t test. ANOVA was used for grouped analyses in (C)-(F). Groups without letters in common are significantly different from each other (P < 0.05). P values of primary interest are denoted as * < 0.05, ** < 0.01, and *** < 0.001