Fig. 2

Fibronectin is involved in activin A-induced cell migration and endothelial-like tube formation in HTR8/SVneo cells. A and B, HTR8/SVneo cells were transfected for 48 h with 20 nM ON-TARGETplus Non-targeting Control Pool (si-Ctrl) or ON-TARGETplus SMARTpool siRNAs targeting human FN1 (si-FN1) prior to treatment with Ctrl or 50 ng/mL activin A for an additional 24 h. The expression levels of FN1 mRNA (A) and fibronectin protein (B) were examined by RT–qPCR and Western blot. C, HTR8/SVneo cell migration was examined by wound healing assay. Representative diagrams of wound healing assays were taken at 0, 12 and 24 h. D, Effects of FN1 siRNA treatment on activin A-induced HTR8/SVneo cell endothelial-like tube formation were examined after transfection for 48 h with si-Ctrl or si-FN1 with or without activin A for 12 h. Left panels show representative images from the endothelial-like tube formation assays. The right panel shows summarized quantitative results from image analysis of total tube length. E and F, HTR8/SVneo cells were given the same interventions as in A and B, and cell proliferation was detected by CCK-8 (E) and EdU (F) at 24 h. The results are presented as the mean ± SEM of three independent experiments. ANOVA was used for quantitative grouped analyses. Columns without letters in common are significantly different (P < 0.05). P values of primary interest are denoted as * < 0.05, ** < 0.01, and *** < 0.001