Fig. 2.

ID1 mediates BMP6-induced trophoblast invasion and vascular mimicry. A-D, BMP6 upregulated ID1 mRNA and protein levels in HTR8/SVneo cells. A and B, HTR8/SVneo cells were treated with different concentrations (0, 6.25, 12.5, 25, 50 or 100 ng/mL) of BMP6, and the ID1 mRNA levels after 6 h of treatment (A) and the ID1 protein levels after 24 h of treatment (B) were examined by RT‒qPCR and Western blot analysis, respectively. C-D, ID1 protein levels in HTR8/SVneo cells (C) and primary EVTs (D) after treatment with vehicle (Ctrl) or 50 ng/mL BMP6 for different durations. E‒I, ID1 mediates BMP6-promoted human trophoblast invasion and vascular mimicry. HTR8/SVneo cells or primary human EVTs were transfected for 48 h with 20 nM control non-targeting siRNA (si-Ctrl) or siRNA targeting ID1 (si-ID1) before treatment with or without 50 ng/mL BMP6. E and F, ID1 mRNA levels were examined by qPCR after BMP6 treatment for 6 h in HTR8/SVneo cells (E) and primary EVTs (F), with GAPDH used as the reference gene. G and H, Transwell assays were employed to examine the invasiveness of HTR8/SVneo cells (G) and primary EVTs (H) with or without BMP6 treatment for 36 h. Representative images from the invasion assay are displayed in the upper panel, while the summarized quantitative results of the invasion assay are shown in the lower panel. Scale bar, 100 μm. I, Endothelial-like tube formation assays were used to assess the acquisition of the endothelial-like phenotype of HTR8/SVneo cells with or without BMP6 treatment for 12 h. Representative images from the endothelial-like tube formation assay are displayed in the upper panel, while the summarized quantitative results of the endothelial-like tube formation assay are shown in the lower panel. Scale bar, 100 μm. The quantitative results are expressed as the means ± SEMs of at least three independent experiments. One-way ANOVA was used for analyses in A and B. Two-way ANOVA was used for grouped analyses in C-I. Groups without letters are significantly different from each other (P < 0.05). BMP6, bone morphogenetic protein 6; ID1, inhibitor of DNA-binding 1; Ctrl, control