Fig. 4.

Both SERPINE2 and PlGF mediate BMP6-induced trophoblast invasion. A-J, HTR8/SVneo cells or primary EVTs were transfected for 48 h with 20 nM control nontargeting siRNA (si-Ctrl), 20 nM siRNA targeting SERPINE2 (si-SERPINE2) or PGF (si-PGF) before treatment with or without 50 ng/mL BMP6 for 24 h. A and B, The protein levels of SERPINE2 in HTR8/SVneo cells (A) and human primary EVTs (B) after 24 h of BMP6 treatment. The upper panel shows a representative Western blot image, and the lower panel shows the summarized quantitative results. C, PlGF accumulation in conditioned medium with or without BMP6 treatment for 24 h was assayed by ELISA in HTR8/SVneo cells. D, PGF mRNA levels in human primary EVTs with or without BMP6 treatment for 6 h were examined by RT‒qPCR, with GAPDH as the reference gene. E-H, Transwell assays were employed to examine the invasiveness of HTR8/SVneo cells (E and G) and primary EVTs (F and H) with or without BMP6 treatment for 36 h. I and J, Endothelial-like tube formation assays were used to assess vascular mimicry 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 above panel; the summarized quantitative results are displayed in the lower panel. Scale bar, 100 μm. The quantitative results are expressed as the means ± SEMs of at least three independent experiments. Two-way ANOVA was used for data comparison. Groups without common letters are significantly different from each other (P < 0.05). BMP6, bone morphogenetic protein 6; SERPINE2, serpin family E member 2; PGF, placental growth factor; Ctrl, control; EVT, extravillous cytotrophoblast