Fig. 3.

ID1 mediates BMP6-induced SERPINE2 and PlGF upregulation in human trophoblasts. A-C, BMP6 upregulates SERPINE2 protein levels in trophoblasts. A, HTR8/SVneo cells were treated with different concentrations (0, 6.25, 12.5, 25, 50, or 100 ng/mL) of BMP6, and the SERPINE2 protein levels after 24 h of treatment were examined by Western blot analysis. The upper panel shows a representative Western blot image, and the lower panel shows the summarized quantitative results. B, SERPINE2 protein levels in HTR8/SVneo cells after treatment with vehicle (Ctrl) or 50 ng/mL BMP6 for different durations (24, 48, and 72 h). The upper panel shows a representative Western blot image, and the lower panel shows the summarized quantitative results. C, SERPINE2 protein levels in human primary EVTs. The left panel shows a representative Western blot image, and the right panel shows the summarized quantitative results. D-E, BMP6 promotes PlGF accumulation in the conditioned medium of trophoblasts. D, HTR8/SVneo cells were treated with or without 50 ng/mL BMP6 for 24–48 h. PlGF accumulation in conditioned medium was measured using ELISA. E, PlGF accumulation in conditioned medium was assayed by ELISA 48 h after BMP6 treatment in primary EVTs. F-J, ID1 mediates BMP6-induced SERPINE2 and PlGF upregulation in human trophoblasts. HTR8/SVneo cells 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. F, ID1 mRNA levels were examined by qPCR 6 h after BMP6 (50 ng/mL) treatment in HTR8/SVneo cells, with GAPDH used as the reference gene. G and H, SERPINE2 and ID1 protein levels in HTR8/SVneo cells (G) and human primary EVTs (H) after transfection with siRNA targeting ID1, followed by treatment with or without BMP6 for 24 h, as assessed by Western blot. The left panel shows a representative Western blot image, and the right panel shows the summarized quantitative results. I, PGF mRNA levels were examined by qPCR 6 h after BMP6 treatment in HTR8/SVneo cells, with GAPDH used as the reference gene. J, PlGF accumulation in conditioned medium was assayed by ELISA 24 h after BMP6 treatment in HTR8/SVneo cells. K, SMAD4 mediates BMP6-induced upregulation of PlGF in human trophoblasts. HTR8/SVneo cells were transfected for 48 h with 20 nM control non-targeting siRNA (si-Ctrl) or siRNA targeting SMAD4 (si-SMAD4) before treatment with or without 50 ng/mL BMP6. PlGF accumulation in conditioned medium was assayed by ELISA 24 h after BMP6 treatment in HTR8/SVneo cells. 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 two-way ANOVA was used for comparisons in B-K. Groups without common letters are significantly different from each other (P < 0.05). BMP6, bone morphogenetic protein 6; SERPINE2, serpin family E member 2; EVT, extravillous cytotrophoblast; Ctrl, control; PlGF, placental growth factor; ID1, inhibitor of DNA-binding 1