Journal: Cellular and Molecular Life Sciences: CMLS
Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry
doi: 10.1007/s00018-025-06040-w
Figure Lengend Snippet: 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
Article Snippet: SMAD1 (D59D7) rabbit monoclonal antibody (#6944), phospho-SMAD1/5/9 (D5B10) rabbit monoclonal antibody (#13820), SMAD2 (D43B4) rabbit monoclonal antibody (#5339), phospho-SMAD2 (E8F3R) rabbit monoclonal antibody (#18338), SMAD3 (C67H9) rabbit monoclonal antibody (#9523), phospho-SMAD3 (C25A9) rabbit monoclonal antibody (# 9520), SMAD4 (D3R4N) rabbit monoclonal antibody (#46535), horseradish peroxidase (HRP)-linked anti-mouse IgG (#7076), and anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA); the mouse monoclonal antibody HLA-G (#11–499-C100) was purchased from EXBIO (Vestec, Czech Republic); CK7-specific rabbit polyclonal antibody (#17513-1-AP), BMP6 rabbit polyclonal antibody (#55421-1-AP), SERPINE2 rabbit polyclonal antibody (#11303-1-AP), and α-Tubulin mouse monoclonal antibody (#66031-1-Ig) were purchased from Proteintech (Wuhan, China); ID1 mouse monoclonal antibody (#sc-133104) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); CD34 rabbit monoclonal antibody (#A19015) and α-SMA rabbit monoclonal antibody (#A2235) were obtained from ABclonal (Wuhan, China); Goat anti-Rabbit IgG secondary antibody, Alexa Fluor 594 (#A-11012) and goat anti-Mouse IgG secondary antibody, Alexa Fluor 488 (#A-11011) were purchased from Thermo Fisher (NY, USA).
Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Control, Binding Assay