human 807 plgf quantikine elisa kit  (R&D Systems)

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: BMP6 signaling enhancement compensates for shallow trophoblast invasion in PE. An examination of clinical samples from PE patients and their gestational age-matched controls revealed that BMP6 was upregulated in preeclamptic placentas. BMP6 enhances trophoblast invasion via ID1-mediated upregulation of SERPINE2 and PlGF. Moreover, BMP6 also intensifies trophoblast vascular mimicry through the ID1-mediated upregulation of PlGF. Both canonical (p-SMAD1/5/9) and noncanonical (p-SMAD2/3) pathways participate in BMP6-induced ID1 expression. Our findings indicate that the increase in BMP6 signaling during late gestation could serve as a compensatory response to shallow trophoblast invasion in PE, which in light of BMP6 and its downstream targets could serve as diagnostic markers and therapeutic target applications in the clinical management of PE. This schematic diagram was created with Biorender.com

Article Snippet: PlGF accumulation in the cell culture supernatant was measured via a Human PlGF Quantikine ELISA Kit (R&D Systems, DPG00) according to the manufacturer’s instructions (both intra-assay and inter-assay with coefficients of variation less than 10%) and was normalized to the total cellular protein concentration.

Techniques: Expressing, Diagnostic Assay

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: PlGF accumulation in the cell culture supernatant was measured via a Human PlGF Quantikine ELISA Kit (R&D Systems, DPG00) according to the manufacturer’s instructions (both intra-assay and inter-assay with coefficients of variation less than 10%) and was normalized to the total cellular protein concentration.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Control, Binding Assay

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: 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

Article Snippet: PlGF accumulation in the cell culture supernatant was measured via a Human PlGF Quantikine ELISA Kit (R&D Systems, DPG00) according to the manufacturer’s instructions (both intra-assay and inter-assay with coefficients of variation less than 10%) and was normalized to the total cellular protein concentration.

Techniques: Transfection, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Tube Formation Assay, Comparison

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: BMP6 is elevated in patients with PE and in PE model rats. A-C , BMP6 is elevated in patients with PE. A , RT‒qPCR analysis of BMP6 mRNA expression levels in the placentas of control women ( n = 10) and PE patients ( n = 10), with GAPDH as the reference gene. B , Western blot analysis of BMP6 protein expression levels in the placentas of control women ( n = 4) and PE patients ( n = 4). C , Spearman correlation analysis between the placental BMP6 mRNA levels and the value of log10 (SBP) of the corresponding patients. The gray area represents the 95% CI. Each dot represents one sample. D , The animal experimental protocol. E and F , BMP6 is elevated in the plasma of PE model rats. Rat plasma levels of BMP6 ( E ) and PlGF ( F ) in the Ad Fc + PBS group ( n = 4) and Ad Flt1 + PBS group ( n = 4). G-M , BMP6 is elevated in the placenta of PE model rats at G13. RNA-seq analysis of rat placentas at G13 in the Ad Fc + PBS group ( n = 3) and Ad Flt1 + PBS group ( n = 3). G , Heatmap depicting DEGs in the two groups. H , Dot plots of significantly enriched GO terms; the dot size represents the number of DEGs associated with a particular GO term. I , KEGG hierarchical network plot of pathways. J , GSEA-KEGG Ridge plot of pathways. K , GSEA plots of cytokine-cytokine receptor interaction pathway. L , Volcano plot of RNA-seq data showing DEGs between the Ad Fc + PBS group and the Ad Flt1 + PBS group. M , Circos graph displaying the coexpression networks of five genes in rat placenta samples. Each sector of the circle represents one gene, and its width indicates the total amount of co-occurrence that connects one gene to the other. The width of each link represents the total number of coexpressed genes among the linked genes. Student’s t-test was used for comparisons between two groups in A , B , E , and F . Groups without common letters are significantly different from each other ( P < 0.05). SD, Sprague–Dawley; BMP6, bone morphogenetic protein 6; PlGF, placental growth factor; PBS, phosphate-buffered saline; Ad Flt1, adenovirus expressing fms-like tyrosine kinase-1; Ad Fc, adenovirus-expressing control IgG2a Fc fragment; FC, fold change; Serpine2, serpin family E member 2; Id1, inhibitor of DNA-binding 1

Article Snippet: PlGF accumulation in the cell culture supernatant was measured via a Human PlGF Quantikine ELISA Kit (R&D Systems, DPG00) according to the manufacturer’s instructions (both intra-assay and inter-assay with coefficients of variation less than 10%) and was normalized to the total cellular protein concentration.

Techniques: Expressing, Control, Western Blot, Clinical Proteomics, RNA Sequencing, Saline, Binding Assay

Journal: bioRxiv

Article Title: Vascular endothelial growth factor receptors 1 and 3 mediate placental trophoblast leptin production in preeclampsia, inducing vascular dysfunction

doi: 10.1101/2025.05.27.656489

Figure Lengend Snippet: Schematic showing immunostaining experiment design ( A) ; immunohistochemistry for BeWo cells treated with a vehicle (top row) or sFlt-1 (bottom row) for 24 hours ( B ); Leptin secretion in media after treatment with vehicle, sFlt-1 (72 hours), VEGF (24 hours), PlGF (24 hours) or combination in Bewo cells (C) ; placental explants from preeclampsia pregnancies (D) or placental explants from normal pregnancies (E); One Way ANOVA, Fisher’s LSD post hoc test, Student’s t-test. *p<0.05, ***p<0.001.

Article Snippet: PLGF, VEGF and Leptin were measured in culture media according to the manufacturer’s protocols (PLGF: #DPG00, R&D Systems; VEGF: #DVR100, R&D Systems; Leptin: RAB0333-1KT, Millipore Sigma).

Techniques: Immunostaining, Immunohistochemistry