Journal: Diabetes Therapy

Article Title: Elevated Serum SERPINE2 Levels are Linked to Impaired Renal Function in Patients with Type 2 Diabetes Mellitus

doi: 10.1007/s13300-025-01742-7

Figure Lengend Snippet: Serum serpin peptidase inhibitor clade E member 2 (SERPINE2) levels were compared between control subjects and patients with type 2 diabetes mellitus (T2DM). a Serum SERPINE2 levels in control subjects and patients with T2DM were detected by enzyme-linked immunosorbent assay (ELISA). The serum SERPINE2 concentration in patients with T2DM ( n = 292) was significantly higher than that in control subjects ( n = 120). Student t test was applied. b Comparison of serum SERPINE2 levels in patients with T2DM with normoalbuminuria, microalbuminuria, and macroalbuminuria. ANOVA was applied. *** P < 0.001

Article Snippet: Serum biomarker levels in control subjects and patients with T2DM were measured using ELISA kits for NGAL (QK1757, R&D Systems), KIM-1 (DSKM100, R&D Systems), TGFβ1 (DY240, R&D Systems), CTGF (DY9190-05, R&D Systems), and antibodies against SERPINE2 (AF2980, R&D Systems) [ ].

Techniques: Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Comparison

Journal: Diabetes Therapy

Article Title: Elevated Serum SERPINE2 Levels are Linked to Impaired Renal Function in Patients with Type 2 Diabetes Mellitus

doi: 10.1007/s13300-025-01742-7

Figure Lengend Snippet: Correlation between serum serpin peptidase inhibitor clade E member 2 (SERPINE2) and clinical indicators. Pearson correlation test was performed between SERPINE2 with a estimated glomerular filtration rate (eGFR), b serum creatinine (Scr), c neutrophil gelatinase-associated lipocalin (NGAL), d kidney injury molecule 1 (KIM-1), e transforming growth factor-β1 (TGFβ1), and f connective tissue growth factor (CTGF) in all patients with type 2 diabetes mellitus (T2DM)

Article Snippet: Serum biomarker levels in control subjects and patients with T2DM were measured using ELISA kits for NGAL (QK1757, R&D Systems), KIM-1 (DSKM100, R&D Systems), TGFβ1 (DY240, R&D Systems), CTGF (DY9190-05, R&D Systems), and antibodies against SERPINE2 (AF2980, R&D Systems) [ ].

Techniques: Filtration

Journal: Diabetes Therapy

Article Title: Elevated Serum SERPINE2 Levels are Linked to Impaired Renal Function in Patients with Type 2 Diabetes Mellitus

doi: 10.1007/s13300-025-01742-7

Figure Lengend Snippet: Receiver operating characteristic (ROC) curve was used to obtain the optimal cutoff value of serum serpin peptidase inhibitor clade E member 2 (SERPINE2) (278.94 pg/mL) that distinguishes the patients with type 2 diabetes mellitus (T2DM) with and without albuminuria. AUC area under the curve

Article Snippet: Serum biomarker levels in control subjects and patients with T2DM were measured using ELISA kits for NGAL (QK1757, R&D Systems), KIM-1 (DSKM100, R&D Systems), TGFβ1 (DY240, R&D Systems), CTGF (DY9190-05, R&D Systems), and antibodies against SERPINE2 (AF2980, R&D Systems) [ ].

Techniques:

Journal: Diabetes Therapy

Article Title: Elevated Serum SERPINE2 Levels are Linked to Impaired Renal Function in Patients with Type 2 Diabetes Mellitus

doi: 10.1007/s13300-025-01742-7

Figure Lengend Snippet: Schematic diagram of serpin peptidase inhibitor clade E member 2 (SERPINE2) in the development of diabetic nephropathy. Increased SERPINE2 in patients with type 2 diabetes mellitus (T2DM) with renal dysfunction promotes podocyte injury and albuminuria. SERPINE2 also enhances renal fibrosis by promoting fibrosis-related cytokines, such as transforming growth factor-β1 (TGFβ1) and connective tissue growth factor (CTGF). Therefore, the increase in SERPINE2 levels plays an aggravating role in injury of glomerular and tubular cells caused by hyperglycemia and advanced glycation end products (AGEs)

Article Snippet: Serum biomarker levels in control subjects and patients with T2DM were measured using ELISA kits for NGAL (QK1757, R&D Systems), KIM-1 (DSKM100, R&D Systems), TGFβ1 (DY240, R&D Systems), CTGF (DY9190-05, R&D Systems), and antibodies against SERPINE2 (AF2980, R&D Systems) [ ].

Techniques:

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: SERPINE2, an inhibitor of plasminogen activators, is highly expressed in the human endometrium during the secretory phase

doi: 10.1186/1477-7827-9-38

Figure Lengend Snippet: Antibody specificity and presence of the SERPINE2 protein in human uterine fluid . (A) Four hundred nanograms of recombinant human SERPINE2 was resolved on 10% SDS-PAGE and followed by Western blotting using anti-mouse SERPINE2 antiserum (lane 1), an anti-human SERPINE2 antibody (R&D) (lane2), or another anti-human SERPINE2 antibody (Abnova) (lane 3). (B) One hundred micrograms of the extract of endometrial curettage was analyzed by anti-mouse SERPINE2 antiserum (lanes 1 and 2) and an anti-human SERPINE2 antibody (R&D) (lanes 3 and 4). (C) Fifty micrograms of uterine fluid proteins collected from each individual patient ( n = 7) was Western-blotted using anti-mouse SERPINE2 antiserum (1:3000) (upper panel). EP, MP, and LP indicate early-, mid-, and late-proliferative phases, and ES and MS indicate early- and mid-secretory phases, respectively. The blot was also overexposed to clearly display the staining signal (lower panel).

Article Snippet: Membranes were blocked with 10% (w/v) skim milk in phosphate-buffered saline (PBS) (blocking solution) for 2 h, and then incubated with our homemade anti-mouse SERPINE2 antiserum (1: 5000) [ , ], anti-human SERPINE2 antibody (1: 1000, catalog no. AF2980, R&D Systems, Minneapolis, MN, USA), or another anti-human SERPINE2 antibody (1: 1000, product no. H00005270-B01, Abnova) in blocking solution for 2 h at 37°C.

Techniques: Recombinant, SDS Page, Western Blot, Staining

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: SERPINE2, an inhibitor of plasminogen activators, is highly expressed in the human endometrium during the secretory phase

doi: 10.1186/1477-7827-9-38

Figure Lengend Snippet: Localization of SERPINE2 in the human uterus . Longitudinal sections of the early secretory phase uterus (n = 5) on the slide were incubated with anti-mouse SERPINE2 antiserum and then treated with biotin-conjugated goat-anti-rabbit IgG and HRP-conjugated streptavidin (brown). For contrast, specimens were further stained with hematoxylin (blue). The representative picture is shown. Magnified pictures of the luminal epithelium (A), glandular epithelium (B), and myometrium (C) are shown. Bar = 50 μm. bv, blood vessel; ge, glandular epithelium; le, luminal epithelium; m, muscle; s, stroma.

Article Snippet: Membranes were blocked with 10% (w/v) skim milk in phosphate-buffered saline (PBS) (blocking solution) for 2 h, and then incubated with our homemade anti-mouse SERPINE2 antiserum (1: 5000) [ , ], anti-human SERPINE2 antibody (1: 1000, catalog no. AF2980, R&D Systems, Minneapolis, MN, USA), or another anti-human SERPINE2 antibody (1: 1000, product no. H00005270-B01, Abnova) in blocking solution for 2 h at 37°C.

Techniques: Incubation, Staining

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: SERPINE2, an inhibitor of plasminogen activators, is highly expressed in the human endometrium during the secretory phase

doi: 10.1186/1477-7827-9-38

Figure Lengend Snippet: Glandular epithelial expression of the SERPINE2 protein in the endometrium during the menstrual cycle . Sections prepared from endometrial curettage during early-proliferative (EP), mid-proliferative (MP), late-proliferative (LP), early-secretory (ES), mid-secretory (MS), and late-secretory (LS) phases were incubated with anti-mouse SERPINE2 antiserum and then treated as described in Figure 2. Bar = 50 μm. bv, blood vessel; ge, glandular epithelium; s, stroma.

Article Snippet: Membranes were blocked with 10% (w/v) skim milk in phosphate-buffered saline (PBS) (blocking solution) for 2 h, and then incubated with our homemade anti-mouse SERPINE2 antiserum (1: 5000) [ , ], anti-human SERPINE2 antibody (1: 1000, catalog no. AF2980, R&D Systems, Minneapolis, MN, USA), or another anti-human SERPINE2 antibody (1: 1000, product no. H00005270-B01, Abnova) in blocking solution for 2 h at 37°C.

Techniques: Expressing, Incubation

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: SERPINE2, an inhibitor of plasminogen activators, is highly expressed in the human endometrium during the secretory phase

doi: 10.1186/1477-7827-9-38

Figure Lengend Snippet: Quantification of SERPINE2 protein expression levels in endometrial glands . Representative samples were analyzed by automated cell acquisition and quantification software (A). The expression signal of a respective glandular gland was quantified using HistoQuest software and is presented as a scattergram. Each spot on the scattergram stands for the intensity of one cell (B). The relative SERPINE2 protein expression levels in patients' glandular glands at various sub-phases of the menstrual cycle are shown as bar diagrams (C). Differences are significant among patients at various groups (χ = 69.32, p < 0.0001).

Article Snippet: Membranes were blocked with 10% (w/v) skim milk in phosphate-buffered saline (PBS) (blocking solution) for 2 h, and then incubated with our homemade anti-mouse SERPINE2 antiserum (1: 5000) [ , ], anti-human SERPINE2 antibody (1: 1000, catalog no. AF2980, R&D Systems, Minneapolis, MN, USA), or another anti-human SERPINE2 antibody (1: 1000, product no. H00005270-B01, Abnova) in blocking solution for 2 h at 37°C.

Techniques: Expressing, Software

Journal: Oncotarget

Article Title: Serpin E2 promotes breast cancer metastasis by remodeling the tumor matrix and polarizing tumor associated macrophages

doi: 10.18632/oncotarget.12927

Figure Lengend Snippet: ( A ) Masson trichrome staining performed on 4T1 Ctrl and shSerpine2 tumors. Representative images show increased collagen encapsulation (blue) of SerpinE2 KD tumors. Scale bar100μm. ( B - C ) Intravital imaging using (IVI-MP) of 4T1 Ctrl and shSerpine2 tumors. (B) Representative images show collagen I fibers detected by the SHG signal (cyan) at the surface of 4T1 tumors; scale bar25μm. (C) Average SHG signal intensity was determined per 100μm z-stack; data are acquired from 19-30 separate z-stacks from 3 mice per cell line. * P < 0.00036. D. IVI-MP performed on mice bearing GFP-labeled 4T1 control and shSerpinE2 tumors. Representative images show GFP-labeled tumor cells (green), phagocytic dextran positive cells (red); SHG imaging identified collagen I fibers (cyan). Scale bars25μm. (E-F) ( E ) GFP-labeled 4T1 tumor-bearing mice were treated with control liposomes or clodronate-containing liposomes until IVI-MP was performed. Representative images are shown as in (D). ( F ) Quantification of SHG (cyan) signal intensity in 100 μm Z-stacks of tumors in treated animals. Data are mean ± SEM of measurements from 40-61 Z-stacks from at least 3 different tumors for each treatment group. * P < 0.016. All data are mean ± SEM.

Article Snippet: Anti-human SerpinE2 Ab (MAB2980) was from R&D systems.

Techniques: Staining, Encapsulation, Imaging, Labeling, Control, Liposomes

Journal: Oncotarget

Article Title: Serpin E2 promotes breast cancer metastasis by remodeling the tumor matrix and polarizing tumor associated macrophages

doi: 10.18632/oncotarget.12927

Figure Lengend Snippet: Conditioned medium (CM) from 4T1 and 168FARN ( A ) or MDA-MB435 ( B ) cultures was incubated overnight with Ab11, then bound serpinE2 was pulled-down with Protein G, and analyzed by western blot with the rodent-specific antibody, 4B3 (A), or a human-specific antibody (B). (A) The right panel shows a short exposure of serpinE2 complexes from 4T1 CM, but none in 168FARN CM (long exposure on the left), as well as an IP of the preformed tPA/serpinE2 complex. ( C ) SFM loaded with purified human serpinE2 or the preformed serpinE2/tPA complex was IP'd with Ab11 as in (A) & (B) and analyzed by western blot with a human serpinE2-specific antibody. The serpinE2 complex and uncomplexed serpinE2 are indicated with arrowheads.

Article Snippet: Anti-human SerpinE2 Ab (MAB2980) was from R&D systems.

Techniques: Incubation, Western Blot, Purification

Journal: Oncotarget

Article Title: Serpin E2 promotes breast cancer metastasis by remodeling the tumor matrix and polarizing tumor associated macrophages

doi: 10.18632/oncotarget.12927

Figure Lengend Snippet: The LRP1 receptor is expressed on tumor cells and on macrophages and we propose that serpinE2 targeting with Ab11 impacts on LRP1 signaling in both cell types. ( A ) In metastatic tumors, serpinE2-LRP1 binding stimulates ERK signaling and secretion of CCL2. Tumor cells display hyperactivation of receptor tyrosine kinases (RTKs); FGFRs, which are active in the 4T1 model, also stimulate ERK pathway activation. We speculate that in macrophages, the combination of high CCL2 levels and serpinE2-activated LRP1 promotes the M2 phenotype. Indeed, there are high levels of phagocytic, Texas-red positive M2 tumor associated macrophages (TAMs) in the metastatic tumors; they are known to be responsible for degrading the matrix during tumor development. ( B ) Ab11 or sepinE2 KD (not drawn in model) lowers ERK pathway activity and the secretion of CCL2, and stimulates TIMP1 secretion. Tyrosine kinase inhibitors (TKIs) block RTK signaling. The matrix-degrading M2 TAMs are decreased in Ab11-treated tumors. Moreover, depleting macrophages with clodronate liposomes results in deposition of a dense collagen matrix, similar to that observed in Ab11-treated tumors. Thus, we propose that the drop in CCL2, which contributes to a decrease in M2 TAMs, as well as blocking serpinE2, which skews the TAMs towards the M1 phenotype, causes the emergence of a dense collagen matrix that inhibits intravasation and metastatic dissemination.

Article Snippet: Anti-human SerpinE2 Ab (MAB2980) was from R&D systems.

Techniques: Binding Assay, Activation Assay, Activity Assay, Blocking Assay, Liposomes

Journal: Oncogene

Article Title: A slow-cycling subpopulation of melanoma cells with highly invasive properties

doi: 10.1038/onc.2017.341

Figure Lengend Snippet: Label-retaining, disseminated melanoma cells express high levels of SerpinE2. ( a ) Table lists proteins identified after whole-cell proteomic analyses of LRC and non-LRC. The last column lists the cell lines with differential expression of the indicated protein. ( b ) Violin plots (left panels) illustrate quantification of SerpinE2 (white spot, top) and BMP1 (white spots, bottom) as determined by mRNA FISH. mRNA counts for single cells are shown on the Y -axes. Note differences in Y -axes. Representative photomicrographs are shown on the right (total magnification: 100 ×). ( c ) Protein expression was analyzed by immunofluorescence and quantified at single-cell levels (scatter plots on left and representative images on right). LRC (red), non-LRC (orange), total cells (gray). ( d ) Flow cytometry of disseminated WM989 melanoma cells detected in three mouse organs (top panel). The percentage of cells double positive for CD146 and SerpinE2 is shown in the upper right quadrants; the lower right quadrants indicate the percentage of cells singly positive for CD146. Micrographs show examples of disseminated SerpinE2-positive cells (arrows, middle and right panels) in mouse lungs. Left panel: negative control.

Article Snippet: Samples were boiled 10 min in NuPAGE LDS sample buffer (Thermo Fisher Scientific), loaded on 10% gels, transferred onto nitrocellulose filters and immunoblotted with mouse anti-human SerpinE2 antibody (R&D), diluted 1:500.

Techniques: Quantitative Proteomics, Expressing, Immunofluorescence, Flow Cytometry, Negative Control

Journal: Oncogene

Article Title: A slow-cycling subpopulation of melanoma cells with highly invasive properties

doi: 10.1038/onc.2017.341

Figure Lengend Snippet: SerpinE2 drives melanoma invasiveness. ( a ) Boyden chamber invasion assay of sorted label-retaining cells (LRC) or non-LRC in presence (blue columns) or absence (gray columns) of human recombinant SerpinE2. Bars show fold change in invasion (mean±s.e.m.) of samples with SerpinE2 over untreated cells. ( b ) Invasion assay in presence of an anti-human SerpinE2 neutralizing antibody or Isotype control (ctrl). The percentage of invading cells after neutralization is shown. Data represent mean±s.e.m. of three independent experiments. ( c ) Invasion assay after SerpinE2 knockdown. Data shown are percentage of invading cells found after silencing with 5 different shRNA (Sh_13-17) relative to Sh_ctrl (ctrl). Bars represent mean±s.e.m.

Article Snippet: Samples were boiled 10 min in NuPAGE LDS sample buffer (Thermo Fisher Scientific), loaded on 10% gels, transferred onto nitrocellulose filters and immunoblotted with mouse anti-human SerpinE2 antibody (R&D), diluted 1:500.

Techniques: Invasion Assay, Recombinant, Control, Neutralization, Knockdown, shRNA

Journal: Oncogene

Article Title: A slow-cycling subpopulation of melanoma cells with highly invasive properties

doi: 10.1038/onc.2017.341

Figure Lengend Snippet: SerpinE2 expression is increased in malignant cells and correlates with tumor progression. ( a ) Western blot analysis of SerpinE2 secretion by melanoma cells ( n =21) and human melanocytes ( n =5) into culture supernatants. Recombinant SerpinE2 protein (500 ng) was used as a positive control and Ponceau’s staining is shown for loading control. Graph shows quantification (right panel). ( b ) SerpinE2 staining of 3D skin reconstructs with melanocytes (left) and melanoma cells (right). ( c ) GEM_1375 data set analysis of SerpinE2 mRNA expression in melanomas, non-malignant nevi and normal skin. ( d ) Examples of immune histochemistry at two different magnifications of: normal skin, benign nevi, in situ , vertical growth phase (VGP) and lymph node metastatic melanomas ( n =15 for each group). SerpinE2 protein expression is indicated by purple staining. Arrows indicate the dotted SerpinE2 expression pattern found in benign nevi only. ( e ) Intensity score of SerpinE2 expression in normal skin, benign nevi and malignant melanomas (radial growth phase (RGP), VGP and metastatic). Bars represent mean±SEM of tissue sections of 15 specimens from each group; P- value after t -student test are given.

Article Snippet: Samples were boiled 10 min in NuPAGE LDS sample buffer (Thermo Fisher Scientific), loaded on 10% gels, transferred onto nitrocellulose filters and immunoblotted with mouse anti-human SerpinE2 antibody (R&D), diluted 1:500.

Techniques: Expressing, Western Blot, Recombinant, Positive Control, Staining, Control, In Situ

Journal: PLoS ONE

Article Title: SERPINE2 Inhibits IL-1α-Induced MMP-13 Expression in Human Chondrocytes: Involvement of ERK/NF-κB/AP-1 Pathways

doi: 10.1371/journal.pone.0135979

Figure Lengend Snippet: A, B . Human SERPINE2 mRNA expression after interleukin IL-1α treatment (0.05–10 ng/mL) during 24 hours in human primary chondrocytes and in T/C-28a2 chondrogenic cells. C, D . Representative western blot of human SERPINE2 protein expression in lysates obtained from human primary chondrocytes and T/C-28a2 chondrogenic cells treated with interleukin IL-1α (0.05–10 ng/mL) for 24 h. β-actin was used to ensure equal sample loading. Data are means ± S.E.M. of at least 3 independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs untreated control cells.

Article Snippet: Blots were incubated with the appropriate antibody: anti-human SERPINE2 (R&D, MN, USA); anti-human MMP-13 (Santa Cruz, CA, USA); anti-phospho ERK1/2 (Millipore, MA, USA); anti-ERK1/2 (Millipore, MA, USA), anti-p65 (Santa Cruz, CA,USA); anti-c-jun (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: SERPINE2 Inhibits IL-1α-Induced MMP-13 Expression in Human Chondrocytes: Involvement of ERK/NF-κB/AP-1 Pathways

doi: 10.1371/journal.pone.0135979

Figure Lengend Snippet: A. Human MMP-13 mRNA expression in T/C-28a2 chondrocytes incubated with SERPINE2 (0.4 ng/mL) in presence or not of IL-1α (0.5 ng/mL) for 24 h. B. Representative western blot of human MMP-13 protein expression in lysates obtained from T/C-28a2 chondrogenic cells treated with with SERPINE2 (0.4 ng/mL) in presence or not of IL-1α (0.5 ng/mL) for 24 h. β-actin was used to ensure equal sample loading. Low panel. Densitometric analysis of at least three independent experiments. C. Human MMP-13 mRNA expression in human primary chondrocytes incubated with SERPINE2 (0.4 ng/mL) in presence or not of IL-1α (0.5 ng/mL) for 24 h. Data are means ± S.E.M. of at least 3 independent experiments. **P<0.01 and ***P<0.001 vs untreated control cells; ## P<0.01 and ### P<0.001 vs IL-1α-stimulated chondrocytes.

Article Snippet: Blots were incubated with the appropriate antibody: anti-human SERPINE2 (R&D, MN, USA); anti-human MMP-13 (Santa Cruz, CA, USA); anti-phospho ERK1/2 (Millipore, MA, USA); anti-ERK1/2 (Millipore, MA, USA), anti-p65 (Santa Cruz, CA,USA); anti-c-jun (Santa Cruz, CA, USA).

Techniques: Expressing, Incubation, Western Blot, Control

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: 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: 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: 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

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