early pregnancy trophoblast cell line htr8 svneo  (ATCC)

Journal: PLOS One

Article Title: Hypoxia regulates epithelial to mesenchymal transition-associated genes in human trophoblast cells by modulating DNA methylation

doi: 10.1371/journal.pone.0325053

Figure Lengend Snippet: The fold change in expression of (A) E-Cadherin (CDH1), (B) N-Cadherin (CDH2), (C) Fibronectin 1 (FN1), (D) Vimentin (VIM), (E) Matrix metalloproteinase 2 (MMP2), and (F) Matrix metalloproteinase 9 (MMP9) mRNAs in HTR8/SVneo cells exposed to hypoxia (1% O 2 ) compared to normoxia (20% O 2 ) as control. **p ≤ 0.01, ***p ≤ 0.001, ns = non-significant.

Article Snippet: Immortalized human trophoblast cell line HTR8/SVneo and choriocarcinoma cell line JEG3 were acquired from ATCC.

Techniques: Expressing, Control

Journal: PLOS One

Article Title: Hypoxia regulates epithelial to mesenchymal transition-associated genes in human trophoblast cells by modulating DNA methylation

doi: 10.1371/journal.pone.0325053

Figure Lengend Snippet: The relative methylation level of the promoter region of (A) E-Cadherin (CDH1), (B) N-Cadherin (CDH2), (C) Fibronectin 1 (FN1), (D) Vimentin (VIM), (E) Matrix metalloproteinase 2 (MMP2), and (F) Matrix metalloproteinase 9 (MMP9) genes in HTR8/SVneo cells exposed to hypoxia (1% O 2 ) compared to normoxia (20% O 2 ) as control. **p ≤ 0.01, ***p ≤ 0.001, ns = non-significant.

Article Snippet: Immortalized human trophoblast cell line HTR8/SVneo and choriocarcinoma cell line JEG3 were acquired from ATCC.

Techniques: Methylation, Control

Journal: PLOS One

Article Title: Hypoxia regulates epithelial to mesenchymal transition-associated genes in human trophoblast cells by modulating DNA methylation

doi: 10.1371/journal.pone.0325053

Figure Lengend Snippet: The fold change in expression of (A) DNMT1, (B) DNMT3A (var 1,2,3, and 6), (C) DNMT3A (var 4 and 5), (D) DNMT3B, (E) DNMT3L, (F) TET1, (G) TET2, and (H) TET3 mRNAs in HTR8/SVneo cells exposed to hypoxia (1% O 2 ) compared to normoxia (20% O 2 ) as control. * p ≤ 0.05, ns = non-significant.

Article Snippet: Immortalized human trophoblast cell line HTR8/SVneo and choriocarcinoma cell line JEG3 were acquired from ATCC.

Techniques: Expressing, Control

Journal: Journal of Nanobiotechnology

Article Title: Endothelial microparticles in high glucose environment: molecular pathways of GDM-associated placental dysfunction and coenzyme Q10-based targeted therapy

doi: 10.1186/s12951-026-04147-1

Figure Lengend Snippet: Vascular endothelial cell damage and placental structural abnormalities in patients with GDM. ( A ) Levels of E-selectin, P-selectin, ICAM-1, and VCAM-1 in the peripheral plasma of healthy pregnant women (negative control, human NC ) and GDM patients (human GDM ) ( n = 30/group). ( B ) Effects of treatment with a high concentration of glucose (25 mM) for 24–48 h on vascular growth and development, as determined by CAM experiments. CAM: Chick chorioallantoic membrane assay ( C ) Absorbance photometric values at 450 nm after 25 mM glucose treatment for 24 h and 48 h in HUVECs determined by CCK-8 assays. ( D ) Images showing the proliferation of HUVECs treated with 25 mM glucose for 24 h and 48 h, as determined by the EdU assay. The nuclei were stained with DAPI (blue), and the proliferating cells were stained with EdU (red). Scale bar, 100 μm. ( E ) Quantitative analysis of the proliferation rates. ( F ) Determination of the percentage of apoptotic HUVECs treated with 25 mM glucose for 24 h and 48 h using flow cytometric assays. ( G ) Histogram analysis of the percentage of apoptotic cells in each group. ( H ) Representative images of HE staining of the maternal side of the placenta in healthy pregnant women and GDM patients. FC: foetal capillaries; M: mesenchymal tissue; green arrows: syncytiotrophoblast; blue arrows: syncytial nodes composed of grouped syncytiotrophoblasts. Scale bar, 50 μm. ( I ) Experimental schedule for the generation of normal wild-type mice (mice WT ) and GDM model mice (mice GDM ). Images of the mice were created using Figdraw. CD: control diet; HFHS: high-fat high-sucrose diet. ( J ) Representative images of HE-stained placentas from the mice WT and mice GDM . D: Decidual zone; J: junctional zone; L: labyrinth zone( n = 6/group). Scale bar, 200 μm. ( K ) Percentage of each region of the placenta in WT mice and mice with GDM. ( L ) Effects of various ratios of HUVEC medium on HTR8/SVneo and JEG-3 cell proliferation, as determined by CCK-8 assays. G-ECM: blank ECM medium containing 25 mM glucose; HUVEC-CM: supernatant of HUVEC medium incubated with ECM containing 25 mM glucose. The data were analyzed using one-way ANOVA with Tukey’s post-hoc test. Data are presented as mean ± SD. ns., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: The human trophoblast cell lines HTR8/SVneo (CRL-3271, ATCC, USA) and JEG-3 (HTB-36, ATCC, USA) were purchased from the American Type Culture Collection.

Techniques: Clinical Proteomics, Negative Control, Concentration Assay, Chick Chorioallantoic Membrane Assay, CCK-8 Assay, EdU Assay, Staining, Control, Incubation

Journal: Journal of Nanobiotechnology

Article Title: Endothelial microparticles in high glucose environment: molecular pathways of GDM-associated placental dysfunction and coenzyme Q10-based targeted therapy

doi: 10.1186/s12951-026-04147-1

Figure Lengend Snippet: Analysis of the effects of G-EMPs on trophoblast biological functions via high-throughput sequencing and experiments. ( A ) Volcano plots showing DEGs between C-EMPs and G-EMPs (|log2(fold change)| > 1; Q value < 0.05). Red, upregulated; blue, downregulated; grey, not significantly different. C-EMPs: normal glucose-cultured HUVEC-derived EMPs; G-EMPs: high glucose (25 mM)-cultured HUVEC-derived EMPs. ( B ) Heatmap showing the top 25 genes significantly upregulated and the top 25 genes significantly downregulated in EMP-treated HTR8 cells. ( C ) Bubble plot displaying the Gene Ontology (GO) biological process analysis of the DEGs. ( D ) Bubble diagram demonstrating the GO molecular function (MF) analysis of the DEGs. ( E ) Histogram analysis of the migration area of HTR8/SVneo and JEG-3 cells in wound healing assays. ( F ) Histogram analysis of the number of invading cells per field. ( G ) Determination of N-cadherin and E-cadherin expression in HTR8/SVneo and JEG-3 cells after treatment with C-EMPs or G-EMPs using western blotting. ( H ) The expression of N-cadherin and E-cadherin in placental tissues from the mice WT and mice GDM was determined using tissue immunofluorescence. N-cadherin and E-cadherin were stained with specific antibodies (green), trophoblasts were stained with CK7-specific antibodies (red), and nuclei were stained with DAPI (blue). Scale bar, 100 μm. The data were analyzed using one-way ANOVA with Tukey’s post-hoc test. Data are presented as mean ± SD. ns., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: The human trophoblast cell lines HTR8/SVneo (CRL-3271, ATCC, USA) and JEG-3 (HTB-36, ATCC, USA) were purchased from the American Type Culture Collection.

Techniques: Next-Generation Sequencing, Cell Culture, Derivative Assay, Migration, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Journal of Nanobiotechnology

Article Title: Endothelial microparticles in high glucose environment: molecular pathways of GDM-associated placental dysfunction and coenzyme Q10-based targeted therapy

doi: 10.1186/s12951-026-04147-1

Figure Lengend Snippet: NGFR is an essential gene for the excessive migration and invasion of trophoblast cells due to G-EMPs. ( A ) Chordal diagram showing the detailed relationship between DEGs (left semicircular perimeter) and their enriched KEGG pathways (right semicircular perimeter). ( B ) Radar plot displaying four genes whose expression was upregulated (upper panel) and five genes whose expression was downregulated (lower panel) after treatment with EMPs in the MAPK pathway. ( C ) Determination of NGFR, p-MEK, MEK, p-ERK and ERK expression in HTR8/SVneo and JEG-3 cells treated with different EMPs via western blotting. ( D ) Cellular immunofluorescence analysis of NGFR expression in HTR8/SVneo cells after treatment with EMPs. NGFR was stained with a specific antibody (green), and nuclei were stained with DAPI (blue). Scale bar, 50 μm. ( E ) western blot analysis of NGFR expression in placental tissues from both wild-type mice and mice with GDM in vivo, as well as in healthy pregnant women and women with GDM in clinical cohorts. ( F ) Tissue immunofluorescence analysis of NGFR expression in placental tissues of the mice WT and mice GDM and the human NC and human GDM . NGFR was stained with a specific antibody (green), trophoblasts were stained with a CK7-specific antibody (red), and nuclei were stained with DAPI (blue). Scale bar, 100 μm. ( G ) Determination of the efficacy of si-NGFR in knocking down NGFR in HTR8/SVneo cells via western blotting. ( H ) NGFR, p-ERK, ERK, N-cadherin, and E-cadherin expression levels in HTR8/SVneo cells in different treatment groups were determined via western blotting. ( I ) Wound healing assays were performed to assess the migration of HTR8/SVneo cells at 0 h and 24 h under different conditions. Scale bar, 200 μm. ( J ) Transwell assays were performed to assess the invasion of HTR8/SVneo cells at 4 h under different conditions. Scale bar, 1 mm. ( K ) Histogram analysis showing the migration area of HTR8/SVneo cells. ( L ) Histogram analysis of the number of invading HTR8/SVneo cells. The data were analyzed using one-way ANOVA with Tukey’s post-hoc test. Data are presented as mean ± SD. ns., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: The human trophoblast cell lines HTR8/SVneo (CRL-3271, ATCC, USA) and JEG-3 (HTB-36, ATCC, USA) were purchased from the American Type Culture Collection.

Techniques: Migration, Expressing, Western Blot, Immunofluorescence, Staining, In Vivo

Journal: Journal of Nanobiotechnology

Article Title: Endothelial microparticles in high glucose environment: molecular pathways of GDM-associated placental dysfunction and coenzyme Q10-based targeted therapy

doi: 10.1186/s12951-026-04147-1

Figure Lengend Snippet: G-EMPs promote NGFR protein stability by inhibiting its ubiquitination by CYLD. ( A ) Western blot assays revealed that the expression levels of NGFR in HTR8/SVneo cells were affected by G-EMPs (100 particles/cell) after different durations of treatment with 100 µmol/L cycloheximide (CHX). G-EMPs: high glucose (25 mM)-cultured HUVEC-derived EMPs. ( B ) western blot analysis of NGFR expression in response to G-EMP treatment was performed to assess the effects of the proteasome inhibitor MG132 (20 µM) and the autophagy inhibitor CQ (45 µM). P21 serves as a positive control for proteasome regulation, whereas LC3B is a marker for autophagy. ( C ) Co-IP analysis of NGFR ubiquitination in HTR8/SVneo cells transfected with the Ub-HA and NGFR-Flag plasmids was performed to assess the effects of C-EMPs and G-EMPs treatment. ( D ) Prediction of deubiquitinating enzymes (DUBs) for NGFR in UniProt ( www.uniprot.orgt ). (DSI: deubiquitinases). ( E ) western blot analysis demonstrating the effects of C-EMPs and G-EMPs on NGFR protein expression levels. ( F ) Expression levels of NGFR in placentas from wild-type mice and GDM mice, as well as in placentas from normal pregnancies and GDM patients. ( G ) Assessment of CYLD and NGFR expression in HTR8/SVneo cells following knockdown with si-CYLD or overexpression via the CYLD plasmid. ( H ) western blot analysis was performed to detect the protein levels of CYLD, NGFR, p-ERK, ERK, N-cadherin, and E-cadherin in HTR8/SVneo cells treated with G-EMPs and subjected to CYLD knockdown using si-CYLD. ( I ) Histogram showing the migration area of HTR8/SVneo cells under various treatment conditions. ( J ) Histogram depicting the number of invading HTR8/SVneo cells under different treatment conditions. ( K , L ) western blot analysis demonstrating the co-IP results of NGFR-Flag and CYLD-His. ( M , N ) Co-IP analysis assessing the ubiquitination status of NGFR following CYLD overexpression and si-CYLD knockdown. ( O , P ) A ubiquitination assay demonstrated that CYLD inhibited NGFR K63-linked ubiquitination in HTR8/SVneo cells but had little effect on NGFR K48-linked ubiquitination. ( Q ) Representative immunoblots showing the interaction between NGFR and truncated CYLD, as indicated by co-IP assays. The CYLD protein is composed of two CAP-Gly domains (CAP), a phosphorylation ( P ) region, a GAP domain and a ubiquitin-specific protease (USP) catalytic domain. ( R ) Representative immunoblots showing the interaction between CYLD and truncated NGFR. NGFR consists of extracellularly cysteine-rich domains (CRDs), a serine/threonine-rich (S/T-rich) stalk, a chopper and a death domain. The data were analyzed using one-way ANOVA with Tukey’s post-hoc test. Data are presented as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: The human trophoblast cell lines HTR8/SVneo (CRL-3271, ATCC, USA) and JEG-3 (HTB-36, ATCC, USA) were purchased from the American Type Culture Collection.

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Cell Culture, Derivative Assay, Positive Control, Marker, Co-Immunoprecipitation Assay, Transfection, Knockdown, Over Expression, Plasmid Preparation, Migration, Phospho-proteomics

Journal: Journal of Nanobiotechnology

Article Title: Endothelial microparticles in high glucose environment: molecular pathways of GDM-associated placental dysfunction and coenzyme Q10-based targeted therapy

doi: 10.1186/s12951-026-04147-1

Figure Lengend Snippet: NGF is an essential component of EMPs that modulates trophoblast function. ( A ) STRING analysis of the protein interaction network of NGFR. ( B ) GeneMANIA prediction of the NGFR protein-related network. ( C ) Determination of NGF, BDNF, RGMA, SC1 expression in C-EMPs and G-EMPs by western blotting. ( D ) Serum levels of NGF in healthy pregnant women and patients with GDM. ( E ) The ROC curve indicates the AUC (0.78) and its 95% CI (0.6608–0.8992). ( F ) The protein expression levels of NGFR, p-ERK and ERK in HTR8/SVneo cells after the addition of 4 µg/mL anti-NGF or EMPs to the culture medium were determined by western blotting. ( G ) Interaction between NGFR and truncated CYLD indicated by Co-IP assays. ( H ) Representative images of the effects of 4 µg/mL anti-NGF or EMPs on the migration of HTR8/SVneo cells in culture medium, as determined by wound healing assays. Scale bar, 200 μm. ( I ) Histogram of the statistical migration area. ( J ) Determination of HTR8/SVneo cell invasion via Transwell assays. (anti-NGF: 4 µg/mL). Scale bar, 1 mm. ( K ) Histograms showing the statistical analysis of the number of invading cells. ( L ) N-cadherin and E-cadherin protein expression levels in different groups were determined via western blotting. ( M ) mRNA expression levels of NGF in the control group and 25 mM glucose group of HUVECs were determined using RT‒qPCR. ( N ) The protein expression levels of NGF in HUVECs in the control group and 25 mM glucose group were determined using western blotting. ( O ) Interaction between NGF and truncated CD63 indicated by Co-IP assays. The data were analyzed using one-way ANOVA with Tukey’s post-hoc test. Data are presented as mean ± SD. ns., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: The human trophoblast cell lines HTR8/SVneo (CRL-3271, ATCC, USA) and JEG-3 (HTB-36, ATCC, USA) were purchased from the American Type Culture Collection.

Techniques: Expressing, Western Blot, Co-Immunoprecipitation Assay, Migration, Control

Journal: Journal of Nanobiotechnology

Article Title: Endothelial microparticles in high glucose environment: molecular pathways of GDM-associated placental dysfunction and coenzyme Q10-based targeted therapy

doi: 10.1186/s12951-026-04147-1

Figure Lengend Snippet: CoQ10 directly binds and inhibits NGFR proteins to alleviate trophoblast dysfunction induced by EMPs. ( A ) Three-dimensional protein structure diagram of NGFR. ( B ) The chemical structure of CoQ10. ( C ) Binding mode 1 of CoQ10 to the NGFR protein is depicted on the left (refer to Supplementary Video 1), whereas the three-dimensional binding structure of the CoQ10 molecule to the NGFR protein is magnified from multiple angles on the right (refer to Supplementary Video 2). ( D ) BLI binding studies of NGFR and CoQ10. Fitted curves and values of the affinity constant. ( E ) Determination of NGFR, p-ERK, ERK, N-cadherin, and E-cadherin protein expression in HTR8/SVneo cells in different treatment groups using western blotting. ( F ) Co-IP analysis of NGFR ubiquitination in HTR8/SVneo cells transfected with the Ub-HA and NGFR-Flag plasmids and treated with G-EMPs/CoQ10. ( G ) Co-IP analysis of the interaction between NGFR and CYLD under the different G-EMP/CoQ10 treatments. ( H ) Histogram statistical analysis of the cell migration area. ( I ) Histogram statistical analysis of the number of invading cells. ( J ) Schematic representation of the mice with GDM treated with CoQ10 (200 mg/kg/day) (drawn from Figdraw). ( K ) Representative images of HE staining of the placentas of the mice with GDM and CoQ10-treated mice with GDM. Scale bar, 200 μm. (D: Decidual zone; J: junctional zone; L: labyrinth zone). ( L ) Area ratios of different regions in the mouse placenta ( n = 30/group). ( M ) Tissue immunofluorescence analysis of NGFR expression in mouse placental tissues. NGFR was stained with a specific antibody (green), trophoblasts were stained with a CK7-specific antibody (red), and nuclei were stained with DAPI (blue). Scale bar, 100 μm. The data were analyzed using one-way ANOVA with Tukey’s post-hoc test. Data are presented as mean ± SD. ns., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: The human trophoblast cell lines HTR8/SVneo (CRL-3271, ATCC, USA) and JEG-3 (HTB-36, ATCC, USA) were purchased from the American Type Culture Collection.

Techniques: Binding Assay, Expressing, Western Blot, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Transfection, Migration, Staining, Immunofluorescence