human extravillous trophoblast cell line htr  (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

Journal: bioRxiv

Article Title: Trophoblast ferroptosis restricts SARS-CoV-2 spread in the placenta

doi: 10.64898/2026.01.26.701742

Figure Lengend Snippet: (A) Viral titers of SARS-CoV-2 Delta variant in supernatants from JEG-3 cells infected over 48 hours with an MOI of 1. Titers were determined via plaque assay. (B-E) Protein expression of iron transport and storage proteins in SARS-CoV-2-infected JEG-3 cells at 6, 24, and 48 hpi, as determined by western blotting. (F) Schematic of regulation of iron transport and storage expression by iron regulatory proteins (IRPs). (G,H) Protein expression of IRPs in infected JEG-3 cells, as determined by western blotting. (I) Impact of excess iron supplementation (FAC) on viral titers in JEG-3 cells, as determined via plaque assay. (J-N) Protein expression of ferroptosis markers and inhibitors in SARS-CoV-2-infected JEG-3 cells over course of infection. Expression was measured by western blotting. (O) Cytotoxicity of SARS-CoV-2 Delta variant in JEG-3 cells as measured by LDH release over course of infection. (P) Cytotoxicity of JEG-3 cells treated with increasing concentrations of the FSP1 inhibitor, iFSP1, with or without infection. Cytotoxicity was determined via LDH assay. (Q) Cytotoxicity of small molecules iFSP1 and RSL3 in JEG-3 cells with or without SARS-CoV-2 infection, as measured by LDH release. (R) Viral titers in supernatants from JEG-3 cells infected with SARS-CoV-2 for 48 hours, following pre-treatment and post-inoculation exposure to iFPS1 and RSL3. Viral titers were determined via plaque assay. Data represents average of n=3 independent replicates per group ± SEM. Significance determined by Student’s unpaired t-test except for P and Q which were determined by two-way ANOVA and R which was determined by one-way ANOVA. For all tests, *p<0.05, **p<0.01, ***p<0.001, ****p<<0.001.

Article Snippet: The human trophoblast cell line JEG-3 (ATCC HTB-36) was cultured in DMEM/F-12 (Gibco, 11330032) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, 16140071).

Techniques: Variant Assay, Infection, Plaque Assay, Expressing, Western Blot, Lactate Dehydrogenase Assay