evt medium  (Thermo Fisher)

Journal: Life Medicine

Article Title: Single-cell analysis reveals essential lncRNAs regulating human trophoblast lineage differentiation

doi: 10.1093/lifemedi/lnag010

Figure Lengend Snippet: Transcriptomic landscape of lncRNAs in human placental trophoblasts. (A) UMAP plot showing first-trimester and term placental single-nucleus transcriptomes (CTB, cytotrophoblast; EVT, extravillous trophoblast; STB, syncytiotrophoblast; STR, stromal cell; e, early; l, late). (B) UMAP plot showing the distribution of first-trimester and term placental samples. (C) Pie plot of the fraction of lncRNAs and mRNAs in snRNA-seq and bulk RNA-seq, respectively. The value “ n ” indicates the average number of detected transcripts per cell for both lncRNAs and mRNAs based on the experimentally measured data. (D) Box plots of transcript counts in per cell type. Statistical significance was determined by two-sided Wilcoxon rank-sum tests (**** P < 0.0001). (E) Volcano plot of cell-type-specific lncRNAs across trophoblast subtypes. The x -axis represents the difference in the percentage of cells expressing each gene between the two compared cell types (Δ percentage = pct.1 − pct.2), while the y -axis indicates the log 2 (fold change) in average gene expression. This representation integrates both expression magnitude and expression prevalence at the single-cell level. (F) Heatmap of cell-type-specific lncRNA expression atlas across trophoblast populations. (G) UMAP plots showing the expression distribution of representative lncRNAs (Color gradient represents the average expression level per cell). (H) GO biological process terms enriched in protein-coding genes (PCGs) associated with stage-specific lncRNAs.

Article Snippet: Cells were cultured in 2 mL of EVT medium [DMEM/F12 supplemented with 0.1 mM 2-mercaptoethanol, 0.5% Penicillin–Streptomycin, 0.3% BSA, 1% ITS-X supplement, 100 ng/mL NRG1 (Sino Biological, 13499-H08H-100), 7.5 mM A83-01, 2.5 mM Y27632, and 4% KnockOut Serum Replacement (KSR) (Thermo Fisher Scientific, 10828028)], and Matrigel (Corning, 356231) was added to the medium containing the cells at a final concentration of 2%.

Techniques: RNA Sequencing, Expressing, Gene Expression, Single Cell

Journal: International Journal of Biological Sciences

Article Title: Excessive Kynurenine Metabolism Impairs Lysosomal acidification and Triggers mtDNA Release via the AHR/CISH/ATP6V1A Axis in Decidual Macrophages Associated with Unexplained Recurrent Pregnancy Loss

doi: 10.7150/ijbs.121947

Figure Lengend Snippet: AHR-activated macrophages impair trophoblast function. ( A ) Heatmap showing expression levels of CGAS , NLRP3 , and TLR9 in various cell types determined by scRNA-seq. ( B ) Bar plots displaying significantly enriched GO terms in EVTs from URPL pregnancies. ( C ) Schematic diagram illustrating the co-culture system. ( D ) Representative images western blot images of TLR9, NLRP3, and cGAS protein expression in primary EVTs co-cultured with KYN-pretreated dMφs or control dMφs (n = 3 per group). ( E ) Representative immunofluorescence images showing co-localization of HLA-G and cGAS in hTSCs-EVTs co-cultured with KYN-pretreated dMφs or control dMφs (n = 5 per group). ( F ) Flow cytometry analysis of apoptosis levels in primary EVTs following co-culture with dMφs (n = 3 per group). ( G ) Scratch wound healing assay evaluating the migration capacity of HTR-8/SVneo cells (n = 3 per group). ( H ) Transwell invasion assay measuring the invasive ability of HTR-8/SVneo cells (n = 3 per group). ( I ) Western blot analysis of proteins associated with NF-κB signaling (p-NF-κB p65, NF-κB p65, p-IκBα, IκBα), apoptosis (BCL2, cleaved Caspase-3), and extracellular matrix remodeling (MMP9, MMP2) in HTR-8/SVneo cells. Data are presented as mean ± SD. Statistical significance was determined using the Student's t-test for two-group comparisons and one-way ANOVA for multiple comparisons; ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To induce hTSCs-derived EVTs (hTSCs-EVTs), hTSCs were seeded onto 6-well plates pre-coated with 1 μg/ml collagen IV and cultured in 2 mL of EVTs medium [DMEM/F12 supplemented with 0.1 mM 2-mercaptoethanol, 0.5% PS, 0.3% BSA, 1% ITS-X, 100 ng/ml NRG1 (CST, Cat#26941), 7.5 μM A83-01, 2.5 μM Y27632, and 4% KnockOut Serum Replacement (KSR, ThermoFisher, Cat#10828010)].

Techniques: Expressing, Co-Culture Assay, Western Blot, Cell Culture, Control, Immunofluorescence, Flow Cytometry, Wound Healing Assay, Migration, Transwell Invasion Assay

Journal: International Journal of Biological Sciences

Article Title: Excessive Kynurenine Metabolism Impairs Lysosomal acidification and Triggers mtDNA Release via the AHR/CISH/ATP6V1A Axis in Decidual Macrophages Associated with Unexplained Recurrent Pregnancy Loss

doi: 10.7150/ijbs.121947

Figure Lengend Snippet: AHR-activated macrophages impair trophoblast function. ( A ) Heatmap showing expression levels of CGAS , NLRP3 , and TLR9 in various cell types determined by scRNA-seq. ( B ) Bar plots displaying significantly enriched GO terms in EVTs from URPL pregnancies. ( C ) Schematic diagram illustrating the co-culture system. ( D ) Representative images western blot images of TLR9, NLRP3, and cGAS protein expression in primary EVTs co-cultured with KYN-pretreated dMφs or control dMφs (n = 3 per group). ( E ) Representative immunofluorescence images showing co-localization of HLA-G and cGAS in hTSCs-EVTs co-cultured with KYN-pretreated dMφs or control dMφs (n = 5 per group). ( F ) Flow cytometry analysis of apoptosis levels in primary EVTs following co-culture with dMφs (n = 3 per group). ( G ) Scratch wound healing assay evaluating the migration capacity of HTR-8/SVneo cells (n = 3 per group). ( H ) Transwell invasion assay measuring the invasive ability of HTR-8/SVneo cells (n = 3 per group). ( I ) Western blot analysis of proteins associated with NF-κB signaling (p-NF-κB p65, NF-κB p65, p-IκBα, IκBα), apoptosis (BCL2, cleaved Caspase-3), and extracellular matrix remodeling (MMP9, MMP2) in HTR-8/SVneo cells. Data are presented as mean ± SD. Statistical significance was determined using the Student's t-test for two-group comparisons and one-way ANOVA for multiple comparisons; ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To induce hTSCs-derived EVTs (hTSCs-EVTs), hTSCs were seeded onto 6-well plates pre-coated with 1 μg/ml collagen IV and cultured in 2 mL of EVTs medium [DMEM/F12 supplemented with 0.1 mM 2-mercaptoethanol, 0.5% PS, 0.3% BSA, 1% ITS-X, 100 ng/ml NRG1 (CST, Cat#26941), 7.5 μM A83-01, 2.5 μM Y27632, and 4% KnockOut Serum Replacement (KSR, ThermoFisher, Cat#10828010)].

Techniques: Expressing, Co-Culture Assay, Western Blot, Cell Culture, Control, Immunofluorescence, Flow Cytometry, Wound Healing Assay, Migration, Transwell Invasion Assay