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