e decidual breg il10 promoter methylation  (Pyrosequencing Inc)

Journal: Cell Communication and Signaling : CCS

Article Title: Reprogramming the epigenetic profile improves the B regulatory cell function of patients with recurrent pregnancy loss

doi: 10.1186/s12964-026-02669-7

Figure Lengend Snippet: Epigenetic dysregulation of the IL10 promoter in regulatory B cells (Bregs) from recurrent pregnancy loss (RPL) patients. Bregs (CD19⁺CD5⁺CD1d⁺) were isolated from peripheral blood of healthy controls (HC, n = 30) and RPL patients ( n = 30). A – D Flow cytometry gating strategy: ( A ) Live cell selection (DAPI⁻), ( B ) doublet exclusion (FSC-A vs. FSC-H), ( C ) CD19⁺ B cell isolation, and ( D ) CD5⁺CD1d⁺ Breg identification. E Peripheral Breg frequency. F – G Global histone modifications in Bregs by ELISA: ( F ) H3K4me3 (transcriptional activation mark) and ( G ) H3K27me3 (transcriptional repression mark). H LPS-induced IL-10 secretion (ELISA). I IL10 promoter methylation quantified by methylation-specific PCR (MSP). J H3K4me3 enrichment at the IL10 promoter (ChIP-ELISA). K - L IL10 expression: ( K ) mRNA (RT-qPCR) and ( L ) secreted protein (ELISA). M Inverse correlation between IL10 promoter methylation and transcriptional activity. The data in bars are mean ± SD. Dots in bars represent biological replicates. Statistics: Mann–Whitney test for two groups, ANOVA + Tukey post hoc test for panel H. Abbreviations: Bregs, regulatory B cells; RPL, recurrent pregnancy loss; HC, healthy control; MSP, methylation-specific PCR; ChIP-ELISA, chromatin immunoprecipitation coupled with ELISA

Article Snippet: E Decidual Breg Il10 promoter methylation (bisulfite pyrosequencing).

Techniques: Isolation, Flow Cytometry, Selection, Cell Isolation, Enzyme-linked Immunosorbent Assay, Activation Assay, Methylation, Expressing, Quantitative RT-PCR, Activity Assay, MANN-WHITNEY, Control, Chromatin Immunoprecipitation

Journal: Cell Communication and Signaling : CCS

Article Title: Reprogramming the epigenetic profile improves the B regulatory cell function of patients with recurrent pregnancy loss

doi: 10.1186/s12964-026-02669-7

Figure Lengend Snippet: IL10 promoter methylation inversely correlates with Breg immunosuppressive capacity. A Gating strategy for proliferating effector T cells (Teffs) by flow cytometry (CFSE-low population). B Proliferating Teff counts in co-cultures with Bregs from healthy controls (HC) or recurrent pregnancy loss (RPL) patients. Data: mean ± SD; *** p < 0.0001 (one-way ANOVA with Tukey’s post hoc test). C – D Correlation between IL10 promoter methylation (quantified by methylation-specific PCR, MSP) and Teff suppression in ( C ) HC ( n = 30) and ( D ) RPL ( n = 30) cohorts. Dots represent individual samples; dashed lines indicate linear regression. Statistics: Pearson’s correlation coefficient (two-tailed). Abbreviations: Bregs, regulatory B cells; Teff, effector T cell; MSP, methylation-specific PCR; HC, healthy control; RPL, recurrent pregnancy loss

Article Snippet: E Decidual Breg Il10 promoter methylation (bisulfite pyrosequencing).

Techniques: Methylation, Flow Cytometry, Two Tailed Test, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Reprogramming the epigenetic profile improves the B regulatory cell function of patients with recurrent pregnancy loss

doi: 10.1186/s12964-026-02669-7

Figure Lengend Snippet: DNMT1 regulates IL10 promoter methylation in regulatory B cells (Bregs). A Quantification of DNMT1 occupancy at the IL10 promoter in Bregs from healthy controls (HC, n = 30) and recurrent pregnancy loss (RPL) patients ( n = 30) by chromatin immunoprecipitation coupled with ELISA (ChIP-ELISA). B - C Correlation analysis between DNMT1 levels and IL10 promoter methylation in HC (B) and RPL ( C ) Bregs. Key: ( A ) DNMT1 levels (mean ± SD) normalized to input DNA. B - C Scatter plots with linear regression lines (Pearson r and p -values indicated). Statistics: Mann–Whitney U test (A); Pearson correlation ( B , C ). P values: *** p < 0.0001. Each dot represents an individual sample. Abbreviations: Bregs, Regulatory B cells; HC, Healthy control; IL10, Interleukin-10; RPL, Recurrent pregnancy loss. IgG: Isotype IgG used as a negative control Ab in ChIP

Article Snippet: E Decidual Breg Il10 promoter methylation (bisulfite pyrosequencing).

Techniques: Methylation, Chromatin Immunoprecipitation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Control, Negative Control

Journal: Cell Communication and Signaling : CCS

Article Title: Reprogramming the epigenetic profile improves the B regulatory cell function of patients with recurrent pregnancy loss

doi: 10.1186/s12964-026-02669-7

Figure Lengend Snippet: DNMT1-mediated suppression of IL-10 expression in B cells. A c-Maf occupancy at the IL10 promoter region measured by ChIP-ELISA. B Correlation analysis between c-Maf and IL10 transcription in regulatory B cells (Bregs). C – F Pan B cells were transfected with DNMT1 or mutant (mu) DNMT1 plasmids, stimulated with CD40L (1 µg/ml; C-D) or LPS (1 µg/ml; E–F) for 24 h, and analyzed for ( C , E ) IL10 mRNA levels (RT-qPCR) and ( D , F ) IL-10 protein in culture supernatants (ELISA). Data are mean ± SD ( n = 3 biological replicates); each dot represents an individual sample. Statistical significance was determined by Student’s t-test (A), Pearson’s r (B), and one-way ANOVA with Tukey’s post-hoc test (C–F). p-values: p < 0.05 ( * ), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****)

Article Snippet: E Decidual Breg Il10 promoter methylation (bisulfite pyrosequencing).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Mutagenesis, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Reprogramming the epigenetic profile improves the B regulatory cell function of patients with recurrent pregnancy loss

doi: 10.1186/s12964-026-02669-7

Figure Lengend Snippet: DNMT1 ubiquitination status and TRIM28-Pol II correlations at the IL10 promoter in Bregs. A – C Ubiquitination levels of DNMT1 at the IL10 promoter in Bregs. ( D ) TRIM28 occupancy at the IL10 promoter in Bregs. E – F Correlation between TRIM28 occupancy and DNMT1 levels. G – H Correlation between DNMT1 occupancy and RNA polymerase II (Pol II) recruitment. I – L B cells transfected with plasmids encoding wild-type (DNMT1p, TRIM28p) or mutant (DNMT1mup, TRIM28mup) proteins. I Quantification of His-DNMT1 by ELISA. J – L Ubiquitination ( J ), K48-linked polyubiquitin chains (K), and proteasomal recruitment ( L ) in His-antibody-precipitated complexes (cross-ELISA). Data are mean ± SD; individual data points represent biological replicates. Statistics: One-way ANOVA with Tukey’s post hoc test ( A – D , I – L ); Pearson’s correlation coefficient ( E – H ). Significance: ** p < 0.01; *** p < 0.001; **** p < 0.0001. Plasmid notation: “p” denotes wild-type plasmid; “mup” denotes catalytically inactive mutant (e.g., DNMT1mup: C1246Y mutation)

Article Snippet: E Decidual Breg Il10 promoter methylation (bisulfite pyrosequencing).

Techniques: Ubiquitin Proteomics, Transfection, Mutagenesis, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Cell Communication and Signaling : CCS

Article Title: Reprogramming the epigenetic profile improves the B regulatory cell function of patients with recurrent pregnancy loss

doi: 10.1186/s12964-026-02669-7

Figure Lengend Snippet: SAHA restores the immunosuppressive capacity of RPL Bregs. A Global TRIM28 protein levels in Bregs. B – C Correlation between TRIM28 expression and the immunosuppressive function of Bregs. D SAHA treatment induces TRIM28 upregulation in RPL Bregs. I – M Levels of indicated molecules at the IL10 promoter region in RPL Bregs. N Teff proliferation rate. O Quantification of proliferating Teff cells. P Effects of TRIM28 knockdown (kd) via RNA interference (RNAi). Q - S Bars show the levels of indicated cytokines in culture supernatant. Individual data points represent independent samples; bar graphs show mean ± SD. Statistical analysis: Mann–Whitney U test (A); Pearson correlation coefficient (B–C); one-way ANOVA with Tukey’s post hoc test (I–M, O, P). Significance: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Abbreviations: IgG, isotype control antibody for ChIP experiments; kd, TRIM28 knockdown; cRNAi, control RNAi (scrambled sequence)

Article Snippet: E Decidual Breg Il10 promoter methylation (bisulfite pyrosequencing).

Techniques: Expressing, Knockdown, MANN-WHITNEY, Control, Sequencing

Journal: Cell Communication and Signaling : CCS

Article Title: Reprogramming the epigenetic profile improves the B regulatory cell function of patients with recurrent pregnancy loss

doi: 10.1186/s12964-026-02669-7

Figure Lengend Snippet: SAHA Rescues Pregnancy Outcomes in RPL Mice via TRIM28-DNMT1-IL10 Axis. A Representative uterine horns (Day 12.5 p.c.): black arrows = resorbed embryos; red arrows = healthy embryos. B Fetal resorption rates. C – D Decidual Breg TRIM28 ( C ) and DNMT1 ( D ) mRNA levels (RT-qPCR). E Decidual Breg Il10 promoter methylation (bisulfite pyrosequencing). F Decidual Breg IL-10 secretion (ELISA). G - H Decidual Breg suppressive effects (Teff co-culture). Data of bar graphs are presented as mean ± SD. Each dot in bars presents one sample. Statistics: One-way ANOVA + Tukey’s test. ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: E Decidual Breg Il10 promoter methylation (bisulfite pyrosequencing).

Techniques: Quantitative RT-PCR, Methylation, Enzyme-linked Immunosorbent Assay, Co-Culture Assay