Journal: Carcinogenesis

Article Title: Estrogen-mediated epigenetic repression of the imprinted gene cyclin-dependent kinase inhibitor 1C in breast cancer cells

doi: 10.1093/carcin/bgr017

Figure Lengend Snippet: 11p15.5 epigenetic and genetic states and expression of CDKN1C in breast cancer cells. (A) The DNA methylation status of the 11p15.5 ICR differentially methylated domain was analyzed by high-resolution bisulfite PCR pyrosequencing in primary tumors (n = 306), breast cancer cell lines (n = 52) and normal breast tissues (n = 19). Methylation levels of individual CpG sites (circles) are shown in a color gradient ranging from 0% (white) to 100% (blue). Left, diagram of CpG sites interrogated by the pyrosequencing assay. Right, results presented in rows of columns. (B) Multiple linear regression analysis of impact of 11p15.5 ICR methylation and genetic status on CDKN1C expression in breast cancer cells. The combined 11p15.5 ICR methylation, copy number and CDKN1C mRNA levels in 16 breast cancer cell lines were assessed by bisulfite PCR pyrosequencing, DNA copy number qPCR and qRT–PCR, respectively. DNA copy number of 11p15.5 ICR was quantified by absolute standard curve of cloned PCR products as described in supplementary Figure S4 (available at Carcinogenesis Online). Relative CDKN1C ΔCt expression values were obtained by normalizing to the 36B4 reference gene. (C) The effect of E2 on CDKN1C expression in breast cancer cells with 11p15.5 ICR hypermethylation. MDA-MB-453 and T47D cells were stimulated with 10 nM E2 or vehicle for 12 h. CDKN1C mRNA levels were assessed as described in Figure 1. Results are the average of two independent experiments. Columns, mean (n = 6); bars, SD.

Article Snippet: Approximately 300 ng of genomic DNA was bisulfite modified with EZ DNA Methylation Kit (Zymo Research, Irivine, CA) according to the manufacturer’s protocol.

Techniques: Expressing, DNA Methylation Assay, Methylation, Pyrosequencing Assay, Quantitative RT-PCR, Clone Assay

Journal: Carcinogenesis

Article Title: Estrogen-mediated epigenetic repression of the imprinted gene cyclin-dependent kinase inhibitor 1C in breast cancer cells

doi: 10.1093/carcin/bgr017

Figure Lengend Snippet: Potential mechanisms causing repression of CDKN1C in breast cancer cells. (A) Proposed model for epigenetic repression of CDKN1C through coordinated loop formation with the 11p15.5 ICR. CTCF binding to the ICR and CDKN1C locus and forms a long-range intrachromosomal loop via dimerization of CTCF. Ligand-bound ERα complex (orange and blue sphere) may mediate silencing through the formation of a secondary loop that serves both to sequester upstream tissue-specific enhancers and to recruit PRC2 and HDAC1 to the 11p15.5 ICR. CTCF serves as a scaffold to secure the PRC2 complex that methylates H3K27, leading to the formation of a repressive chromatin state at the CDKN1C locus. (B) Proposed regulatory mechanism of CDKN1C-AS. The formation of a double-stranded RNA may negatively regulate stability, transport and/or translation of the sense CDKN1C transcript. (C) Summary of three potential mechanisms causing CDKN1C repression in breast cancer cells. DNA methylation status of 11p15.5 ICR is indicated by large oval: white (unmethylated), black (methylated). Upper left, in the normal imprinted domain unmethylated 11p15.5 ICR on the paternal allele (♂) functions as a silencer and a promoter for KCNQ1OT1 transcription, repressing CDKN1C expression. The methylated maternal allele (♀) cannot function as a silencer or a promoter for KCNQ1OT1, thus permitting expression of CDKN1C. Upper right, the CDKN1C-AS transcript represses CDKN1C in trans, potentially through a double-stranded RNA mechanism. Under certain cellular conditions, this may be induced by estrogen-mediated upregulation of CDKN1C-AS. Lower left, DNA hypomethylation resulting from genetic loss of the methylated 11p15.5 ICR allele leads to aberrant domain silencer activity mediated by unrestricted CTCF binding and KCNQ1OT1 transcription, repressing CDKN1C expression. Lower right, estrogen induces KCNQ1OT1 transcription and CTCF recruitment to mediate ICR silencer activity, which in turn direct epigenetic repression of the CDKN1C locus.

Article Snippet: Approximately 300 ng of genomic DNA was bisulfite modified with EZ DNA Methylation Kit (Zymo Research, Irivine, CA) according to the manufacturer’s protocol.

Techniques: Binding Assay, DNA Methylation Assay, Methylation, Expressing, Activity Assay

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