Journal: bioRxiv

Article Title: Foxp3 and BATF cooperatively direct cis -regulatory programs and gene expression for functional differentiation of Treg cells

doi: 10.1101/2025.08.13.669552

Figure Lengend Snippet: (A) Survival of cKO ( Batf 6l / 6l Foxp3 YFPCre/Y or Foxp3 YFPCre/YFPCre ) and control (Ctrl; Batf 6l /+ or Batf +/+ Foxp3 YFPCre/Y or Foxp3 YFPCre/YFPCre ) mice. (B) Histological analysis of indicated organs by hematoxylin and eosin staining. Inflammation scores are summarized. (C) Numbers of leukocytes isolated from indicated organs ( n =6–10). (D) FCM of Foxp3 + (Treg) and Foxp3 - (Tconv) CD4 + TCRβ + cells. Percentages of CD44 high CCR7 low cells are summarized ( n =5). (E) FCM of CD4 + TCRβ + cells. Percentages of cytokine-producing cells among CD4 + TCRβ + cells are summarized ( n =3–8). Mice aged 5–6 (D) or 5–8 (B, C, E) weeks were analyzed. Each dot represents an individual mouse. Data analyzed by the log-rank test (A) or two-way ANOVA with Sidak’s multiple comparisons test (B–E).

Article Snippet: Unconjugated anti-BATF rabbit mAb (D7C5, Cell Signaling Technology) was revealed using PE F(ab’) 2 donkey anti-rabbit IgG (Thermo Fisher Scientieic) in the presence of mouse IgG, rat IgG, Syrian hamster IgG (Jackson ImmunoResearch or Sigma-Aldrich) to block non-specific binding.

Techniques: Control, Staining, Isolation

Journal: bioRxiv

Article Title: Foxp3 and BATF cooperatively direct cis -regulatory programs and gene expression for functional differentiation of Treg cells

doi: 10.1101/2025.08.13.669552

Figure Lengend Snippet: (A) FCM of CD4 + T cell subsets from indicated mice. gMFI values of BATF and Foxp3 are normalized to mean values of Tconv cells in each experiment and summarized in graphs ( n =4). (B) Sorting of cTreg, eTreg, Tn, and Teff cells from pooled spleens and LNs of Foxp3 hCD 2 mice. FCM of Treg and Tconv cell subsets before and after sorting. Sorted cells were subjected to RNA-seq or ATAC-seq analysis ( n =2). (C) PCA of RNA-seq data using the 1,000 most variable genes with the lowest FDR by the likelihood ratio test. (D) Comparison of gene expression between BATF cKO and BATF KO Treg cells. hCD2 + CD4 + Treg cells from pooled spleens and LNs of Batf −/– or Batf +/+ Foxp3 hCD 2 mice were subjected to RNA-seq analysis ( n =2). FC vs. FC plot comparing the effects of Treg cell-specific BATF deficiency vs. germline BATF deficiency on gene expression. BATF-induced (red) and BATF-repressed (blue) (defined in ) genes are highlighted. (E) Percentages of cells expressing indicated molecules or gMFI of ICOS or CTLA-4 in indicated subsets ( n =3–7). gMFI values are normalized to mean values in Tconv cells in each experiment. (F) Over-representation analysis of BATF-induced genes and genes near BATF induced OCRs within the Molecular Signatures Database (MSigDB) gene sets. ‘Count’ refers to the number of genes in the MSigDB gene set, and ‘GeneRatio’ represents the ratio of genes in the MSigDB gene set to the analyzed gene set. Data analyzed by one-way ANOVA with Tukey’s multiple comparisons test (A) or two-way ANOVA with Sidak’s multiple comparisons test (E).

Article Snippet: Unconjugated anti-BATF rabbit mAb (D7C5, Cell Signaling Technology) was revealed using PE F(ab’) 2 donkey anti-rabbit IgG (Thermo Fisher Scientieic) in the presence of mouse IgG, rat IgG, Syrian hamster IgG (Jackson ImmunoResearch or Sigma-Aldrich) to block non-specific binding.

Techniques: RNA Sequencing, Comparison, Gene Expression, Expressing

Journal: bioRxiv

Article Title: Foxp3 and BATF cooperatively direct cis -regulatory programs and gene expression for functional differentiation of Treg cells

doi: 10.1101/2025.08.13.669552

Figure Lengend Snippet: (A) FCM of BATF cKO and WT Treg cells. Graphs display log2 ratios of YFP + to hCD2 + cells within CCR7 high or CCR7 low CD4 + TCRβ + populations ( n =2–8; N.D., not detected in sufficient numbers). (B) RNA-seq analysis of cKO and WT Treg cells. Volcano plot comparing gene expression, highlighting BATF-induced (red) and BATF-repressed (blue) genes. (C) Heatmap illustrating expression levels of BATF-induced genes across CD4 + T cell subsets. Genes are categorized as indicated, with the percentage of each category summarized in stacked bar graphs. (D) ATAC-seq analysis of BATF KO and WT Treg cells. Volcano plot comparing chromatin accessibility, highlighting BATF-induced (red) and BATF-repressed (blue) OCRs. (E) Top three de novo motifs differentially enriched in each OCR category. (F) Heatmap showing accessibility of BATF-induced OCRs across CD4 + T cell subsets. Stacked bar graphs represent the percentage of each OCR category, defined as in (C). (G) FCM of cKO and WT Treg cells. Graphs show percentages of cells expressing indicated molecules or normalized geometric MFI (gMFI) of ICOS or CTLA4 in Foxp3 + hCD2 - Treg cells ( n =3–7). Data analyzed by two-way ANOVA with Sidak’s multiple comparisons test (A) or unpaired t -test (G). See also and .

Article Snippet: Unconjugated anti-BATF rabbit mAb (D7C5, Cell Signaling Technology) was revealed using PE F(ab’) 2 donkey anti-rabbit IgG (Thermo Fisher Scientieic) in the presence of mouse IgG, rat IgG, Syrian hamster IgG (Jackson ImmunoResearch or Sigma-Aldrich) to block non-specific binding.

Techniques: RNA Sequencing, Gene Expression, Expressing

Journal: bioRxiv

Article Title: Foxp3 and BATF cooperatively direct cis -regulatory programs and gene expression for functional differentiation of Treg cells

doi: 10.1101/2025.08.13.669552

Figure Lengend Snippet: (A) FCM of R397W and WT Treg cells. Graphs display log2 ratios of hCD2 + to GFP + cells within CCR7 high or CCR7 low CD4 + TCRβ + populations ( n =4). (B) RNA-seq and ATAC-seq analysis of R397W and WT Treg cells. Volcano plots comparing gene expression and chromatin accessibility, highlighting Foxp3-induced (red) and Foxp3-repressed (blue) genes and OCRs. (C) Heatmaps of expression levels and chromatin accessibility of Foxp3-induced genes and OCRs across CD4 + T cell subsets. Stacked bar graphs show the percentage of each gene or OCR category, defined as in and . (D) FC vs. FC plots of RNA-seq and ATAC-seq data, comparing effects of BATF deficiency and the R397W mutation on gene expression and chromatin accessibility in Treg cells, highlighting BATF-induced (red) and BATF-repressed genes and OCRs (blue). (E) Log2 FC of gene expression and chromatin accessibility in WT vs. R397W Treg cells for indicated gene and OCR categories. (F) FCM of R397W and WT Treg cells. Graphs show percentages of cells expressing indicated molecules or normalized gMFI of ICOS or CTLA-4 in hCD2 + Treg cells ( n =3– 7). Treg cells were gated as Foxp3 + hCD2 + cells for intracellular CTLA-4 staining or as hCD2 + GFP - cells for other analyses. (G) Top three de novo motifs differentially enriched in each OCR category. Data analyzed by two-way ANOVA with Sidak’s multiple comparisons test (A), Mann-Whitney U test (E), or unpaired t -test (F). See also .

Article Snippet: Unconjugated anti-BATF rabbit mAb (D7C5, Cell Signaling Technology) was revealed using PE F(ab’) 2 donkey anti-rabbit IgG (Thermo Fisher Scientieic) in the presence of mouse IgG, rat IgG, Syrian hamster IgG (Jackson ImmunoResearch or Sigma-Aldrich) to block non-specific binding.

Techniques: RNA Sequencing, Gene Expression, Expressing, Mutagenesis, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Foxp3 and BATF cooperatively direct cis -regulatory programs and gene expression for functional differentiation of Treg cells

doi: 10.1101/2025.08.13.669552

Figure Lengend Snippet: (A, B) hCD2 + CD4 + Treg cells from pooled spleens and LNs of indicated mice were retrovirally transduced with the indicated RV. (A) GFP + and GFP - cells were sorted and analyzed by FCM. FCM of GFP + Thy1.1 + hCD2 + CD4 + TCRβ + cells, with gMFI values of Foxp3 or BATF summarized ( n =3). gMFI values are normalized to mock RV GFP-Thy1.1-CD4 + TCRβ + cells from Foxp3 R 397 W:hCD 2 /Y Batf −/– mice in each experiment. (B) CPD eFluor 450-labeled Ly5.1 hCD2-CD4 + T cells were mixed with sorted Ly5.2 GFP + Thy1.1 + hCD2 + CD4 + cells at indicated ratios and stimulated. Representative CPD histograms of Ly5.1 + Ly5.2-CD4 + T cells and percentages of divided cells are shown ( n =3). (C) hCD2 + CD4 + cells from Ly5.2 Foxp3 R 397 W:hCD 2 /Y Batf −/– mice were retrovirally transduced, labeled with CPD eFluor 450, and transferred into irradiated Ly5.1 or Ly5.1/5.2 Foxp3 hCD 2 /Y mice. FCM of GFP + Thy1.1 + donor cells on day 5 post-transfer, with percentages of CD103 + cells and normalized gMFI of indicated molecules summarized ( n =4–7). gMFI values are normalized to mock-transduced cells. Data analyzed by one-way ANOVA with Tukey’s multiple comparisons test (A, C).

Article Snippet: Unconjugated anti-BATF rabbit mAb (D7C5, Cell Signaling Technology) was revealed using PE F(ab’) 2 donkey anti-rabbit IgG (Thermo Fisher Scientieic) in the presence of mouse IgG, rat IgG, Syrian hamster IgG (Jackson ImmunoResearch or Sigma-Aldrich) to block non-specific binding.

Techniques: Transduction, Labeling, Irradiation

Journal: bioRxiv

Article Title: Foxp3 and BATF cooperatively direct cis -regulatory programs and gene expression for functional differentiation of Treg cells

doi: 10.1101/2025.08.13.669552

Figure Lengend Snippet: (A, B) Ly5.2 Batf −/– Foxp3 R 397 W Treg cells were transduced with the indicated GFP and Thy1.1 retroviruses (RV), labeled with Cell Proliferation Dye (CPD) eFluor 450, and transferred into irradiated Ly5.1 mice. On day 5 post-transfer, leukocytes were analyzed by FCM. (C, D) Ly5.2 OTII TCR transgenic Batf −/– Rag1 −/– Tn cells were retrovirally transduced, CPD labeled, and transferred into non-irradiated Ly5.1 mice. On day 1, OVA or PBS was administered intranasally. On day 4, leukocytes were analyzed by FCM (medLN: mediastinal lymph node). (A, C) FCM of donor CD4 + T cells before and after transfer. Graphs show numbers of GFP + Thy1.1 + donor cells relative to transferred GFP + Thy1.1 + cells ( n =8–18 for A, n =3–6 for C). (B, D) FCM of GFP + Thy1.1 + donor cells post-transfer. Graphs show percentages of cells with indicated phenotypes ( n =7–11 for B, n =3–6 for D). Data analyzed by two-way ANOVA with Tukey’s multiple comparisons test (C) or one-way ANOVA with Tukey’s multiple comparisons test (A, B, D). See also .

Article Snippet: Unconjugated anti-BATF rabbit mAb (D7C5, Cell Signaling Technology) was revealed using PE F(ab’) 2 donkey anti-rabbit IgG (Thermo Fisher Scientieic) in the presence of mouse IgG, rat IgG, Syrian hamster IgG (Jackson ImmunoResearch or Sigma-Aldrich) to block non-specific binding.

Techniques: Transduction, Labeling, Irradiation, Transgenic Assay

Journal: bioRxiv

Article Title: Foxp3 and BATF cooperatively direct cis -regulatory programs and gene expression for functional differentiation of Treg cells

doi: 10.1101/2025.08.13.669552

Figure Lengend Snippet: (A) Merged UMAP of integrated scATAC-seq data. (B) UMAP showing individual samples from two independent experiments. Graph summarizes cluster percentages. (C) Clusters color-coded by the log2 ratio of their average frequency in WT Treg cells vs. WT Tconv, BATF KO Treg, or R397W Treg cells, visualized on UMAP. Graphs show average proportions of WT Treg cells (red) and other populations (blue) per cluster. (D) Accessibility scores for CD4 + T cell subset-specific OCRs in single cells (UMAPs) or their distribution per cluster (violin plots). (E) Cluster-level expression of indicated genes. (F-I) Re-clustering and analysis of eTreg and Teff cell clusters 3 and 5–7. (F) Merged UMAP of re-clustered scATAC-seq data. Stacked bar graphs show subcluster proportions per cluster. (G) Accessibility scores for CD4 + T cell subset-specific OCRs. (H) Single-cell expression of indicated genes. (I) Subclusters color-coded and visualized, with graphs summarized as in (C). (J) FCM of BATF cKO and R397W Treg cells and their WT controls. Graphs display log2 ratios of Foxp3 + hCD2 - to Foxp3 + hCD2 + cells (BATF cKO) or Foxp3 + hCD2 + to Foxp3 + hCD2 - cells (R397W) in indicated subsets ( n =4–7). Data analyzed by two-way ANOVA with Tukey’s multiple comparisons test (J). See also .

Article Snippet: Unconjugated anti-BATF rabbit mAb (D7C5, Cell Signaling Technology) was revealed using PE F(ab’) 2 donkey anti-rabbit IgG (Thermo Fisher Scientieic) in the presence of mouse IgG, rat IgG, Syrian hamster IgG (Jackson ImmunoResearch or Sigma-Aldrich) to block non-specific binding.

Techniques: Expressing

Journal: bioRxiv

Article Title: Foxp3 and BATF cooperatively direct cis -regulatory programs and gene expression for functional differentiation of Treg cells

doi: 10.1101/2025.08.13.669552

Figure Lengend Snippet: (A) Distribution of distances between OCRs and TSSs for OCR-gene links. (B) Frequencies of numbers of genes linked to each OCR or OCRs linked to each gene. (C) Distribution of OCR-gene links per gene for all detected genes, Treg SE genes, and Tconv SE genes, as defined by H3K27ac ChIP-seq . CDF plot shows the cumulative distribution of OCR-gene links for these gene categories. (D) Percentages of OCR-gene links or control OCR-gene pairs overlapping with enhancer-promoter loops (EPLs) detected in Treg and/or Tconv cells, as defined by H3K27ac HiChIP . Control pairs are randomly selected and matched for OCRs-TSS distances. (E) Accessibility scores for topic-associated CREs in eTreg and Teff cell subclusters, visualized on UMAPs. (F) FC vs. FC plots comparing the effect of BATF deficiency and the R397W mutation on chromatin accessibility of topic-associated CREs. Each dot represents a CRE. (G, H) Percentages of CREs with the canonical Foxp3 motif (MA0850.1) (G) or indicated combinations of TF motifs (H) among topic-associated or all CREs. (I) Percentages of OCRs with indicated combinations of TF motifs in CREs and all OCRs. Data analyzed by Kolmogorov-Smirnov test (C) or Fisher’s exact test (D, I).

Article Snippet: Unconjugated anti-BATF rabbit mAb (D7C5, Cell Signaling Technology) was revealed using PE F(ab’) 2 donkey anti-rabbit IgG (Thermo Fisher Scientieic) in the presence of mouse IgG, rat IgG, Syrian hamster IgG (Jackson ImmunoResearch or Sigma-Aldrich) to block non-specific binding.

Techniques: ChIP-sequencing, Control, HiChIP, Mutagenesis

Journal: bioRxiv

Article Title: Foxp3 and BATF cooperatively direct cis -regulatory programs and gene expression for functional differentiation of Treg cells

doi: 10.1101/2025.08.13.669552

Figure Lengend Snippet: (A) Numbers of OCR-gene links per gene, ranked by abundance. Treg and Tconv SE genes are highlighted. Aggregated scATAC-seq tracks for indicated populations are shown at specific loci, with Treg SEs (red lines) and OCR-gene links (arcs). (B) Percentages of indicated OCR groups (defined in ) among linked OCRs (CREs) or all OCRs. (C) Accessibility scores for topic-associated CREs, visualized on UMAPs for all cells. (D, E) Heatmaps of accessibility scores for CREs associated with variable topics, averaged per cluster (D) or subcluster (E). (F) Percentages of indicated CRE groups in topic-associated or all CREs. (G) Aggregated scATAC-seq tracks for indicated clusters or populations at specific loci, with OCR positions (red lines) and OCR-gene links (arcs). BATF - and Foxp3-induced CREs associated with specific topics are highlighted in gray. (H) Heatmap of normalized frequency of CREs containing indicated TF motifs in topic-associated CREs. (I, J) Percentages of CREs with indicated (combinations of) TF motifs among topic associated or all CREs. (K) Percentages of OCRs with indicated combinations of TF motifs in CREs or all OCRs. Data analyzed by Fisher’s exact test (B, K). See also .

Article Snippet: Unconjugated anti-BATF rabbit mAb (D7C5, Cell Signaling Technology) was revealed using PE F(ab’) 2 donkey anti-rabbit IgG (Thermo Fisher Scientieic) in the presence of mouse IgG, rat IgG, Syrian hamster IgG (Jackson ImmunoResearch or Sigma-Aldrich) to block non-specific binding.

Techniques:

Journal: bioRxiv

Article Title: Foxp3 and BATF cooperatively direct cis -regulatory programs and gene expression for functional differentiation of Treg cells

doi: 10.1101/2025.08.13.669552

Figure Lengend Snippet: (A) Volcano plots comparing normalized read concentrations of BATF binding sites between Treg and Tconv cells, or Foxp3 binding sites between eTreg and cTreg cells. Differential binding sites are highlighted (red or blue). (B) Percentages of BATF or Foxp3 binding sites overlapping with CREs, other OCRs, or non-OCRs. (C) Percentages of BATF or Foxp3 binding sites overlapping with indicated categories of Foxp3 or BATF binding sites, respectively. (D) Top four de novo motifs differentially enriched at indicated binding sites. (E) Log2 normalized read concentrations of Foxp3 binding sites, comparing WT and BATF KO Treg cells in indicated categories. (F) BATF and Foxp3 ChIP-seq tracks. BATF - and Foxp3-induced CREs overlapping with Treg-specific BATF and eTreg-specific Foxp3 binding sites are highlighted in gray. (G) Nuclear proteins from TCR-stimulated or unstimulated Treg cells were immunoprecipitated (IP) with anti-BATF or isotype-matched mAbs, followed by immunoblotting (IB). Representative results from two independent experiments. (H) Venn diagram showing overlap between Treg-specific BATF binding sites and eTreg-specific Foxp3 binding sites among CREs. (I, J) Percentages of CREs overlapping with specified categories of BATF and/or Foxp3 binding sites in indicated CRE categories (I) or topic-associated or all CREs (J). (K) Cumulative distribution function (CDF) plots showing effects of BATF deficiency or the R397W mutation on accessibility of indicated CRE categories and expression of associated genes. Data analyzed by Mann-Whitney U test (E), hypergeometric test (H), or Kolmogorov-Smirnov test (K). See also .

Article Snippet: Unconjugated anti-BATF rabbit mAb (D7C5, Cell Signaling Technology) was revealed using PE F(ab’) 2 donkey anti-rabbit IgG (Thermo Fisher Scientieic) in the presence of mouse IgG, rat IgG, Syrian hamster IgG (Jackson ImmunoResearch or Sigma-Aldrich) to block non-specific binding.

Techniques: Binding Assay, ChIP-sequencing, Immunoprecipitation, Western Blot, Mutagenesis, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Foxp3 and BATF cooperatively direct cis -regulatory programs and gene expression for functional differentiation of Treg cells

doi: 10.1101/2025.08.13.669552

Figure Lengend Snippet: (A, B) Top five de novo motifs significantly enriched in all BATF or Foxp3 binding sites (A) or in BATF or Foxp3 binding sites overlapping with CREs (B). (C) CDF plots showing effects of BATF deficiency or the Foxp3 R397W mutation on accessibility of CREs or other OCRs overlapping with indicated categories of BATF or Foxp3 binding sites, and on expression of associated genes. (D) Percentages of CREs overlapping with indicated categories of BATF and/or Foxp3 binding sites in CREs with specified TF motif combinations. (E) Percentages of CREs overlapping with indicated categories of BATF and/or Foxp3 binding sites in topic-associated or all CREs. Data analyzed by Kolmogorov-Smirnov test (C).

Article Snippet: Unconjugated anti-BATF rabbit mAb (D7C5, Cell Signaling Technology) was revealed using PE F(ab’) 2 donkey anti-rabbit IgG (Thermo Fisher Scientieic) in the presence of mouse IgG, rat IgG, Syrian hamster IgG (Jackson ImmunoResearch or Sigma-Aldrich) to block non-specific binding.

Techniques: Binding Assay, Mutagenesis, Expressing

Journal: Journal of Assisted Reproduction and Genetics

Article Title: Histone demethylase KDM4A and KDM4B expression in granulosa cells from women undergoing in vitro fertilization

doi: 10.1007/s10815-018-1151-3

Figure Lengend Snippet: Immunolocalization of KDM4A and KDM4B in human ovaries. KDM4B was immunolocalized to granulosa cells and theca in large and small follicles, corpora lutea, and granulosa and oocyte in primordial and primary follicles. In all cells, localization of KDM4B was confined to the nucleus. KDM4A immunostaining was localized to granulosa and luteal cells and was weakly and diffusely noted throughout the ovary. Due to the weaker level of staining, ×2 magnified images are presented for KDM4A. Control immunostaining using IgG-stained sections are presented. The figure includes results from four different ovaries and is representative of eight ovaries analyzed. Black arrowheads denote staining for KDM4B and KDM4A. Bar indicates 50 μm

Article Snippet: Rabbit monoclonal KDM4A (#5328, Cell Signaling) and KDM4B antibody (#8638, Cell Signaling) were each used at dilutions of 1:50 and 1:100, respectively.

Techniques: Immunostaining, Staining, Control

Journal: Journal of Assisted Reproduction and Genetics

Article Title: Histone demethylase KDM4A and KDM4B expression in granulosa cells from women undergoing in vitro fertilization

doi: 10.1007/s10815-018-1151-3

Figure Lengend Snippet: Correlation of KDM4A and KDM4B mRNA expression in cumulus and mural granulosa cells. The correlation of the fold change values for expression of KDM4A and KDM4B mRNA in the cumulus granulosa (a) and mural granulosa (b) was determined for patients in the not pregnant (red) and pregnant-live birth (green) groups. Spearman’s rs values are indicated for each correlation. Spearman’s correlation (rs) = 1.0–0.8 very strong, 0.79–0.6 strong, 0.59–0.4 moderate

Article Snippet: Rabbit monoclonal KDM4A (#5328, Cell Signaling) and KDM4B antibody (#8638, Cell Signaling) were each used at dilutions of 1:50 and 1:100, respectively.

Techniques: Expressing

Journal: Journal of Assisted Reproduction and Genetics

Article Title: Histone demethylase KDM4A and KDM4B expression in granulosa cells from women undergoing in vitro fertilization

doi: 10.1007/s10815-018-1151-3

Figure Lengend Snippet: Expression of KDM4A and KDM4B mRNA in cumulus and mural granulosa. Granulosa cells were collected at the time of oocyte retrieval; RNA was isolated and expression of KDM4A and KDM4B was assessed by qRT-PCR. Normalized data (∆CT) is expressed as fold change from the mean of the pregnant cohort. The median line ± 95% confidence intervals are shown. Pregnant-live birth (n = 31) versus not pregnant (n = 53) were compared by Mann-Whitney. *p < 0.04 pregnant-live birth versus not pregnant

Article Snippet: Rabbit monoclonal KDM4A (#5328, Cell Signaling) and KDM4B antibody (#8638, Cell Signaling) were each used at dilutions of 1:50 and 1:100, respectively.

Techniques: Expressing, Isolation, Quantitative RT-PCR, MANN-WHITNEY

Journal: Journal of Assisted Reproduction and Genetics

Article Title: Histone demethylase KDM4A and KDM4B expression in granulosa cells from women undergoing in vitro fertilization

doi: 10.1007/s10815-018-1151-3

Figure Lengend Snippet: Correlation of KDM4 mRNA expression in cumulus and mural granulosa cells with patient age. The fold change values for expression of KDM4A and KDM4B mRNA in the cumulus and mural granulosa in pregnant-live birth (green) and not pregnant (red) groups did not correlate with patient age. Spearman’s correlation values are indicated (rs) = 1.0–0.8 very strong, 0.79–0.6 strong, 0.59–0.4 moderate, 0.39–0.2 weak, 0.19–0 very weak correlation

Article Snippet: Rabbit monoclonal KDM4A (#5328, Cell Signaling) and KDM4B antibody (#8638, Cell Signaling) were each used at dilutions of 1:50 and 1:100, respectively.

Techniques: Expressing

Journal: Nature Communications

Article Title: Inflammation-induced Id2 promotes plasticity in regulatory T cells

doi: 10.1038/s41467-018-07254-2

Figure Lengend Snippet: Id2 is induced by IL-1β and IL-6 mediated signaling and downstream transcription factors STAT3, IRF4 and BATF in T reg cells. a Strategy for sorting T Naive , iT reg , and ex-Foxp3 T H 17 cells from Foxp3 Thy1.1 reporter mice (left). Flow cytometry analysis of IL-1r1 and IL-6rα among the T Naive , iT reg , and ex-Foxp3 T H 17 cells (right). b , c FACS-sorted iT reg cells were re-stimulated with cytokines as indicated and analyzed for Id2 and Foxp3 mRNA expression by RT-qPCR ( b ) as well as for Id2 protein levels by flow cytometry ( c ). d Cartoon depicting highly putative STAT3, IRF4, and BATF binding motifs upstream of Id2 transcription start site (TSS). e Cartoon depicting Id2 promoter constructs used for luciferase reporter assay. f Id2 promoter-luciferase construct (−582/+36) were co-transfected with the combination of STAT3, IRF4 and BATF expressing vectors in HEK-293 T cells. Lysates were prepared 30 h after transfection, and luciferase activities were measured with the reporter activities normalized to renilla luciferase activity. *P < 0.05, **P < 0.005, ***P < 0.001 (Student’s t -test). All data are representative three independent experiments with similar results (error bars, s.d.)

Article Snippet: 0.5 μg of the Id2 promoter-luciferase constructs or pGL4.17 empty vector was co-transfected with STAT3 (MR227265; Origene), IRF4 (MR226642; Origene) and BATF (MR222114; Origene) expression plasmids (HEK-293 T) and 1 μg of the Foxp3 promoter-luciferase construct was co-transfected with E2A (MG209745; Origene) and Id2 (MR200792; Origene) expression plasmids (Jurkat).

Techniques: Flow Cytometry, Expressing, Quantitative RT-PCR, Binding Assay, Construct, Luciferase, Reporter Assay, Transfection, Activity Assay

Journal: Immunity

Article Title: Negative costimulation constrains T cell differentiation by imposing boundaries on possible cell states

doi: 10.1016/j.immuni.2019.03.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-BATF 154Sm , Fluidigm , Cat #:3154012A; Clone:D7C5.

Techniques: Virus, Recombinant, Adjuvant, Polymer, Conjugation Assay, Staining, Mass Cytometry, Sequencing, Transgenic Assay, Double Knockout, Software

Journal: Scientific Reports

Article Title: Immune system development-related signature predicts prognosis and sorafenib-treatment resistance of hepatocellular carcinoma by intergrating machine learning and single-cell analyses

doi: 10.1038/s41598-025-93187-y

Figure Lengend Snippet: BATF in relation to prognosis and sorafenib response in MCHH cohort. ( A ) Boxplot shows the distribution of BATF expression between resistant and sensitive samples. ( B , C ) IHC images and BATF-score based on IHC images showing the distribution of BATF expression between sorafenib resistant and sensitive samples (Red arrows: BATF + immune cells; black arrows: BATF- immune cells). ( D ) Paired plot shows the distribution of BATF expression between HCC and paracancer samples. ( E , F ) IHC images and BATF-score based on IHC images shows the distribution of BATF expression between HCC and paracancer samples (Black arrows: immune cells). ( G ) Kaplan-Meier curves of OS with high- and low-BATF expression groups. ( H ) Time-dependent ROC curve of BATF expression analysis for predicting OS at 1-, 2-, and 3-years. ( I ). Univariate and multivariate Cox regression analysis of BATF expression in HCC samples. ( J ) Kaplan-Meier curves of OS with high- and low-BATF score groups. ( K ) Time-dependent ROC curve of BATF-score analysis for predicting OS at 1-, 2-, and 3-years. ( M ) Univariate and multivariate Cox regression analysis of BATF-score expression in HCC samples. MCHH: Mengchao Hepatobiliary Hospital; IHC: Immunohistochemistry; HCC: Hepatocellular carcinoma; OS: Overall survival; ROC: Receiver operating characteristic; *: P < 0.05; **: P < 0.01.

Article Snippet: IHC staining were performed using the antibodies against BATF (1:15000, CSB-PA619074LA01HU, CUSABIO) as described previously.

Techniques: Expressing, Immunohistochemistry

Journal: Mucosal immunology

Article Title: A role for BATF3 in T H 9 differentiation and T-cell-driven mucosal pathologies.

doi: 10.1038/s41385-018-0122-4

Figure Lengend Snippet: Fig. 2 TL1A upregulates BATF and BATF3 expression during TH9 differentiation. a Transcriptional profiling of TH9 and TH9-TL1A cells by RNA sequencing. Heat map displaying RNA-sequencing data using the top 100 genes with the largest IQR (interquartile range). The dendrograms to the left and above the heat map represent clustering of genes (rows) and samples (columns). b Naive CD4+ T cells were differentiated into different TH subsets and Tregs for 3 days. Relative Batf mRNA expression was analyzed by qPCR. c Naive CD4+ T cells were differentiated under TH0- or TH9-polarizing conditions with or without TL1A for the indicated time periods. QPCR analysis of relative Batf mRNA expression. d Co- staining of BATF and IRF4 (top). Quantitative analysis of the percentages of BATF+IRF4+ cells (bottom). e Co-staining of BATF and IL-9. f–g Naive human CD4+ T cells were isolated from PBMCs of healthy volunteers and differentiated under TH9-polarizing conditions with or without TL1A. f Representative intracellular staining of IL-9 and BATF at 48 h. g Frequency of BATF+IL-9+ T cells differentiated under TH9 and TH9-TL1A conditions. Means are shown. Each symbol represents an individual donor. N = 4. h Relative Batf3 mRNA expression in different murine TH subsets and Tregs was analyzed by qPCR. i Relative Batf3 mRNA expression at indicated time points. j Representative co-staining of BATF3 and IL-9. k Frequency of BATF3+IL-9+ T cells differentiated under TH9 and TH9-TL1A conditions. Means are shown. Each symbol represents an individual experiment. N = 3. l–m Naive human CD4+ T cells were differentiated under TH9-polarizing conditions with or without TL1A. l Representative intracellular staining of IL-9 and BATF3 at 48 h. m Frequency of BATF3+IL-9+ T cells differentiated under TH9 and TH9-TL1A conditions. Means are shown. Each symbol represents an individual donor. N = 4. Data represent means ± SD of one independent experiment out of two (a) or at least three (b–d, i, k) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.005 as determined by Student’s t test

Article Snippet: Intracellular staining Cells were restimulated with 50 ng/ml PMA (phorbol 12-myristate 13-acetate), 500 ng/ml ionomycin, and monensin (eBioscience) for 4 h, stained with anti-CD4 (RM4-5, eBioscience), fixed and permeabilized using the FoxP3 staining buffer set (eBioscience), and stained with antibodies against murine IL-9 (RM9A4, BioLegend), IL-10 (JES5-16E3), IL-13 (13A), IL-17A (eBio17B7), IL-17F (eBio18F10), IFN-γ (XMG1.2), IL-4 (BVD6-24G2), IL-22 (1H8PWSR), Ki67 (SolA15), BATF (MBM7C7), IRF4 (3E4), human IL-9 (MH9A4, all eBioscience), and BATF3 (841792, R&D Systems).

Techniques: Expressing, RNA Sequencing, Staining, Isolation

Journal: Mucosal immunology

Article Title: A role for BATF3 in T H 9 differentiation and T-cell-driven mucosal pathologies.

doi: 10.1038/s41385-018-0122-4

Figure Lengend Snippet: Fig. 4 Effects of BATF and BATF3 deficiencies on TH9 differentiation. a–d Naive CD4+ T cells from Batf−/−or WT mice were differentiated under TH0- or TH9-polarizing conditions with or without TL1A for 3 days. a Intracellular staining of IL-9 and IL-10. b ELISA analysis of IL-9 production. c ELISA analysis of IL-10 production. d ELISA analysis of IL-13 production. e–h Naive CD4+ T cells from Batf3−/−or WT mice were differentiated under TH9-polarizing conditions with or without TL1A for 3 days. e Intracellular staining of IL-9 and IL-10. f ELISA analysis of IL-9 production. g ELISA analysis of IL-10 production. h ELISA analysis of IL-13 production. i Transcriptional profiling of WT and Batf3−/−TH9-TL1A cells by RNA sequencing. Heat map displaying RNA-sequencing data of genes differentially expressed with p < 0.01. The dendrograms to the left and above the heat map represent clustering of genes (rows) and samples (columns). Data represent means ± SD of one independent experiment out of two (i) or three (a–h) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.005 determined by Student’s t test

Article Snippet: Intracellular staining Cells were restimulated with 50 ng/ml PMA (phorbol 12-myristate 13-acetate), 500 ng/ml ionomycin, and monensin (eBioscience) for 4 h, stained with anti-CD4 (RM4-5, eBioscience), fixed and permeabilized using the FoxP3 staining buffer set (eBioscience), and stained with antibodies against murine IL-9 (RM9A4, BioLegend), IL-10 (JES5-16E3), IL-13 (13A), IL-17A (eBio17B7), IL-17F (eBio18F10), IFN-γ (XMG1.2), IL-4 (BVD6-24G2), IL-22 (1H8PWSR), Ki67 (SolA15), BATF (MBM7C7), IRF4 (3E4), human IL-9 (MH9A4, all eBioscience), and BATF3 (841792, R&D Systems).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, RNA Sequencing