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Abcam foxp3 rabbit polyclonal ab
Foxp3 Rabbit Polyclonal Ab, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/foxp3 rabbit polyclonal ab/product/Abcam
Average 90 stars, based on 1 article reviews
Price from $9.99 to $1999.99
foxp3 rabbit polyclonal ab - by Bioz Stars, 2020-09
90/100 stars

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Article Title: Intrahepatic Tissue Implantation Represents a Favorable Approach for Establishing Orthotopic Transplantation Hepatocellular Carcinoma Mouse Models
Article Snippet: .. Immunohistochemistry To examine the presence of T lymphocytes and regulatory T cells in tumors, mouse tumors and human HCC tissues (kindly provided by biobank from Cancer Institute and Hospital of Tianjin Medical University) were fixed with Bouin’s solution (Sigma, US) and embedded with paraffin followed by staining with CD3 rabbit polyclonal Ab (Novas, US) and Foxp3+ rabbit polyclonal Ab (abcam, UK) with a dilution of 1 in 250, detected with goat-anti-rabbit secondary Ab. .. Magnetic resonance imaging (MRI) The magnetic resonance images of orthotopic HCC mice were acquired using a 3.0 Tesia MR scanner (Signa Excite HDx; GE healthcare, Milwaukee, WI, USA) with a small animal coil in Tianjin Medical University General Hospital.

Immunohistochemistry:

Article Title: Intrahepatic Tissue Implantation Represents a Favorable Approach for Establishing Orthotopic Transplantation Hepatocellular Carcinoma Mouse Models
Article Snippet: .. Immunohistochemistry To examine the presence of T lymphocytes and regulatory T cells in tumors, mouse tumors and human HCC tissues (kindly provided by biobank from Cancer Institute and Hospital of Tianjin Medical University) were fixed with Bouin’s solution (Sigma, US) and embedded with paraffin followed by staining with CD3 rabbit polyclonal Ab (Novas, US) and Foxp3+ rabbit polyclonal Ab (abcam, UK) with a dilution of 1 in 250, detected with goat-anti-rabbit secondary Ab. .. Magnetic resonance imaging (MRI) The magnetic resonance images of orthotopic HCC mice were acquired using a 3.0 Tesia MR scanner (Signa Excite HDx; GE healthcare, Milwaukee, WI, USA) with a small animal coil in Tianjin Medical University General Hospital.

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  • 99
    Abcam rabbit anti foxp3
    HBZ inhibites <t>Foxp3-mediated</t> CTLA-4 and GITR expression in vitro . (A) Mouse CD4 + CD25 − T cells co-transduced with the retroviral vectors were stained with the indicated antibodies. Mean fluorescence intensity (MFI) of CD25, GITR, and CTLA-4 in GFP/NGFR double-positive cells are shown as mean ± SD. for triplicate culture. *, P
    Rabbit Anti Foxp3, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti foxp3/product/Abcam
    Average 99 stars, based on 63 article reviews
    Price from $9.99 to $1999.99
    rabbit anti foxp3 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    98
    Abcam anti foxp3 antibody
    Tcf1 and Lef1 share common and distinct targets in T cell genome. (A) Venn diagram showing distinct and overlapping Tcf1 and Lef1 binding peaks in T conv CD4 + T cells. The value of 741 in the intersection denotes the number of Lef1 peaks that overlapped with 675 of 4,505 Tcf1 peaks. (B) Violin plots showing Lef1 binding strength (log 2 (1+RPKM)) in Tcf1-Lef1 common peaks and Lef1-only peaks (left), and Tcf1 binding strength (log 2 (1+RPKM)) in Tcf1-Lef1 common peaks and Tcf1-only peaks. The P values were determined using two-sided Mann–Whitney U test. (C and D) Lef1 binding events at T reg–up signature genes (C) and cell cycle/antigen response genes (D). Lef1 peaks were identified within ±50 kb of the gene bodies defined in Figs. 6 C and 7 (A and B) . Tcf1-Lef1 common or Lef1-only sites were further fractioned into groups where these sites showed cooccupancy with <t>Foxp3,</t> nonoverlapping but associated with Foxp3 peaks, or no connection with Foxp3 peaks. The number and identity of genes in each group are shown. (E) Visualization of Tcf1, Lef1, and Foxp3 binding peaks at select gene loci. Displayed on UCSC genome browser are ChIP-seq tracks at the Bub1b, Ccr2 , and Ccr5 gene loci. Refer to Fig. 6 D for Lef1 binding peaks at Izumo1r and Fig. 7 C for Lef1 binding peaks at Prdm1 and Birc5 loci. (F) Proposed model to explain Tcf1 and Lef1 functional redundancy in T cells. See text for details.
    Anti Foxp3 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti foxp3 antibody/product/Abcam
    Average 98 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    anti foxp3 antibody - by Bioz Stars, 2020-09
    98/100 stars
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    HBZ inhibites Foxp3-mediated CTLA-4 and GITR expression in vitro . (A) Mouse CD4 + CD25 − T cells co-transduced with the retroviral vectors were stained with the indicated antibodies. Mean fluorescence intensity (MFI) of CD25, GITR, and CTLA-4 in GFP/NGFR double-positive cells are shown as mean ± SD. for triplicate culture. *, P

    Journal: PLoS Pathogens

    Article Title: HTLV-1 bZIP Factor Induces T-Cell Lymphoma and Systemic Inflammation In Vivo

    doi: 10.1371/journal.ppat.1001274

    Figure Lengend Snippet: HBZ inhibites Foxp3-mediated CTLA-4 and GITR expression in vitro . (A) Mouse CD4 + CD25 − T cells co-transduced with the retroviral vectors were stained with the indicated antibodies. Mean fluorescence intensity (MFI) of CD25, GITR, and CTLA-4 in GFP/NGFR double-positive cells are shown as mean ± SD. for triplicate culture. *, P

    Article Snippet: Antibodies and reagents The following antibodies were used for immunoprecipitation and Western blotting: mouse anti-Flag (clone M2; Sigma, Saint Louis, MO), mouse anti-c-myc (clone 9E10; Sigma), mouse anti-HA (clone HA-7; Sigma), rabbit anti-His polyclonal antibody (MBL), rabbit anti-FOXP3 (polyclonal antibody; Abcam), and rabbit anti-HBZ polyclonal antisera .

    Techniques: Expressing, In Vitro, Transduction, Staining, Fluorescence

    HBZ directly induces Foxp3 expression in CD4 + T cells. (A) Schematic diagrams of retrovirus vectors used in this study. (B) Mouse CD4 + CD25 − T cells transduced with retrovirus vector encoding HBZ or empty vector with or without TGF-β were stained with anti-Foxp3 antibody and analyzed by flow cytometry. (C) To study the effect of HBZ on promoter activity of the Foxp3 gene, EL4 cells were transfected with Foxp3 reporter plasmid and/or HBZ expressing plasmid. Representative data shown are firefly luciferase activities normalized to those of renilla luciferase (mean ± SD).

    Journal: PLoS Pathogens

    Article Title: HTLV-1 bZIP Factor Induces T-Cell Lymphoma and Systemic Inflammation In Vivo

    doi: 10.1371/journal.ppat.1001274

    Figure Lengend Snippet: HBZ directly induces Foxp3 expression in CD4 + T cells. (A) Schematic diagrams of retrovirus vectors used in this study. (B) Mouse CD4 + CD25 − T cells transduced with retrovirus vector encoding HBZ or empty vector with or without TGF-β were stained with anti-Foxp3 antibody and analyzed by flow cytometry. (C) To study the effect of HBZ on promoter activity of the Foxp3 gene, EL4 cells were transfected with Foxp3 reporter plasmid and/or HBZ expressing plasmid. Representative data shown are firefly luciferase activities normalized to those of renilla luciferase (mean ± SD).

    Article Snippet: Antibodies and reagents The following antibodies were used for immunoprecipitation and Western blotting: mouse anti-Flag (clone M2; Sigma, Saint Louis, MO), mouse anti-c-myc (clone 9E10; Sigma), mouse anti-HA (clone HA-7; Sigma), rabbit anti-His polyclonal antibody (MBL), rabbit anti-FOXP3 (polyclonal antibody; Abcam), and rabbit anti-HBZ polyclonal antisera .

    Techniques: Expressing, Transduction, Plasmid Preparation, Staining, Flow Cytometry, Cytometry, Activity Assay, Transfection, Luciferase

    HBZ physically interacts with Foxp3 and NFAT. (A) The expression vectors of the indicated proteins were co-transfected into 293FT cells, and their interactions were analyzed by immunoprecipitation (IP). (B) Nuclear extract of ATL-43T cells was subjected to IP with anti-HBZ antibody or control IgG, and detected by anti-FoxP3 antibody. (C and D) The interactions of HBZ and Foxp3 were analyzed by IP using HBZ mutants (C) or Foxp3 mutants (D). A schematic diagram of Foxp3 mutants is shown. ZnF, zinc finger; LZ, leucine zipper; FH, forkhead domain. Asterisks (* or **) show responsible region for each molecular interaction. (E) The interactions among HBZ, Foxp3 and NFATc2 were analyzed with or without EtBr.

    Journal: PLoS Pathogens

    Article Title: HTLV-1 bZIP Factor Induces T-Cell Lymphoma and Systemic Inflammation In Vivo

    doi: 10.1371/journal.ppat.1001274

    Figure Lengend Snippet: HBZ physically interacts with Foxp3 and NFAT. (A) The expression vectors of the indicated proteins were co-transfected into 293FT cells, and their interactions were analyzed by immunoprecipitation (IP). (B) Nuclear extract of ATL-43T cells was subjected to IP with anti-HBZ antibody or control IgG, and detected by anti-FoxP3 antibody. (C and D) The interactions of HBZ and Foxp3 were analyzed by IP using HBZ mutants (C) or Foxp3 mutants (D). A schematic diagram of Foxp3 mutants is shown. ZnF, zinc finger; LZ, leucine zipper; FH, forkhead domain. Asterisks (* or **) show responsible region for each molecular interaction. (E) The interactions among HBZ, Foxp3 and NFATc2 were analyzed with or without EtBr.

    Article Snippet: Antibodies and reagents The following antibodies were used for immunoprecipitation and Western blotting: mouse anti-Flag (clone M2; Sigma, Saint Louis, MO), mouse anti-c-myc (clone 9E10; Sigma), mouse anti-HA (clone HA-7; Sigma), rabbit anti-His polyclonal antibody (MBL), rabbit anti-FOXP3 (polyclonal antibody; Abcam), and rabbit anti-HBZ polyclonal antisera .

    Techniques: Expressing, Transfection, Immunoprecipitation

    Transgenic expression of HBZ in CD4 + T cells increases Foxp3 + T reg cells with impaired suppressive function. (A and B) Mouse splenocytes were stained with the indicated antibodies, and analyzed by flow cytometry. Representative dot plots gated on the CD4 + population are shown. For these experiments, HBZ -Tg mice without any symptoms were used. Tables show the mean ± SD (n = 5 for A, n = 3 for B). (C) The absolute number of Foxp3 + or Foxp3 − CD4 + T cells in HBZ -Tg and non-Tg mice. The results shown are the mean ± SD (n = 3). (D) Flow cytometric analysis for the Foxp3 expression in CD4 single positive thymocytes. Representative dot plots gated on the CD4 single positive population are shown from three independent analyses. (E) Flow cytometric analyses of CD4 + T cells for T reg related molecules. Numbers in dot plots indicate mean fluorescence intensity (MFI) of each molecule in the rectangular gates. (F) Suppressive activity of T reg cells from HBZ- or non-Tg mice on T-cell proliferation. Sorted Foxp3 + T cells were cultured with CD4 + CD25 − cells of non-Tg mice as responder cells for 72 h with ConA or soluble anti-CD3 antibody and x-irradiated antigen presenting cells (APCs), and [ 3 H] thymidine incorporation during the last 6 hours was measured. Results are means ± SD for triplicate cultures. (G) In vivo BrdU incorporation in total CD4 + , Foxp3 + CD4 + , or Foxp3 − CD4 + T cells. The results shown are the mean ± SD (n = 3). (H) Sorted Foxp3 + cells were labeled with CFSE and cultured with anti-CD3 antibody and x-irradiated APCs. After 96 hours, the cells were stained with anti-Foxp3, and CFSE dilution was analyzed for Foxp3 + cells. *, P

    Journal: PLoS Pathogens

    Article Title: HTLV-1 bZIP Factor Induces T-Cell Lymphoma and Systemic Inflammation In Vivo

    doi: 10.1371/journal.ppat.1001274

    Figure Lengend Snippet: Transgenic expression of HBZ in CD4 + T cells increases Foxp3 + T reg cells with impaired suppressive function. (A and B) Mouse splenocytes were stained with the indicated antibodies, and analyzed by flow cytometry. Representative dot plots gated on the CD4 + population are shown. For these experiments, HBZ -Tg mice without any symptoms were used. Tables show the mean ± SD (n = 5 for A, n = 3 for B). (C) The absolute number of Foxp3 + or Foxp3 − CD4 + T cells in HBZ -Tg and non-Tg mice. The results shown are the mean ± SD (n = 3). (D) Flow cytometric analysis for the Foxp3 expression in CD4 single positive thymocytes. Representative dot plots gated on the CD4 single positive population are shown from three independent analyses. (E) Flow cytometric analyses of CD4 + T cells for T reg related molecules. Numbers in dot plots indicate mean fluorescence intensity (MFI) of each molecule in the rectangular gates. (F) Suppressive activity of T reg cells from HBZ- or non-Tg mice on T-cell proliferation. Sorted Foxp3 + T cells were cultured with CD4 + CD25 − cells of non-Tg mice as responder cells for 72 h with ConA or soluble anti-CD3 antibody and x-irradiated antigen presenting cells (APCs), and [ 3 H] thymidine incorporation during the last 6 hours was measured. Results are means ± SD for triplicate cultures. (G) In vivo BrdU incorporation in total CD4 + , Foxp3 + CD4 + , or Foxp3 − CD4 + T cells. The results shown are the mean ± SD (n = 3). (H) Sorted Foxp3 + cells were labeled with CFSE and cultured with anti-CD3 antibody and x-irradiated APCs. After 96 hours, the cells were stained with anti-Foxp3, and CFSE dilution was analyzed for Foxp3 + cells. *, P

    Article Snippet: Antibodies and reagents The following antibodies were used for immunoprecipitation and Western blotting: mouse anti-Flag (clone M2; Sigma, Saint Louis, MO), mouse anti-c-myc (clone 9E10; Sigma), mouse anti-HA (clone HA-7; Sigma), rabbit anti-His polyclonal antibody (MBL), rabbit anti-FOXP3 (polyclonal antibody; Abcam), and rabbit anti-HBZ polyclonal antisera .

    Techniques: Transgenic Assay, Expressing, Staining, Flow Cytometry, Cytometry, Mouse Assay, Fluorescence, Activity Assay, Cell Culture, Irradiation, In Vivo, BrdU Incorporation Assay, Labeling

    The frequency of FoxP3 + Tregs was increased in the lesions of DSS-induced colitis in KO mice. Immunohistochemical staining of the lesions of DSS-induced colitis for FoxP3 (A). For the counting of FoxP3 + cells, 5 high-power fields selected at random in each slide were examined by three different pathologists (B). KO, GPx1 −/− × Cat −/− . Scale bar is 50 µm. Data are mean ± SE of six separate experiments.

    Journal: PLoS ONE

    Article Title: Attenuation of Experimental Colitis in Glutathione Peroxidase 1 and Catalase Double Knockout Mice through Enhancing Regulatory T Cell Function

    doi: 10.1371/journal.pone.0095332

    Figure Lengend Snippet: The frequency of FoxP3 + Tregs was increased in the lesions of DSS-induced colitis in KO mice. Immunohistochemical staining of the lesions of DSS-induced colitis for FoxP3 (A). For the counting of FoxP3 + cells, 5 high-power fields selected at random in each slide were examined by three different pathologists (B). KO, GPx1 −/− × Cat −/− . Scale bar is 50 µm. Data are mean ± SE of six separate experiments.

    Article Snippet: Endogenous peroxidase was blocked with hydrogen peroxide (5% in PBS) for 30 min and the sections were subjected to heat-induced antigen retrieval step before incubation with a universal blocking solution (Dako, Glostrup, Denmark) for 30 min. Then, the sections were incubated with anti-phosphotyrosine (pY)-Stat3 (Tyr705) (clone D3A7, Cell Signaling Technology, Danvers, MA), anti-indoleamine 2,3,-dioxygenase (IDO, rabbit polyclonal antibody, Abcam, Cambridge, UK) or anti-FoxP3 (rabbit polyclonal antibody, Abcam).

    Techniques: Mouse Assay, Immunohistochemistry, Staining

    Tcf1 and Lef1 share common and distinct targets in T cell genome. (A) Venn diagram showing distinct and overlapping Tcf1 and Lef1 binding peaks in T conv CD4 + T cells. The value of 741 in the intersection denotes the number of Lef1 peaks that overlapped with 675 of 4,505 Tcf1 peaks. (B) Violin plots showing Lef1 binding strength (log 2 (1+RPKM)) in Tcf1-Lef1 common peaks and Lef1-only peaks (left), and Tcf1 binding strength (log 2 (1+RPKM)) in Tcf1-Lef1 common peaks and Tcf1-only peaks. The P values were determined using two-sided Mann–Whitney U test. (C and D) Lef1 binding events at T reg–up signature genes (C) and cell cycle/antigen response genes (D). Lef1 peaks were identified within ±50 kb of the gene bodies defined in Figs. 6 C and 7 (A and B) . Tcf1-Lef1 common or Lef1-only sites were further fractioned into groups where these sites showed cooccupancy with Foxp3, nonoverlapping but associated with Foxp3 peaks, or no connection with Foxp3 peaks. The number and identity of genes in each group are shown. (E) Visualization of Tcf1, Lef1, and Foxp3 binding peaks at select gene loci. Displayed on UCSC genome browser are ChIP-seq tracks at the Bub1b, Ccr2 , and Ccr5 gene loci. Refer to Fig. 6 D for Lef1 binding peaks at Izumo1r and Fig. 7 C for Lef1 binding peaks at Prdm1 and Birc5 loci. (F) Proposed model to explain Tcf1 and Lef1 functional redundancy in T cells. See text for details.

    Journal: The Journal of Experimental Medicine

    Article Title: Tcf1 and Lef1 are required for the immunosuppressive function of regulatory T cells

    doi: 10.1084/jem.20182010

    Figure Lengend Snippet: Tcf1 and Lef1 share common and distinct targets in T cell genome. (A) Venn diagram showing distinct and overlapping Tcf1 and Lef1 binding peaks in T conv CD4 + T cells. The value of 741 in the intersection denotes the number of Lef1 peaks that overlapped with 675 of 4,505 Tcf1 peaks. (B) Violin plots showing Lef1 binding strength (log 2 (1+RPKM)) in Tcf1-Lef1 common peaks and Lef1-only peaks (left), and Tcf1 binding strength (log 2 (1+RPKM)) in Tcf1-Lef1 common peaks and Tcf1-only peaks. The P values were determined using two-sided Mann–Whitney U test. (C and D) Lef1 binding events at T reg–up signature genes (C) and cell cycle/antigen response genes (D). Lef1 peaks were identified within ±50 kb of the gene bodies defined in Figs. 6 C and 7 (A and B) . Tcf1-Lef1 common or Lef1-only sites were further fractioned into groups where these sites showed cooccupancy with Foxp3, nonoverlapping but associated with Foxp3 peaks, or no connection with Foxp3 peaks. The number and identity of genes in each group are shown. (E) Visualization of Tcf1, Lef1, and Foxp3 binding peaks at select gene loci. Displayed on UCSC genome browser are ChIP-seq tracks at the Bub1b, Ccr2 , and Ccr5 gene loci. Refer to Fig. 6 D for Lef1 binding peaks at Izumo1r and Fig. 7 C for Lef1 binding peaks at Prdm1 and Birc5 loci. (F) Proposed model to explain Tcf1 and Lef1 functional redundancy in T cells. See text for details.

    Article Snippet: To determine enriched Foxp3 binding, rabbit anti-mouse Foxp3 polyclonal antibody (ab150743; Abcam) was used in ChIP experiments on sorted T reg cells following a published protocol, with Gmpr and Ctla4 as negative and positive controls, respectively ( ).

    Techniques: Binding Assay, MANN-WHITNEY, Chromatin Immunoprecipitation, Functional Assay

    Tcf1 and Foxp3 cooccupy at select genes regulating cell cycle and antigen responses. (A and B) Tcf1 and Foxp3 binding events at genes regulating cell cycle and antigen responses. Tcf1 and Foxp3 binding peaks were identified within ±50 kb of the gene bodies of 203 antigen response genes (A) and 185 cell cycle genes (B) that showed increased expression in Tcf1/Lef1-deficient T reg cells (as defined in Fig. 5 B and Fig. 5 A , respectively). These peaks were further fractioned into groups harboring Tcf1-only, Foxp3-only, Tcf1 and Foxp3 associated, and Tcf1-Foxp3 cooccupied peaks. The number and identity of genes in each group are shown. (C) Visualization of Tcf1, Lef1, and Foxp3 binding peaks at select cell cycle or antigen response genes. Displayed on UCSC genome browser are ChIP-seq tracks at the Brca1 , Kif11 , Ifng , Prdm1 , and Birc5 gene loci, in the same format as in Fig. 6 D . Lef1 ChIP-seq tracks are not shown for the three genes in top panels because they did not harbor evident Lef1 binding peaks. Highlighted in yellow at the Prdm1 locus is the –24-kb upstream region that was targeted by CRISPR/Cas9 in Fig. 7 E . (D) Tcf1 occupies the same sites in select genes regulating cell cycle and antigen responses in T conv and T reg cells. T conv and T reg cells were analyzed with ChIP-qPCR for enriched binding of Tcf1 to the indicated genomic locations. Dotted horizontal line denotes a no-enrichment level at the Hprt locus. Data are means ± SD from two to three independent experiments with each sample measured in duplicate or triplicate. The enriched Tcf1 binding at each genomic location has P

    Journal: The Journal of Experimental Medicine

    Article Title: Tcf1 and Lef1 are required for the immunosuppressive function of regulatory T cells

    doi: 10.1084/jem.20182010

    Figure Lengend Snippet: Tcf1 and Foxp3 cooccupy at select genes regulating cell cycle and antigen responses. (A and B) Tcf1 and Foxp3 binding events at genes regulating cell cycle and antigen responses. Tcf1 and Foxp3 binding peaks were identified within ±50 kb of the gene bodies of 203 antigen response genes (A) and 185 cell cycle genes (B) that showed increased expression in Tcf1/Lef1-deficient T reg cells (as defined in Fig. 5 B and Fig. 5 A , respectively). These peaks were further fractioned into groups harboring Tcf1-only, Foxp3-only, Tcf1 and Foxp3 associated, and Tcf1-Foxp3 cooccupied peaks. The number and identity of genes in each group are shown. (C) Visualization of Tcf1, Lef1, and Foxp3 binding peaks at select cell cycle or antigen response genes. Displayed on UCSC genome browser are ChIP-seq tracks at the Brca1 , Kif11 , Ifng , Prdm1 , and Birc5 gene loci, in the same format as in Fig. 6 D . Lef1 ChIP-seq tracks are not shown for the three genes in top panels because they did not harbor evident Lef1 binding peaks. Highlighted in yellow at the Prdm1 locus is the –24-kb upstream region that was targeted by CRISPR/Cas9 in Fig. 7 E . (D) Tcf1 occupies the same sites in select genes regulating cell cycle and antigen responses in T conv and T reg cells. T conv and T reg cells were analyzed with ChIP-qPCR for enriched binding of Tcf1 to the indicated genomic locations. Dotted horizontal line denotes a no-enrichment level at the Hprt locus. Data are means ± SD from two to three independent experiments with each sample measured in duplicate or triplicate. The enriched Tcf1 binding at each genomic location has P

    Article Snippet: To determine enriched Foxp3 binding, rabbit anti-mouse Foxp3 polyclonal antibody (ab150743; Abcam) was used in ChIP experiments on sorted T reg cells following a published protocol, with Gmpr and Ctla4 as negative and positive controls, respectively ( ).

    Techniques: Binding Assay, Expressing, Chromatin Immunoprecipitation, CRISPR, Real-time Polymerase Chain Reaction

    Impact of Tcf1/Lef1 deficiency on T reg cell transcriptome. (A–C) GSEA of transcriptomic changes of Tcf1/Lef1-deficient T reg cells. CD25 + YFP + T reg cells were sorted from the spleens of male Foxp3 Cre control or Foxp3 Cre Tcf7 fl/fl Lef1 fl/fl (dKO) mice at 16 wk of age and analyzed by RNA-seq, and the data were analyzed by GSEA C2-curated gene sets including “REACTOME_CELL_CYCLE” (A) and “GOLDRATH_ANTIGEN_RESPONSE” (B), and custom T reg–up signature gene set (C). Blue and red rectangles denote the leading edges of enrichment plots showing negative and positive enrichment in control T reg cells, respectively. Heat maps of gene expression levels within the leading edge (top 50 for A and B, and all for C) are shown. NES, normalized enrichment score; NOM p-val, nominal P values; FDR q-val, FDR q values generated from the GSEA algorithm. (D and E) Validation of expression changes in T reg–up signature genes (D) and cell cycle and antigen response-related genes (E) in Tcf1/Lef1-deficient T reg cells. T reg cells were sorted from old (≥24 wk) or young (6–8 wk) mice and analyzed by quantitative RT-PCR. The expression of each gene in T reg cells from the old Foxp3 Cre control mice was set at 1, and that in cells from other groups was normalized accordingly. Data are means ± SD from two independent experiments ( n = 3 for each group). Statistical significance was determined by one way ANOVA, followed by Student’s t test for indicated pairwise comparisons. ns, not statistically significant; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Tcf1 and Lef1 are required for the immunosuppressive function of regulatory T cells

    doi: 10.1084/jem.20182010

    Figure Lengend Snippet: Impact of Tcf1/Lef1 deficiency on T reg cell transcriptome. (A–C) GSEA of transcriptomic changes of Tcf1/Lef1-deficient T reg cells. CD25 + YFP + T reg cells were sorted from the spleens of male Foxp3 Cre control or Foxp3 Cre Tcf7 fl/fl Lef1 fl/fl (dKO) mice at 16 wk of age and analyzed by RNA-seq, and the data were analyzed by GSEA C2-curated gene sets including “REACTOME_CELL_CYCLE” (A) and “GOLDRATH_ANTIGEN_RESPONSE” (B), and custom T reg–up signature gene set (C). Blue and red rectangles denote the leading edges of enrichment plots showing negative and positive enrichment in control T reg cells, respectively. Heat maps of gene expression levels within the leading edge (top 50 for A and B, and all for C) are shown. NES, normalized enrichment score; NOM p-val, nominal P values; FDR q-val, FDR q values generated from the GSEA algorithm. (D and E) Validation of expression changes in T reg–up signature genes (D) and cell cycle and antigen response-related genes (E) in Tcf1/Lef1-deficient T reg cells. T reg cells were sorted from old (≥24 wk) or young (6–8 wk) mice and analyzed by quantitative RT-PCR. The expression of each gene in T reg cells from the old Foxp3 Cre control mice was set at 1, and that in cells from other groups was normalized accordingly. Data are means ± SD from two independent experiments ( n = 3 for each group). Statistical significance was determined by one way ANOVA, followed by Student’s t test for indicated pairwise comparisons. ns, not statistically significant; *, P

    Article Snippet: To determine enriched Foxp3 binding, rabbit anti-mouse Foxp3 polyclonal antibody (ab150743; Abcam) was used in ChIP experiments on sorted T reg cells following a published protocol, with Gmpr and Ctla4 as negative and positive controls, respectively ( ).

    Techniques: Mouse Assay, RNA Sequencing Assay, Expressing, Generated, Quantitative RT-PCR

    Tcf1/Lef1-deficient T reg cells are functionally impaired. (A–C) DSS-induced acute colitis model. Foxp3 Cre Tcf7 fl/fl Lef1 fl/fl and Foxp3 Cre control mice at 6–12 wk were given DSS at 1.75 g/100 ml (1.75% wt/vol) in drinking water for 8 d and the body weight was tracked daily (A). On day 9, the mesenteric LNs were harvested, and T conv YFP – CD4 + and CD8 + T cells were stimulated with anti-CD3 and anti-CD28 and measured for IFN-γ production. Representative data are in B, and cumulative data are in C. Data in A and C are means ± SD from two independent experiments ( n = 8 for control, and 5 for Foxp3 Cre Tcf7 fl/fl Lef1 fl/fl mice). *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Tcf1 and Lef1 are required for the immunosuppressive function of regulatory T cells

    doi: 10.1084/jem.20182010

    Figure Lengend Snippet: Tcf1/Lef1-deficient T reg cells are functionally impaired. (A–C) DSS-induced acute colitis model. Foxp3 Cre Tcf7 fl/fl Lef1 fl/fl and Foxp3 Cre control mice at 6–12 wk were given DSS at 1.75 g/100 ml (1.75% wt/vol) in drinking water for 8 d and the body weight was tracked daily (A). On day 9, the mesenteric LNs were harvested, and T conv YFP – CD4 + and CD8 + T cells were stimulated with anti-CD3 and anti-CD28 and measured for IFN-γ production. Representative data are in B, and cumulative data are in C. Data in A and C are means ± SD from two independent experiments ( n = 8 for control, and 5 for Foxp3 Cre Tcf7 fl/fl Lef1 fl/fl mice). *, P

    Article Snippet: To determine enriched Foxp3 binding, rabbit anti-mouse Foxp3 polyclonal antibody (ab150743; Abcam) was used in ChIP experiments on sorted T reg cells following a published protocol, with Gmpr and Ctla4 as negative and positive controls, respectively ( ).

    Techniques: Mouse Assay

    Ablating T-bet in T reg cells exacerbates inflammatory responses in the absence of Tcf1 and Lef1. (A) Detection of CXCR3 (surface) and T-bet (intracellular) expression in splenic T reg cells from mice of indicated genotypes at ≥24 wk. Values denote gMFI and those in percentages denote the frequency of gated population. Data are representative from three experiments with similar results. (B) Detection of Foxp3-YFP + T reg cells in the spleens from mice of indicated genotypes at ≥24 wk of age. Data are means ± SD ( n ≥ 8 for each group). (C) Detection of CD44 hi CD62L – effector and CD44 lo CD62L + resting T reg cells in spleens from mice of indicated genotypes at ≥24 wk of age. Representative data from three experiments are on the left, and cumulative data on subset frequency on the right are means ± SD from three experiments ( n ≥ 8 for each group). (D) IFN-γ production by splenic T reg cells, with representative data on the left and cumulative data on the right, showing means ± SD from three independent experiments ( n ≥ 6 for each group). (E and F) Detection of activation status and IFN-γ production by splenic YFP – CD4 + and CD8 + T cells. Representative data are shown in left panels, and cumulative data are in right panels, showing the frequency of CD44 hi CD62L – effector and CD44 lo CD62L + naive subsets in YFP – CD4 + and CD8 + T cells (top and bottom panels in E, respectively). The cumulative data are means ± SD from three independent experiments ( n ≥ 8 for each group). Statistical significance in B–F was determined by one way ANOVA, followed by Student’s t test for indicated pairwise comparisons. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Tcf1 and Lef1 are required for the immunosuppressive function of regulatory T cells

    doi: 10.1084/jem.20182010

    Figure Lengend Snippet: Ablating T-bet in T reg cells exacerbates inflammatory responses in the absence of Tcf1 and Lef1. (A) Detection of CXCR3 (surface) and T-bet (intracellular) expression in splenic T reg cells from mice of indicated genotypes at ≥24 wk. Values denote gMFI and those in percentages denote the frequency of gated population. Data are representative from three experiments with similar results. (B) Detection of Foxp3-YFP + T reg cells in the spleens from mice of indicated genotypes at ≥24 wk of age. Data are means ± SD ( n ≥ 8 for each group). (C) Detection of CD44 hi CD62L – effector and CD44 lo CD62L + resting T reg cells in spleens from mice of indicated genotypes at ≥24 wk of age. Representative data from three experiments are on the left, and cumulative data on subset frequency on the right are means ± SD from three experiments ( n ≥ 8 for each group). (D) IFN-γ production by splenic T reg cells, with representative data on the left and cumulative data on the right, showing means ± SD from three independent experiments ( n ≥ 6 for each group). (E and F) Detection of activation status and IFN-γ production by splenic YFP – CD4 + and CD8 + T cells. Representative data are shown in left panels, and cumulative data are in right panels, showing the frequency of CD44 hi CD62L – effector and CD44 lo CD62L + naive subsets in YFP – CD4 + and CD8 + T cells (top and bottom panels in E, respectively). The cumulative data are means ± SD from three independent experiments ( n ≥ 8 for each group). Statistical significance in B–F was determined by one way ANOVA, followed by Student’s t test for indicated pairwise comparisons. *, P

    Article Snippet: To determine enriched Foxp3 binding, rabbit anti-mouse Foxp3 polyclonal antibody (ab150743; Abcam) was used in ChIP experiments on sorted T reg cells following a published protocol, with Gmpr and Ctla4 as negative and positive controls, respectively ( ).

    Techniques: Expressing, Mouse Assay, Activation Assay

    Loss of Tcf1 and/or Lef1 does not detectably perturb T reg cell homeostasis. (A) Detection of Tcf1 and Lef1 expression levels. Splenocytes from Foxp3 Cre control and Foxp3 Cre Tcf7 fl/fl Lef1 fl/fl mice (5 wk old) were stained to identify TCRβ + CD4 + CD25 – Foxp3 – T conv and TCRβ + CD4 + CD25 + Foxp3 + T reg cells. Tcf1 and Lef1 were determined by intracellular staining in CD44 lo CD62L + naive T conv cells, CD44 lo CD62L + resting T reg, and CD44 hi CD62L – effector T reg cell subsets. Values denote geometric mean fluorescent intensity (gMFI), and dotted green lines mark gMFI of isotype control staining. Data are representative from two experiments with similar results. (B and C) Detection of T reg cells in the spleen. Spleens were harvested from mice of indicated genotypes at 12 or 24 wk of age, and Foxp3-YFP + cells were detected in TCRβ + CD4 + T cells, with representative data in B and cumulative data on the frequency (top) and numbers (bottom) of T reg cells in C. (D and E) Detection of key molecules on T reg cells. Splenic T reg cells from mice of indicated genotypes at ≥24 wk were analyzed for expression of GITR, CD28, and Nrp1 by cell surface staining and that of Helios and CTLA4 by intracellular staining. CD25 expression was detected on T reg cells from LNs. Values denote gMFI, and those in percentages denote the frequency of gated population. Cumulative data on gMFI of total CTLA and CD25 are in E. (F and G) Detection of effector and resting T reg cells. Splenic T reg cells were subfractionated to CD44 hi CD62L – effector T reg and CD44 lo CD62L + resting T reg subsets, with representative data in F and cumulative data on subset frequency in G. Data are means ± SD from more than four independent experiments ( n ≥ 9 for each group). Statistical significance in C, E, and G was determined by one-way ANOVA, followed by Student’s t test for indicated pairwise comparisons. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Tcf1 and Lef1 are required for the immunosuppressive function of regulatory T cells

    doi: 10.1084/jem.20182010

    Figure Lengend Snippet: Loss of Tcf1 and/or Lef1 does not detectably perturb T reg cell homeostasis. (A) Detection of Tcf1 and Lef1 expression levels. Splenocytes from Foxp3 Cre control and Foxp3 Cre Tcf7 fl/fl Lef1 fl/fl mice (5 wk old) were stained to identify TCRβ + CD4 + CD25 – Foxp3 – T conv and TCRβ + CD4 + CD25 + Foxp3 + T reg cells. Tcf1 and Lef1 were determined by intracellular staining in CD44 lo CD62L + naive T conv cells, CD44 lo CD62L + resting T reg, and CD44 hi CD62L – effector T reg cell subsets. Values denote geometric mean fluorescent intensity (gMFI), and dotted green lines mark gMFI of isotype control staining. Data are representative from two experiments with similar results. (B and C) Detection of T reg cells in the spleen. Spleens were harvested from mice of indicated genotypes at 12 or 24 wk of age, and Foxp3-YFP + cells were detected in TCRβ + CD4 + T cells, with representative data in B and cumulative data on the frequency (top) and numbers (bottom) of T reg cells in C. (D and E) Detection of key molecules on T reg cells. Splenic T reg cells from mice of indicated genotypes at ≥24 wk were analyzed for expression of GITR, CD28, and Nrp1 by cell surface staining and that of Helios and CTLA4 by intracellular staining. CD25 expression was detected on T reg cells from LNs. Values denote gMFI, and those in percentages denote the frequency of gated population. Cumulative data on gMFI of total CTLA and CD25 are in E. (F and G) Detection of effector and resting T reg cells. Splenic T reg cells were subfractionated to CD44 hi CD62L – effector T reg and CD44 lo CD62L + resting T reg subsets, with representative data in F and cumulative data on subset frequency in G. Data are means ± SD from more than four independent experiments ( n ≥ 9 for each group). Statistical significance in C, E, and G was determined by one-way ANOVA, followed by Student’s t test for indicated pairwise comparisons. *, P

    Article Snippet: To determine enriched Foxp3 binding, rabbit anti-mouse Foxp3 polyclonal antibody (ab150743; Abcam) was used in ChIP experiments on sorted T reg cells following a published protocol, with Gmpr and Ctla4 as negative and positive controls, respectively ( ).

    Techniques: Expressing, Mouse Assay, Staining

    Tcf1 and Foxp3 cooccupy at select T reg–up signature genes. (A) Venn diagram showing distinct and overlapping Tcf1 and Foxp3 binding peaks in T reg cells. Tcf1 ChIP-seq was performed on WT T reg cells, and high-confidence Tcf1 binding peaks were compared with the top 5,039 reported Foxp3 peaks. Note that the value of 1,033 in the intersection denotes the number of Tcf1 peaks that overlapped with 1,042 of 5,039 Foxp3 peaks. (B) Violin plot showing Tcf1 binding strength (log 2 (1+RPKM)) at regions cobound by Tcf1 and Foxp3 and those bound by Tcf1-only. The P value was determined using two-sided Mann–Whitney U test. (C) Tcf1 and Foxp3 binding events at the T reg–up signature genes. Tcf1 and Foxp3 binding peaks were identified within ±50 kb of the gene bodies of 75 T reg–up signature genes that showed diminished expression in Tcf1/Lef1-deficient T reg cells (as defined in Fig. 5 C , and further fractioned into groups harboring Tcf1-only, Foxp3-only, nonoverlapping Tcf1 and Foxp3 peaks [called Tcf1 and Foxp3 associated], and overlapping Tcf1 and Foxp3 peaks [called Tcf1-Foxp3 cooccupied]). The number and identity of genes in each group are shown. (D) Visualization of Tcf1, Lef1, and Foxp3 binding peaks at select T reg–up signature genes. Displayed on UCSC genome browser are ChIP-seq tracks at the Izumo1r , Ikzf4 , and Lrig1 gene loci in the following order: Tcf1 ChIP-seq tracks in WT T reg and Tcf1-deficient CD4 + T cells, Foxp3 ChIP-seq, and corresponding input track from Kitagawa et al. (2017) . Tcf1 ChIP-seq tracks in WT CD4 + T cells and Lef1 ChIP-seq tracks in WT and Lef1-deficient CD4 + T cells are shown for Izumo1r , but not Ikzf4 and Lrig1 because the latter two genes did not harbor evident Lef1 binding peaks. MACS2-called Tcf1 and Lef1 peaks are shown as filled black bars on top of corresponding tracks; and for Foxp3 peaks, only the top 5,000 defined in Kwon et al. (2017) are marked with black bars. Y-axis denotes normalized read counts (fragments pile-up per million reads). The whole or partial gene structure and gene transcription orientations are also shown, with Tcf1 or Tcf1-Foxp3–cooccupied sites marked with red rectangles. (E) Tcf1 occupies the same sites in select T reg–up signature genes in T conv and T reg cells. T conv and T reg cells were sorted from Foxp3 Cre WT mice and analyzed with ChIP-qPCR for enriched binding of Tcf1 to the indicated genomic locations. Dotted horizontal line denotes a no-enrichment level at the Hprt locus. Data are means ± SD from two to three independent experiments with each sample measured in duplicate or triplicate. Although not marked for consideration of clarity, the enriched Tcf1 binding at each genomic location has P

    Journal: The Journal of Experimental Medicine

    Article Title: Tcf1 and Lef1 are required for the immunosuppressive function of regulatory T cells

    doi: 10.1084/jem.20182010

    Figure Lengend Snippet: Tcf1 and Foxp3 cooccupy at select T reg–up signature genes. (A) Venn diagram showing distinct and overlapping Tcf1 and Foxp3 binding peaks in T reg cells. Tcf1 ChIP-seq was performed on WT T reg cells, and high-confidence Tcf1 binding peaks were compared with the top 5,039 reported Foxp3 peaks. Note that the value of 1,033 in the intersection denotes the number of Tcf1 peaks that overlapped with 1,042 of 5,039 Foxp3 peaks. (B) Violin plot showing Tcf1 binding strength (log 2 (1+RPKM)) at regions cobound by Tcf1 and Foxp3 and those bound by Tcf1-only. The P value was determined using two-sided Mann–Whitney U test. (C) Tcf1 and Foxp3 binding events at the T reg–up signature genes. Tcf1 and Foxp3 binding peaks were identified within ±50 kb of the gene bodies of 75 T reg–up signature genes that showed diminished expression in Tcf1/Lef1-deficient T reg cells (as defined in Fig. 5 C , and further fractioned into groups harboring Tcf1-only, Foxp3-only, nonoverlapping Tcf1 and Foxp3 peaks [called Tcf1 and Foxp3 associated], and overlapping Tcf1 and Foxp3 peaks [called Tcf1-Foxp3 cooccupied]). The number and identity of genes in each group are shown. (D) Visualization of Tcf1, Lef1, and Foxp3 binding peaks at select T reg–up signature genes. Displayed on UCSC genome browser are ChIP-seq tracks at the Izumo1r , Ikzf4 , and Lrig1 gene loci in the following order: Tcf1 ChIP-seq tracks in WT T reg and Tcf1-deficient CD4 + T cells, Foxp3 ChIP-seq, and corresponding input track from Kitagawa et al. (2017) . Tcf1 ChIP-seq tracks in WT CD4 + T cells and Lef1 ChIP-seq tracks in WT and Lef1-deficient CD4 + T cells are shown for Izumo1r , but not Ikzf4 and Lrig1 because the latter two genes did not harbor evident Lef1 binding peaks. MACS2-called Tcf1 and Lef1 peaks are shown as filled black bars on top of corresponding tracks; and for Foxp3 peaks, only the top 5,000 defined in Kwon et al. (2017) are marked with black bars. Y-axis denotes normalized read counts (fragments pile-up per million reads). The whole or partial gene structure and gene transcription orientations are also shown, with Tcf1 or Tcf1-Foxp3–cooccupied sites marked with red rectangles. (E) Tcf1 occupies the same sites in select T reg–up signature genes in T conv and T reg cells. T conv and T reg cells were sorted from Foxp3 Cre WT mice and analyzed with ChIP-qPCR for enriched binding of Tcf1 to the indicated genomic locations. Dotted horizontal line denotes a no-enrichment level at the Hprt locus. Data are means ± SD from two to three independent experiments with each sample measured in duplicate or triplicate. Although not marked for consideration of clarity, the enriched Tcf1 binding at each genomic location has P

    Article Snippet: To determine enriched Foxp3 binding, rabbit anti-mouse Foxp3 polyclonal antibody (ab150743; Abcam) was used in ChIP experiments on sorted T reg cells following a published protocol, with Gmpr and Ctla4 as negative and positive controls, respectively ( ).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, MANN-WHITNEY, Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    FOXP3 cysteine 232 to glycine (FOXP3–C232G) mutation implicated in loss of Treg-suppressor function and early onset IBD disrupts EZH2 interaction and its gene co-repressor function. ( A ) Schematic depicts structural domains within 50 kilodalton human FOXP3 (1–431 amino acids), detailing genetic mutations associated with IBD and IPEX syndrome. Structural domains include the following repressor domain (RD), zinc finger (ZnF), LZ, and forkhead (FKH)-DNA binding. Red and black arrows indicate amino acids mutated in IBD and IPEX patients. C232G, cysteine 232 to glycine; L242P, leucine 242 to proline; K250Δ, lysine 250 deletion. ( B ) Jurkat cells transfected with IL2 firefly and renilla luciferase plasmids plus either empty vector or His–FOXP3 plasmids were treated with 0.4% dimethyl sulfoxide (DMSO)–vehicle control or indicated concentrations of EZH2 inhibitor GSK126. Thirty-six hours later, cells were treated with 0.4% DMSO–vehicle control or PMA/ionomycin for 12 hours to activate the IL2 promoter as measured by firefly luciferase expression normalized to the renilla internal control. Bottom panel : Whole-cell lysates from transfected and GSK126-treated cells were immunoblotted for H3K27me3; the same membrane was stripped and reblotted for H3 as control. Red horizontal bar denotes the mean IL2 firefly/renilla ratio ± SD from 3 replicates. **** P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Disruption of FOXP3–EZH2 Interaction Represents a Pathobiological Mechanism in Intestinal Inflammation

    doi: 10.1016/j.jcmgh.2018.08.009

    Figure Lengend Snippet: FOXP3 cysteine 232 to glycine (FOXP3–C232G) mutation implicated in loss of Treg-suppressor function and early onset IBD disrupts EZH2 interaction and its gene co-repressor function. ( A ) Schematic depicts structural domains within 50 kilodalton human FOXP3 (1–431 amino acids), detailing genetic mutations associated with IBD and IPEX syndrome. Structural domains include the following repressor domain (RD), zinc finger (ZnF), LZ, and forkhead (FKH)-DNA binding. Red and black arrows indicate amino acids mutated in IBD and IPEX patients. C232G, cysteine 232 to glycine; L242P, leucine 242 to proline; K250Δ, lysine 250 deletion. ( B ) Jurkat cells transfected with IL2 firefly and renilla luciferase plasmids plus either empty vector or His–FOXP3 plasmids were treated with 0.4% dimethyl sulfoxide (DMSO)–vehicle control or indicated concentrations of EZH2 inhibitor GSK126. Thirty-six hours later, cells were treated with 0.4% DMSO–vehicle control or PMA/ionomycin for 12 hours to activate the IL2 promoter as measured by firefly luciferase expression normalized to the renilla internal control. Bottom panel : Whole-cell lysates from transfected and GSK126-treated cells were immunoblotted for H3K27me3; the same membrane was stripped and reblotted for H3 as control. Red horizontal bar denotes the mean IL2 firefly/renilla ratio ± SD from 3 replicates. **** P

    Article Snippet: Primary antibodies used were rabbit monoclonal anti-FOXP3 (1:500, cat. ab54501, lot GR285227-2; Abcam, Cambridge, MA), mouse monoclonal anti-EZH2 (1:100, cat. 3147; Cell Signaling, Beverly, MA), mouse monoclonal anti-myc (1:500, cat. 2276, lot 24; Cell Signaling), rabbit monoclonal anti-His (1:500, cat. sc-499, lot 11214; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-phospho (p)-tyrosine (1:100, cat. sc-7020, lot L2916; Santa Cruz Biotechnology), mouse monoclonal anti-STAT3 (1:1000, cat. 9139, lot 7; Cell Signaling), rabbit monoclonal anti-pSTAT3-Y705 (1:100, cat. 9131, lot 30; Cell Signaling), rabbit monoclonal anti-pSTAT3-S727 (1:100, cat. 9134, lot 20; Cell Signaling).

    Techniques: Mutagenesis, Binding Assay, Transfection, Luciferase, Plasmid Preparation, Expressing

    IBD-associated IL6-induced membrane-to-nucleus signaling pathway similarly disrupts FOXP3–EZH2 interaction in a manner reversible by JAK1/2 inhibition. ( A ) Confocal microscopic PLA images shows endogenous FOXP3–EZH2 interaction (red signals) in the nucleus of CD4 + CD25 ++ cells (Tregs) before (0.2%–0.4% dimethyl sulfoxide [DMSO]), after IL6 (50 or 100 ng/mL, 2×) or after IL6 and JAK1/2 inhibitor ruxolitinib pretreatment (10 μmol/L). CD4 + CD25 - cells were used as negative controls. Scale bar : 5 μm. Result is representative of 3 independent experiments using freshly isolated PBMC-derived Tregs from 3 different donors. ( B ) Quantitation of nuclear PLA signals in images from panel A (rows 2–7). n = number of cells imaged. *** P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Disruption of FOXP3–EZH2 Interaction Represents a Pathobiological Mechanism in Intestinal Inflammation

    doi: 10.1016/j.jcmgh.2018.08.009

    Figure Lengend Snippet: IBD-associated IL6-induced membrane-to-nucleus signaling pathway similarly disrupts FOXP3–EZH2 interaction in a manner reversible by JAK1/2 inhibition. ( A ) Confocal microscopic PLA images shows endogenous FOXP3–EZH2 interaction (red signals) in the nucleus of CD4 + CD25 ++ cells (Tregs) before (0.2%–0.4% dimethyl sulfoxide [DMSO]), after IL6 (50 or 100 ng/mL, 2×) or after IL6 and JAK1/2 inhibitor ruxolitinib pretreatment (10 μmol/L). CD4 + CD25 - cells were used as negative controls. Scale bar : 5 μm. Result is representative of 3 independent experiments using freshly isolated PBMC-derived Tregs from 3 different donors. ( B ) Quantitation of nuclear PLA signals in images from panel A (rows 2–7). n = number of cells imaged. *** P

    Article Snippet: Primary antibodies used were rabbit monoclonal anti-FOXP3 (1:500, cat. ab54501, lot GR285227-2; Abcam, Cambridge, MA), mouse monoclonal anti-EZH2 (1:100, cat. 3147; Cell Signaling, Beverly, MA), mouse monoclonal anti-myc (1:500, cat. 2276, lot 24; Cell Signaling), rabbit monoclonal anti-His (1:500, cat. sc-499, lot 11214; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-phospho (p)-tyrosine (1:100, cat. sc-7020, lot L2916; Santa Cruz Biotechnology), mouse monoclonal anti-STAT3 (1:1000, cat. 9139, lot 7; Cell Signaling), rabbit monoclonal anti-pSTAT3-Y705 (1:100, cat. 9131, lot 30; Cell Signaling), rabbit monoclonal anti-pSTAT3-S727 (1:100, cat. 9134, lot 20; Cell Signaling).

    Techniques: Inhibition, Proximity Ligation Assay, Isolation, Derivative Assay, Quantitation Assay

    IL6-induced disruption of FOXP3–EZH2 protein interaction correlates with increased STAT3 activation and FOXP3 tyrosine phosphorylation. ( A ) HEK293T cells ectopically expressing plasmids encoding His–FOXP3 and myc–EZH2 were treated with IL6 (50 ng/mL) for the indicated duration under reduced-serum conditions. Whole-cell lysates were subjected to immunoprecipitation with IgG or FOXP3 antibody and immunoblotted for His–FOXP3. The same membrane was stripped and reblotted for myc–EZH2. For input, lysates were immunoblotted for His–FOXP3, myc–EZH2, STAT3, and p-STAT3 (Y705) with their corresponding antibodies. ( B ) Reverse co-immunoprecipitation of experiment in panel A using myc antibody for EZH2. Data are representative of 3 independent experiments. ( C ) Representative confocal PLA images of human CD4 + CD25 ++ cells (Tregs) shows tyrosine phosphorylated FOXP3 (red) in response to IL6 (50 ng/mL) alone or in combination with ruxolitinib (Ruxo.) (10 μmol/L) for the indicated time points. To detect FOXP3 tyrosine phosphorylation, cells were stained with pan p-Tyr antibody and specific FOXP3 antibody as indicated. Data are representative of 3 independent experiments. Scale bar : 2 µ m. ( D ) Quantitation of nuclear PLA signals in images from panel C . n = number of cells imaged. *** P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Disruption of FOXP3–EZH2 Interaction Represents a Pathobiological Mechanism in Intestinal Inflammation

    doi: 10.1016/j.jcmgh.2018.08.009

    Figure Lengend Snippet: IL6-induced disruption of FOXP3–EZH2 protein interaction correlates with increased STAT3 activation and FOXP3 tyrosine phosphorylation. ( A ) HEK293T cells ectopically expressing plasmids encoding His–FOXP3 and myc–EZH2 were treated with IL6 (50 ng/mL) for the indicated duration under reduced-serum conditions. Whole-cell lysates were subjected to immunoprecipitation with IgG or FOXP3 antibody and immunoblotted for His–FOXP3. The same membrane was stripped and reblotted for myc–EZH2. For input, lysates were immunoblotted for His–FOXP3, myc–EZH2, STAT3, and p-STAT3 (Y705) with their corresponding antibodies. ( B ) Reverse co-immunoprecipitation of experiment in panel A using myc antibody for EZH2. Data are representative of 3 independent experiments. ( C ) Representative confocal PLA images of human CD4 + CD25 ++ cells (Tregs) shows tyrosine phosphorylated FOXP3 (red) in response to IL6 (50 ng/mL) alone or in combination with ruxolitinib (Ruxo.) (10 μmol/L) for the indicated time points. To detect FOXP3 tyrosine phosphorylation, cells were stained with pan p-Tyr antibody and specific FOXP3 antibody as indicated. Data are representative of 3 independent experiments. Scale bar : 2 µ m. ( D ) Quantitation of nuclear PLA signals in images from panel C . n = number of cells imaged. *** P

    Article Snippet: Primary antibodies used were rabbit monoclonal anti-FOXP3 (1:500, cat. ab54501, lot GR285227-2; Abcam, Cambridge, MA), mouse monoclonal anti-EZH2 (1:100, cat. 3147; Cell Signaling, Beverly, MA), mouse monoclonal anti-myc (1:500, cat. 2276, lot 24; Cell Signaling), rabbit monoclonal anti-His (1:500, cat. sc-499, lot 11214; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-phospho (p)-tyrosine (1:100, cat. sc-7020, lot L2916; Santa Cruz Biotechnology), mouse monoclonal anti-STAT3 (1:1000, cat. 9139, lot 7; Cell Signaling), rabbit monoclonal anti-pSTAT3-Y705 (1:100, cat. 9131, lot 30; Cell Signaling), rabbit monoclonal anti-pSTAT3-S727 (1:100, cat. 9134, lot 20; Cell Signaling).

    Techniques: Activation Assay, Expressing, Immunoprecipitation, Proximity Ligation Assay, Staining, Quantitation Assay

    FOXP3 interacts with EZH2 in murine induced Tregs (iTreg) and freshly isolated PBMC-derived human Tregs. ( A ) Sketch depicts PLA to detect and quantify protein–protein interactions [A] and [B]

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Disruption of FOXP3–EZH2 Interaction Represents a Pathobiological Mechanism in Intestinal Inflammation

    doi: 10.1016/j.jcmgh.2018.08.009

    Figure Lengend Snippet: FOXP3 interacts with EZH2 in murine induced Tregs (iTreg) and freshly isolated PBMC-derived human Tregs. ( A ) Sketch depicts PLA to detect and quantify protein–protein interactions [A] and [B]

    Article Snippet: Primary antibodies used were rabbit monoclonal anti-FOXP3 (1:500, cat. ab54501, lot GR285227-2; Abcam, Cambridge, MA), mouse monoclonal anti-EZH2 (1:100, cat. 3147; Cell Signaling, Beverly, MA), mouse monoclonal anti-myc (1:500, cat. 2276, lot 24; Cell Signaling), rabbit monoclonal anti-His (1:500, cat. sc-499, lot 11214; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-phospho (p)-tyrosine (1:100, cat. sc-7020, lot L2916; Santa Cruz Biotechnology), mouse monoclonal anti-STAT3 (1:1000, cat. 9139, lot 7; Cell Signaling), rabbit monoclonal anti-pSTAT3-Y705 (1:100, cat. 9131, lot 30; Cell Signaling), rabbit monoclonal anti-pSTAT3-S727 (1:100, cat. 9134, lot 20; Cell Signaling).

    Techniques: Isolation, Derivative Assay, Proximity Ligation Assay