Journal: Genome Medicine

Article Title: The IL-4/STAT6 signaling axis establishes a conserved microRNA signature in human and mouse macrophages regulating cell survival via miR-342-3p

doi: 10.1186/s13073-016-0315-y

Figure Lengend Snippet: IL-4Rα and Stat6-dependency of IL-4-regulated miRNA expression in mouse macrophages. a miR-342-3p, miR-99b, and miR-125a-5p expression in IL-4-stimulated or unstimulated wild-type ( WT ) and IL-4Rα-defficient ( IL-4Rα KO ) mouse bone marrow-derived macrophages as measured by stem-loop RT-qPCR. Each data point represents the mean and standard deviation of four or three (WT or IL-4Rα KO, respectively) individual animals. *P ˂ 0.05, **P ˂ 0.01, ***P ˂ 0.001. b The number of miRNAs showing significant (FDR ˂0.1) IL-4-dependent regulation in wild-type ( WT ) and Stat6-deficient ( STAT6 KO ) macrophages. c Stem-loop RT-qPCR-based quantification of miR-342-3p, miR-99b, and miR-125a-5p expression in IL-4-stimulated or unstimulated WT and Stat6 KO mouse bone marrow-derived macrophages. Each data point represents the mean and standard deviation of three individual animals. *P ˂ 0.05, **P ˂ 0.01, ***P ˂ 0.001

Article Snippet: Chromatin was immunoprecipitated with antibodies against pre-immune IgG (Millipore, 12–370), H3K27ac (Abcam, ab4729) and STAT6 (Santa Cruz, sc-981).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Standard Deviation

Journal: Genome Medicine

Article Title: The IL-4/STAT6 signaling axis establishes a conserved microRNA signature in human and mouse macrophages regulating cell survival via miR-342-3p

doi: 10.1186/s13073-016-0315-y

Figure Lengend Snippet: Mechanism of IL-4-dependent co-regulation of miR-342-3p and EVL expression in human and mouse macrophages. a Strand-specific GRO-Seq, CTCF, and Rad21-specific ChIP-seq signals in unstimulated as well as H3K4m3, H3K4m1, H3K27Ac, Pu.1, and Stat6 ChIP-Seq signals in IL-4-stimulated and unstimulated mouse macrophages at the EVL locus visualized by the Integrative Genomics Viewer. b RT-qPCR-based measurement of Evl expression in bone marrow ( BM ) cells and IL-4 stimulated or unstimulated bone marrow-derived macrophages ( BMDM ) in wild-type ( WT ) or Stat6-deficient ( Stat6 KO ) mice. Each data point represents the mean and standard deviation (SD) of three individual animals. **P ˂ 0.01. c Stem-loop RT-qPCR-based quantification of miR-342-3p expression in bone marrow cells and IL-4 stimulated or unstimulated bone marrow-derived macrophages in WT or Stat6 KO mice. Each data point represents the mean and SD of three individual animals. **P ˂ 0.01. d IL-4-induced Stat6 binding of two regulatory regions of the mouse EVL gene in WT and Stat6 KO mouse macrophages measured by ChIP-qPCR. Columns represent mean arbitrary units described in the “Methods” section ± SD. e RT-qPCR-based measurement of EVL expression during human macrophage differentiation in the absence or presence of IL-4 (a representative example of three independent human donors is shown). f Stem-loop RT-qPCR-based quantification of miR-342-3p expression during human macrophage differentiation in the absence or presence of IL-4 (a representative example of three independent human donors is shown). g IL-4-induced recruitment of STAT6 to the human EVL ( hEVL ) locus in macrophages obtained from two independent human donors ( D1 and D2 ) measured by ChIP-qPCR. Data are expressed as mean ± SD

Article Snippet: Chromatin was immunoprecipitated with antibodies against pre-immune IgG (Millipore, 12–370), H3K27ac (Abcam, ab4729) and STAT6 (Santa Cruz, sc-981).

Techniques: Expressing, ChIP-sequencing, Quantitative RT-PCR, Derivative Assay, Standard Deviation, Binding Assay, ChIP-qPCR

Journal: Genome Medicine

Article Title: The IL-4/STAT6 signaling axis establishes a conserved microRNA signature in human and mouse macrophages regulating cell survival via miR-342-3p

doi: 10.1186/s13073-016-0315-y

Figure Lengend Snippet: IL-4-dependent and Stat6-mediated repression of the miR-99b-125a miRNA polycistron. a Strand-specific GRO-Seq, CTCF, and Rad21-specific ChIP-seq signals in unstimulated as well as H3K4m3, H3K4m1, H3K27Ac, Pu.1, and Stat6 ChIP-Seq signals in IL-4-stimulated and unstimulated mouse macrophages at the miR-99b-125a and Spaca6 loci visualized by the Integrative Genomics Viewer. b Pri-miR-99b-125a, miR-99b, and miR-125a-5p expression in bone marrow cells ( BM ), MCSF, and MCSF + IL-4-treated BMDMs. Each data point represents the mean and standard deviation (SD) of three individual animals. *P ˂ 0.05, **P ˂ 0.01. c Expression of the pri-miR-99b-125a polycistron in IL-4-stimulated or unstimulated BMDMs derived from wild-type ( WT ) or Stat6-defficient ( Stat6 KO ) mice. Each data point represents the mean and SD of three individual animals. *P ˂ 0.05. d H3K27Ac ChIP-qPCR in IL-4-treated or nontreated WT or Stat6 KO mouse BMDMs at the potential TSS of pri-miR-99b-125a. Data are expressed as mean ± SD. e RT-PCR-based detection of a common transcript of pri-miR-99b-125a and Spaca6 in IL-4-treated or nontreated WT mouse BMDMs. Cyclophilin A (cyclo) serves as normalization control

Article Snippet: Chromatin was immunoprecipitated with antibodies against pre-immune IgG (Millipore, 12–370), H3K27ac (Abcam, ab4729) and STAT6 (Santa Cruz, sc-981).

Techniques: ChIP-sequencing, Expressing, Standard Deviation, Derivative Assay, ChIP-qPCR, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Nature Communications

Article Title: Modulation of M2 macrophage polarization by the crosstalk between Stat6 and Trim24

doi: 10.1038/s41467-019-12384-2

Figure Lengend Snippet: Stat6 is acetylated at Lys383. a – c Immunoblot analysis of the lysine acetylation of Stat6 in primary murine macrophages ( a ) or 293T cells transfected with the indicated expression vectors ( b ), or in control and CBP -knockdown iBMDMs ( c ) that were (+) or were not (−) stimulated with IL-4 for 30 min; lysates were assessed by immunoprecipitation (IP) with anti-Stat6 or anti-Flag and immunoblotting with anti-Ac-Lys and anti-Stat6, or anti-Flag, and immunoblot of the lysates to detect input proteins and loading controls. d Luciferase assay showing Stat6 transcriptional activity in 293T cells transfected with the indicated vector that were pretreated with DMSO (DM) or trichostatin A (TSA, 5 μM) plus nicotinamide (NAM, 1 mM) (T/N) and then stimulated with (+) or without (−) IL-4 for 4 h before detection. Lysates were immunoblotted for acetylated (Ac−), phosphorylated (P−) Stat6, HA, Flag, and Hsp60 as controls. e , f Immunoblot of Stat6 and Lamin B in nuclear extracts and phosphorylated (P)-Stat6 and Hsp60 in whole-cell lysates ( e ) and ChIP–QPCR analysis of Stat6 binding to the promoters of Arg1 or Ym1 ( f ) in murine primary macrophages that were pretreated with DMSO or TSA plus NAM (T/N) and then stimulated with (+) or without (−) IL-4 for 1 h. g Mass spectrometry analysis showing potential acetylation sites in Stat6 after the immunoprecipitation of Stat6 in 293T cells transfected with Stat6 and CBP. h Schematic representation of the mouse Stat6 protein showing the DNA-binding domain (DBD) and its amino acid sequence with all lysine (K) residues highlighted in red. i Immunoblot analysis of the lysine acetylation of wild-type (WT) and KR mutant Stat6 in 293T cells transfected with the indicated expression vectors; lysates were assessed by immunoprecipitation (IP) with anti-Flag and immunoblotting with anti-Ac-Lys and anti-Flag. j Amino acid sequence alignment of Stat6 among the indicated species and different mouse STAT proteins showing Lys383 that are highlighted in red. Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 as determined by the unpaired Student’s t test. Source data are provided as a Source Data file

Article Snippet: Antibodies for Arg1 (9819), Mrc1 (91992), Jnk (9252), Tbk1 (3013), CBP (7389), Stat6 (5397), Acetylated-Lysine (9441), Normal Rabbit IgG (2729), P-Jnk (4668), P-Stat6 (9361), P-Akt (2965, 4060), P-p65 (3033), P-IκBα (2859), P-p38 (9215), P-Stat1 (7649), and P-Tbk1 (5483) were from Cell Signaling.

Techniques: Western Blot, Transfection, Expressing, Control, Knockdown, Immunoprecipitation, Luciferase, Activity Assay, Plasmid Preparation, ChIP-qPCR, Binding Assay, Mass Spectrometry, Sequencing, Mutagenesis

Journal: Nature Communications

Article Title: Modulation of M2 macrophage polarization by the crosstalk between Stat6 and Trim24

doi: 10.1038/s41467-019-12384-2

Figure Lengend Snippet: Lys383 acetylation in Stat6 suppressed its transcriptional activity. a Dot-blot analysis to detect the efficiency of the antibody against Stat6 site-specific (Lys383) acetylation. b , c Immunoblot analysis of the Lys383 acetylation of Stat6 in 293T cells transfected with the indicated expression vectors; lysates were assessed by immunoprecipitation (IP) with anti-Flag and immunoblotting with anti-Ac-Stat6 (K383) and anti-Flag; lysates were immunoblotted to detect input proteins and loading controls before immunoprecipitation. d , e Flow cytometric analysis of the endogenous Stat6 acetylation at Lys383 in murine primary peritoneal macrophages isolated from naive C57BL/6 mice treated with DMSO or trichostatin A (TSA) (5 μM) plus nicotinamide (NAM) (1 mM) (T/N) for 2 h. Data are presented as a representative histography ( d ) and summary bar graph ( e ). f – h Luciferase assay to assess Stat6 transcriptional activity in 293T cells transfected with wild-type (WT) or KR mutant Stat6 ( f ), 293T cells transfected with WT or the K383R mutant Stat6 pretreated with DMSO (−) or with NAM plus TSA ( g ), and 293T cells transfected with WT or K383A or the K383Q mutant Stat6 ( h ) and then stimulated with (+) or without (−) IL-4 (20 ng mL −1 ) for 4 h before detection. Immunoblots showing the protein expression levels in transfected cells are presented below each bar graph. i , j QPCR analysis of Arg1 , Mrc1 , and Ccl24 mRNA in Stat6- knockdown iBMDMs that were reconstituted with empty vector (EV), WT, or the K383R mutant Stat6, and then were left unstimulated (−) or stimulated (+) with IL-4 for 6 h ( i ). Immunoblot of Flag-Stat6, phosphorylated (P)-Stat6, and Hsp60 showing the reconstitution efficiency of Flag-Stat6 ( j ). Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 as determined by the unpaired Student’s t test. Source data are provided as a Source Data file

Article Snippet: Antibodies for Arg1 (9819), Mrc1 (91992), Jnk (9252), Tbk1 (3013), CBP (7389), Stat6 (5397), Acetylated-Lysine (9441), Normal Rabbit IgG (2729), P-Jnk (4668), P-Stat6 (9361), P-Akt (2965, 4060), P-p65 (3033), P-IκBα (2859), P-p38 (9215), P-Stat1 (7649), and P-Tbk1 (5483) were from Cell Signaling.

Techniques: Activity Assay, Dot Blot, Western Blot, Transfection, Expressing, Immunoprecipitation, Isolation, Luciferase, Mutagenesis, Knockdown, Plasmid Preparation

Journal: Nature Communications

Article Title: Modulation of M2 macrophage polarization by the crosstalk between Stat6 and Trim24

doi: 10.1038/s41467-019-12384-2

Figure Lengend Snippet: Trim24 promoted CBP-induced Stat6 acetylation. a Venn diagram showing the number of genes up/downregulated in M2-polarized macrophages and those that encode CBP-associated proteins. b Confocal microscopy images showing the intracellular localization of Trim24 and CBP in peritoneal macrophages that were left nontreated (NT) or stimulated with IL-4 for 30 min, with DAPI staining used to indicate the nuclei of the cells. Scale bars: 5 μm. c , d Immunoblot analysis of the lysine acetylation of Stat6 in 293T cells transfected with the indicated expression vectors assessed by immunoprecipitation (IP) with anti-Flag and immunoblotting with anti-Ac-Lys and anti-Flag. e , f Interaction between CBP, Stat6, and Trim24 in 293T cells transfected with the indicated expression vectors ( e ) and control and Trim24- knockdown iBMDMs that were left unstimulated (−) or stimulated with IL-4 (+) for 30 min ( f ) assessed by immunoprecipitation (IP) with anti-Flag or anti-CBP and immunoblotting with anti-HA and anti-Flag, or anti-CBP, anti-Stat6, and anti-Trim24. g Immunoblot analysis of endogenous lysine acetylation of Stat6 in control and Trim24 -knockdown iBMDMs stimulated with IL-4 for 30 min assessed by immunoprecipitation (IP) with anti-Stat6 and immunoblotting with anti-Ac-Stat6 (K383) and anti-Stat6. h , i Luciferase assay to assess Stat6 transcriptional activity in 293T cells transfected with the indicated expression vectors ( h ) and 293T cells transfected with scramble (siCtrl) or si TRIM24 ( i ) stimulated with (+) or without (−) IL-4 (50 ng mL −1 ) for 4 h before detection. Immunoblot of acetylated (Ac−) Stat6 after immunoprecipitation; phosphorylated (P−) Stat6, HA-CBP, HA-Trim24, or Hsp60 in whole-cell lysates were applied as controls. j QPCR and immunoblot analysis of M2 gene expression in control and Trim24- knockdown iBMDMs that were left nontreated (NT) or stimulated with IL-4, and the immunoblot of Trim24 and Hsp60 to detect the knockdown efficiency. Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 as determined by the unpaired Student’s t test. Source data are provided as a Source Data file

Article Snippet: Antibodies for Arg1 (9819), Mrc1 (91992), Jnk (9252), Tbk1 (3013), CBP (7389), Stat6 (5397), Acetylated-Lysine (9441), Normal Rabbit IgG (2729), P-Jnk (4668), P-Stat6 (9361), P-Akt (2965, 4060), P-p65 (3033), P-IκBα (2859), P-p38 (9215), P-Stat1 (7649), and P-Tbk1 (5483) were from Cell Signaling.

Techniques: Confocal Microscopy, Staining, Western Blot, Transfection, Expressing, Immunoprecipitation, Control, Knockdown, Luciferase, Activity Assay, Gene Expression

Journal: Nature Communications

Article Title: Modulation of M2 macrophage polarization by the crosstalk between Stat6 and Trim24

doi: 10.1038/s41467-019-12384-2

Figure Lengend Snippet: Trim24-mediated Lys63-linked ubiquitination of CBP at Lys119. a Structure schematic of full-length (FL) Trim24 and its truncations. b Immunoblot analysis of the lysine acetylation of Stat6 in 293T cells transfected with the indicated expression vectors assessed by immunoprecipitation (IP) with anti-Flag and immunoblotting with anti-Ac-Lys and anti-Flag; lysates were immunoblotted to detect input proteins and loading controls. c Endogenous ubiquitination of CBP in control and Trim24- knockdown iBMDMs that were unstimulated (−) or stimulated (+) with IL-4 for 30 min assessed by immunoblot analysis with anti-ubiquitin after immunoprecipitation with anti-CBP (top) and immunoblot analysis with input proteins and loading controls (below). d Ubiquitination of CBP in 293T cells transfected with the indicated expression vectors assessed by immunoblot analysis with anti-HA after immunoprecipitation with anti-Flag (top) or immunoblot analysis with input proteins in lysates without immunoprecipitation (below). e In vitro ubiquitination assay to determine CBP ubiquitination after a mixture reaction of ubiquitin-charged E2 (UbcH5a) and in vitro-translated Flag-CBP with or without HA-Trim24 proteins was assessed by immunoblot analysis with anti-CBP and anti-HA. f Mass spectrometry analysis of the CBP ubiquitination site after the immunoprecipitation of overexpressed Flag-CBP in 293T cells transfected with Flag-CBP and HA-Trim24 showing that Lys119 is the site of CBP ubiquitination by Trim24. g Ubiquitination of CBP in 293T cells transfected with the indicated expression vectors assessed by immunoblot analysis with anti-HA after immunoprecipitation with anti-Flag (top) or immunoblot analysis with input proteins in lysates without immunoprecipitation (below). h In vitro ubiquitination assay to determine CBP ubiquitination after a mixture reaction of ubiquitin-charged E2 (UbcH5a) and in vitro-translated wild-type (WT) or the K119R mutant Flag-CBP with or without HA-Trim24 proteins was assessed by immunoblot analysis with anti-CBP and anti-HA. i , j Immunoblot analysis of the lysine acetylation of Stat6 and the interaction of wild-type (WT) or the K119R mutant CBP with Stat6 in 293T cells transfected with the indicated expression vectors, assessed by immunoprecipitation with anti-Flag and immunoblot with anti-Ac-Lys, anti-Ac-Stat6 (K383), anti-HA, and anti-Flag. Each panel is a representative experiment of at least three independent biological replicates. Source data are provided as a Source Data file

Article Snippet: Antibodies for Arg1 (9819), Mrc1 (91992), Jnk (9252), Tbk1 (3013), CBP (7389), Stat6 (5397), Acetylated-Lysine (9441), Normal Rabbit IgG (2729), P-Jnk (4668), P-Stat6 (9361), P-Akt (2965, 4060), P-p65 (3033), P-IκBα (2859), P-p38 (9215), P-Stat1 (7649), and P-Tbk1 (5483) were from Cell Signaling.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Expressing, Immunoprecipitation, Control, Knockdown, In Vitro, Mass Spectrometry, Mutagenesis

Journal: Nature Communications

Article Title: Modulation of M2 macrophage polarization by the crosstalk between Stat6 and Trim24

doi: 10.1038/s41467-019-12384-2

Figure Lengend Snippet: Trim24 deficiency enhanced DNA-binding activity of Stat6 in M2 gene promoters. a Endogenous ubiquitination of CBP in WT and Trim24 -deficient macrophages that were unstimulated (−) or stimulated (+) with IL-4 for 30 min assessed by immunoblot analysis with anti-ubiquitin after immunoprecipitation with anti-CBP (top) and immunoblot analysis with input proteins and loading controls (below). b Immunoblot analysis of endogenous lysine acetylation of Stat6 in WT and Trim24 -deficient peritoneal macrophages (PMs) that were unstimulated (−) or stimulated (+) with IL-4 for 30 min assessed by immunoprecipitation (IP) with anti-Stat6 and immunoblotting with anti-Ac-Lys, anti-Ac-Stat6 (K383), and anti-Stat6. c Electrophoretic mobility shift assay (EMSA) of nuclear extracts from WT and Trim24 -deficient macrophages that were untreated (−) or stimulated (+) with IL-4 for 1 h assessed with 32 P-radiolabeled WT or mutant probes for Stat6. Immunoblotting of phosphorylated (P−) Stat6 and Lamin B in the nuclear extract were applied for control. d Average Stat6 ChIP signals in the promoter regions of M2 cluster genes in WT and Trim24 -deficient macrophages stimulated with IL-4 for 1 h. e Snapshot of the Stat6 ChIP-Seq signals at the Irf4 gene loci in WT and Trim24 -deficient macrophages stimulated with IL-4 for 1 h. f , g ChIP–QPCR analysis of Stat6 binding to the promoters of Arg1 and Irf4 in WT and Trim24 -deficient macrophages that were left nontreated or stimulated with IL-4 for the indicated time points ( f ) or Stat6 binding to the promoters of Arg1 and Ym1 in Stat6- knockdown iBMDMs that were reconstituted with empty vector (EV), WT, or the K383R mutant Stat6 and then were left nontreated (NT) or stimulated with IL-4 for 1 h ( g ). Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 as determined by the unpaired Student’s t test. Source data are provided as a Source Data file

Article Snippet: Antibodies for Arg1 (9819), Mrc1 (91992), Jnk (9252), Tbk1 (3013), CBP (7389), Stat6 (5397), Acetylated-Lysine (9441), Normal Rabbit IgG (2729), P-Jnk (4668), P-Stat6 (9361), P-Akt (2965, 4060), P-p65 (3033), P-IκBα (2859), P-p38 (9215), P-Stat1 (7649), and P-Tbk1 (5483) were from Cell Signaling.

Techniques: Binding Assay, Activity Assay, Ubiquitin Proteomics, Western Blot, Immunoprecipitation, Electrophoretic Mobility Shift Assay, Mutagenesis, Control, ChIP-sequencing, ChIP-qPCR, Knockdown, Plasmid Preparation

Journal: Nature Communications

Article Title: Modulation of M2 macrophage polarization by the crosstalk between Stat6 and Trim24

doi: 10.1038/s41467-019-12384-2

Figure Lengend Snippet: Trim24 negatively regulated macrophage M2 polarization. a RNA-sequencing analysis showing differentiated gene expression profiles in WT and Trim24 -deficient macrophages stimulated with IL-4 for 6 h. Representative M2 genes that were upregulated in Trim24 -deficient macrophages are indicated in the scatter plot. b Gene set enrichment analysis (GSEA) of the M2 gene set in WT and Trim24 -deficient macrophages stimulated with IL-4 for 6 h by using the RNA-sequencing data from ( a ). c QPCR and immunoblot analysis of M2 gene expression in WT and Trim24 -deficient macrophages that were left nontreated (NT) or stimulated with IL-4. d , e Flow cytometric analysis of endogenous Stat6 acetylation at Lys383 in control and TRIM24- knockdown human peripheral blood mononuclear cell (PBMC)-derived primary macrophages that were stimulated with IL-4 for 30 min. Data are presented as a representative histogram ( d ) and summary bar graph ( e ). f QPCR analysis of TRIM24 , TGM2 , CCL17 , and IRF4 mRNA in control and TRIM24- knockdown human PBMC-derived primary macrophages that were left nontreated (−) or stimulated (+) with IL-4 for 6 h. Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 as determined by the unpaired Student’s t test. Source data are provided as a Source Data file

Article Snippet: Antibodies for Arg1 (9819), Mrc1 (91992), Jnk (9252), Tbk1 (3013), CBP (7389), Stat6 (5397), Acetylated-Lysine (9441), Normal Rabbit IgG (2729), P-Jnk (4668), P-Stat6 (9361), P-Akt (2965, 4060), P-p65 (3033), P-IκBα (2859), P-p38 (9215), P-Stat1 (7649), and P-Tbk1 (5483) were from Cell Signaling.

Techniques: RNA Sequencing, Gene Expression, Western Blot, Control, Knockdown, Derivative Assay

Journal: Nature Communications

Article Title: Modulation of M2 macrophage polarization by the crosstalk between Stat6 and Trim24

doi: 10.1038/s41467-019-12384-2

Figure Lengend Snippet: Trim24 deficiency in macrophages impaired antitumor immunity. a – c Tumor growth ( a ), representative tumor pictures ( b ), and tumor weight ( c ) of WT and Trim24 M−/− mice ( n = 8) that were injected s.c. with B16 melanoma cells. d Confocal microscopy images showing Lys383-acetylated Stat6 in the nucleus of tumor-associated macrophages (TAMs) that isolated from B16 melanoma of WT and Trim24 -deficient mice; DAPI staining indicated the nucleus of the cells. Scale bars: 5 μm. e , f Flow cytometric analysis of endogenous Stat6 acetylation at Lys383 in WT and Trim24 -deficient TAM as described in ( d ). Data are presented as a representative histogram ( e ) and summary bar graph ( f ). g Tumor weight of WT (n = 6) and Trim24 M−/− mice ( n = 7) that were injected s.c. with MC-38 colon adenocarcinoma cells. h – j RNA-sequencing analysis of gene profiles ( h ), M2 gene enrichment ( i ), and QPCR verification of M2 genes ( j ) in WT and Trim24 M−/− TAM that were isolated from B16 melanoma. The M2 signature genes were indicated in the plot. k , l Flow cytometric analysis of CD45 + CD11b + F4/80 + TAM ( k ) and IFNγ-producing CD4 + and CD8 + T cells ( l ) in B16 melanoma of WT and Trim24 M−/− mice. Data are presented as a representative plot (left panel) and summary graph (right panel). m , n Tumor growth ( m ) and weight ( n ) in WT or Trim24 M−/− mice ( n = 4) that were injected s.c. with B16 melanoma cells, and with intravenous injection of the anti-CSF1R antibody to deplete macrophages in vivo. Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 as determined by the unpaired Student’s t test. Source data are provided as a Source Data file

Article Snippet: Antibodies for Arg1 (9819), Mrc1 (91992), Jnk (9252), Tbk1 (3013), CBP (7389), Stat6 (5397), Acetylated-Lysine (9441), Normal Rabbit IgG (2729), P-Jnk (4668), P-Stat6 (9361), P-Akt (2965, 4060), P-p65 (3033), P-IκBα (2859), P-p38 (9215), P-Stat1 (7649), and P-Tbk1 (5483) were from Cell Signaling.

Techniques: Injection, Confocal Microscopy, Isolation, Staining, RNA Sequencing, In Vivo

Journal: Nature Communications

Article Title: Modulation of M2 macrophage polarization by the crosstalk between Stat6 and Trim24

doi: 10.1038/s41467-019-12384-2

Figure Lengend Snippet: Stat6-mediated direct transcriptional suppression of Trim24. a QPCR analysis of TRIM24 , IRF4 , or CCL17 mRNA expression in sorted CD11b + macrophages that isolated from six pairs of breast tumor and adjacent normal tissues from six breast cancer patients. b – e QPCR and immunoblot analysis of Trim24 gene expression in murine primary macrophages or in human PBMC-derived macrophages or in human THP-1 cells that were left nontreated (−) or stimulated (+) with a conditioned medium of B16 melanoma cells or IL-4 for 6 h. f , g Structure schema of the constructed luciferase reporters by using different truncated promoter sequences of TRIM24 gene, which were then used to test the luciferase activity. h Immunoblot of Trim24, phosphorylated (P), and total Stat6 and Hsp60 (loading control) expression in macrophages isolated from wild-type (WT) and Stat6 -deficient ( Stat6- KO) mice. i Schematic representation showing a Stat6-conserved binding site located in the promoter region of TRIM24 gene between –1167 and –380. j ChIP–QPCR analysis of the binding of STAT6 in the promoter region of TRIM24 gene in the THP-1 cells that were stimulated with (+) or without (−) IL-4 for 1 h. k Luciferase assay of Stat6-induced transcriptional suppression of TRIM24 in 293T cells that were transfected with the indicated expression vector, and then were stimulated with (+) or without (−) IL-4 for 4 h. Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 as determined by the unpaired Student’s t test. Source data are provided as a Source Data file

Article Snippet: Antibodies for Arg1 (9819), Mrc1 (91992), Jnk (9252), Tbk1 (3013), CBP (7389), Stat6 (5397), Acetylated-Lysine (9441), Normal Rabbit IgG (2729), P-Jnk (4668), P-Stat6 (9361), P-Akt (2965, 4060), P-p65 (3033), P-IκBα (2859), P-p38 (9215), P-Stat1 (7649), and P-Tbk1 (5483) were from Cell Signaling.

Techniques: Expressing, Isolation, Western Blot, Gene Expression, Derivative Assay, Construct, Luciferase, Activity Assay, Control, Binding Assay, ChIP-qPCR, Transfection, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements

doi: 10.1128/mcb.00920-10

Figure Lengend Snippet: FIG. 6. Activation-specific BRG1 binding at specific genes. (A and D) ChIP-seq profiles for resting (uTh1 and uTh2) and activated (Th1S and Th2S) cells are shown. The BRG1 occupancy values (y axis) are identical for all graphs to allow direct comparison (minimum tag frequency of 0, maximum tag frequency of 1.14 105). Asterisks indicate statistically significant BRG1 binding regions identified using CisGenome. Genes are indicated as boxes below the profiles, and the arrows inside the boxes indicate the direction of transcription. (A) For the Egr1 locus, Chr18, nt 35010000 to 35030000 (MM9) are shown. A scale bar indicates 2 kb along the x axis. Downward pointing arrows indicate regions analyzed by standard ChIP as shown in panels B and C. (D) For the Pdcd4 locus, Chr19, nt 53950000 to 54020000 (MM9) are shown. A scale bar indicates 10 kb along the x axis. (B) BRG1 binding was measured by standard ChIP, and the results were normalized to the input samples. Control IgG ChIP results were similar to those obtained with Nfm, a negative-control locus. The y axis labels indicate distances from the Egr1 TSS in kilobases. This experiment was a biological replicate of the ChIP-seq experiment whose results are depicted in Fig. 6A (but performed with different mice, with cells harvested on different days, etc.). (C) H3S10pK14ac levels were measured by standard ChIP and normalized to the input samples. Control IgG IP results were similar to those obtained with Nfm (Nefm exon 1), a negative-control locus. The locus position is indicated with respect to distance from the Egr1 TSS in kilobases. This experiment was performed using the same chromatin used for the BRG1 ChIP whose results are shown in panel B.

Article Snippet: Antibodies used in ChIP experiments were specific to BRG1 (J1; Weidong Wang), H3K9,14 acetylated histones (catalog no. 06-599; Upstate/Millipore), H3K4Me3 (catalog no. 04-745; Millipore), and Stat6 (catalog no. M-200; Santa Cruz Biotech). mRNA quantitation.

Techniques: Activation Assay, Binding Assay, ChIP-sequencing, Comparison, Control, Negative Control

Journal: Molecular and Cellular Biology

Article Title: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements

doi: 10.1128/mcb.00920-10

Figure Lengend Snippet: FIG. 7. Lineage-specific BRG1 binding. Signature cytokines (A, C, and E) and transcription factors (B, D, and F) are shown. Th1-specific (A and B), Th2-specific (C and D), and Th17-specific (E and F) genes were compared. ChIP-seq profiles for naïve, Th1S, Th2S, and Th17S cells are shown for each gene; the uTh1 profile is shown for Th1 genes; and the uTh2 profile is shown for Th2 genes. BRG1 occupancy range values (y axis) are identical for all graphs to allow direct comparisons (minimum tag frequency of 0, maximum tag frequency of 1.14 105). Vertical arrows indicate lineage-specific BRG1 binding identified using CisGenome. Asterisks indicate statistically significant BRG1 binding regions identified using CisGenome. Each panel contains a scale bar for the x axis (genomic location). Genes are indicated as boxes with names below, and arrows inside the boxes indicate the direction of transcription. Note that the Rorc gene produces the ROR- mRNA; the ROR-T (T-cell specific) transcript initiates from an internal, downstream promoter within the Rorc gene. Gata3 promoter A is 10 kb upstream of the indicated Gata3 gene. The genomic coordinates represented (MM9 assembly) are as follows: (A) for Ifn, Chr10, nt 117820000 to 117960000; (B) for Tbx21, Chr11, nt 96930000 to 97020000; (C) for Il4, Il5, and Il13, Chr11, nt 53370000 to 53570000; (D) for Gata3, Chr2, nt 8700000 to 10000000; (E) for Il17a and Il17f, Chr1, nt 20680000 to 20820000; (F) for Rorc, Chr3, nt 94140000 to 94230000.

Article Snippet: Antibodies used in ChIP experiments were specific to BRG1 (J1; Weidong Wang), H3K9,14 acetylated histones (catalog no. 06-599; Upstate/Millipore), H3K4Me3 (catalog no. 04-745; Millipore), and Stat6 (catalog no. M-200; Santa Cruz Biotech). mRNA quantitation.

Techniques: Binding Assay, ChIP-sequencing

Journal: Molecular and Cellular Biology

Article Title: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements

doi: 10.1128/mcb.00920-10

Figure Lengend Snippet: FIG. 9. BRG1 binding to miRNA loci is specific with respect to lineage and activation. BRG1 binding to miRNA loci was measured by ChIP-seq, and the results obtained for T helper subsets were com- pared. The BRG1 occupancy range values (y axis) are identical for all graphs to allow direct comparison (minimum tag frequency of 0, max- imum tag frequency of 1.14 105). Asterisks indicate statistically significant BRG1 binding regions identified using CisGenome. Each panel contains a scale bar for the x axis (genomic location). Genes are indicated as boxes with names below, and arrows inside the boxes indicate the direction of transcription. Mature miRNA locations are marked as bars; the direction of transcription is indicated with arrows. Predicted transcriptional start sites are indicated with arrows (“T” represents T cell predictions [11], “C” represents cancer cell predic- tions [59]). CpG islands are marked as vertical bars. (A) For miR-146a, Chr11, nt 43157899 to 43217963. (B) For miR-155, Chr16, nt 84684385 to 84744449. (C) For miR-326, Chr7, nt 106670000 to 106730000. (D) For miRNA 181c, 181d, 23a, 27a, and 24-2 clusters, Chr8, nt 86690000 to 86750000.

Article Snippet: Antibodies used in ChIP experiments were specific to BRG1 (J1; Weidong Wang), H3K9,14 acetylated histones (catalog no. 06-599; Upstate/Millipore), H3K4Me3 (catalog no. 04-745; Millipore), and Stat6 (catalog no. M-200; Santa Cruz Biotech). mRNA quantitation.

Techniques: Binding Assay, Activation Assay, ChIP-sequencing, Comparison

Journal: Molecular and Cellular Biology

Article Title: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements

doi: 10.1128/mcb.00920-10

Figure Lengend Snippet: FIG. 10. BRG1 binding occurs at STAT binding sites. ChIP-seq profiles from Th2S cells for BRG1, STAT6, STAT5A, and STAT5B are shown. BRG1 data are from this study, while Stat6 data are from reference 86 and Stat5 data are from reference 42. Occupancy range values (y axis) are identical for all graphs to allow direct comparison (minimum tag frequency of 0, maximum tag frequency of 1.14 105). Asterisks indicate statistically significant binding regions identified using CisGenome. Each panel contains a scale bar for the x axis (genomic location). Gene exons are indicated as vertical bars with names on the side, and arrows indicate the direction of transcription. Gata3 promoter A is 10 kb upstream (rightward) of the indicated Gata3 gene. The BRG1 data from Fig. 6 are repeated here to facilitate direct comparison. The genomic coordinates represented (MM9 assembly) are as follows: (A) for Gata3, Chr2, nt 8700000 to 10000000; (B) for Il4, Il5, and Il13, Chr11, nt 53370000 to 53570000.

Article Snippet: Antibodies used in ChIP experiments were specific to BRG1 (J1; Weidong Wang), H3K9,14 acetylated histones (catalog no. 06-599; Upstate/Millipore), H3K4Me3 (catalog no. 04-745; Millipore), and Stat6 (catalog no. M-200; Santa Cruz Biotech). mRNA quantitation.

Techniques: Binding Assay, ChIP-sequencing, Comparison

Journal: Nature immunology

Article Title: Opposing macrophage polarization programs show extensive epigenomic and transcriptional cross talks

doi: 10.1038/ni.3710

Figure Lengend Snippet: ( a ) Scatter plot showing the identified groups of IFN-γ-induced acetylated regions highly sensitive (orange dots, n=773) or completely resistant (blue dots, n=736) to IL-4 inhibition. ( b ) Box plots showing the quantification of histone acetylation changes in the IL-4-sensitive and resistant groups defined in a). *** p = 3.53e-128. ( c ) Over-representation analyses of transcription factor motifs within IL-4-sensitive and resistant regions either relative to the FANTOM5 collection of active enhancers or in the cross-comparison. The complete analyses are reported in and . ( d ) mRNA expression (from RNA-seq data) of selected TFs belonging to IRF, AP-1 and CEBP families. Bars indicate means +/- SE. ( e ) Genomic snapshots of the JunB and Cebpb loci showing binding of both STAT6 and STAT1 as well as RNA levels. Adjacent genes are shown for comparison.

Article Snippet: Antibodies used in ChIP experiments against STAT1 (sc-592), STAT6 (sc-981), MYC (sc-764), JUNB (sc-46x), C/EBPβ (sc-150X) were all from Santa Cruz.

Techniques: Inhibition, Comparison, Expressing, RNA Sequencing, Binding Assay

Journal: Nature immunology

Article Title: Opposing macrophage polarization programs show extensive epigenomic and transcriptional cross talks

doi: 10.1038/ni.3710

Figure Lengend Snippet: ( a-b ) Scatter plots reporting ChIP-seq data for JUNB ( a ) and C/EBPβ ( b ) in macrophages stimulated for 4h with IFN-γ in the presence or absence of IL-4. Red and light blue dots indicate peaks that were highly sensitive or relatively resistant to IL-4-mediated inhibition, respectively. ( c-d ) For each TF, data were divided in two groups based on sensitivity or resistance of transcription factor binding to IL-4-mediated inhibition. In each group, the box plot on the left shows transcription factor ChIP-seq read counts in the three indicated conditions. The box plot on the right shows histone acetylation read counts in untreated or IFN-γ-treated cells transduced with scrambled or TF-specific lentiviral shRNA. ( e-f ) Left: JUNB and C/EBPβ peaks in proximity of IFN-γ-inducible genes that are either sensitive or resistant to IL-4-mediated inhibition. Right: empiric cumulative distribution functions (ECDFs) showing the effects of Junb ( e ) or Cebpb ( f ) depletion on the induction by IFN-γ of IL-4-sensitive and resistant genes.

Article Snippet: Antibodies used in ChIP experiments against STAT1 (sc-592), STAT6 (sc-981), MYC (sc-764), JUNB (sc-46x), C/EBPβ (sc-150X) were all from Santa Cruz.

Techniques: ChIP-sequencing, Inhibition, Binding Assay, Transduction, shRNA