14-6988-37

antibody from Invitrogen Antibodies

Targeting: RORC NR1F3, RORG, RZRG, TOR

Provider product page for 14-6988-37

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Antibody data

Antibody Data
Antigen structure
References [84]
Comments [0]
Validations
Western blot [1]
Other assay [38]

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Product number: 14-6988-37 - Provider product page
Provider: Invitrogen Antibodies
Product name: ROR gamma (t) Monoclonal Antibody (AFKJS-9), eBioscience™
Antibody type: Monoclonal
Antigen: Other
Reactivity: Human, Mouse
Host: Rat
Isotype: IgG
Antibody clone number: AFKJS-9
Vial size: 2 mg
Concentration: 0.5 mg/mL
Storage: 4° C

RORC protein structure - 14-6988-37 shown in red.

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Experimental details: Figure 6 Splenic Th17 cells and pro-inflammatory cytokine expression. The lymphocytes were isolated from mice subjected to various treatments. (A) Flow cytometry was used to evaluate RORgammat + CD3 + CD4 + Th17 cells among splenic CD4 + cells obtained from mice. Quantification of (B) RORgamma and (C) IL-17 from the flow cytometry results. (D) The mRNA levels of the cytokines IL-6, IFN-gamma, TNF-alpha, IL-1beta, and MCP-1beta in the spleen tissue samples were assessed. (E) IL-17 mRNA and protein levels were evaluated by reverse transcription-quantitative polymerase chain reaction, western blot and ELISA. Values are expressed as the mean +- standard error of the mean (n=10 in each group). *** P

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Experimental details: Figure 1 Presence and frequency of ILC subtypes within the meninges of severe TBI patients. ( A ) Dura was collected from consecutive, severe TBI patients undergoing decompressive craniectomy to alleviate elevated intracranial pressure. ILCs were sorted using forward scatter (FSC)/side scatter (SSC) and identified as CD45 + , lineage-negative (Lin - ), CD127 + lymphoid cells. ILCs subtypes were further defined as ILC1:, CD45 + Lin - CD127 + CD161 + NKp44 + ; ILC2, CD45 + Lin - CD127 + GATA3 + CRTH2 + ; and ILC3, CD45 + Lin - CD127 + RORgammat + AhR + , as shown in representative flow cytometry scatterplots. Gray shaded areas indicate isotype controls. To demonstrate functionality, ILCs were further stimulated with cytokine cocktails, and production of signature cytokines was assessed (ILC1, IFN-gamma; ILC2, IL-5/IL-13; ILC3, IL-17). ( B and C ) Frequency of ILC subtypes from individual patients, expressed as total cell number ( B ) and % leukocytes ( C ) ( n = 5). Scatterplots depict mean +- SD. ( D ) Computed tomography scan of a TBI patient before (Pre-) and after (Post-) decompressive craniectomy surgery. The dura was collected during surgery at the time of bone flap removal.

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Experimental details: Further certainty, the regulation of cyp26a1 on Th17 cells at uterine implantation sites by uterine horn injection of anti-cyp26a1 antibody during mice peri-implantation. ( A ) Decrease in cyp26a1 levels at D5 uterine implantation sites following uterine horn injection of cyp26a1 antibody on D3 of pregnancy. Cyp26a1 levels were reduced at uterine implantation sites based on Western blotting and immunohistochemistry. beta-actin was used as a loading control. The bars represent the SD of the mean of the relative value in grey (cyp26a1/beta-actin). A paired t -test was used to assess the significance of differences. Pairwise comparisons between each treatment group, * P < 0.05, ** P < 0.01; bars = 50 mum. The data were derived from three separate samples from pregnant mice. At least, three independent experiments were repeated for this time-point. A total of nine samples from pregnant mice were assessed. ( B ) Th17cell increased in the peripheral blood and the spleen following uterine horn injection of cyp26a1 antibody. Flow cytometry histogram of Th17 cells is on the left side. Th17 cells are CD 4 + ROR gammat + . Th17 cell ratio was evaluated by flow cytometry analysis. The data are expressed as the% of CD 4 + ROR gammat + double positive cells. The bars represent the SD of the mean. A paired t -test was used to assess the significance of differences. Pairwise comparisons between each treatment group, * P < 0.05. Three independent experiments were repeated for this time-point.

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Experimental details: 7 Inhibition of CK2 activity modulates mRNA expression of IRFs in MoDCs. Immature MoDCs were pretreated for 1 h with DMSO 0.1% or CX-4945 (CX, 5 uM) and then stimulated for 6 h with (A) NiSO 4 500 uM or (B) DNCB 10 uM. IRF-1, -8, and -4 mRNAs were quantified by real-time RT-PCR. Naive CD4 + T lymphocytes were coincubated with NiSO4-stimulated MoDCs (C) in the absence or presence of CX-4945 (5 uM). On day 5, cells were restimulated with anti-CD3/CD2/CD28 beads for 48 h and stained with CD4-Alexa Fluor 700, CD3-FITC, RorgammaT-APC, GATA3-PE, and T-Bet Brilliant Violet 605. Living cells (ZombieAqua negative cells) were analyzed by flow cytometry. Data represent the results of 5 independent experiments and are normalized to the control. * P < 0.05; ** P < 0.01 (Mann-Whitney test).

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Experimental details: Figure 4. Flow cytometric mapping of the expression of forkhead box P3 and retinoic acid-related orphan receptor gammat (ROR-gammat) molecules in CD4 + cells of peripheral blood.

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Experimental details: Figure 5 Successful transfection of mcCTLA4Ig into hepatocytes. (a) GFP expression of mcMock plasmids in CIA mice liver is shown as positive fluorescence. The minicircular form showed a relatively high positive expression in hepatocytes compared to the parental form. (b) Splenocytes of CIA were analyzed by flow cytometry. (c) mcCTLA4Ig-treated mice had more Foxp3 + cells compared with mice given a low dose of mcCTLA4Ig or ppCTLA4Ig. (d) Fewer RORgammat + cells were seen in mice given mcCTLA4Ig compared with those given mcMock, low-dose mcCTLA4Ig, or ppCTLA4Ig. (** P < 0.005 ).

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Experimental details: Figure 2. Rorgammat, IRF4, and STAT3 bind the Procr promoter and induce PROCR expression. Naive CD4 + T cells from Rorgammat KO mice (A), CD4 Cre xIRF4 fl/fl mice (B), or CD4 Cre xSTAT3 fl/fl mice (C) and control mice were polarized under Th17 conditions (TGF-beta1 and IL-6) or without cytokines (Th0) as control. On day 3, PROCR expression was determined by flow cytometry. (D) Purified WT naive CD4 + T cells were differentiated into Th17 cells in the presence of TGF-beta1 and IL-6. After 4 d, binding of Rorgammat, IRF4, or STAT3 to the Procr and IL23r promoters was assessed by ChIP-PCR (TF, transcription factor; mean +- SD of triplicates). (E) Procr promoter activity was measured in HEK293T cells transfected with a luciferase vector driven by 1,040 bp of the Procr promoter (pGL4-Procr), and the indicated transcription factor expression constructs (mean +- SD of triplicates). Representative plots of two to three independent experiments are shown.

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Experimental details: Figure 2. ILC3s limit CD4 + T cell responses to allergens in the airway (A) Representative immunofluorescence staining for ILC3s and T cells in the MedLNs of HDM-exposed mice (representative of 2 independent experiments and n = 3 mice per group). Images taken at 20x magnification; scale bars, 50 mum (left panel) and 10 mum (inserts; right panel). Dashed line indicates the B cell follicle border. (B) Histograms demonstrating CD80 and CD86 expression in MedLN cells following HDM exposure (representative of 2 independent assays containing 8 mice each). FMO, fluorescence minus one. (C) Graphs quantifying CD25 staining or IL-2 binding in gated MedLN cells following HDM exposure (pooled from 2 independent assays, n = 10). Naive T cells were gated as CD44 - CD62L + , effector T cells were gated as CD44 + CD62L - , and Tregs were gated as FoxP3 + . (D and E) Representative plots of MHC class II expression in designated immune cells (D) and quantification of MHC class II staining on those cell types in the MedLN following HDM exposure (E) (representative of 2 independent assays, n = 4). (F) Representative plots and quantitative graphs of the frequency and numbers of 2W1S-specific CD4 + T cells in lung parenchyma from H2-Ab1 fl/fl control mice and MHC class II DeltaILC3 mice after exposure to papain plus 2W1S peptide or PBS control. Cells were gated on live CD45 + CD19 - TCRbeta + CD4 + (data pooled from 2 independent assays). (G) Representative plots and quantitative graphs of the fre

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Experimental details: Figure 3 Interleukin (IL)-22 expression is reduced in Citrobacter rodentium -infected CX 3 CR1 GFP/GFP animals. ( a ) From the proximal colon of noninfected and C. rodentium -infected CX 3 CR1 GFP/GFP and wild-type littermate controls mRNA was isolated. cDNA was prepared by reverse transcription and quantitative real-time PCR was performed with cyber green and the indicated primers. beta-actin was used as a housekeeping gene to normalize cDNA input between samples. Normalized ct-values of the untreated samples (baseline) are set to 1 and values are plotted as fold expression of the baseline. The assays were carried out in triplicates. As per indicated, group 4 animals were analyzed. ( b ) IL-22 expression by colonic lamina propria (cLP) cell isolates from noninfected and C. rodentium -infected B6, (age- and sex-matched) and CX 3 CR1 GFP/+ and CX 3 CR1 GFP/GFP animals was analyzed by multicolor flow cytometry. Numbers indicate the percentage of IL-22-positive cells. ( c ) cLP IL22 + cells from noninfected and C. rodentium- infected B6, (age- and sex-matched), and CX 3 CR1 GFP/+ and CX 3 CR1 GFP/GFP animals were stained for CD3e and CD4. Numbers indicate the percentage of CD3e + , CD3e + CD4 + , CD4 + , and CD3e - CD4 - cells within the IL-22 + cell population. Data from an individual representative mouse per group (of five to seven individual mice analyzed per group) are shown. ( d ) cLP IL-22 + CD3e - CD4 + cells were (intracellular) stained for related orphan receptor gamma-

Supportive validation

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Experimental details: Figure 2 Th17 cell increased in the peripheral blood and the spleen following pCR3.1-cyp26a1 immunization during mice peri-implantation, especially on D5 of mice pregnancy. (A) Flow cytometry scatterplot of Th17 cells. Th17 cells are CD4 + RORgammat + . (B) The Th17 cell proportion increased significantly in the peripheral blood and the spleen under the influence of recombinant plasmid, especially on D5 of pregnancy. The CD4 + RORgammat + Th17 cell ratio was evaluated by flow cytometry analysis. The data are expressed as the% of CD4 + RORgammat + double positive cells. The bars represent the SD of the mean. A paired t -test was used to assess the significance of differences. Pairwise comparisons between each treatment group, * P < 0.05. At least, three independent experiments were repeated for each time-point. A total of 49 samples from pregnant mice were assessed.

Supportive validation

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Experimental details: Figure 3 Decrease in Th17 cells at uterine implantation sites following pCR3.1-cyp26a1 immunization during mice peri-implantation. (A) Th17 levels were significantly reduced at uterine implantation sites based on Western blotting, especially on D5 of mice pregnancy. RORgammat is the specific marker of Th17 cell, IL-17 is the specific cytokines secreted by Th17 cells. beta-actin was used as a loading control. The bars represent the SD of the mean of the relative value in grey (RORgammat/beta-actin or IL-17/beta-actin). A paired t -test was used to assess the significance of differences. Pairwise comparisons between each treatment group, * P < 0.05, ** P < 0.01. At least, three independent experiments were repeated for each time-point. A total of 36 samples from pregnant mice were assessed. (B) Th17 levels were obviously reduced at implantation sites of D5 pregnancy based on RORgammat and IL-17 immunohistochemistry stain; bars = 200 and 25 mum. At least, three independent experiments were repeated for this time-point. A total of nine samples from pregnant mice were assessed. E, embryo; S, uterine stroma; LE, Luminal epithelium; GE, Glandular epithelium.

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Experimental details: Figure 4 Further certainty, the regulation of cyp26a1 on Th17 cells at uterine implantation sites by uterine horn injection of anti-cyp26a1 antibody during mice peri-implantation. (A) Decrease in cyp26a1 levels at D5 uterine implantation sites following uterine horn injection of cyp26a1 antibody on D3 of pregnancy. Cyp26a1 levels were reduced at uterine implantation sites based on Western blotting and immunohistochemistry. beta-actin was used as a loading control. The bars represent the SD of the mean of the relative value in grey (cyp26a1/beta-actin). A paired t -test was used to assess the significance of differences. Pairwise comparisons between each treatment group, * P < 0.05, ** P < 0.01; bars = 50 mum. The data were derived from three separate samples from pregnant mice. At least, three independent experiments were repeated for this time-point. A total of nine samples from pregnant mice were assessed. (B) Th17cell increased in the peripheral blood and the spleen following uterine horn injection of cyp26a1 antibody. Flow cytometry histogram of Th17 cells is on the left side. Th17 cells are CD4 + RORgammat + . Th17 cell ratio was evaluated by flow cytometry analysis. The data are expressed as the% of CD4 + RORgammat + double positive cells. The bars represent the SD of the mean. A paired t -test was used to assess the significance of differences. Pairwise comparisons between each treatment group, * P < 0.05. Three independent experiments were repeated for this time-point

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Experimental details: Figure 5 Further certainty, the regulation of cyp26a1 on Th17 cells at uterine implantation sites by tail vein injection of cyp26a1 lentiviral interference segment (No. 306) during mice peri-implantation. (A) Decrease in cyp26a1 levels at uterine implantation sites of D5 following tail vein injection of cyp26a1 lentiviral interference segment (306) on D3 of pregnancy. Cyp26a1 levels were reduced at uterine implantation sites based on Western blotting and immunohistochemistry; bars = 25 mum. The data were derived from two separate samples from pregnant mice. At least, three independent experiments were repeated for this time-point. A total of six samples from pregnant mice were assessed. (B) Th17 cell increased in the peripheral blood, but the spleen following tail vein injection of cyp26a1 lentiviral interference segment (No. 306). Flow cytometry histogram of Th17 cells is on the left side. Th17 cells are CD4 + RORgammat + . Th17 cell ratio was evaluated by flow cytometry analysis. The data are expressed as the% of CD4 + RORgammat + double positive cells. The bars represent the SD of the mean. A paired t -test was used to assess the significance of differences. Pairwise comparisons between each treatment group, * P < 0.05. Three independent experiments were repeated. A total of nine samples from pregnant mice were assessed. (C) Th17 levels were significantly reduced at uterine implantation sites based on Western blotting of D5 pregnancy. beta-actin was used as a loading contr

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Experimental details: Figure 7 Increase in RARalpha at uterine implantation sites following pCR3.1-cyp26a1 immunization during mice peri-implantation and further certainty, the influence of RARalpha on Th17 cells at uterine implantation sites by tail vein injection of RARalpha specific agonist and antagonist during mice peri-implantation. (A) RARalpha levels were significantly increased at uterine implantation sites based on Western blotting. beta-actin was used as a loading control. (B) RARalpha levels were obviously increased at implantation sites of D5 pregnancy based on immunohistochemistry following pCR3.1-cyp26a1 immunization; bars = 200 and 25 mum. The data were derived from three separate samples from pregnant mice. At least, three independent experiments were repeated for this time-point. A total of nine samples from pregnant mice were assessed. E, embryo; S, uterine stroma; LE, Luminal epithelium; GE, Glandular epithelium. (C) RARalpha and RORgammat protein expression changes in uterus on D5 of pregnancy in response to administration of agonist (Am-580) and antagonist (Ro-41-5253) of RARalpha. RARalpha levels were significantly increased by the activation of Am-580 and significantly reduced by the inhibition of Ro-41-5253 at uterine implantation sites based on western blotting of pregnancy D5. RORgammat levels just took the opposite changes on RARalpha. beta-actin was used as a loading control. The administration (125 mug/mouse) of RARalpha agonist (Am-580) or antagonist (Ro-41-5253) was

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Experimental details: Figure 2. Th17-polarized CD4 + T cells are sufficient to promote hippocampal precursor cell proliferation in adult TCRalpha -/- mice. ( A - C ) Th17 polarization in vitro . ( A ) Flow cytometry of encephalitogenic CD4 + T cells. Dot plots show pre-sort (left) and post-sort (right) analysis of naive T cells (CD4 + CD62L high CD25 - Foxp3 GFP- ) from pooled spleen and lymph nodes of 2D2 x Foxp3 GFP mice. FACS-purified T cell populations were cultured under Th17-polarizing conditions, as described in the Methods section. On day 7, efficiency of Th17 cell differentiation was confirmed by intracellular flow cytometry of ( B ) the Th17 transcription factor ROR-gammat and ( C ) the Th17 and Th1 signature cytokines IL-17 and IFN-gamma, respectively. ( D - I ) Impact of adoptive Th17 cell transfer on hippocampal precursor cell proliferation in TCRalpha -/- mice. ( D ) On day 7, 4 x 10 6 total cells from Th17 polarization cultures were injected i.v. into adult TCRalpha -/- mice. (D1) Dot plots show representative flow cytometry of CD4 + T cells in peripheral blood of recipient mice that express the Valpha3.2 subunit of the transgenic 2D2 TCR, two weeks after adoptive transfer. (D2) and (D3) Graphs show composite percentages of total CD4 + T cells (D2) and MOG 35-55 -reactive Valpha3.2 + T cells among gated CD4 + T cell populations (D3) from peripheral blood of recipient mice. The arrowheads in (D2) and (D3) highlight an individual mouse that exhibited immune cell infiltrations in the b

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Experimental details: 2 FIGURE Iron chelation inhibited metabolic activities and modulated the differentiation of CD4 + T-cells. (A)-(E) CD4 + T-cells isolated from healthy controls (HC) were stimulated with anti-CD3/CD28 beads for 3 d. Ciclopirox (CPX) (1 muM), deferasirox (DFX) (2 muM), deferoxamine (DFO) (2 muM) and transferrin (TF, 50 mug/ml) were included in some of the experiments as indicated. For metabolic activities evaluation of CD4 + T-cells, Glycolysis Stress Test Kit and Mito Stress Test Kit were used to test extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively, by a Seahorse XF96 analyser. (A and B) Glucose, oligomycin (Oligo) and 2-deoxyglucose (2-DG) were injected sequentially and ECAR were recorded. ECAR tracing curves and parameters of glycolysis, glycolysis capacity and glycolysis reserve were summarized ( n = 5). (C and D) Oligo, carbonyl cyanide- p -trifluoromethoxyphenylhydrazone (FCCP) and rotenone/antimycin A (R/A) were injected sequentially. OCR of basal respiration, respiration coupled to ATP production, maximal respiration and respiratory spare capacity were summarized from four independent samples. (E) Energy map showing metabolic potential of CD4 + T-cells. (F and G) CD4 + T-cells were stimulated with anti-CD3/CD28 beads in the presence of CPX (1 muM), DFX (2 muM), DFO (2 muM) and TF (50 mug/ml) for 5 d. CD4 + T-cells were stained with Brilliant Violet 421-conjugated anti-interferon gamma (IFNgamma), APC-conjugated anti-interleukin (I

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Experimental details: 5 FIGURE Iron promotes glucose metabolism in CD4 + T-cells through PFKFB4 in idiopathic inflammatory myopathies (IIM). (A) Double staining of CD4 (dark blue) and PFKFB4 (red) of skin sections from patients with IIM by immunohistochemistry. Original magnification: 200x. (B)-(I) CD4 + T-cells were stimulated with anti-CD3/CD28 beads for 72 h in the presence of ciclopirox (CPX) (1 muM), deferasirox (DFX) (2 muM), deferoxamine (DFO) (2 muM) or rapamycin (10 nM) as indicated. PFKFB4 expression in CD4 + T-cells from IIM patients (p) or healthy controls (HC) ( n ) (B), or CD4 + T-cells from HC treated with iron chelators (C), or CD4 + T-cells from IIM treated with DFX (D) was measured by western blot. Representative bands of three independent samples. (E) Phosphorylation of AKT (p-AKT) and p-S6 (ribosomal protein) expression in CD4 + T-cells from HC was measured by western blot. (F and G) CD4 + T-cells from HC were stained with primary antibodies against p-AKT and p-S6, followed by Alexa Fluor 594-conjugated goat anti-rabbit IgG and measured by flow cytometry ( n = 6). (H) Expression of phosphorylation of mTOR (p-mTOR), mTOR and Raptor in CD4 + T-cells from IIM or HC treated with or without DFX was measured by western blot. Representative bands of three independent samples. (I) PFKFB4 expression in CD4 + T-cells from HC treated with rapamycin was measured by western blot ( n = 3). (J)-(O) CD4 + T-cells from HC or IIM were electrotransfected with Pfkfb4 - or scramble siRNA. (J) Pfkfb

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Experimental details: Effect of p70 S6K1 on the status of transcription factors involved in the differentiation of Th1, Th2, and Th17 cells. Naive splenic CD4 + T cells (CD4 + CD62L + ) from WT and p70 S6K1 KO mice were activated by plate-bound anti-CD3 and anti-CD28 under either Th1, Th2, or Th17 skewing conditions. (A) Flow cytometry and RT-PCR analyses were performed for retinoic acid receptor-related orphan receptor gamma T (RORgammaT) using in vitro-differentiated Th17 cells. Following in vitro differentiation, Th17 cells were stimulated with PMA and ionomycin for 4 h, last 2 h with monensin. Stimulated cells were either stained with antibodies against CD4 and RORgammaT and analyzed by flow cytometry (left), or used for total RNA isolation (right). The cDNA was then analyzed by quantitative PCR to measure RORgammaT transcript levels (right). These experiments were first normalized to a housekeeping gene (either beta actin or GAPDH). Then the normalized results were compared to wild-type Th17 cells to determine the fold change. (B) RT-PCR analyses of T-bet (left) and GATA3 (right) levels were done using in vitro-differentiated Th1 and Th2 cells, respectively. The amount of T-bet and GATA3 mRNA expression in WT and KO differentiated cells were normalized first to beta actin, and then the normalized values were compared to wild type to determine the fold change. All data shown as mean + SEM from two independent experiments with total six samples per group.

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Experimental details: Fig. 7 Cytokine signaling regulated TRIM28 recruitment. a Expression of TRIM28 in different T-cell subsets and naive CD4 + T cells. b TRIM28 ChIP-qPCR results in WT Th0, WT Th17, STAT3 KO Th17 or RORgammat KO Th17 cells. c RORgammat overexpression in WT or STAT3 KO CD4 + T cells polarized under neutral (Th0) condition. d Overlay of p300 binding peaks in WT/STAT3KO(Upper) or WT/RORgammaKO (lower) Th17 cells over Th17-specific SEs with decreased p300 intensity. e 3C-PCR experiments were performed in WT and TRIM28KO Th17 cells cultured for 3 days and the PCR products were acquired by nest PCR. M is short for marker, and the whole DNA gels are shown in Supplementary Fig. 8 . f Immunoblot results of TRIM28 immunoprecipitated protein complexes as detected by RORgammat and STAT3 antibodies in Th17 cells. The whole WB gels are shown in Supplementary Fig. 9 . Those experiments were repeated twice with consistent results, and Student's t test was used for the statistical test (ns = not significant; * p < 0.05, ** p < 0.01, *** p < 0.005). All error bars represent SDs

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Experimental details: Figure 6 Rest-time food and alcohol access promote tumor-associated inflammation. ( A ) Colon permeability increased in WTE + alcohol mice using urine excretion ratios of sucralose. ( B and C ) By using immunofluorescence, ( B ) polyp-associated CD3 + cell density per surface area as well as ( C ) the CD3 + /CD4 + cell density ratio was determined for each polyp and compared between the groups. ( D and E ) The density of ( D ) FOXP3 + and ( E ) RORgammat + cells in the peripolyp surface areas was lower and higher, respectively, in the WTE + alcohol mice vs the control water group (N = 99 polyp images were used for quantification). Representative stained slide images are shown for ( B ) CD3, ( D ) FOXP3, and ( E ) RORgammat at 20x magnification, with the inset at 40x magnification. Between-group comparisons of the RORgammat + /FOXP3 + ratio in the ( F ) mucosa and ( G ) mesenteric lymph nodes as determined by flow cytometry are shown. Representative gating scheme of 1 mouse for CD4 + T-cell intracellular FOXP3 and RORgammat cytometry is shown. No significant differences in FOXP3 + and RORgammat + T-cell abundance were observed among the tested groups. ( H ) Correlation between the urine excretion ratio of sucralose and the polyp-associated RORgammat + /FOXP3 + ratio. ( I ) TD160445 treatment increased mucosal FOXP3 + and decreased RORgammat + cell density, as well as the RORgammat/FOXP3 + ratio in WTE + alcohol-treated mice (N = 48 polyp images were used for t

Supportive validation

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Experimental details: Figure 7. Cooperative regulation of IL-17 and IL-10 by Trim33, Smad2, and ROR-gamma. (A) Trim33 binding was assessed by ChIP-PCR analysis. PCR amplification was conducted with input DNA and I.P. DNA prepared from chromatin precipitated with anti-Trim33 antibodies, with C57BL/6 CD4 + T cells stimulated by anti-CD3 and anti-CD28 antibodies under indicated conditions. Primer for the H19 ICR locus was used as a negative control. Data are representative of three independent experiments. (B) Trim33 ChIP was performed with WT and Smad2 KO CD4 + T cells cultured under Th17 skewing conditions. (C) Physical association of Trim33 with Smad2 and ROR-gamma in Th17 cells. Lysates of Th17 cells were subjected to immunoprecipitation with anti-Trim33 antibody or isotype Ig, followed by immunoblot analysis with anti-Trim33, Smad2, and ROR-gamma antibodies. Data are representative of four independent experiments. Error bars show means +- SD. *, P < 0.05; **, P < 0.01.