resource source identifier anti human antibodies anti human ccr6  (fluidigm)

Journal: bioRxiv

Article Title: Mapping the MIF-2 Chemokine Interactome Reveals MIF-2–CCL20 Complex Formation in Liver Fibrosis

doi: 10.64898/2026.03.02.708954

Figure Lengend Snippet: Shown are gene expression data re-analyzed from the GSE135251 dataset, focusing on CCL20, MIF, and MIF-2 (DDT) together with their corresponding receptors CCR6, CXCR4, and CD74 in NASH across increasing fibrosis stages (F0–F4; shades of magenta) compared with NAFL (grey) ( Govaere et al ., 2020 ). Statistical comparisons between groups were performed using the Mann–Whitney U test, with P values adjusted for multiple testing using the Holm–Bonferroni correction. NASH: non-alcoholic steatohepatitis; NAFL: non-alcoholic fatty liver disease. Statistical significance is indicated by actual adjusted P values.

Article Snippet: Antibodies included APC-conjugated anti-CXCR4 (BioLegend; Cat# 306510), FITC-conjugated anti-CD74 (BD Biosciences; Cat# 555540), and anti-CCR6 antibodies (PE-conjugated, Miltenyi; Cat# 130-100-377; APC/Cy7-conjugated, BioLegend; Cat# 353432).

Techniques: Gene Expression, MANN-WHITNEY

Journal: bioRxiv

Article Title: Z-TAC enables custom and combinatorial degradation of cell surface proteins

doi: 10.64898/2026.04.03.716357

Figure Lengend Snippet: (a) Schematic comparing TransTACs and Z-TACs. TransTACs are bispecific molecules that co-engage a POI antibody and TfR1 to induce lysosomal degradation. Z-TACs replace the engineered anti-POI arm with a compact Fc-binding Z-domain derived from Staphylococcal protein A, enabling receptor-mediated internalization and lysosomal degradation of the bound POI. (b) Purification of recombinant Z-TAC protein from Expi293 cells. SDS-PAGE analysis under non-reducing and reducing conditions shows the expected bands corresponding to monomeric Z-TAC (22.2 kDa). (c) Quantification of cell-surface PD-L1 levels by flow cytometry in MDA-MB-231 cells after 2 h treatment with indicated constructs and concentrations. Data represents means ± s.d. from n = 2 replicates. (d) Western blot analysis of PD-L1 degradation in MDA-MB-231 cells following treatment with atezolizumab + Z-TAC (1:2 stoichiometry) for 16 h compared with TransTAC and untreated controls. β-actin serves as a loading control. Remaining PD-L1 (% of untreated) is indicated below lanes. (e) Western blot analysis of EGFR degradation and TfR1 levels in PC9 cells following treatment with EGFR Affibody-Fc + VHHA F51I Z-TAC (1:2 stoichiometry) for 16 h compared with EGFR Affibody-Fc and untreated controls. β-actin serves as a loading control. Remaining EGFR (% of untreated) is indicated below lanes. (f) Western blot analysis of CCR6 degradation and TfR1 levels CCR6 + Jurkat cells following treatment with CCR6 antibody + VHHA F51I Z-TAC (1:2 stoichiometry) for 16 h compared with CCR6 antibody control. β-actin serves as a loading control. Remaining CCR6 (% of untreated) is indicated below lanes. (g) Time-course flow cytometry quantification of cell-surface PD-L1 and TfR1 following treatment with the Y96A Z-TAC variant and atezolizumab. Data represents means ± s.d. from n = 2 replicates. (h) Schematic illustrating a covalent conjugation strategy to generate Z-TAC-IgGs. (i) Reducing SDS-PAGE analysis of cetuximab and covalently conjugated Q32C Z-TAC-cetuximab.

Article Snippet: Supernatants were mixed with 4× Laemmli Sample Buffer (Bio-Rad) and 2-mercaptoethanol and heated at 95 °C for 5 min. For CCR6, 2-mercaptoethanol was not added and samples were not heated.

Techniques: Binding Assay, Derivative Assay, Purification, Recombinant, SDS Page, Flow Cytometry, Construct, Western Blot, Control, Variant Assay, Conjugation Assay

Journal: bioRxiv

Article Title: Z-TAC enables custom and combinatorial degradation of cell surface proteins

doi: 10.64898/2026.04.03.716357

Figure Lengend Snippet: (a) Western blot analysis of EGFR degradation and TfR1 levels in PC9 cells following treatment with EGFR Affibody-Fc + VHHA Z-TAC (1:2 stoichiometry) for 16 h compared with EGFR Affibody-Fc and untreated controls. β-actin serves as a loading control. Remaining EGFR (% of untreated) is indicated below lanes. (b) Western blot analysis of EGFR degradation and TfR1 levels in PC9 cells following treatment with EGFR Affibody-Fc + VHHA Y96A Z-TAC (1:2 stoichiometry) for 16 h compared with EGFR Affibody-Fc and untreated controls. β-actin serves as a loading control. Remaining EGFR (% of untreated) is indicated below lanes. (c) Western blot analysis of EGFR degradation and TfR1 levels in PC9 cells following treatment with cetuximab + VHHA Z-TAC (1:2 stoichiometry) for 16 h compared with cetuximab and untreated controls. β-actin serves as a loading control. Remaining EGFR (% of untreated) is indicated below lanes. (d) Western blot analysis of CCR6 degradation and TfR1 levels CCR6 + Jurkat cells following treatment with CCR6 antibody + VHHA Y96A Z-TAC (1:2 stoichiometry) for 16 h compared with CCR6 antibody control. β-actin serves as a loading control. Remaining CCR6 (% of untreated) is indicated below lanes. (e) Flow cytometry dose-response quantification of cell-surface TfR1 levels in CCR6 + Jurkat cells after 2 h treatment with indicated constructs and concentrations. Data represents means ± s.d. from n = 2 replicates. (f) Bar graph representation of cell-surface CCR6 across the indicated Z-TAC and antibody pre-incubation times, measured by flow cytometry following treatment with CCR6 antibody plus VHHA Z-TAC (1:2 stoichiometry) for 2 h. Data represent means ± s.d. from n = 2 replicates.

Article Snippet: Supernatants were mixed with 4× Laemmli Sample Buffer (Bio-Rad) and 2-mercaptoethanol and heated at 95 °C for 5 min. For CCR6, 2-mercaptoethanol was not added and samples were not heated.

Techniques: Western Blot, Control, Flow Cytometry, Construct, Incubation

Journal: bioRxiv

Article Title: Z-TAC enables custom and combinatorial degradation of cell surface proteins

doi: 10.64898/2026.04.03.716357

Figure Lengend Snippet: (a) Schematic illustrating that Z-TACs can be deployed to simultaneously co-degrade multiple targets when more than one IgG is present. (b) Western blot and quantifications showing Z-TAC mediated EGFR and PD-L1 co-degradation in MDA-MB-231 cells. Cells are treated with cetuximab (anti-EGFR), atezolizumab (anti-PD-L1), and VHHA F51I Z-TAC (1:2 stoichiometry) or controls for 16 h. β-actin serves as a loading control. (c) Schematic illustrating Z-TAC mediated functional inhibition of CCR6 via targeted degradation. (d) Flow cytometry dose-response quantification of cell-surface CCR6 levels in CCR6 + Jurkat cells after 2 h treatment with indicated constructs and concentrations. Data represents means ± s.d. from n = 2 replicates. (e) Microscopy images of HeLa cells expressing LAMP-mCherry and CCR6-GFP with 16 h treatment of 10 nM anti-CCR6 antibody and 20nM VHHA Z-TAC (f) Flow cytometry quantification of total F-actin in CCR6 + Jurkat cells with fixation and phalloidin staining. Cells were treated with varying the indicated constructs for 2 h, followed by 1 min stimulation with 50 nM CCL20. Data represents means ± s.d. from n = 3 replicates. (g) Chemotaxis of CCR6 + luciferase + Jurkat cells measured by Transwell migration assay after 2 h treatment & 2 h migration with CCL20 as the chemoattractant. Data represents means ± s.d. from n = 3 replicates.

Article Snippet: Supernatants were mixed with 4× Laemmli Sample Buffer (Bio-Rad) and 2-mercaptoethanol and heated at 95 °C for 5 min. For CCR6, 2-mercaptoethanol was not added and samples were not heated.

Techniques: Western Blot, Control, Functional Assay, Inhibition, Flow Cytometry, Construct, Microscopy, Expressing, Staining, Chemotaxis Assay, Luciferase, Transwell Migration Assay, Migration

Journal: Biochemistry and Biophysics Reports

Article Title: Comparative analysis of activation of macrophages/microglia in diabetic retinopathy and Familial Exudative Vitreoretinopathy

doi: 10.1016/j.bbrep.2025.102396

Figure Lengend Snippet: FZD4 knockout mice develop FEVR features. FZD4 knockout mice (FZD4KO) was used as an FEVR model. (A) Western blot for FZD4 in the retinal tissue of FZD4 knockout mice, compared to wildtype controls (WT). (B–C) Electroretinogram (ERG) assessments at 16 weeks, shown by representative chars (B) and by quantification (C). Blue circle: b-wave; green circle: a-wave. (D) Retinal vascular density by CD31 + area quantification. N = 5. ∗p < 0.05. NS: no significant.

Article Snippet: Knockout of FZD4 in these mice were validated by Western blot for FZD4 in the retinal tissue, using monoclonal antibody against mouse FZD4 (MAB195–050, R&D Systems).

Techniques: Knock-Out, Western Blot

Journal: NeuroSci

Article Title: Inflammatory Mediators of Alzheimer’s Disease Characterized in a Mouse Model (APP/PS1)

doi: 10.3390/neurosci7010023

Figure Lengend Snippet: Protein expression of CCR6 and its chemokine CCL20 in the cortex of transgenic (TG) and wild-type (WT) mice. Data are presented as relative mRNA expression (2 −ΔΔCt ). Values represent the mean ± SD of five independent biological replicates ( n = 5 per group), with each data point corresponding to the average of technical replicates. * p < 0.05 vs. WT.

Article Snippet: The following primers were obtained from Thermo Fisher Scientific: CCL6 (Mm01302419_m1), CCL8 (Mm01297183_m1), CCL24 (Mm00444701_m1), CCL20 (Mm01268754_m1), CCL19 (Mm00839967_g1), CCL27a (Mm00441257_g1), CCR6 (Mm99999114_s1), CCR7 (Mm99999130_s1), CCR10 (Mm01946242_m1), and the endogenous reference gene β-actin (Mm00607939_s1).

Techniques: Expressing, Transgenic Assay

Journal: iScience

Article Title: CCL20-CCR6 signaling alters the metabolic reprogramming to promote the pathogenic Th17 cell differentiation

doi: 10.1016/j.isci.2025.114385

Figure Lengend Snippet: Patients with ulcerative colitis show increased CCR6 + Th1-like Th17 cells in PBMC (A) A representative colonoscopy image of a healthy and a grade III ulcerative colitis patient (upper left panel). Peripheral blood was obtained from patients with ulcerative colitis (six women, thirteen men; age 39 ± 9.3 years) and healthy individuals (six women, seven men; age 63 ± 8 years). Peripheral blood mononuclear cells (PBMCs) were analyzed for CCR6 expression after gating on CD4 + cells, as shown in the dot plots (lower panel). Data from individual healthy and patients with UC are shown (right panel). (B) RORγt expression was analyzed after gating on CD4 + CCR6 + or CD4 + CCR6 - T cells. The dot plots shown are gated on CD4 + T cells. (C) The dot plot illustrates the expression of RORγt and T-bet on CCR6 − CD4 + or CCR6 + CD4 + gated T cell population (left panel) and provides the corresponding statistics (right panel). Each symbol represents data from an individual. (D) PBMCs were stimulated with PMA/ionomycin in the presence of brefeldin A for 6 h. Intracellular IL-17A and IFN-γ expression was analyzed after gating on CD4 + cells. (E) Purified human CD4 + CD8 − CD25 − CD45RA + T cells were in vitro differentiated into non-pathogenic (IL-6 plus TGF-β) or pathogenic conditions (IL-23 plus IL-1β) in the presence or absence of recombinant human CCL20 at 37°C for 4 days. On day 4, cells were stained with the indicated molecules and analyzed using flow cytometry. RORγt expression in CD4 + T cells is shown after gating on CD4 + CD25 + cells. (F) Expression of RORγt and T-bet in CD4 + CD25 + cells is shown. Mann-Whitney test (C). The error bar represents ±standard error of the mean (SEM). Student’s t test (A, D, E, F). Data are representative of three independent experiments (E, F). The p -value for the comparison between the two groups is indicated in the graphs. n.s., not significant): p > 0.05.

Article Snippet: Wild-type C57BL/6, CCR6 -/- , Foxp3-egfp, or RAG1 -/- (C57BL/6 background) mice were procured from Jackson Laboratory (Bar Harbor, ME) and maintained in the National Centre for Cell Science (NCCS) experimental animal facility.

Techniques: Expressing, Purification, In Vitro, Recombinant, Staining, Flow Cytometry, MANN-WHITNEY, Comparison

Journal: iScience

Article Title: CCL20-CCR6 signaling alters the metabolic reprogramming to promote the pathogenic Th17 cell differentiation

doi: 10.1016/j.isci.2025.114385

Figure Lengend Snippet: CCR6 + CD4 + T cells show an increased Th1-like Th17 phenotype during inflammatory colitis (A and B) Acute colitis was induced in wild-type C57BL/6 mice by administering 2% DSS in their drinking water, and body weight loss was monitored. On day 10, spleen (SP), mesenteric lymph nodes (mLN), Peyer’s patches (PPs), and lamina propria (LP) cells were harvested, and cells were analyzed using flow cytometry. (A) Body weight was monitored and plotted (upper panel). CCL20 expression in colon tissue sections was analyzed by immunofluorescence staining (lower panel). Original magnification 400x, scale bars 100 μm. (B) The dot plot represents the expression of RORγt on CD4 + CCR6 - and CD4 + CCR6 + T cells (upper left), and IL-17A + and IFN-γ+ expression (upper right). Mean percentages of RORγt + cells (lower left), mean percentages of IL-17A + cells (lower middle), and IL-17 and IFN-γ expression (lower right) on CD4 + CCR6 - and CD4 + CCR6 + T cells. (C) Representative immunofluorescence images of PP of wild-type mice treated with or without DSS are shown (upper right). Original magnification 400x, scale bars 100 μm. (D) CCR6 −/− or CCR6 +/+ mice were given 2% DSS in the drinking water. The body weight changes in mice were monitored (left), and RORγt + T-bet + cells were analyzed in the indicated organs by flow cytometry (right). (E) CCR6 −/− splenocytes (CD45.2 congenic; 10 × 10 6 cells/mouse) were adoptively transferred into CD45.1 congenic mice (CCR6 +/+ genotype), and the mice were administered 2% DSS in their drinking water. On day 12, the mice were sacrificed. (F) The immunofluorescence staining of CD45.2 and CD4 markers was performed on the colon tissue. Original magnification 400x, scale bars 100 μm. (G and H) RORγt + and RORγt + T-bet + cells were analyzed after gating on CD45.2 + CD4 + or CD45.2 − CD4 + T cells. (I) IL-17A + IFN-γ + cells were analyzed after gating on CD45.2 + CD4 + or CD45.2 − CD4 + T cells. (J) Naive CD4 + T cells (CD4 + CD25 − CD44 lo CD45RB hi CD62L hi Foxp3rfp − T cells; 0.5 × 10 6 cells/mouse) from CCR6 gfp/+ Foxp3 rfp/rfp mice were adoptively transferred into RAG1 −/− mice. The mean percentage in body weight change from the initial weight was plotted. ( n = 4 mice/group). The error bar represents ±SD. (K) On day 21, RORγt + T-bet + cells were analyzed after gating on CCR6gfp + and CCR6gfp − CD4 + T cells in the SP, mLN, and lamina propria (LP) (left panel). The mean percentage and MFI of RORγt + cells were analyzed after gating on CD4 + CCR6gfp − T cells or CD4 + CCR6gfp + cells (right panel). The bar represents the mean ± SEM; each symbol represents data from an individual mouse, with n = 5–6 mice/group (A–K). The p -value for the comparison between the two groups is indicated in the graphs. The data shown are representative of three independent experiments (A–K). Student’s t test was used, not significant (ns): p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: Wild-type C57BL/6, CCR6 -/- , Foxp3-egfp, or RAG1 -/- (C57BL/6 background) mice were procured from Jackson Laboratory (Bar Harbor, ME) and maintained in the National Centre for Cell Science (NCCS) experimental animal facility.

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Staining, Comparison

Journal: iScience

Article Title: CCL20-CCR6 signaling alters the metabolic reprogramming to promote the pathogenic Th17 cell differentiation

doi: 10.1016/j.isci.2025.114385

Figure Lengend Snippet: CCL20 promotes the differentiation of the pathogenic Th1-like Th17 cells (A) Naive CD4 + CD25 − CD44 − T cells were in vitro differentiated into the Th17 lineage (IL-1β + IL-23 condition) in the presence or absence of CCL20. On day 4, the percentages of CD25 + RORγt + cells or RORγt + T-bet + T cells were analyzed after gating on CD4 + cells (left panel). The mean percentage of RORγt + CD4 + T cells is shown (right panel). Each symbol represents data from a single experiment. n = 9–10 experiments. (B) IL-17A secretion in the cultured supernatant was analyzed using ELISA. n = 3–4 experiments. (C) Purified CD4 + CD25 − CD44 − T cells from C57BL/6 or CCR6 −/− mice were differentiated into the Th17 lineage. The mean percentage of RORγt + or RORγt + T-bet + cells was analyzed after gating on CD25 + CD4 + T cells. Intracellular IL-17A expression was analyzed after gating on CD4 + CD25 + cells. n = 3–4 experiments. The error bar represents the ±SEM, and each dots represent data from an individual experiment. Student’s t test. (D) Purified CD4 + CD25 − CD44 − T cells were differentiated into the Th17 lineage in the presence or absence of CCL20. On day 4, the proliferation of RORγt + Th17 cells was analyzed by Ki-67 expression in CD4 + T cells. n = 2 experiments. (E) Ki67 expression in the various lymphoid organs was analyzed from the experiments given in D. (F) On day 4, cells were harvested, and the expression of the indicated molecules was analyzed using quantitative real-time PCR (qRT-PCR). Data were normalized to the Cyclophilin A gene. The error bar represents ±SD. n = 2–4 independent experiments. The p -value for the comparison between the two groups is indicated in the graphs. not significant (ns): p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: Wild-type C57BL/6, CCR6 -/- , Foxp3-egfp, or RAG1 -/- (C57BL/6 background) mice were procured from Jackson Laboratory (Bar Harbor, ME) and maintained in the National Centre for Cell Science (NCCS) experimental animal facility.

Techniques: In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay, Purification, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Comparison

Journal: iScience

Article Title: CCL20-CCR6 signaling alters the metabolic reprogramming to promote the pathogenic Th17 cell differentiation

doi: 10.1016/j.isci.2025.114385

Figure Lengend Snippet: CCR6 signaling regulates the expression of various genes involved in the activation and differentiation of Th17 cells CD4 + CD25 − CD44 − naive T cells were in vitro differentiated into Th17-lineage (IL-1β and IL-23 condition) cells in the presence or absence of CCL20 (100 ng/mL), and total mRNA was analyzed by RNA-seq using the Illumina Mi-Seq platform. (A) A Venn diagram illustrating the overlapping gene signatures between the Th17 culture condition in the presence or absence of CCL20. (B) Heatmap of the fold change (log2) for differentially expressed genes in Th17 plus CCL20 Vs. Th17 cells. Each line represents one gene, and each column represents one sample. Different colors represent the expression levels (from green to red: increased expression). (C) Volcano plot showing various differentially regulated genes in Th17 plus CCL20 Vs. Th17 cells (a few selected genes were listed in the plot) (Log2FC for upregulated genes ≥1, downregulated genes ≤ −1, pval≤0.05). (D–F) Functional enrichment of the differential genes (with log2FC > 1<−1) in Th17 plus CCL20 cells by the GO annotation of (D) molecular functions (E) biological process, (F) GSEA enrichment plots of gene sets in Th17 plus CCL20 cells compared with Th17 cells, showing enrichment scores of GO categories including Th17 cell differentiation, JAK-STAT signaling pathway, phosphatidylinositol, and ErbB signaling pathway. CCR6 signaling upregulates important phenotypic proteins of Th17 lineages. (G) Whole differentially expressed proteins were analyzed using mass spectrometry. A Venn diagram showing the overlapping proteins in Th17 cells with CCL20 versus those in Th17 cells. (H) Heatmap of the fold change (log2) for differentially expressed proteins in Th17 plus CCL20 Vs. Th17 cells. Each line represents one gene, and each column represents one sample. Different colors represent expression levels (from blue to dark red, with increased expression). Functional enrichment of the differential proteins (with log2FC > 1<−1) comparing Th17 plus CCL20 Vs. Th17 cells. (I) The volcano plot shows the differentially expressed protein. Some of the proteins are indicated within the plot. (J) Proteins enriched in the top 10 biological functions are shown. (K) Western blot shows the expression of selected proteins.

Article Snippet: Wild-type C57BL/6, CCR6 -/- , Foxp3-egfp, or RAG1 -/- (C57BL/6 background) mice were procured from Jackson Laboratory (Bar Harbor, ME) and maintained in the National Centre for Cell Science (NCCS) experimental animal facility.

Techniques: Expressing, Activation Assay, In Vitro, RNA Sequencing, Functional Assay, Cell Differentiation, Mass Spectrometry, Western Blot

Journal: iScience

Article Title: CCL20-CCR6 signaling alters the metabolic reprogramming to promote the pathogenic Th17 cell differentiation

doi: 10.1016/j.isci.2025.114385

Figure Lengend Snippet: CCR6 intrinsic signaling induces the phosphorylation of the PI3K/Akt/mTORC1/STAT3 pathway and promotes RORγt binding on IL-17A regulatory elements (A) CCR6 + and CCR6 - CD4 T cells were sorted based on the expression of eGFP and stimulated with 100 ng/mL CCL20 at different time points (0, 5, 10, 15, 30, and 60 min). They were then immunoblotted with antibodies against various kinases in the downstream signaling pathways of Akt/mTOR/STAT3. (B) Sorted CCR6 + cells were rested overnight with or without rapamycin (50 ng/mL) and, the next day, stimulated with recombinant CCL20 (100 ng/mL) at two time points, 0 and 30 min. The phosphorylation status of STAT3 was analyzed by immunoblotting. Cyclophilin B and total STAT3 were used as loading controls. Blots are from 2 to 3 independent experiments. (C) The schematic presentation of IL-17A promoter (IL-17AP), IL-17F promoter (IL-17FP), conserved non-coding sequences (CNSs), and binding of RORγt. (D) CCR6gfp + Jurkat cells were transfected with the pGL4 plasmid vector containing the mouse IL-17 promoter (2 Kbp) and CNS5 enhancer elements. Transfected cells were stimulated with purified recombinant CCL20 (100 ng/mL) or TGF-β1 plus IL-6 at 37°C for 72 h. Then, cells were lysed, and luciferase activity was measured. The data shown are normalized to the Renilla luciferase activity. Each dot represents an independent experiment. Student’s t test. p -values are shown. (E) CCR6gfp + Jurkat cells were transfected with the pGL4 plasmid containing the IL-17 promoter (2 Kb) or IL-17 promoter (2 Kb) having a mutation in the RORγt binding site. Cells were cultured as given above, and luciferase activity was measured and plotted. The data shown are representative of one of the two independent experiments. The error bar represents ±SD. (F) Naive CD4 T cells were differentiated into the Th17 lineage condition (TGF-β + IL-6) in the presence or absence of CCL20 and with or without rapamycin (25 ng/mL) for 4 days. Expression of RORγt was analyzed after gating on CD4 + T cells. n = 3 experiments. (G) The schematic CCR6-CCL20 signaling pathways are shown. The p -value for the comparison between the two groups is indicated in the graphs. not significant (ns): p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: Wild-type C57BL/6, CCR6 -/- , Foxp3-egfp, or RAG1 -/- (C57BL/6 background) mice were procured from Jackson Laboratory (Bar Harbor, ME) and maintained in the National Centre for Cell Science (NCCS) experimental animal facility.

Techniques: Phospho-proteomics, Binding Assay, Expressing, Protein-Protein interactions, Recombinant, Western Blot, Transfection, Plasmid Preparation, Purification, Luciferase, Activity Assay, Mutagenesis, Cell Culture, Comparison

Journal: iScience

Article Title: CCL20-CCR6 signaling alters the metabolic reprogramming to promote the pathogenic Th17 cell differentiation

doi: 10.1016/j.isci.2025.114385

Figure Lengend Snippet: CCR6 signaling alters the energy metabolism in Th17 cells (A) Mouse naive T cells (CD4 + CD25 − CD44 lo ) were in vitro differentiated into pathogenic and non-pathogenic Th17 cells. Transcriptomic analysis of Th17 cells cultured in the presence or absence of CCL20 was performed. A heatmap of the fold change (log2) for differentially expressed genes that participate in glycolysis and oxidative phosphorylation is shown. Each line represents one gene, and each column represents one sample. Different colors represent the expression levels (from red to green: increased expression). (B) GSEA enrichment plots of gene sets in Th17 + CCL20 cells compared with Th17 cells, and showing the enrichment scores of oxidative phosphorylation. (C) Glucose uptake was monitored using 2-NBDG by using Flow cytometry and shown as a stacked histogram (left), and the MFI of 2-NBDG uptake (right). (D) After four days of culture of naive CD4 T cells, lactate secretion in the cultured media was measured. The error bar represents ±SD. Experiments were performed with four technical replicates. (E) The extracellular acidification rate (ECAR) and (F) the oxygen consumption rate (OCR) were measured. Error bar represents ± S.E.M. Data shown represents one of the three independent experiments. One-way ANOVA followed by Tukey’s test was used. ∗ p < 0.01, ∗∗ p < 0.001, ∗∗∗ p < 0.0001, ∗∗∗∗ p < 0.0001. (G) The expression of enzymes involved in glycolysis, and (H) a few of the enzymes involved in the TCA cycle in Th17 (non-pathogenic) and Th17 + CCL20 (pathogenic) cells were monitored using Western blot. The data shown represents one of the two independent experiments.

Article Snippet: Wild-type C57BL/6, CCR6 -/- , Foxp3-egfp, or RAG1 -/- (C57BL/6 background) mice were procured from Jackson Laboratory (Bar Harbor, ME) and maintained in the National Centre for Cell Science (NCCS) experimental animal facility.

Techniques: In Vitro, Cell Culture, Phospho-proteomics, Expressing, Flow Cytometry, Western Blot