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Proteintech anti ncoa6
Anti Ncoa6, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ncoa6/NCOA6+Antibody/pmc12821289-136-5-8
Average 93 stars, based on 7 article reviews

anti ncoa6 - by Bioz Stars, 2026-09

93/100 stars

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Article Title: Alterations of Histone Modifications Contribute to Pregnane X Receptor-Mediated Induction of CYP3A4 by Rifampicin
Article Snippet: Penicillin, streptomycin, puromycin, ampicillin, dimethylsulfoxide (DMSO), and all other chemicals were purchased from Solarbio Company (Beijing, China), unless otherwise noted. .. LS174T cells that were grown on coverslip-coated 24-well plates and treated with rifampicin or solvent for 48 hours were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100, then blocked with 1% bovine serum albumin for 1.5 hours followed by overnight incubation of a primary antibody at 4°C and 1.5-hour incubation of a secondary antibody at room temperature: For PXR localization, an anti-PXR (1:50; Santa Cruz Biotechnology) and a Alexa Fluor 647-conjugated rabbit secondary antibody (1:200; Abcam) were used; for NCOA6 or p300 localization, an anti-NCOA6 (1:50; Proteintech, Wuhan, China) or anti-p300 (1:100; Abcam) with a Alexa Fluor 488-conjugated mouse secondary antibody (1:200; Abcam) were used. .. Human NCOA6 expression vector, pCMV2-hNCOA6:V5, was a gift from Kenneth Irvine (Addgene plasmid no. 63585) ( Oh et al., 2014 ).

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NCOA6 expression in monocytes and macrophages is upregulated by proinflammatory stimuli. A THP-1 cells were induced to differentiate into macrophages by treatment with PMA, and NCOA6 expression was determined using flow cytometry after 3 days. A representative plot is shown in the upper panel ( n = 5). B THP-1 cells were left untreated or stimulated with LPS, IL-1β, or TNFα for the indicated duration, and cell lysates were immunoblotted with an anti-NCOA6 Ab. C Primary CD14 + monocytes isolated from the peripheral blood of healthy donors were stimulated with M-CSF, LPS, IL-1β or TNFα. NCOA6 was detected using flow cytometry. D Bone marrow-derived monocytes (BMMos) and bone marrow-derived macrophages (BMDMs) were left untreated or stimulated with LPS, IL-1β or TNFα, and N COA6 mRNA expression was analyzed using qPCR. Peritoneal exudate cells were obtained from C57BL/6 mice intraperitoneally injected with MSU crystals ( E ), and synovial tissues were isolated from DBA/1 mice with CIA ( F ). Ncoa6 mRNA levels in peritoneal exudate cells and synovial tissues were analyzed using qPCR. G Representative immunofluorescence staining for NCOA6, CD68, and CD55 in the synovium of rheumatoid arthritis (RA) patients. The white arrows indicate CD68 + NCOA6 + cells. The rectangular area in the merged image is magnified in the lower right panel. Scale bar=100 μm for the middle and 25 μm for the right. H Venn diagram showing the overlap between transcription factors (TFs) that interact with NCOA6 and TFs enriched in the differentially expressed genes (DEGs) in synovial macrophages from RA patients compared to healthy macrophages differentiated from peripheral monocytes (GSE49604). The immunoblotting and immunofluorescence results are representative of three independent experiments. β-Actin was detected as an internal control. Data in the bar graphs are shown as the mean ± SEM. Each dot represents an individual experiment (A) or mouse ( D , E , F ). * P < 0.05, ** P < 0.01, and *** P < 0.001 compared to the controls by unpaired two-tailed t test ( A , F ) and one-way ANOVA ( D , E )

Journal: Cellular and Molecular Immunology

Article Title: Nuclear receptor coactivator 6 is a critical regulator of NLRP3 inflammasome activation and gouty arthritis

doi: 10.1038/s41423-023-01121-x

Figure Lengend Snippet: NCOA6 expression in monocytes and macrophages is upregulated by proinflammatory stimuli. A THP-1 cells were induced to differentiate into macrophages by treatment with PMA, and NCOA6 expression was determined using flow cytometry after 3 days. A representative plot is shown in the upper panel ( n = 5). B THP-1 cells were left untreated or stimulated with LPS, IL-1β, or TNFα for the indicated duration, and cell lysates were immunoblotted with an anti-NCOA6 Ab. C Primary CD14 + monocytes isolated from the peripheral blood of healthy donors were stimulated with M-CSF, LPS, IL-1β or TNFα. NCOA6 was detected using flow cytometry. D Bone marrow-derived monocytes (BMMos) and bone marrow-derived macrophages (BMDMs) were left untreated or stimulated with LPS, IL-1β or TNFα, and N COA6 mRNA expression was analyzed using qPCR. Peritoneal exudate cells were obtained from C57BL/6 mice intraperitoneally injected with MSU crystals ( E ), and synovial tissues were isolated from DBA/1 mice with CIA ( F ). Ncoa6 mRNA levels in peritoneal exudate cells and synovial tissues were analyzed using qPCR. G Representative immunofluorescence staining for NCOA6, CD68, and CD55 in the synovium of rheumatoid arthritis (RA) patients. The white arrows indicate CD68 + NCOA6 + cells. The rectangular area in the merged image is magnified in the lower right panel. Scale bar=100 μm for the middle and 25 μm for the right. H Venn diagram showing the overlap between transcription factors (TFs) that interact with NCOA6 and TFs enriched in the differentially expressed genes (DEGs) in synovial macrophages from RA patients compared to healthy macrophages differentiated from peripheral monocytes (GSE49604). The immunoblotting and immunofluorescence results are representative of three independent experiments. β-Actin was detected as an internal control. Data in the bar graphs are shown as the mean ± SEM. Each dot represents an individual experiment (A) or mouse ( D , E , F ). * P < 0.05, ** P < 0.01, and *** P < 0.001 compared to the controls by unpaired two-tailed t test ( A , F ) and one-way ANOVA ( D , E )

Article Snippet: Human NCOA6 siRNA (sc-61401), control siRNA (sc-37007), and febuxostat (144060-53-7) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Flow Cytometry, Isolation, Derivative Assay, Injection, Immunofluorescence, Staining, Western Blot, Control, Two Tailed Test

$Nuclear-to cytoplasmic translocalization of NCOA6 in macrophages and its potential role in innate immunity and inflammation. A – F THP-1 cells were left untreated or treated with PMA. The PMA-treated THP-1 cells were left unstimulated or stimulated with LPS for 4 h and with ATP for an additional 30 min. Representative immunofluorescence staining for NCOA6, actin, and DAPI in resting cells ( A ) and PMA-treated THP-1 cells ( B ). The dotted circle indicates the nuclear margin. Scale bars, 20 μm. The cellular localization of NCOA6 was analyzed by immunoblotting of nuclear and cytoplasmic fractions ( C ) and by immunofluorescence staining ( D , left panel). Scale bars, 50 μm. The localization and cytoplasmic form of NCOA6 were analyzed in at least 200 cells for each group ( D , bar graphs in the right panel). Representative immunofluorescence staining showing the colocalization of NCOA6, ASC, vimentin, and NLRP3 ( E , F ). Cells with colocalized staining (merge) were rotated by 90° along the dashed line and imaged from the side view (bottom). Scale bars, 20 μm. G Venn diagram illustrating the upregulated DEGs (Up-DEGs) and downregulated DEGs (Down-DEGs) in Ncoa6 -KO BMDMs ( n = 2) compared to wild-type (WT) BMDMs ( n = 2) stimulated without or with LPS + MSU. H Heatmap showing the six groups formed by the 847 DEGs in Ncoa6 -KO BMDMs compared to their respective WT BMDMs. The letters U, D, and X in the right colored box denote upregulation, downregulation, and no expression change, respectively. The UX and DX groups indicate up- and downregulation only with medium alone, the XU and XD groups up- and downregulation only with LPS + MSU, and the UU and DD groups up- and downregulation both with media alone and with LPS + MSU, respectively. The UD group was excluded from gene ontology (GO) enrichment analysis because it only has 6 genes and lacks biological relevance. The number of DEGs is shown in parentheses in the colored box. I Heat plot visualizing the GOBP terms enriched by the genes in the UU, UX, XU, DD, DX, and XD groups; the UD group containing the 6 DEGs was excluded. J GSEA plot demonstrating significant enrichment between downregulated DEGs in Ncoa6 -KO BMDMs and upregulated genes in WT BMDMs stimulated with LPS + MSU compared to media alone. The horizontal and vertical dotted red lines indicate the highest enrichment score and leading edge, respectively$

Journal: Cellular and Molecular Immunology

Article Title: Nuclear receptor coactivator 6 is a critical regulator of NLRP3 inflammasome activation and gouty arthritis

doi: 10.1038/s41423-023-01121-x

Figure Lengend Snippet: Nuclear-to cytoplasmic translocalization of NCOA6 in macrophages and its potential role in innate immunity and inflammation. A – F THP-1 cells were left untreated or treated with PMA. The PMA-treated THP-1 cells were left unstimulated or stimulated with LPS for 4 h and with ATP for an additional 30 min. Representative immunofluorescence staining for NCOA6, actin, and DAPI in resting cells ( A ) and PMA-treated THP-1 cells ( B ). The dotted circle indicates the nuclear margin. Scale bars, 20 μm. The cellular localization of NCOA6 was analyzed by immunoblotting of nuclear and cytoplasmic fractions ( C ) and by immunofluorescence staining ( D , left panel). Scale bars, 50 μm. The localization and cytoplasmic form of NCOA6 were analyzed in at least 200 cells for each group ( D , bar graphs in the right panel). Representative immunofluorescence staining showing the colocalization of NCOA6, ASC, vimentin, and NLRP3 ( E , F ). Cells with colocalized staining (merge) were rotated by 90° along the dashed line and imaged from the side view (bottom). Scale bars, 20 μm. G Venn diagram illustrating the upregulated DEGs (Up-DEGs) and downregulated DEGs (Down-DEGs) in Ncoa6 -KO BMDMs ( n = 2) compared to wild-type (WT) BMDMs ( n = 2) stimulated without or with LPS + MSU. H Heatmap showing the six groups formed by the 847 DEGs in Ncoa6 -KO BMDMs compared to their respective WT BMDMs. The letters U, D, and X in the right colored box denote upregulation, downregulation, and no expression change, respectively. The UX and DX groups indicate up- and downregulation only with medium alone, the XU and XD groups up- and downregulation only with LPS + MSU, and the UU and DD groups up- and downregulation both with media alone and with LPS + MSU, respectively. The UD group was excluded from gene ontology (GO) enrichment analysis because it only has 6 genes and lacks biological relevance. The number of DEGs is shown in parentheses in the colored box. I Heat plot visualizing the GOBP terms enriched by the genes in the UU, UX, XU, DD, DX, and XD groups; the UD group containing the 6 DEGs was excluded. J GSEA plot demonstrating significant enrichment between downregulated DEGs in Ncoa6 -KO BMDMs and upregulated genes in WT BMDMs stimulated with LPS + MSU compared to media alone. The horizontal and vertical dotted red lines indicate the highest enrichment score and leading edge, respectively

Article Snippet: Human NCOA6 siRNA (sc-61401), control siRNA (sc-37007), and febuxostat (144060-53-7) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Immunofluorescence, Staining, Western Blot, Expressing

NCOA6 promotes NLRP3 inflammasome activity. A – C Human monocytes from healthy donors transfected with NCOA6 siRNA or control siRNA were primed with LPS for 6 h, followed by stimulation with ATP for 30 min or MSU crystals for 2 h. Cell lysates and culture supernatants were immunoblotted with the indicated antibodies. Sups indicates supernatants ( A ). NCOA6 mRNA levels were analyzed by real-time qRT–PCR ( B left panel). The amounts of secreted IL-1β, TNFα, and active caspase-1 in the supernatant were measured using ELISA ( B middle and right panel). Each dot represents an individual donor. Representative immunofluorescence staining for NCOA6, ASC, and DAPI ( C ). The white arrows (Merge) indicate ASC specks, and the red arrows (DIC) indicate phagocytosed MSU crystals. DIC, differential interference contrast. Scale bars, 20 μm. D – H BMDMs derived from Ncoa6 fl/fl ( fl/fl ) mice and Ncoa6 fl/fl LysM CRE ( Δ / Δ ) mice ( n = 6 per group) were primed with LPS for 4 h, followed by stimulation with ATP for 30 min, MSU crystals for 2 h, or nigericin (Nig) for 2 h. Cell lysates and culture supernatants were immunoblotted with the indicated antibodies. Sups denotes supernatants. D NCOA6 mRNA levels were analyzed using real-time qPCR ( E , left panel). The levels of secreted IL-1β, TNFα, and active caspase-1 in the supernatant were determined using ELISA ( E middle and right panels). Each dot indicates an individual mouse. Representative immunofluorescence images for ASC and DAPI ( F upper panel). The white arrows indicate ASC specks. Scale bars, 20 μm. The ratio of cells with ASC specks was calculated by randomly counting at least 20 cells per field, and the results are presented as a bar graph ( F lower panel). To detect NLRP3 oligomerization or ASC oligomerization, cells were crosslinked using disuccinimidyl suberate. Cell lysates as input controls were immunoblotted with antibodies against NLRP3, ASC and β-actin ( G , H ). The immunoblotting and immunocytochemistry results are representative of at least three independent experiments with similar results. * P < 0.05, ** P < 0.01, and **** P < 0.0001 by paired two-tailed t test

Journal: Cellular and Molecular Immunology

Article Title: Nuclear receptor coactivator 6 is a critical regulator of NLRP3 inflammasome activation and gouty arthritis

doi: 10.1038/s41423-023-01121-x

Figure Lengend Snippet: NCOA6 promotes NLRP3 inflammasome activity. A – C Human monocytes from healthy donors transfected with NCOA6 siRNA or control siRNA were primed with LPS for 6 h, followed by stimulation with ATP for 30 min or MSU crystals for 2 h. Cell lysates and culture supernatants were immunoblotted with the indicated antibodies. Sups indicates supernatants ( A ). NCOA6 mRNA levels were analyzed by real-time qRT–PCR ( B left panel). The amounts of secreted IL-1β, TNFα, and active caspase-1 in the supernatant were measured using ELISA ( B middle and right panel). Each dot represents an individual donor. Representative immunofluorescence staining for NCOA6, ASC, and DAPI ( C ). The white arrows (Merge) indicate ASC specks, and the red arrows (DIC) indicate phagocytosed MSU crystals. DIC, differential interference contrast. Scale bars, 20 μm. D – H BMDMs derived from Ncoa6 fl/fl ( fl/fl ) mice and Ncoa6 fl/fl LysM CRE ( Δ / Δ ) mice ( n = 6 per group) were primed with LPS for 4 h, followed by stimulation with ATP for 30 min, MSU crystals for 2 h, or nigericin (Nig) for 2 h. Cell lysates and culture supernatants were immunoblotted with the indicated antibodies. Sups denotes supernatants. D NCOA6 mRNA levels were analyzed using real-time qPCR ( E , left panel). The levels of secreted IL-1β, TNFα, and active caspase-1 in the supernatant were determined using ELISA ( E middle and right panels). Each dot indicates an individual mouse. Representative immunofluorescence images for ASC and DAPI ( F upper panel). The white arrows indicate ASC specks. Scale bars, 20 μm. The ratio of cells with ASC specks was calculated by randomly counting at least 20 cells per field, and the results are presented as a bar graph ( F lower panel). To detect NLRP3 oligomerization or ASC oligomerization, cells were crosslinked using disuccinimidyl suberate. Cell lysates as input controls were immunoblotted with antibodies against NLRP3, ASC and β-actin ( G , H ). The immunoblotting and immunocytochemistry results are representative of at least three independent experiments with similar results. * P < 0.05, ** P < 0.01, and **** P < 0.0001 by paired two-tailed t test

Article Snippet: Human NCOA6 siRNA (sc-61401), control siRNA (sc-37007), and febuxostat (144060-53-7) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activity Assay, Transfection, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Derivative Assay, Western Blot, Immunocytochemistry, Two Tailed Test

Association of NCOA6 with the NACHT domain of NLRP3. A , B THP−1 cells were stimulated with PMA for the indicated duration, primed with LPS, and treated with ATP. Cell lysates were immunoblotted with the indicated antibodies ( A ). Lysates were analyzed by NLRP3 pulldown and immunoblotting with antibodies against NCOA6 and NLRP3 ( B ). C Proximity ligation assay (PLA) in PMA-stimulated THP-1 cells primed with LPS (100 ng ml −1 ) for 4 h, followed by ATP (5 mM) treatment for 30 min. Red fluorescent dots indicate sites of interactions between NCOA6 and NLRP3 proteins. The rectangular area in the middle panel is magnified to the right panel. Scale bars, 20 μm. D Schematic of full-length NLRP3 and the truncated domain constructs. The number refers to the human NLRP3 sequence. E , F Wild-type or mutant NLRP3 (PYD, NACHT or LRR domain) and/or Myc-tagged ASC were overexpressed in HEK293T cells. Cell lysates were immunoprecipitated with the NCOA6 antibody and then immunoblotted with the indicated antibodies: FL=full length. G Decrease in the NCOA6-NLRP3 interaction by Walker A and B motif point mutations in the NACHT domain. HEK293T cells were transfected with the DNA constructs for wild-type NLRP3 (WT) and mutant NLRP3 (Mutant) and then subjected to immunoprecipitation with anti-NCOA6 Ab, followed by immunoblotting with the indicated antibodies. A schematic of the NLRP3 Walker mutant is shown in the top panel. H Immortalized BMDMs (iBMDMs) expressing wild-type NLRP3 (NLRP3 WT) or NLRP3 R258W were primed with LPS followed by ATP treatment. Cell lysates and culture supernatants were immunoblotted with the indicated antibodies. I , J Upstream regulatory effect of NCOA6 on NLRP3 inflammasome activity. NLRP3 R258W iBMDMs were transfected with Ncoa6 siRNA or control siRNA for 24 h and then stimulated with LPS for 6 h. Ncoa6 mRNA expression was analyzed by real-time qPCR ( I left panel). Cell lysates and culture supernatants were immunoblotted with the indicated antibodies ( I right panel). Immunoprecipitation and immunoblotting results are representative of at least three independent experiments. ELISA for IL-1β and TNF-α in culture supernatants ( J ). K ASC oligomerization in LPS-stimulated NLRP3 R258W iBMDMs transfected with Ncoa6 siRNA or control siRNA. Lysates or cross-linked pellets were immunoblotted with the indicated antibodies. The results are representative of three independent experiments. *** P < 0.01 by unpaired two-tailed t test (H) and two-way ANOVA corrected for multiple comparisons

Journal: Cellular and Molecular Immunology

Article Title: Nuclear receptor coactivator 6 is a critical regulator of NLRP3 inflammasome activation and gouty arthritis

doi: 10.1038/s41423-023-01121-x

Figure Lengend Snippet: Association of NCOA6 with the NACHT domain of NLRP3. A , B THP−1 cells were stimulated with PMA for the indicated duration, primed with LPS, and treated with ATP. Cell lysates were immunoblotted with the indicated antibodies ( A ). Lysates were analyzed by NLRP3 pulldown and immunoblotting with antibodies against NCOA6 and NLRP3 ( B ). C Proximity ligation assay (PLA) in PMA-stimulated THP-1 cells primed with LPS (100 ng ml −1 ) for 4 h, followed by ATP (5 mM) treatment for 30 min. Red fluorescent dots indicate sites of interactions between NCOA6 and NLRP3 proteins. The rectangular area in the middle panel is magnified to the right panel. Scale bars, 20 μm. D Schematic of full-length NLRP3 and the truncated domain constructs. The number refers to the human NLRP3 sequence. E , F Wild-type or mutant NLRP3 (PYD, NACHT or LRR domain) and/or Myc-tagged ASC were overexpressed in HEK293T cells. Cell lysates were immunoprecipitated with the NCOA6 antibody and then immunoblotted with the indicated antibodies: FL=full length. G Decrease in the NCOA6-NLRP3 interaction by Walker A and B motif point mutations in the NACHT domain. HEK293T cells were transfected with the DNA constructs for wild-type NLRP3 (WT) and mutant NLRP3 (Mutant) and then subjected to immunoprecipitation with anti-NCOA6 Ab, followed by immunoblotting with the indicated antibodies. A schematic of the NLRP3 Walker mutant is shown in the top panel. H Immortalized BMDMs (iBMDMs) expressing wild-type NLRP3 (NLRP3 WT) or NLRP3 R258W were primed with LPS followed by ATP treatment. Cell lysates and culture supernatants were immunoblotted with the indicated antibodies. I , J Upstream regulatory effect of NCOA6 on NLRP3 inflammasome activity. NLRP3 R258W iBMDMs were transfected with Ncoa6 siRNA or control siRNA for 24 h and then stimulated with LPS for 6 h. Ncoa6 mRNA expression was analyzed by real-time qPCR ( I left panel). Cell lysates and culture supernatants were immunoblotted with the indicated antibodies ( I right panel). Immunoprecipitation and immunoblotting results are representative of at least three independent experiments. ELISA for IL-1β and TNF-α in culture supernatants ( J ). K ASC oligomerization in LPS-stimulated NLRP3 R258W iBMDMs transfected with Ncoa6 siRNA or control siRNA. Lysates or cross-linked pellets were immunoblotted with the indicated antibodies. The results are representative of three independent experiments. *** P < 0.01 by unpaired two-tailed t test (H) and two-way ANOVA corrected for multiple comparisons

Article Snippet: Human NCOA6 siRNA (sc-61401), control siRNA (sc-37007), and febuxostat (144060-53-7) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Western Blot, Proximity Ligation Assay, Construct, Sequencing, Mutagenesis, Immunoprecipitation, Transfection, Expressing, Activity Assay, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Amelioration of acute tubular necrosis and MSU crystal-induced arthritis in mice by Ncoa6 deficiency. A – D Folic acid (FA) or vehicle (Veh) was injected into the peritoneal cavity of WT and Ncoa6 + /– mice. Kidney samples were obtained 36 h after FA injection. The body weight ( A upper panel) and survival ( A lower panel) of WT mice (n = 6 for Veh, n = 16 for FA) and Ncoa6 +/− mice ( n = 5 for Veh, n = 14 for FA) were monitored for the indicated duration. Representative H&E ( B upper panel) and periodic acid-Schiff images ( B lower panel) of the kidney; the black arrows indicate cast formation, and the red arrows indicate tubular necrosis. Immunohistochemistry of kidney tissues ( C ) using anti-F4/80 Abs (for macrophages), anti-NIMP-14 Abs (for neutrophils), anti-mature IL-1β (p17) Abs, and anti-active caspase-1 (p20) Abs. The number of infiltrated cells in the corticomedullary junction was counted in randomly selected images (40x magnification) on a slide scanner ( C bar graphs in the upper panel). IL-1β and active caspase-1 levels were assessed based on the ratio of the stained area per field using ImageJ software ( C bar graphs in the lower panel). For ( C ), at least five fields were counted per slide. n.d.=not detected. Kidney homogenates were immunoblotted with Abs for mature IL-1β, active caspase-1, and GAPDH ( D ). MSU crystals ( E left panel) or CFA ( E right panel) were injected into the footpads of WT and Ncoa6 + /– mice. Paw and ankle edema was monitored for the indicated duration ( E ). Representative H&E and immunohistochemical staining of MSU-injected joint tissues using Abs for F4/80, NIMP-14, mature IL-1β, and active caspase-1 ( F ). G–J Ncoa6 fl/fl ( n = 8) and Ncoa6 fl/fl LysM CRE ( n = 8) mice were subcutaneously treated with MSU crystals. Paw and ankle edema was monitored for 2 days ( G ). Representative macroscopic images of hind limbs ( H upper panel) and H&E-stained images of footpads ( H lower panel). The bar graph in the lower panel of H shows the inflammation grade of the affected joints. Representative immunohistochemistry images ( I ). Representative immunoblotting images ( J upper panel); the bar graph shows the mean intensity of caspase-1 ( J lower panel), which was summed from two blot images. Each dot represents an individual mouse. K IL-1β-induced loss of the decrease in arthritis severity in Ncoa6 -KO mice. Recombinant IL-1β or PBS was subcutaneously injected around crystals 1 h and 6 h after administering MSU to Ncoa6 fl/fl ( n = 8) and Ncoa6 fl/fl LysM CRE mice ( n = 8). Paw and ankle edema was monitored for 24 h ( K top panel): representative images of H&E staining ( K bottom panel). Each “C” in panels ( F , H , I , K ) indicates an MSU crystal. Scale bars = 50 μm for ( F , I ); 25 μm for ( B , C , H ); and 20 μm for ( K ). * P < 0.05, ** P < 0.01, and *** P < 0.001 by log rank t test ( A ), unpaired two-tailed t test ( C ), and two-way ANOVA corrected for multiple comparisons ( E , G , J , K )

Journal: Cellular and Molecular Immunology

Article Title: Nuclear receptor coactivator 6 is a critical regulator of NLRP3 inflammasome activation and gouty arthritis

doi: 10.1038/s41423-023-01121-x

Figure Lengend Snippet: Amelioration of acute tubular necrosis and MSU crystal-induced arthritis in mice by Ncoa6 deficiency. A – D Folic acid (FA) or vehicle (Veh) was injected into the peritoneal cavity of WT and Ncoa6 + /– mice. Kidney samples were obtained 36 h after FA injection. The body weight ( A upper panel) and survival ( A lower panel) of WT mice (n = 6 for Veh, n = 16 for FA) and Ncoa6 +/− mice ( n = 5 for Veh, n = 14 for FA) were monitored for the indicated duration. Representative H&E ( B upper panel) and periodic acid-Schiff images ( B lower panel) of the kidney; the black arrows indicate cast formation, and the red arrows indicate tubular necrosis. Immunohistochemistry of kidney tissues ( C ) using anti-F4/80 Abs (for macrophages), anti-NIMP-14 Abs (for neutrophils), anti-mature IL-1β (p17) Abs, and anti-active caspase-1 (p20) Abs. The number of infiltrated cells in the corticomedullary junction was counted in randomly selected images (40x magnification) on a slide scanner ( C bar graphs in the upper panel). IL-1β and active caspase-1 levels were assessed based on the ratio of the stained area per field using ImageJ software ( C bar graphs in the lower panel). For ( C ), at least five fields were counted per slide. n.d.=not detected. Kidney homogenates were immunoblotted with Abs for mature IL-1β, active caspase-1, and GAPDH ( D ). MSU crystals ( E left panel) or CFA ( E right panel) were injected into the footpads of WT and Ncoa6 + /– mice. Paw and ankle edema was monitored for the indicated duration ( E ). Representative H&E and immunohistochemical staining of MSU-injected joint tissues using Abs for F4/80, NIMP-14, mature IL-1β, and active caspase-1 ( F ). G–J Ncoa6 fl/fl ( n = 8) and Ncoa6 fl/fl LysM CRE ( n = 8) mice were subcutaneously treated with MSU crystals. Paw and ankle edema was monitored for 2 days ( G ). Representative macroscopic images of hind limbs ( H upper panel) and H&E-stained images of footpads ( H lower panel). The bar graph in the lower panel of H shows the inflammation grade of the affected joints. Representative immunohistochemistry images ( I ). Representative immunoblotting images ( J upper panel); the bar graph shows the mean intensity of caspase-1 ( J lower panel), which was summed from two blot images. Each dot represents an individual mouse. K IL-1β-induced loss of the decrease in arthritis severity in Ncoa6 -KO mice. Recombinant IL-1β or PBS was subcutaneously injected around crystals 1 h and 6 h after administering MSU to Ncoa6 fl/fl ( n = 8) and Ncoa6 fl/fl LysM CRE mice ( n = 8). Paw and ankle edema was monitored for 24 h ( K top panel): representative images of H&E staining ( K bottom panel). Each “C” in panels ( F , H , I , K ) indicates an MSU crystal. Scale bars = 50 μm for ( F , I ); 25 μm for ( B , C , H ); and 20 μm for ( K ). * P < 0.05, ** P < 0.01, and *** P < 0.001 by log rank t test ( A ), unpaired two-tailed t test ( C ), and two-way ANOVA corrected for multiple comparisons ( E , G , J , K )

Article Snippet: Human NCOA6 siRNA (sc-61401), control siRNA (sc-37007), and febuxostat (144060-53-7) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Injection, Immunohistochemistry, Staining, Software, Immunohistochemical staining, Western Blot, Recombinant, Two Tailed Test

Pathological and clinical importance of NCOA6 in patients with gout arthritis. A Heatmap of 3894 DEGs (|log 2 fold change| > 1) in synovial macrophages from patients with gouty arthritis (GA, n = 4) compared to CD14 + peripheral blood monocytes of healthy controls (HC, n = 4). B Venn diagram showing the overlap of the three sets of DEGs generated by the comparisons of gout macrophages versus healthy macrophages (blue), Ncoa6 -KO BMDMs versus unstimulated WT BMDMs (green), and Ncoa6 -KO BMDMs versus WT BMDMs stimulated with LPS plus MSU (yellow). P values were computed by chi-squared test. C GOBP terms related to inflammasome activation that were enriched in the overlapping genes (178 genes for medium alone and 277 genes for medium or LPS + MSU) between DEGs in Ncoa6 -KO BMDMs and DEGs in gout macrophages. D qRT–PCR analysis of NCOA6 mRNA levels in MSU- or LPS + MSU-stimulated human CD14 + monocytes . E , F Representative immunofluorescence images of NCOA6 and ASC in CD14 + monocytes , which were isolated from the peripheral blood of healthy controls and joint fluid of patients with gouty arthritis ( E ) or CPPD ( F ). Nuclei were stained with DAPI. G Immunohistochemistry analysis of NCOA6 in synovial tissues from osteoarthritis patients or gout arthritis patients; the red arrows indicate crystal deposits. H Images of immunohistochemical staining of NCOA6 in the corona and fibrovascular zones of the synovium of gout arthritis patients. C = MSU crystal. I Triple immunofluorescence staining for NCOA6, CD68 (macrophages), and IL-1β in the gouty arthritis synovium. Nuclei were stained with DAPI. The percentage of CD68 + NCOA6 + cells (middle panel) and the ratios of IL-1β + cells among CD68 + NCOA6 + cells or CD68 + NCOA6 – cells (right panel) were calculated from at least three fields per slide. Each dot represents one field. C = MSU crystal, V = blood vessel. J THP-1 cells were stimulated with LPS (10 ng ml −1 ) for 48 h in the presence of the indicated drugs. Cell lysates were immunoblotted with antibodies against NCOA6 and β-actin. K Differentiated THP-1 cells were primed with LPS (100 ng ml −1 ) for 4 h, followed by exposure to MSU crystals in the presence of allopurinol and colchicine. Representative immunofluorescence images are shown in the left panel. The ratio of cells with NCOA6 specks was calculated by randomly counting at least 50 cells per field (5 fields/group) (right panel). L Hypothetical model of the NCOA6-induced activation of the NLRP3 inflammasome in gout arthritis. Following the deposition of MSU crystals, monocytes and macrophages migrate to these deposits and attempt to phagocytize them. In macrophages stimulated by MSU and/or other proinflammatory stimuli, NCOA6 can translocate from the nucleus into the cytoplasm. Cytoplasmic NCOA6 incorporates into the NLRP3 inflammasome by binding the NACHT domain of NLRP3 and then activates the NLRP3 inflammasome and induces ASC oligomerization. As a result, activated macrophages secrete the mature form of IL-1β, which in turn may trigger the production of other inflammatory mediators, including TNFα, IL-6, CCL2, and prostaglandins, by stromal cells; these events lead to the recruitment of more monocytes and neutrophils to the inflamed sites embedded with MSU crystals, thus generating the vicious cycle of gouty inflammation. Treatment with colchicine might block this self-perpetuating cycle by repressing NCOA6 expression. Scale bars = 20 μm for ( E , F ); 50 μm for ( G , H , K ); and 100 μm for ( I ). * P < 0.05, ** P < 0.01, and *** P < 0.001 by paired two-tailed t test ( D ), unpaired two-tailed t test ( I ) and one-way ANOVA corrected for multiple comparisons ( K )

Journal: Cellular and Molecular Immunology

Article Title: Nuclear receptor coactivator 6 is a critical regulator of NLRP3 inflammasome activation and gouty arthritis

doi: 10.1038/s41423-023-01121-x

Figure Lengend Snippet: Pathological and clinical importance of NCOA6 in patients with gout arthritis. A Heatmap of 3894 DEGs (|log 2 fold change| > 1) in synovial macrophages from patients with gouty arthritis (GA, n = 4) compared to CD14 + peripheral blood monocytes of healthy controls (HC, n = 4). B Venn diagram showing the overlap of the three sets of DEGs generated by the comparisons of gout macrophages versus healthy macrophages (blue), Ncoa6 -KO BMDMs versus unstimulated WT BMDMs (green), and Ncoa6 -KO BMDMs versus WT BMDMs stimulated with LPS plus MSU (yellow). P values were computed by chi-squared test. C GOBP terms related to inflammasome activation that were enriched in the overlapping genes (178 genes for medium alone and 277 genes for medium or LPS + MSU) between DEGs in Ncoa6 -KO BMDMs and DEGs in gout macrophages. D qRT–PCR analysis of NCOA6 mRNA levels in MSU- or LPS + MSU-stimulated human CD14 + monocytes . E , F Representative immunofluorescence images of NCOA6 and ASC in CD14 + monocytes , which were isolated from the peripheral blood of healthy controls and joint fluid of patients with gouty arthritis ( E ) or CPPD ( F ). Nuclei were stained with DAPI. G Immunohistochemistry analysis of NCOA6 in synovial tissues from osteoarthritis patients or gout arthritis patients; the red arrows indicate crystal deposits. H Images of immunohistochemical staining of NCOA6 in the corona and fibrovascular zones of the synovium of gout arthritis patients. C = MSU crystal. I Triple immunofluorescence staining for NCOA6, CD68 (macrophages), and IL-1β in the gouty arthritis synovium. Nuclei were stained with DAPI. The percentage of CD68 + NCOA6 + cells (middle panel) and the ratios of IL-1β + cells among CD68 + NCOA6 + cells or CD68 + NCOA6 – cells (right panel) were calculated from at least three fields per slide. Each dot represents one field. C = MSU crystal, V = blood vessel. J THP-1 cells were stimulated with LPS (10 ng ml −1 ) for 48 h in the presence of the indicated drugs. Cell lysates were immunoblotted with antibodies against NCOA6 and β-actin. K Differentiated THP-1 cells were primed with LPS (100 ng ml −1 ) for 4 h, followed by exposure to MSU crystals in the presence of allopurinol and colchicine. Representative immunofluorescence images are shown in the left panel. The ratio of cells with NCOA6 specks was calculated by randomly counting at least 50 cells per field (5 fields/group) (right panel). L Hypothetical model of the NCOA6-induced activation of the NLRP3 inflammasome in gout arthritis. Following the deposition of MSU crystals, monocytes and macrophages migrate to these deposits and attempt to phagocytize them. In macrophages stimulated by MSU and/or other proinflammatory stimuli, NCOA6 can translocate from the nucleus into the cytoplasm. Cytoplasmic NCOA6 incorporates into the NLRP3 inflammasome by binding the NACHT domain of NLRP3 and then activates the NLRP3 inflammasome and induces ASC oligomerization. As a result, activated macrophages secrete the mature form of IL-1β, which in turn may trigger the production of other inflammatory mediators, including TNFα, IL-6, CCL2, and prostaglandins, by stromal cells; these events lead to the recruitment of more monocytes and neutrophils to the inflamed sites embedded with MSU crystals, thus generating the vicious cycle of gouty inflammation. Treatment with colchicine might block this self-perpetuating cycle by repressing NCOA6 expression. Scale bars = 20 μm for ( E , F ); 50 μm for ( G , H , K ); and 100 μm for ( I ). * P < 0.05, ** P < 0.01, and *** P < 0.001 by paired two-tailed t test ( D ), unpaired two-tailed t test ( I ) and one-way ANOVA corrected for multiple comparisons ( K )

Article Snippet: Human NCOA6 siRNA (sc-61401), control siRNA (sc-37007), and febuxostat (144060-53-7) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Generated, Activation Assay, Quantitative RT-PCR, Immunofluorescence, Isolation, Staining, Immunohistochemistry, Immunohistochemical staining, Binding Assay, Blocking Assay, Expressing, Two Tailed Test

Fig. 3 NCOA6 promotes NLRP3 inﬂammasome activity. A–C Human monocytes from healthy donors transfected with NCOA6 siRNA or control siRNA were primed with LPS for 6 h, followed by stimulation with ATP for 30 min or MSU crystals for 2 h. Cell lysates and culture supernatants were immunoblotted with the indicated antibodies. Sups indicates supernatants (A). NCOA6 mRNA levels were analyzed by real-time qRT–PCR (B left panel). The amounts of secreted IL-1β, TNFα, and active caspase-1 in the supernatant were measured using ELISA (B middle and right panel). Each dot represents an individual donor. Representative immunoﬂuorescence staining for NCOA6, ASC, and DAPI (C). The white arrows (Merge) indicate ASC specks, and the red arrows (DIC) indicate phagocytosed MSU crystals. DIC, differential interference contrast. Scale bars, 20 μm. D–H BMDMs derived from Ncoa6ﬂ/ﬂ(ﬂ/ﬂ) mice and Ncoa6ﬂ/ﬂLysMCRE (Δ/Δ) mice (n = 6 per group) were primed with LPS for 4 h, followed by stimulation with ATP for 30 min, MSU crystals for 2 h, or nigericin (Nig) for 2 h. Cell lysates and culture supernatants were immunoblotted with the indicated antibodies. Sups denotes supernatants. D NCOA6 mRNA levels were analyzed using real-time qPCR (E, left panel). The levels of secreted IL-1β, TNFα, and active caspase-1 in the supernatant were determined using ELISA (E middle and right panels). Each dot indicates an individual mouse. Representative immunoﬂuorescence images for ASC and DAPI (F upper panel). The white arrows indicate ASC specks. Scale bars, 20 μm. The ratio of cells with ASC specks was calculated by randomly counting at least 20 cells per ﬁeld, and the results are presented as a bar graph (F lower panel). To detect NLRP3 oligomerization or ASC oligomerization, cells were crosslinked using disuccinimidyl suberate. Cell lysates as input controls were immunoblotted with antibodies against NLRP3, ASC and β-actin (G, H). The immunoblotting and immunocytochemistry results are representative of at least three independent experiments with similar results. *P < 0.05, **P < 0.01, and ****P < 0.0001 by paired two-tailed t test

Journal: Cellular & molecular immunology

Article Title: Nuclear receptor coactivator 6 is a critical regulator of NLRP3 inflammasome activation and gouty arthritis.

doi: 10.1038/s41423-023-01121-x

Figure Lengend Snippet: Fig. 3 NCOA6 promotes NLRP3 inﬂammasome activity. A–C Human monocytes from healthy donors transfected with NCOA6 siRNA or control siRNA were primed with LPS for 6 h, followed by stimulation with ATP for 30 min or MSU crystals for 2 h. Cell lysates and culture supernatants were immunoblotted with the indicated antibodies. Sups indicates supernatants (A). NCOA6 mRNA levels were analyzed by real-time qRT–PCR (B left panel). The amounts of secreted IL-1β, TNFα, and active caspase-1 in the supernatant were measured using ELISA (B middle and right panel). Each dot represents an individual donor. Representative immunoﬂuorescence staining for NCOA6, ASC, and DAPI (C). The white arrows (Merge) indicate ASC specks, and the red arrows (DIC) indicate phagocytosed MSU crystals. DIC, differential interference contrast. Scale bars, 20 μm. D–H BMDMs derived from Ncoa6ﬂ/ﬂ(ﬂ/ﬂ) mice and Ncoa6ﬂ/ﬂLysMCRE (Δ/Δ) mice (n = 6 per group) were primed with LPS for 4 h, followed by stimulation with ATP for 30 min, MSU crystals for 2 h, or nigericin (Nig) for 2 h. Cell lysates and culture supernatants were immunoblotted with the indicated antibodies. Sups denotes supernatants. D NCOA6 mRNA levels were analyzed using real-time qPCR (E, left panel). The levels of secreted IL-1β, TNFα, and active caspase-1 in the supernatant were determined using ELISA (E middle and right panels). Each dot indicates an individual mouse. Representative immunoﬂuorescence images for ASC and DAPI (F upper panel). The white arrows indicate ASC specks. Scale bars, 20 μm. The ratio of cells with ASC specks was calculated by randomly counting at least 20 cells per ﬁeld, and the results are presented as a bar graph (F lower panel). To detect NLRP3 oligomerization or ASC oligomerization, cells were crosslinked using disuccinimidyl suberate. Cell lysates as input controls were immunoblotted with antibodies against NLRP3, ASC and β-actin (G, H). The immunoblotting and immunocytochemistry results are representative of at least three independent experiments with similar results. *P < 0.05, **P < 0.01, and ****P < 0.0001 by paired two-tailed t test

Article Snippet: Cells were fixed in 4% paraformaldehyde and processed for indirect immunofluorescence staining using the following Abs: mouse anti-human NLRP3 (1:100, ab16097, Abcam, Cambridge, MA, UK), mouse anti-human ASC (1:50, sc-514414, Santa Cruz Biotechnology), rabbit antihuman NCOA6 (1:500, A300-411A, Bethyl Laboratories, Montgomery, TX, Cellular & Molecular Immunology USA), mouse anti-vimentin (1:100, ab8987, Abcam), and appropriate secondary Abs, such as donkey anti-mouse IgG Cy3 (1:200, 715-166-151, Jackson laboratory, Bar Harbor, MA, USA) and donkey anti-rabbit IgG Alexa 488 (1:1000, A21206, Invitrogen).

Techniques: Activity Assay, Transfection, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Derivative Assay, Western Blot, Immunocytochemistry, Two Tailed Test

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