elisa kits  (Thermo Fisher)


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    Structured Review

    Thermo Fisher elisa kits
    Analyzing function and cellular localization of ADAM17 mutants in primary immune cells. <t>TNF-α–</t> ( A ) and TNFR II – ( B ) shedding <t>ELISA</t> of cell supernatant of stably ADAM17-reconstituted ADAM17 ex/ex MØP, M-MØ, and GM-MØ after stimulation with LPS (light gray) and zymosan (dark gray). Results are shown normalized to the mock-transfected control, and dotted lines indicate baseline shedding activity ( n = 9–12, derived from three to four independent rounds of differentiation). Representative immunoblot of the ADAM17 substrates TNFR II ( C ) and pro–TNF-α ( D ) in MØP lysates without and after LPS stimulation. For detection of substrates, Abs targeted against cytoplasmic epitopes were used. ( E ) Densitometric analysis of the ∼70-kDa-sized band of TNFR II as well as the main form of pro–TNF-α (∼35 kDa) normalized to loading control β-actin indicate no differences of substrate level after LPS stimulation compared with unstimulated conditions ( n = 3). ( F and G ) Cell surface staining of ADAM17 in stably ADAM17-transfected ADAM17 ex/ex MØP by FACS analysis using an N-terminal ADAM17 Ab (10.1). (F) Gating strategy for analysis: single cells were gated (FSC-A × FSC-H), then live cells (7-AAD negative) were separated and plotted for secondary Ab signal (Alexa Fluor 488). (G) Median fluorescence intensity normalized to mock-transfected cells. Dotted line indicates baseline fluorescence ( n = 3). ( H ) Immunoblot of iRhom2 in M-MØ and GM-MØ in ADAM17-deficient background and after reconstitution with ADAM17 wt. β-Actin was used as loading control. ( I ) Representative immunofluorescence staining of iRhom2 and KDEL as ER marker in M-MØ and GM-MØ. The white box shows magnification of cell surface structures. Scale bar, 10 μm. If not indicated differently, statistical significance is shown in comparison with the respective ADAM17 wt (A and B) or mock control (G), using a one-way ANOVA, followed by a Tukey multiple comparison test in (A), (B), and (G). A two-sided Student t test was applied in (E), exhibiting no significant differences. * p
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    Images

    1) Product Images from "Structural and Functional Analyses of the Shedding Protease ADAM17 in HoxB8-Immortalized Macrophages and Dendritic-like Cells"

    Article Title: Structural and Functional Analyses of the Shedding Protease ADAM17 in HoxB8-Immortalized Macrophages and Dendritic-like Cells

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1701556

    Analyzing function and cellular localization of ADAM17 mutants in primary immune cells. TNF-α– ( A ) and TNFR II – ( B ) shedding ELISA of cell supernatant of stably ADAM17-reconstituted ADAM17 ex/ex MØP, M-MØ, and GM-MØ after stimulation with LPS (light gray) and zymosan (dark gray). Results are shown normalized to the mock-transfected control, and dotted lines indicate baseline shedding activity ( n = 9–12, derived from three to four independent rounds of differentiation). Representative immunoblot of the ADAM17 substrates TNFR II ( C ) and pro–TNF-α ( D ) in MØP lysates without and after LPS stimulation. For detection of substrates, Abs targeted against cytoplasmic epitopes were used. ( E ) Densitometric analysis of the ∼70-kDa-sized band of TNFR II as well as the main form of pro–TNF-α (∼35 kDa) normalized to loading control β-actin indicate no differences of substrate level after LPS stimulation compared with unstimulated conditions ( n = 3). ( F and G ) Cell surface staining of ADAM17 in stably ADAM17-transfected ADAM17 ex/ex MØP by FACS analysis using an N-terminal ADAM17 Ab (10.1). (F) Gating strategy for analysis: single cells were gated (FSC-A × FSC-H), then live cells (7-AAD negative) were separated and plotted for secondary Ab signal (Alexa Fluor 488). (G) Median fluorescence intensity normalized to mock-transfected cells. Dotted line indicates baseline fluorescence ( n = 3). ( H ) Immunoblot of iRhom2 in M-MØ and GM-MØ in ADAM17-deficient background and after reconstitution with ADAM17 wt. β-Actin was used as loading control. ( I ) Representative immunofluorescence staining of iRhom2 and KDEL as ER marker in M-MØ and GM-MØ. The white box shows magnification of cell surface structures. Scale bar, 10 μm. If not indicated differently, statistical significance is shown in comparison with the respective ADAM17 wt (A and B) or mock control (G), using a one-way ANOVA, followed by a Tukey multiple comparison test in (A), (B), and (G). A two-sided Student t test was applied in (E), exhibiting no significant differences. * p
    Figure Legend Snippet: Analyzing function and cellular localization of ADAM17 mutants in primary immune cells. TNF-α– ( A ) and TNFR II – ( B ) shedding ELISA of cell supernatant of stably ADAM17-reconstituted ADAM17 ex/ex MØP, M-MØ, and GM-MØ after stimulation with LPS (light gray) and zymosan (dark gray). Results are shown normalized to the mock-transfected control, and dotted lines indicate baseline shedding activity ( n = 9–12, derived from three to four independent rounds of differentiation). Representative immunoblot of the ADAM17 substrates TNFR II ( C ) and pro–TNF-α ( D ) in MØP lysates without and after LPS stimulation. For detection of substrates, Abs targeted against cytoplasmic epitopes were used. ( E ) Densitometric analysis of the ∼70-kDa-sized band of TNFR II as well as the main form of pro–TNF-α (∼35 kDa) normalized to loading control β-actin indicate no differences of substrate level after LPS stimulation compared with unstimulated conditions ( n = 3). ( F and G ) Cell surface staining of ADAM17 in stably ADAM17-transfected ADAM17 ex/ex MØP by FACS analysis using an N-terminal ADAM17 Ab (10.1). (F) Gating strategy for analysis: single cells were gated (FSC-A × FSC-H), then live cells (7-AAD negative) were separated and plotted for secondary Ab signal (Alexa Fluor 488). (G) Median fluorescence intensity normalized to mock-transfected cells. Dotted line indicates baseline fluorescence ( n = 3). ( H ) Immunoblot of iRhom2 in M-MØ and GM-MØ in ADAM17-deficient background and after reconstitution with ADAM17 wt. β-Actin was used as loading control. ( I ) Representative immunofluorescence staining of iRhom2 and KDEL as ER marker in M-MØ and GM-MØ. The white box shows magnification of cell surface structures. Scale bar, 10 μm. If not indicated differently, statistical significance is shown in comparison with the respective ADAM17 wt (A and B) or mock control (G), using a one-way ANOVA, followed by a Tukey multiple comparison test in (A), (B), and (G). A two-sided Student t test was applied in (E), exhibiting no significant differences. * p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Stable Transfection, Transfection, Activity Assay, Derivative Assay, Staining, FACS, Fluorescence, Immunofluorescence, Marker

    2) Product Images from "The Novel PKC Activator 10-Methyl-Aplog-1 Combined with JQ1 Induced Strong and Synergistic HIV Reactivation with Tolerable Global T Cell Activation"

    Article Title: The Novel PKC Activator 10-Methyl-Aplog-1 Combined with JQ1 Induced Strong and Synergistic HIV Reactivation with Tolerable Global T Cell Activation

    Journal: Viruses

    doi: 10.3390/v13102037

    Pro-inflammatory cytokine production induced by the combined treatment of LRAs. PBMCs were treated with increasing concentrations of PMA (0.1 nM, 1 nM, or 10 nM), JQ1 (10 nM, 100 nM, or 1 µM), 10MA-1 (1 nM, 10 nM, 100 nM, or 1 µM), or JQ1 (10 nM, 100 nM, or 1 µM) and 10MA-1 (1 nM, 10 nM, 100 nM, or 1 µM) for 24 h. IL-8 ( A ) and TNF-α ( B ) in the culture supernatants were measured by ELISA. Experiments were performed in triplicate; the averages and standard deviations of the results obtained from the treated PBMCs are shown.
    Figure Legend Snippet: Pro-inflammatory cytokine production induced by the combined treatment of LRAs. PBMCs were treated with increasing concentrations of PMA (0.1 nM, 1 nM, or 10 nM), JQ1 (10 nM, 100 nM, or 1 µM), 10MA-1 (1 nM, 10 nM, 100 nM, or 1 µM), or JQ1 (10 nM, 100 nM, or 1 µM) and 10MA-1 (1 nM, 10 nM, 100 nM, or 1 µM) for 24 h. IL-8 ( A ) and TNF-α ( B ) in the culture supernatants were measured by ELISA. Experiments were performed in triplicate; the averages and standard deviations of the results obtained from the treated PBMCs are shown.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    3) Product Images from "Yin Yang 1 (YY1)-induced long intergenic non-protein coding RNA 472 (LINC00472) aggravates sepsis-associated cardiac dysfunction via the micro-RNA-335-3p (miR-335-3p)/Monoamine oxidase A (MAOA) cascade"

    Article Title: Yin Yang 1 (YY1)-induced long intergenic non-protein coding RNA 472 (LINC00472) aggravates sepsis-associated cardiac dysfunction via the micro-RNA-335-3p (miR-335-3p)/Monoamine oxidase A (MAOA) cascade

    Journal: Bioengineered

    doi: 10.1080/21655979.2021.2017589

    LINC00472 knockdown alleviates LPS-induced cardiac dysfunction in vitro. (a) The viability of AC-16 cardiomyocytes was measured by CCK-8 assay. (b and c) Levels of TNF-α (b) and IL-1β (c) in the supernatants were measured by ELISA. (d) LINC00472 expression in AC-16 cells from Control group and LPS group was detected by RT-qPCR. (e) RT-qPCR analysis was used to assess the transfection efficiency of LINC00472 knockdown. (f) CCK-8 assay was adopted to detect the viability of AC-16 cardiomyocytes from Control group, LPS group, LPS+si-NC group, or LPS+si-LINC00472 group, respectively. (g) Flow cytometry was performed to detect AC-16 cardiomyocyte in each group. (h and i) TNF-α (h) and IL-1β (i) expression levels in the supernatants from each group were detected by ELISA. All data were performed at least three independent experiments. Data were expressed as mean ± SD. * P
    Figure Legend Snippet: LINC00472 knockdown alleviates LPS-induced cardiac dysfunction in vitro. (a) The viability of AC-16 cardiomyocytes was measured by CCK-8 assay. (b and c) Levels of TNF-α (b) and IL-1β (c) in the supernatants were measured by ELISA. (d) LINC00472 expression in AC-16 cells from Control group and LPS group was detected by RT-qPCR. (e) RT-qPCR analysis was used to assess the transfection efficiency of LINC00472 knockdown. (f) CCK-8 assay was adopted to detect the viability of AC-16 cardiomyocytes from Control group, LPS group, LPS+si-NC group, or LPS+si-LINC00472 group, respectively. (g) Flow cytometry was performed to detect AC-16 cardiomyocyte in each group. (h and i) TNF-α (h) and IL-1β (i) expression levels in the supernatants from each group were detected by ELISA. All data were performed at least three independent experiments. Data were expressed as mean ± SD. * P

    Techniques Used: In Vitro, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Transfection, Flow Cytometry

    YY1-induced LINC00472 upregulation promotes LPS-induced cardiomyocyte dysfunction. (a) RT-qPCR analysis was used to assess the transfection efficiency of LINC00472 overexpression. (b) LPS-challenged AC-16 cardiomyocytes were transfected with si-NC, si-YY1, or si-YY1+ oe-LINC00472. RT-qPCR was utilized to detect LINC00472 expression in AC-16 cardiomyocytes from each group. (c) CCK-8 assay was adopted to detect the viability of AC-16 cardiomyocytes in each group. (d) Flow cytometry was performed to detect AC-16 cardiomyocyte in each group. (e and f) TNF-α (e) and IL-1β (f) expression levels in the supernatants from each group were detected by ELISA. All data were performed at least three independent experiments. Data were expressed as mean ± SD. * P
    Figure Legend Snippet: YY1-induced LINC00472 upregulation promotes LPS-induced cardiomyocyte dysfunction. (a) RT-qPCR analysis was used to assess the transfection efficiency of LINC00472 overexpression. (b) LPS-challenged AC-16 cardiomyocytes were transfected with si-NC, si-YY1, or si-YY1+ oe-LINC00472. RT-qPCR was utilized to detect LINC00472 expression in AC-16 cardiomyocytes from each group. (c) CCK-8 assay was adopted to detect the viability of AC-16 cardiomyocytes in each group. (d) Flow cytometry was performed to detect AC-16 cardiomyocyte in each group. (e and f) TNF-α (e) and IL-1β (f) expression levels in the supernatants from each group were detected by ELISA. All data were performed at least three independent experiments. Data were expressed as mean ± SD. * P

    Techniques Used: Quantitative RT-PCR, Transfection, Over Expression, Expressing, CCK-8 Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

    MiR-335-3p inhibition or MAOA overexpression reverses the improvement of AC-16 cardiomyocyte functions induced by LINC00472 knockdown. (a) The efficiency of MAOA overexpression was tested by RT-qPCR analysis. (b) LPS-challenged AC-16 cardiomyocytes were transfected with si-NC, si-LINC00472, si-LINC00472+ miR-335-3p inhibitor, or si-LINC00472+ oe-MAOA. CCK-8 assay was adopted to detect the viability of AC-16 cardiomyocytes in each group. (c) Flow cytometry was performed to detect AC-16 cardiomyocyte in each group. (d and e) TNF-α (d) and IL-1β (e) expression levels in the supernatants from each group were detected by ELISA. All data were performed at least three independent experiments. Data were expressed as mean ± SD. * P
    Figure Legend Snippet: MiR-335-3p inhibition or MAOA overexpression reverses the improvement of AC-16 cardiomyocyte functions induced by LINC00472 knockdown. (a) The efficiency of MAOA overexpression was tested by RT-qPCR analysis. (b) LPS-challenged AC-16 cardiomyocytes were transfected with si-NC, si-LINC00472, si-LINC00472+ miR-335-3p inhibitor, or si-LINC00472+ oe-MAOA. CCK-8 assay was adopted to detect the viability of AC-16 cardiomyocytes in each group. (c) Flow cytometry was performed to detect AC-16 cardiomyocyte in each group. (d and e) TNF-α (d) and IL-1β (e) expression levels in the supernatants from each group were detected by ELISA. All data were performed at least three independent experiments. Data were expressed as mean ± SD. * P

    Techniques Used: Inhibition, Over Expression, Quantitative RT-PCR, Transfection, CCK-8 Assay, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

    Upregulated LINC00472 expression was observed in the in vivo SICD model. (a) The myocardial structure of mice was observed via H E staining. (b and c) The cTnI (b) and CK-MB (c) levels in mouse serum were measured with ELISA. (d and e) TNF-α (d) and IL-1β (e) expression levels in serum samples from mice were detected by ELISA. (f) The expression levels of apoptosis-associated proteins (Bcl-2 and cleaved Caspase-3) were detected by Western blotting. (g) LINC00472 expression in myocardial tissue of mice from Sham group and LPS group was detected by RT-qPCR. All data were performed at least three independent experiments. Data were expressed as mean ± SD. * P
    Figure Legend Snippet: Upregulated LINC00472 expression was observed in the in vivo SICD model. (a) The myocardial structure of mice was observed via H E staining. (b and c) The cTnI (b) and CK-MB (c) levels in mouse serum were measured with ELISA. (d and e) TNF-α (d) and IL-1β (e) expression levels in serum samples from mice were detected by ELISA. (f) The expression levels of apoptosis-associated proteins (Bcl-2 and cleaved Caspase-3) were detected by Western blotting. (g) LINC00472 expression in myocardial tissue of mice from Sham group and LPS group was detected by RT-qPCR. All data were performed at least three independent experiments. Data were expressed as mean ± SD. * P

    Techniques Used: Expressing, In Vivo, Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR

    4) Product Images from "Extracellular Vesicles Derived from Acidified Metastatic Melanoma Cells Stimulate Growth, Migration, and Stemness of Normal Keratinocytes"

    Article Title: Extracellular Vesicles Derived from Acidified Metastatic Melanoma Cells Stimulate Growth, Migration, and Stemness of Normal Keratinocytes

    Journal: Biomedicines

    doi: 10.3390/biomedicines10030660

    Effect of “normal” and “acidified” EVs on secretion of the different cytokines and adhesion factors by the keratinocytes. Het1-A cells were incubated with “normal” and “acidified” EVs for 48 h, and the concentration of IL5 ( a ), IL10 ( d ), IL12 ( e ), GM-CSF ( p ), and TRAIL ( r ) was assayed by ELISA. Concentration of IL6 ( b ), IL8 ( c ), sVCAM-1 ( f ), sICAM-1 ( g ), sICAM-3 ( h ), sPECAM-1 ( i ), sE-selectin ( k ), sP-selectin ( l ), MCP-1 ( m ), t-PA ( n ), and sCD40L ( o ) was analyzed by the Flow Cytomix kits. Data presented as the normalized protein concentration ± SEM ( n = 6). Control corresponds to the untreated cells. * ( p
    Figure Legend Snippet: Effect of “normal” and “acidified” EVs on secretion of the different cytokines and adhesion factors by the keratinocytes. Het1-A cells were incubated with “normal” and “acidified” EVs for 48 h, and the concentration of IL5 ( a ), IL10 ( d ), IL12 ( e ), GM-CSF ( p ), and TRAIL ( r ) was assayed by ELISA. Concentration of IL6 ( b ), IL8 ( c ), sVCAM-1 ( f ), sICAM-1 ( g ), sICAM-3 ( h ), sPECAM-1 ( i ), sE-selectin ( k ), sP-selectin ( l ), MCP-1 ( m ), t-PA ( n ), and sCD40L ( o ) was analyzed by the Flow Cytomix kits. Data presented as the normalized protein concentration ± SEM ( n = 6). Control corresponds to the untreated cells. * ( p

    Techniques Used: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Protein Concentration

    5) Product Images from "Doxorubicin and CpG loaded liposomal spherical nucleic acid for enhanced Cancer treatment"

    Article Title: Doxorubicin and CpG loaded liposomal spherical nucleic acid for enhanced Cancer treatment

    Journal: Journal of Nanobiotechnology

    doi: 10.1186/s12951-022-01353-5

    A Cell viability of HUVEC after incubation with various concentrations of hNPs or free DOX and CpG. B Quantification of the cellular uptake of DOX by E.G7-OVA cells. C Representative CLSM images of E.G7-OVA cells after incubation with DOPE-DOX NPs or free DOX for 2 h, 4 h and 6 h, respectively. The molecule expression of D CD40 and E MHC-II was analyzed by flow cytometry. The cytokine expression of F TNF-α, G IFN-γ and H IL-1β were analyzed by ELISA assays. Results represent mean ± SD (n = 6; *P
    Figure Legend Snippet: A Cell viability of HUVEC after incubation with various concentrations of hNPs or free DOX and CpG. B Quantification of the cellular uptake of DOX by E.G7-OVA cells. C Representative CLSM images of E.G7-OVA cells after incubation with DOPE-DOX NPs or free DOX for 2 h, 4 h and 6 h, respectively. The molecule expression of D CD40 and E MHC-II was analyzed by flow cytometry. The cytokine expression of F TNF-α, G IFN-γ and H IL-1β were analyzed by ELISA assays. Results represent mean ± SD (n = 6; *P

    Techniques Used: Incubation, Confocal Laser Scanning Microscopy, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

    6) Product Images from "Carbonic anhydrase XII mediates the survival and prometastatic functions of macrophages in human hepatocellular carcinoma"

    Article Title: Carbonic anhydrase XII mediates the survival and prometastatic functions of macrophages in human hepatocellular carcinoma

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI153110

    Glycolysis induces CA12 upregulation in monocytes and macrophages via HIF1α and autocrine cytokine-dependent pathways. CD14 + cells were purified from the peripheral blood of healthy donors. ( A and B ) Cells were left untreated or treated with TSN in the presence or absence of 2DG and 3PO. CA12 expression were determined by qPCR ( A ) and immunoblotting ( B ). n = 4. ( C ) Cells were left untreated or treated with TSN for the indicated times. Levels of CA12 and HIF1α were determined by immunoblotting ( n = 5). ( D ) Cells were left untreated or treated with TSN in the presence or absence of DMSO, TEPP-46 (TEPP) or 2DG. CA12 and HIF1α expressions were determined by immunoblotting ( n = 5). ( E ) Cells were transfected with siNC or si HIF1A before being treated with or without TSN. CA12 and HIF1α expressions were determined by immunoblotting ( n = 4). ( F–H ) Cells were left untreated or treated with TSN in the presence or absence of echinomycin, DMOG or 2DG. CA12 and HIF1α expressions were determined by immunoblotting ( F and G ) ( n = 4), and levels of cytokines production were measured by ELISA ( H ) ( n = 8). ( I and J ) Cells were left untreated or treated with recombinant human cytokines or indicated neutralizing antibodies of cytokines, CA12 expression were determined by immunoblotting ( n = 3). CA12 or β-actin lanes in I were run on the same gel, but were noncontiguous. ( K ) Cells were left untreated or treated with TSN in the presence or absence of anti-CKs (anti–TNF-α, anti–IL-10, and anti–IL-1β neutralizing antibodies) or/and echinomycin. CA12 expression were determined by immunoblotting ( n = 3). Results shown in A and H are represented as mean ± SEM. P values were obtained by 1-way ANOVA ( A ) or 2-way ANOVA ( H ). * P
    Figure Legend Snippet: Glycolysis induces CA12 upregulation in monocytes and macrophages via HIF1α and autocrine cytokine-dependent pathways. CD14 + cells were purified from the peripheral blood of healthy donors. ( A and B ) Cells were left untreated or treated with TSN in the presence or absence of 2DG and 3PO. CA12 expression were determined by qPCR ( A ) and immunoblotting ( B ). n = 4. ( C ) Cells were left untreated or treated with TSN for the indicated times. Levels of CA12 and HIF1α were determined by immunoblotting ( n = 5). ( D ) Cells were left untreated or treated with TSN in the presence or absence of DMSO, TEPP-46 (TEPP) or 2DG. CA12 and HIF1α expressions were determined by immunoblotting ( n = 5). ( E ) Cells were transfected with siNC or si HIF1A before being treated with or without TSN. CA12 and HIF1α expressions were determined by immunoblotting ( n = 4). ( F–H ) Cells were left untreated or treated with TSN in the presence or absence of echinomycin, DMOG or 2DG. CA12 and HIF1α expressions were determined by immunoblotting ( F and G ) ( n = 4), and levels of cytokines production were measured by ELISA ( H ) ( n = 8). ( I and J ) Cells were left untreated or treated with recombinant human cytokines or indicated neutralizing antibodies of cytokines, CA12 expression were determined by immunoblotting ( n = 3). CA12 or β-actin lanes in I were run on the same gel, but were noncontiguous. ( K ) Cells were left untreated or treated with TSN in the presence or absence of anti-CKs (anti–TNF-α, anti–IL-10, and anti–IL-1β neutralizing antibodies) or/and echinomycin. CA12 expression were determined by immunoblotting ( n = 3). Results shown in A and H are represented as mean ± SEM. P values were obtained by 1-way ANOVA ( A ) or 2-way ANOVA ( H ). * P

    Techniques Used: Purification, Expressing, Real-time Polymerase Chain Reaction, Transfection, Enzyme-linked Immunosorbent Assay, Recombinant

    7) Product Images from "miR‐34b‐5p inhibition attenuates lung inflammation and apoptosis in an LPS‐induced acute lung injury mouse model by targeting progranulin, et al. miR‐34b‐5p inhibition attenuates lung inflammation and apoptosis in an LPS‐induced acute lung injury mouse model by targeting progranulin"

    Article Title: miR‐34b‐5p inhibition attenuates lung inflammation and apoptosis in an LPS‐induced acute lung injury mouse model by targeting progranulin, et al. miR‐34b‐5p inhibition attenuates lung inflammation and apoptosis in an LPS‐induced acute lung injury mouse model by targeting progranulin

    Journal: Journal of Cellular Physiology

    doi: 10.1002/jcp.26274

    The protective effects of miR‐34b‐5p are dependent on PGRN. Mice were subjected to the miR‐34b‐5p antagomir after administration of Ad‐PGRN‐shRNA or the NC (Ad‐GFP) for 7 days. After 48 hr, the mice were administered with LPS by intratracheal injection for 24 hr. (a) Representative images of the lung lesions in the lung sections by HE staining. Scale bar = 50 µm, n = 4. (b) Quantitation of the lung injury scores in the different treatment groups. (c) The caspase‐3 activity of the lungs in the different treatment groups, n = 4. The levels of (d)IL‐1β, (e)TNF‐α, and (f) IL‐6 in the BALF were assessed by ELISA. * p
    Figure Legend Snippet: The protective effects of miR‐34b‐5p are dependent on PGRN. Mice were subjected to the miR‐34b‐5p antagomir after administration of Ad‐PGRN‐shRNA or the NC (Ad‐GFP) for 7 days. After 48 hr, the mice were administered with LPS by intratracheal injection for 24 hr. (a) Representative images of the lung lesions in the lung sections by HE staining. Scale bar = 50 µm, n = 4. (b) Quantitation of the lung injury scores in the different treatment groups. (c) The caspase‐3 activity of the lungs in the different treatment groups, n = 4. The levels of (d)IL‐1β, (e)TNF‐α, and (f) IL‐6 in the BALF were assessed by ELISA. * p

    Techniques Used: Mouse Assay, shRNA, Injection, Staining, Quantitation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

    miR‐34b‐5p inhibition significantly decreases inflammatory mediator levels in the BALF and improves the survival ratio during ALI. Mice were subjected to LPS after administration of the miR‐34b‐5p antagomir or NC for 48 hr. (a) MPO in the lungs was assessed by ELISA. (b) The total number of BALF cells and neutrophils was determined with cytospin and a hemocytometer. (c) Lung W/D ratios. (d) The protein concentrations in the BALF were determined with BCA assays. The levels of (e) TNF‐α, (f) IL‐6, (g) IL‐1βin the BALF. (h) Mouse survival ( n = 10) was monitored at different times for 48 hr. * p
    Figure Legend Snippet: miR‐34b‐5p inhibition significantly decreases inflammatory mediator levels in the BALF and improves the survival ratio during ALI. Mice were subjected to LPS after administration of the miR‐34b‐5p antagomir or NC for 48 hr. (a) MPO in the lungs was assessed by ELISA. (b) The total number of BALF cells and neutrophils was determined with cytospin and a hemocytometer. (c) Lung W/D ratios. (d) The protein concentrations in the BALF were determined with BCA assays. The levels of (e) TNF‐α, (f) IL‐6, (g) IL‐1βin the BALF. (h) Mouse survival ( n = 10) was monitored at different times for 48 hr. * p

    Techniques Used: Inhibition, Mouse Assay, Enzyme-linked Immunosorbent Assay

    8) Product Images from "Resveratrol Alleviates Dextran Sulfate Sodium-Induced Acute Ulcerative Colitis in Mice by Mediating PI3K/Akt/VEGFA Pathway"

    Article Title: Resveratrol Alleviates Dextran Sulfate Sodium-Induced Acute Ulcerative Colitis in Mice by Mediating PI3K/Akt/VEGFA Pathway

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2021.693982

    RSV can bring positive results to UC. (A) Schematic diagram of animal experiment. (B) The colon length in MG, NG, RG, and PG mice was compared. (C) The curve of weight change of mice in different treatment groups was analyzed. (D) DAI disease scores of mice in different treatment groups were evaluated. (E) The pathological sections of the colon of mice in different treatment groups were observed using HE staining (100 ×, scale bar = 100 µm). (F) PCNA expression in the colon of mice in different treatment groups was measured using IHC (100 ×, scale bar = 100 µm). (G–K) The expression of IL-6, IL-8, IL-1β, TNF-α, and IL-10 in the colon tissues of mice in different treatment groups was monitored using ELISA. n = 20. Measurement data were expressed by mean ± SD. One-way ANOVA or repeated measures ANOVA was conducted for multiple group comparison, followed by Tukey’s post hoc test. * p
    Figure Legend Snippet: RSV can bring positive results to UC. (A) Schematic diagram of animal experiment. (B) The colon length in MG, NG, RG, and PG mice was compared. (C) The curve of weight change of mice in different treatment groups was analyzed. (D) DAI disease scores of mice in different treatment groups were evaluated. (E) The pathological sections of the colon of mice in different treatment groups were observed using HE staining (100 ×, scale bar = 100 µm). (F) PCNA expression in the colon of mice in different treatment groups was measured using IHC (100 ×, scale bar = 100 µm). (G–K) The expression of IL-6, IL-8, IL-1β, TNF-α, and IL-10 in the colon tissues of mice in different treatment groups was monitored using ELISA. n = 20. Measurement data were expressed by mean ± SD. One-way ANOVA or repeated measures ANOVA was conducted for multiple group comparison, followed by Tukey’s post hoc test. * p

    Techniques Used: Mouse Assay, Staining, Expressing, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

    Inhibition of PI3K/Akt pathway alleviates DSS-induced UC in mice. (A–E) The expression of IL-6, IL-8, IL-1β, TNF-α, and IL-10 in the colon tissues of mice in different treatment groups was determined using ELISA. (F) The expression of PI3K in colon tissues was examined using RT-qPCR. (G) The expression of PI3K, p-Akt, and Akt protein in colon tissues was examined using Western blot. (H) The expression of VEGFA and Akt protein in colon tissues was detected using IHC. * p
    Figure Legend Snippet: Inhibition of PI3K/Akt pathway alleviates DSS-induced UC in mice. (A–E) The expression of IL-6, IL-8, IL-1β, TNF-α, and IL-10 in the colon tissues of mice in different treatment groups was determined using ELISA. (F) The expression of PI3K in colon tissues was examined using RT-qPCR. (G) The expression of PI3K, p-Akt, and Akt protein in colon tissues was examined using Western blot. (H) The expression of VEGFA and Akt protein in colon tissues was detected using IHC. * p

    Techniques Used: Inhibition, Mouse Assay, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Immunohistochemistry

    9) Product Images from "LCZ696 Attenuated Doxorubicin-Induced Chronic Cardiomyopathy Through the TLR2-MyD88 Complex Formation"

    Article Title: LCZ696 Attenuated Doxorubicin-Induced Chronic Cardiomyopathy Through the TLR2-MyD88 Complex Formation

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2021.654051

    LCZ696 treatment and TLR2 knockdown attenuated doxorubicin-induced cardiac inflammation in mice. (A) Representative images of anti-TNF-α staining in the hearts of each group (200×). (B) TNF-α levels in mouse heart tissue homogenates determined by ELISA. (C) Western blot analysis of IκBα levels in heart tissue. GAPDH was used as a loading control. (D) Nuclei were isolated from mouse heart tissue, and NF-κB in the nucleus was detected by western blot. Lamin B and GAPDH were used as controls. (E–G) Real-time PCR was used to determine the mRNA levels of Tnfa (E) , Mcp1 (F) , and Il-6 (G) in mouse heart tissue (data were normalized to Actb ). ( n = 7; *** P
    Figure Legend Snippet: LCZ696 treatment and TLR2 knockdown attenuated doxorubicin-induced cardiac inflammation in mice. (A) Representative images of anti-TNF-α staining in the hearts of each group (200×). (B) TNF-α levels in mouse heart tissue homogenates determined by ELISA. (C) Western blot analysis of IκBα levels in heart tissue. GAPDH was used as a loading control. (D) Nuclei were isolated from mouse heart tissue, and NF-κB in the nucleus was detected by western blot. Lamin B and GAPDH were used as controls. (E–G) Real-time PCR was used to determine the mRNA levels of Tnfa (E) , Mcp1 (F) , and Il-6 (G) in mouse heart tissue (data were normalized to Actb ). ( n = 7; *** P

    Techniques Used: Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Isolation, Real-time Polymerase Chain Reaction

    10) Product Images from "Deguelin Attenuates Allergic Airway Inflammation via Inhibition of NF-κb Pathway in Mice"

    Article Title: Deguelin Attenuates Allergic Airway Inflammation via Inhibition of NF-κb Pathway in Mice

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.17238

    Effects of deguelin treatment on systemic immunoglobulin production in the serum of OVA-sensitized asthmatic mice. Serum was collected 24 h after the last OVA aerosol challenge. The levels of total IgE, IgG1 and IgG2a were analyzed using ELISA. Values are shown as mean ± SEM (n = 6 for each group). Deg 1 = Deguelin 1mg/kg; Deg 4 = Deguelin 4mg/kg; DXM = dexamethasone. # P
    Figure Legend Snippet: Effects of deguelin treatment on systemic immunoglobulin production in the serum of OVA-sensitized asthmatic mice. Serum was collected 24 h after the last OVA aerosol challenge. The levels of total IgE, IgG1 and IgG2a were analyzed using ELISA. Values are shown as mean ± SEM (n = 6 for each group). Deg 1 = Deguelin 1mg/kg; Deg 4 = Deguelin 4mg/kg; DXM = dexamethasone. # P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    11) Product Images from "Underlying Mechanism and Active Ingredients of Tianma Gouteng Acting on Cerebral Infarction as Determined via Network Pharmacology Analysis Combined With Experimental Validation"

    Article Title: Underlying Mechanism and Active Ingredients of Tianma Gouteng Acting on Cerebral Infarction as Determined via Network Pharmacology Analysis Combined With Experimental Validation

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2021.760503

    Intervention of quercetin on cerebral infarction. Methods used for quercetin (40 μM) administration and modeling were equivalent to corresponding TMGT methods. (A, B) Effect of quercetin on cell viability and LDH leakage from OGD-induced PC12 cells. (C–E) After quercetin pretreatment, the levels of IL-6, IL-1β, and TNF-α in supernatants of PC12 cells and BV2 cells were detected using ELISA kits. (F, G) Western blot analysis of protein expression of HIF-1α, PPARγ, nuclear and cytosolic p65, IκBα, and p -IκBα with β-actin as the respective internal standards. ∗∗ p
    Figure Legend Snippet: Intervention of quercetin on cerebral infarction. Methods used for quercetin (40 μM) administration and modeling were equivalent to corresponding TMGT methods. (A, B) Effect of quercetin on cell viability and LDH leakage from OGD-induced PC12 cells. (C–E) After quercetin pretreatment, the levels of IL-6, IL-1β, and TNF-α in supernatants of PC12 cells and BV2 cells were detected using ELISA kits. (F, G) Western blot analysis of protein expression of HIF-1α, PPARγ, nuclear and cytosolic p65, IκBα, and p -IκBα with β-actin as the respective internal standards. ∗∗ p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

    Detection of inflammatory factors. (A–C) After PC12 cells were pretreated with TMGT for 24 h, they were cultured under OGD conditions for 2 h, then levels of IL-6, IL-1β, and TNF-α in the supernatants of PC12 cells were detected by ELISA kits. (D–F) After the pretreatment with different concentrations of TMGT for 24 h followed stimulation with 100 ng/ml LPS for 6 h, the levels of IL-6, IL-1β, and TNF-α in the supernatant liquid of BV2 cells were detected by ELISA kits. ∗ p
    Figure Legend Snippet: Detection of inflammatory factors. (A–C) After PC12 cells were pretreated with TMGT for 24 h, they were cultured under OGD conditions for 2 h, then levels of IL-6, IL-1β, and TNF-α in the supernatants of PC12 cells were detected by ELISA kits. (D–F) After the pretreatment with different concentrations of TMGT for 24 h followed stimulation with 100 ng/ml LPS for 6 h, the levels of IL-6, IL-1β, and TNF-α in the supernatant liquid of BV2 cells were detected by ELISA kits. ∗ p

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    12) Product Images from "Trazodone regulates neurotrophic/growth factors, mitogen-activated protein kinases and lactate release in human primary astrocytes"

    Article Title: Trazodone regulates neurotrophic/growth factors, mitogen-activated protein kinases and lactate release in human primary astrocytes

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-015-0446-x

    Effect of TDZ on cytokine release. Human astrocytes were treated with different concentrations of TDZ (1 nM-10 μM), or FLUOX (10 μM) or 5-HT (10 μM) for 24 h ( a , c , e ) or 72 h ( b , d , f ). At the end of treatment, culture supernatants were collected, and the amounts of IL-6 ( a , b ), IFN-γ ( c , d ) and IL-10 ( e, f ) released were measured using ELISA kits following the manufacturer’s instructions. The data are expressed as percentages relative to untreated cells (control), which were set at 100 %, and represent the mean ± SEM of two independent experiments, each performed in duplicate. Statistical significance was determined using a one-way ANOVA-Tukey HSD post hoc test: * P
    Figure Legend Snippet: Effect of TDZ on cytokine release. Human astrocytes were treated with different concentrations of TDZ (1 nM-10 μM), or FLUOX (10 μM) or 5-HT (10 μM) for 24 h ( a , c , e ) or 72 h ( b , d , f ). At the end of treatment, culture supernatants were collected, and the amounts of IL-6 ( a , b ), IFN-γ ( c , d ) and IL-10 ( e, f ) released were measured using ELISA kits following the manufacturer’s instructions. The data are expressed as percentages relative to untreated cells (control), which were set at 100 %, and represent the mean ± SEM of two independent experiments, each performed in duplicate. Statistical significance was determined using a one-way ANOVA-Tukey HSD post hoc test: * P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Cytokine release in an experimental model of inflammation. Human astrocytes were treated with medium alone (control), or different concentrations of TDZ (1 nM-10 μM), or FLUOX (10 μM) or 5-HT (10 μM) for 24 h ( a , c , e ) or 72 h ( b , d , f ); after drug removal, cells were incubated with 50 μg/ml LPS and 50 ng/ml TNF-α for an additional 24 h. At the end of treatments, supernatants were collected, and the amounts of IL-6 ( a, b ), IFN-γ ( c, d ) and IL-10 ( e, f ) were measured using ELISA kits following the manufacturer’s instructions. The data are expressed as percentages relative to untreated cells (control), which were set at 100 %, and represent the mean ± SEM of two independent experiments, each performed in duplicate. Statistical significance was determined using a one-way ANOVA-Tukey HSD post hoc test: * P
    Figure Legend Snippet: Cytokine release in an experimental model of inflammation. Human astrocytes were treated with medium alone (control), or different concentrations of TDZ (1 nM-10 μM), or FLUOX (10 μM) or 5-HT (10 μM) for 24 h ( a , c , e ) or 72 h ( b , d , f ); after drug removal, cells were incubated with 50 μg/ml LPS and 50 ng/ml TNF-α for an additional 24 h. At the end of treatments, supernatants were collected, and the amounts of IL-6 ( a, b ), IFN-γ ( c, d ) and IL-10 ( e, f ) were measured using ELISA kits following the manufacturer’s instructions. The data are expressed as percentages relative to untreated cells (control), which were set at 100 %, and represent the mean ± SEM of two independent experiments, each performed in duplicate. Statistical significance was determined using a one-way ANOVA-Tukey HSD post hoc test: * P

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

    13) Product Images from "Tumor cell-released autophagosomes (TRAPs) promote immunosuppression through induction of M2-like macrophages with increased expression of PD-L1"

    Article Title: Tumor cell-released autophagosomes (TRAPs) promote immunosuppression through induction of M2-like macrophages with increased expression of PD-L1

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1186/s40425-018-0452-5

    TRAPs derived from malignant pleural effusions or ascites of cancer patients regulate monocytes polarization. a , b The MFIs of ( a) CD163 and ( b ) PD-L1 on CD14 + monocytes from peripheral blood of cancer patients (CPs) ( n = 14) and healthy donors (HDs) ( n = 8) were assessed by flow cytometry. c PBMCs from CPs and HDs were stimulated with LPS (100 ng/ml) for 18 h, breferdin A plus monensin was added for the last 6 h of culture, and the frequency of CD14 + IL-10 + was determined by flow cytometry. d , e The graphs showed the correlation between concentration of TRAP and MFI of PD-L1 on CD14 + monocytes and total IL-10 in malignant pleural effusions or ascites of cancer patients ( n = 20). f-h Purified CD14 + monocytes from healthy donors were treated with TRAPs (5 μg/ml) derived from pleural effusions or ascites of cancer patients ( n = 10) for 3 d. f , g The expression of CD163, PD-L1, CD86 and HLA-DR was detected by flow cytometry. h The amount of IL-10 in the supernatant was assessed by ELISA. i Analysis of T cell proliferation by flow cytometry. Monocytes were left untreated or pretreated with TRAPs for 3 d, then incubated with CFSE-labeled autologous T cells (1:3) for 5 d in the presence of anti-CD3 and anti-CD28, or monocytes and T cells were separated by a transwell chamber. j , k Monocytes were left untreated or pretreated with TRAPs from MDA-MB-231 cells (c-TRAP) and cancer patient (p-TRAP) for 3 d, then incubated with autologous T cells (1:3) for 20 h in the presence of immobilized anti-CD3. j CD25 expression on T cells was examined by flow cytometry. k Breferdin A plus monensin was added during the last 8 h of culture. The percentage of CD4 + IFN-γ + and CD8 + IFN-γ + T cells and total IFN-γ secretion in the supernatant was detected by flow cytometry and ELISA, respectively. Data (mean ± SEM) are representative of three independent experiments. * p
    Figure Legend Snippet: TRAPs derived from malignant pleural effusions or ascites of cancer patients regulate monocytes polarization. a , b The MFIs of ( a) CD163 and ( b ) PD-L1 on CD14 + monocytes from peripheral blood of cancer patients (CPs) ( n = 14) and healthy donors (HDs) ( n = 8) were assessed by flow cytometry. c PBMCs from CPs and HDs were stimulated with LPS (100 ng/ml) for 18 h, breferdin A plus monensin was added for the last 6 h of culture, and the frequency of CD14 + IL-10 + was determined by flow cytometry. d , e The graphs showed the correlation between concentration of TRAP and MFI of PD-L1 on CD14 + monocytes and total IL-10 in malignant pleural effusions or ascites of cancer patients ( n = 20). f-h Purified CD14 + monocytes from healthy donors were treated with TRAPs (5 μg/ml) derived from pleural effusions or ascites of cancer patients ( n = 10) for 3 d. f , g The expression of CD163, PD-L1, CD86 and HLA-DR was detected by flow cytometry. h The amount of IL-10 in the supernatant was assessed by ELISA. i Analysis of T cell proliferation by flow cytometry. Monocytes were left untreated or pretreated with TRAPs for 3 d, then incubated with CFSE-labeled autologous T cells (1:3) for 5 d in the presence of anti-CD3 and anti-CD28, or monocytes and T cells were separated by a transwell chamber. j , k Monocytes were left untreated or pretreated with TRAPs from MDA-MB-231 cells (c-TRAP) and cancer patient (p-TRAP) for 3 d, then incubated with autologous T cells (1:3) for 20 h in the presence of immobilized anti-CD3. j CD25 expression on T cells was examined by flow cytometry. k Breferdin A plus monensin was added during the last 8 h of culture. The percentage of CD4 + IFN-γ + and CD8 + IFN-γ + T cells and total IFN-γ secretion in the supernatant was detected by flow cytometry and ELISA, respectively. Data (mean ± SEM) are representative of three independent experiments. * p

    Techniques Used: Derivative Assay, Flow Cytometry, Cytometry, Concentration Assay, Purification, Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Labeling, Multiple Displacement Amplification

    TRAPs polarize macrophages toward an M2-like phenotype in vitro and in vivo. a Confocal images of BMDMs treated with CFSE-labeled TRAPs (green). After incubation with TRAPs (10 μg/ml) for 0.5 h, BMDMs were stained with PE-F4/80 antibody (red) and DAPI (blue). Scale bar, 10 μm. b Expression analysis of CD206, PD-L1, CD86 and MHC II by flow cytometry. BMDMs were stimulated with LPS (100 ng/ml) + IFN-γ (20 ng/ml), IL-4 (20 ng/ml) or TRAPs (10 μg/ml) for 48, 48 or 72 h, respectively. c Flow cytometry analysis of PD-L2, B7-H2, B7-H3, B7-H4, Tim-4 and VISTA for BMDMs after incubating with TRAPs for 48 h. d Expression analysis of NOS2 and Arg1 mRNA in BMDMs treated with TRAPs (10 μg/ml) for 6 h by qRT-PCR. e ELISA detection of IL-1β, IL-6, IL-10 and IL-12p70 produced by BMDMs exposed to TRAPs (10 μg/ml) for 72 h. f-h Mice ( n = 3) were intraperitoneally injected with four different doses of TRAPs (0, 10, 30 and 100 μg) at day 0 and the phenotype of peritoneal macrophages was analyzed at day 3. Expression of CD206 ( f ) and PD-L1 ( g ) on macrophages (F4/80 + CD11b + ) was determined by flow cytometry. h mRNA expression level of IL-10 and Arg1 in purified macrophages was detected by qRT-PCR. Results are representative of three independent experiments. * p
    Figure Legend Snippet: TRAPs polarize macrophages toward an M2-like phenotype in vitro and in vivo. a Confocal images of BMDMs treated with CFSE-labeled TRAPs (green). After incubation with TRAPs (10 μg/ml) for 0.5 h, BMDMs were stained with PE-F4/80 antibody (red) and DAPI (blue). Scale bar, 10 μm. b Expression analysis of CD206, PD-L1, CD86 and MHC II by flow cytometry. BMDMs were stimulated with LPS (100 ng/ml) + IFN-γ (20 ng/ml), IL-4 (20 ng/ml) or TRAPs (10 μg/ml) for 48, 48 or 72 h, respectively. c Flow cytometry analysis of PD-L2, B7-H2, B7-H3, B7-H4, Tim-4 and VISTA for BMDMs after incubating with TRAPs for 48 h. d Expression analysis of NOS2 and Arg1 mRNA in BMDMs treated with TRAPs (10 μg/ml) for 6 h by qRT-PCR. e ELISA detection of IL-1β, IL-6, IL-10 and IL-12p70 produced by BMDMs exposed to TRAPs (10 μg/ml) for 72 h. f-h Mice ( n = 3) were intraperitoneally injected with four different doses of TRAPs (0, 10, 30 and 100 μg) at day 0 and the phenotype of peritoneal macrophages was analyzed at day 3. Expression of CD206 ( f ) and PD-L1 ( g ) on macrophages (F4/80 + CD11b + ) was determined by flow cytometry. h mRNA expression level of IL-10 and Arg1 in purified macrophages was detected by qRT-PCR. Results are representative of three independent experiments. * p

    Techniques Used: In Vitro, In Vivo, Labeling, Incubation, Staining, Expressing, Flow Cytometry, Cytometry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Produced, Mouse Assay, Injection, Purification

    14) Product Images from "Functional IKK/NF-κB signaling in pancreatic stellate cells is essential to prevent autoimmune pancreatitis"

    Article Title: Functional IKK/NF-κB signaling in pancreatic stellate cells is essential to prevent autoimmune pancreatitis

    Journal: Communications Biology

    doi: 10.1038/s42003-022-03371-3

    Spontaneous pancreatitis develops in NEMO ΔCol1a2 mice after long-term NEMO inhibition. a Scheme of treatment with tamoxifen diet (400 mg/kg) for 18 weeks. b Body weight changes during the 18 weeks of treatment period. Mean±SD (NEMO WT : n = 9; NEMO ΔCol1a2 : n = 8). Area under the curve was compared. c Spontaneous pancreatitis and infiltration of immune cells observed in NEMO ΔCol1a2 pancreata. Scale bar: 100 µm. d Staining of CD45 and αSMA indicated an increase in activated pancreatic stellate cells at area where abundant immune cells were present. Scale bar: 50 µm. e , f Circulating IgM, IgG and ANA levels measured by ELISA on serum samples (NEMO WT : n = 6; NEMO ΔCol1a2 : n = 5). Whiskers: Min to Max. T -test (two-tailed): * p
    Figure Legend Snippet: Spontaneous pancreatitis develops in NEMO ΔCol1a2 mice after long-term NEMO inhibition. a Scheme of treatment with tamoxifen diet (400 mg/kg) for 18 weeks. b Body weight changes during the 18 weeks of treatment period. Mean±SD (NEMO WT : n = 9; NEMO ΔCol1a2 : n = 8). Area under the curve was compared. c Spontaneous pancreatitis and infiltration of immune cells observed in NEMO ΔCol1a2 pancreata. Scale bar: 100 µm. d Staining of CD45 and αSMA indicated an increase in activated pancreatic stellate cells at area where abundant immune cells were present. Scale bar: 50 µm. e , f Circulating IgM, IgG and ANA levels measured by ELISA on serum samples (NEMO WT : n = 6; NEMO ΔCol1a2 : n = 5). Whiskers: Min to Max. T -test (two-tailed): * p

    Techniques Used: Mouse Assay, Inhibition, Staining, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Th2-predominant response promotes tissue/peripheral eosinophilia and humoral response. a CD3, CD4 and CD8 stainings on pancreatic tissues. Scale bar: 50 µm. b Expression of Th1, Th2 and Th17 cytokines analyzed by qPCR ( n = 8). c FACS analysis showing an increase in the CD45 + CD3 + CD4 + IL17A + population in the NEMO ΔCol1a2 pancreata. d B220 and CD138 stainings showing increased B cells/plasma cells in the NEMO ΔCol1a2 pancreata. Scale bar: 50 µm. e Quantification of B220 + cells per field ( n = 3). f Measurement of the circulating IgM (3 weeks: n = 8; 6 weeks: n = 6) and IgG ( n = 7 for all time points) levels by ELISA on serum and the absorbance was determined. g Serum ANA levels measured by ELISA ( n = 8). Whiskers: Min to Max. T -test (two-tailed): * p
    Figure Legend Snippet: Th2-predominant response promotes tissue/peripheral eosinophilia and humoral response. a CD3, CD4 and CD8 stainings on pancreatic tissues. Scale bar: 50 µm. b Expression of Th1, Th2 and Th17 cytokines analyzed by qPCR ( n = 8). c FACS analysis showing an increase in the CD45 + CD3 + CD4 + IL17A + population in the NEMO ΔCol1a2 pancreata. d B220 and CD138 stainings showing increased B cells/plasma cells in the NEMO ΔCol1a2 pancreata. Scale bar: 50 µm. e Quantification of B220 + cells per field ( n = 3). f Measurement of the circulating IgM (3 weeks: n = 8; 6 weeks: n = 6) and IgG ( n = 7 for all time points) levels by ELISA on serum and the absorbance was determined. g Serum ANA levels measured by ELISA ( n = 8). Whiskers: Min to Max. T -test (two-tailed): * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, FACS, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    15) Product Images from "Anticolitic Effect of the Rhizome Mixture of Anemarrhena asphodeloides and Coptidis chinensis (AC-mix) in Mice"

    Article Title: Anticolitic Effect of the Rhizome Mixture of Anemarrhena asphodeloides and Coptidis chinensis (AC-mix) in Mice

    Journal: Biomolecules & Therapeutics

    doi: 10.4062/biomolther.2013.048

    Anti-inflammatory effect of 80% ethanol extracts of Anemarrhenae Rhizoma (AA) and Coptidis rhizoma (CC) in LPSstimulated intestinal macrophages. (A) Effects of AA and CC on the expression of TNF-α and IL-1β (A). (B) Effect of AA on IRAK1, IKKβ and IκBα phosphorylation and NF-κB activation. Colonic macrophages (0.5×10 6 cells) were treated with 50 ng/ml LPS in the absence or presence of test agents (10 and 20 μM) for 20 h. The normal control group was treated with vehicle alone. The levels of these cytokines in culture supernatants were measured by ELISA and p65, p-p65, IRAK1, p-IRAK1, p-IKK β , I κ B α , and p-I κ B α were measured by immunoblotting. * Significantly different vs. group treated with LPS alone ( p
    Figure Legend Snippet: Anti-inflammatory effect of 80% ethanol extracts of Anemarrhenae Rhizoma (AA) and Coptidis rhizoma (CC) in LPSstimulated intestinal macrophages. (A) Effects of AA and CC on the expression of TNF-α and IL-1β (A). (B) Effect of AA on IRAK1, IKKβ and IκBα phosphorylation and NF-κB activation. Colonic macrophages (0.5×10 6 cells) were treated with 50 ng/ml LPS in the absence or presence of test agents (10 and 20 μM) for 20 h. The normal control group was treated with vehicle alone. The levels of these cytokines in culture supernatants were measured by ELISA and p65, p-p65, IRAK1, p-IRAK1, p-IKK β , I κ B α , and p-I κ B α were measured by immunoblotting. * Significantly different vs. group treated with LPS alone ( p

    Techniques Used: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay

    Inhibitory effect of 80% ethanol extract of Anemarrhenae Rhizoma on the activation of NF-κB and the expression of COX-2, iNOS, TNF-α, IL-1β, IL-6 and IL-10 in TNBS-induced colitic mice. TNBS, except in the control group, was intrarectally administered to mice treated with saline or test agents. Test compounds [80% ethanol extract of Anemarrhenae Rhizoma (A, 10 or 20 mg/kg), mesalazine (M, 10 mg/kg), or saline] were orally administered for 3 days after TNBS treatment. The mice were sacrificed at 20 h after the final administration of test agents. (A) NF-κB, IRAK1, COX-2, iNOS, and β-actin were analyzed by immunoblotting. (B) TNF-α (a), IL-1β (b), IL-6 (c), IL-10 (d) were analyzed by ELISA kits. All values are the mean ± S.D. (n=6). # p
    Figure Legend Snippet: Inhibitory effect of 80% ethanol extract of Anemarrhenae Rhizoma on the activation of NF-κB and the expression of COX-2, iNOS, TNF-α, IL-1β, IL-6 and IL-10 in TNBS-induced colitic mice. TNBS, except in the control group, was intrarectally administered to mice treated with saline or test agents. Test compounds [80% ethanol extract of Anemarrhenae Rhizoma (A, 10 or 20 mg/kg), mesalazine (M, 10 mg/kg), or saline] were orally administered for 3 days after TNBS treatment. The mice were sacrificed at 20 h after the final administration of test agents. (A) NF-κB, IRAK1, COX-2, iNOS, and β-actin were analyzed by immunoblotting. (B) TNF-α (a), IL-1β (b), IL-6 (c), IL-10 (d) were analyzed by ELISA kits. All values are the mean ± S.D. (n=6). # p

    Techniques Used: Activation Assay, Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Inhibitory effect of AC-mix on the activation of NF-κB and the expression of COX-2, iNOS, TNF-α, IL-1β, IL-6 and IL-10 in TNBS-induced colitic mice. TNBS, except in the control group, was intrarectally administered to mice treated with saline, AC mixture or mesalazine. Test compounds [AC-mix (10 or 20 mg/kg), mesalazine (MS; 10 mg/kg), or saline] were orally administered for 3 days after TNBS treatment. The mice were sacrificed at 20 h after the final administration of test agents. (A) NF-κB, COX-2, iNOS, and β-actin were analyzed by immunoblotting. (B) TNF-α (a), IL-1β (b), IL-6 (c), IL-10 (d) were analyzed by ELISA kits. All values are the mean ± S.D. (n=6). # p
    Figure Legend Snippet: Inhibitory effect of AC-mix on the activation of NF-κB and the expression of COX-2, iNOS, TNF-α, IL-1β, IL-6 and IL-10 in TNBS-induced colitic mice. TNBS, except in the control group, was intrarectally administered to mice treated with saline, AC mixture or mesalazine. Test compounds [AC-mix (10 or 20 mg/kg), mesalazine (MS; 10 mg/kg), or saline] were orally administered for 3 days after TNBS treatment. The mice were sacrificed at 20 h after the final administration of test agents. (A) NF-κB, COX-2, iNOS, and β-actin were analyzed by immunoblotting. (B) TNF-α (a), IL-1β (b), IL-6 (c), IL-10 (d) were analyzed by ELISA kits. All values are the mean ± S.D. (n=6). # p

    Techniques Used: Activation Assay, Expressing, Mouse Assay, Mass Spectrometry, Enzyme-linked Immunosorbent Assay

    Inhibitory effect of AC-mix on the activation of NF-κB and IRAK1 and the expression of COX-2, iNOS, TNF-α, IL-1β, IL-6 and IL-10 in oxazolone-induced colitic mice. Oxazolone, except in normal control group, was intrarectally administered to mice treated with saline, AC mixture or mesalazine. Test compounds [AC mixure (AC, 10 or 20 mg/kg), mesalazine (M, 10 mg/kg), or saline] were orally administered for 3 days after TNBS treatment. The mice were sacrificed at 20 h after the final administration of test agents. (A) NF-κB, COX-2, iNOS, and β-actin were analyzed by immunoblotting. (B) TNF-α (a), IL-1β (b), IL-6 (c), IL-10 (d) were analyzed by ELISA kits. All values are the mean ± S.D. (n=6). # p
    Figure Legend Snippet: Inhibitory effect of AC-mix on the activation of NF-κB and IRAK1 and the expression of COX-2, iNOS, TNF-α, IL-1β, IL-6 and IL-10 in oxazolone-induced colitic mice. Oxazolone, except in normal control group, was intrarectally administered to mice treated with saline, AC mixture or mesalazine. Test compounds [AC mixure (AC, 10 or 20 mg/kg), mesalazine (M, 10 mg/kg), or saline] were orally administered for 3 days after TNBS treatment. The mice were sacrificed at 20 h after the final administration of test agents. (A) NF-κB, COX-2, iNOS, and β-actin were analyzed by immunoblotting. (B) TNF-α (a), IL-1β (b), IL-6 (c), IL-10 (d) were analyzed by ELISA kits. All values are the mean ± S.D. (n=6). # p

    Techniques Used: Activation Assay, Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay

    16) Product Images from "The Protective Effect of Recombinant FomA-expressing Lactobacillus acidophilus Against Periodontal Infection"

    Article Title: The Protective Effect of Recombinant FomA-expressing Lactobacillus acidophilus Against Periodontal Infection

    Journal: Inflammation

    doi: 10.1007/s10753-013-9651-x

    Levels of IL-1β in oral mucosal tissues of mice. After 3 days of injection, the mandibular and tongue mucosal tissues of the mice were collected, and 100 mg/ml PBS buffer was added. After homogenisation, the supernatant was collected by centrifugation at 20,000× g at 4 °C for 20 min. The IL-1β levels in the serum and oral mucosal tissues were determined using ELISA kits (Thermo Scientific, USA). The IL-1β levels in the experimental group of mice were lower than in the control group following injections with both F. nucleatum alone and with F. nucleatum plus P. gingivalis (*, # p
    Figure Legend Snippet: Levels of IL-1β in oral mucosal tissues of mice. After 3 days of injection, the mandibular and tongue mucosal tissues of the mice were collected, and 100 mg/ml PBS buffer was added. After homogenisation, the supernatant was collected by centrifugation at 20,000× g at 4 °C for 20 min. The IL-1β levels in the serum and oral mucosal tissues were determined using ELISA kits (Thermo Scientific, USA). The IL-1β levels in the experimental group of mice were lower than in the control group following injections with both F. nucleatum alone and with F. nucleatum plus P. gingivalis (*, # p

    Techniques Used: Mouse Assay, Injection, Homogenization, Centrifugation, Enzyme-linked Immunosorbent Assay

    Levels of IL-1β in mouse serum samples. After 3 days of injection, blood was collected from the heart, and the serum was isolated. The levels of IL-1β in the mouse serum were determined using ELISA kits in accordance with the manufacturer's instructions (Thermo Scientific, USA). Following injection with both F. nucleatum alone and F. nucleatum plus P. gingivalis , the mean IL-1β levels in the experimental group of mice were lower than in the control group (*, # p
    Figure Legend Snippet: Levels of IL-1β in mouse serum samples. After 3 days of injection, blood was collected from the heart, and the serum was isolated. The levels of IL-1β in the mouse serum were determined using ELISA kits in accordance with the manufacturer's instructions (Thermo Scientific, USA). Following injection with both F. nucleatum alone and F. nucleatum plus P. gingivalis , the mean IL-1β levels in the experimental group of mice were lower than in the control group (*, # p

    Techniques Used: Injection, Isolation, Enzyme-linked Immunosorbent Assay, Mouse Assay

    17) Product Images from "Patient-derived avian influenza A (H5N6) virus is highly pathogenic in mice but can be effectively treated by anti-influenza polyclonal antibodies"

    Article Title: Patient-derived avian influenza A (H5N6) virus is highly pathogenic in mice but can be effectively treated by anti-influenza polyclonal antibodies

    Journal: Emerging Microbes & Infections

    doi: 10.1038/s41426-018-0113-2

    Pro-inflammatory cytokines and chemokines in the lungs of H5N6/GZ14-infected mice. The lung tissues were collected and homogenized from H5N6/GZ14-infected mice at 1, 3, and 5 dpi ( n = 3). The levels of cytokines and chemokines in the lung homogenates (pg/g of lung tissue) were determined by ELISA. The results from each time point are expressed as the mean ± SD. *A significant difference (* P
    Figure Legend Snippet: Pro-inflammatory cytokines and chemokines in the lungs of H5N6/GZ14-infected mice. The lung tissues were collected and homogenized from H5N6/GZ14-infected mice at 1, 3, and 5 dpi ( n = 3). The levels of cytokines and chemokines in the lung homogenates (pg/g of lung tissue) were determined by ELISA. The results from each time point are expressed as the mean ± SD. *A significant difference (* P

    Techniques Used: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay

    18) Product Images from "Arctigenin Effectively Ameliorates Memory Impairment in Alzheimer's Disease Model Mice Targeting Both β-Amyloid Production and Clearance"

    Article Title: Arctigenin Effectively Ameliorates Memory Impairment in Alzheimer's Disease Model Mice Targeting Both β-Amyloid Production and Clearance

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4790-12.2013

    Arctigenin reduced Aβ production with BACE1 translation inhibition in HEK293-APP swe cells. HEK293-APP swe cells were cultured with 0.1% DMSO (as a control) or different concentrations of arctigenin (ATG) for 24 h. A , Chemical structure of arctigenin. B , ELISA assay of arctigenin-decreased Aβ 40 . C , ELISA assay of arctigenin-reduced sAPPβ. D , Arctigenin inhibited BACE1 activity in cells. E , FRET-based assay demonstrated that arctigenin failed to inhibit BACE1 activity at protein level. F , Western blot assay indicated that arctigenin decreased BACE1 protein level and phosphorylation of eIF2α or PERK. G , RT-PCR assay indicated that arctigenin had no effect on BACE1 transcription. The results shown are representative of three independent experiments. ** p
    Figure Legend Snippet: Arctigenin reduced Aβ production with BACE1 translation inhibition in HEK293-APP swe cells. HEK293-APP swe cells were cultured with 0.1% DMSO (as a control) or different concentrations of arctigenin (ATG) for 24 h. A , Chemical structure of arctigenin. B , ELISA assay of arctigenin-decreased Aβ 40 . C , ELISA assay of arctigenin-reduced sAPPβ. D , Arctigenin inhibited BACE1 activity in cells. E , FRET-based assay demonstrated that arctigenin failed to inhibit BACE1 activity at protein level. F , Western blot assay indicated that arctigenin decreased BACE1 protein level and phosphorylation of eIF2α or PERK. G , RT-PCR assay indicated that arctigenin had no effect on BACE1 transcription. The results shown are representative of three independent experiments. ** p

    Techniques Used: Inhibition, Cell Culture, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

    19) Product Images from "A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection"

    Article Title: A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1615422114

    Detection of cytokines and the effect of HRV infection on host translation. ( A ) HIEs from four individuals (j2, j3, j6, and j11) were treated with increasing doses of IFN-λ1, and ISG expression was assessed after 3 h by RT-qPCR. Data are of HIEs from four individuals and are a compilation of two independent experiments. Results are expressed as geometric mean ± 95% CI of the geometric mean ( n = 4 biological replicates). ( B ) HIEs (j3) were mock-inoculated, HRV-inoculated (MOI of 10), or treated with poly(I:C) (30 μg/mL). After 1.5 hpi, HIEs were washed and resuspended in new medium, which was analyzed at 8 hpi for secreted IFN-λ1 by ELISA. ( C ) HIEs (j2) were mock-inoculated, HRV-inoculated (MOI of 10), treated with poly(I:C) (100 μg/mL), or HRV-inoculated (MOI of 10) and treated 10 min later with poly(I:C) (100 μg/mL). After 1.5 hpi, HIEs were washed and resuspended in new medium, which was analyzed at 20 hpi for secreted IFN-λ1 by ELISA. In B and C , data from one representative experiment of three independent experiments is presented as arithmetic mean ± SD of three technical replicates. Statistical analyses in C were performed by Student’s t test.
    Figure Legend Snippet: Detection of cytokines and the effect of HRV infection on host translation. ( A ) HIEs from four individuals (j2, j3, j6, and j11) were treated with increasing doses of IFN-λ1, and ISG expression was assessed after 3 h by RT-qPCR. Data are of HIEs from four individuals and are a compilation of two independent experiments. Results are expressed as geometric mean ± 95% CI of the geometric mean ( n = 4 biological replicates). ( B ) HIEs (j3) were mock-inoculated, HRV-inoculated (MOI of 10), or treated with poly(I:C) (30 μg/mL). After 1.5 hpi, HIEs were washed and resuspended in new medium, which was analyzed at 8 hpi for secreted IFN-λ1 by ELISA. ( C ) HIEs (j2) were mock-inoculated, HRV-inoculated (MOI of 10), treated with poly(I:C) (100 μg/mL), or HRV-inoculated (MOI of 10) and treated 10 min later with poly(I:C) (100 μg/mL). After 1.5 hpi, HIEs were washed and resuspended in new medium, which was analyzed at 20 hpi for secreted IFN-λ1 by ELISA. In B and C , data from one representative experiment of three independent experiments is presented as arithmetic mean ± SD of three technical replicates. Statistical analyses in C were performed by Student’s t test.

    Techniques Used: Infection, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    IFN response to inactivated HRV. HIEs (j3) were inoculated with replication-competent HRV (MOI of 10 FFU) or with an equivalent amount (in micrograms) of inactivated HRV particles. ( A ) Expression of type III IFN ( IFNL1 ), type I IFN ( IFNB1 ), and the ISG IFI44L was assessed at 6 hpi by RT-qPCR. Each data bar represents the geometric mean ± 95% CI of the geometric mean ( n = 4 technical replicates). Statistical analyses were performed by Mann–Whitney U test. ( B ) HIEs were infected as described in A and, after 1 h, HIEs were washed and resuspended in medium containing type III IFN receptor-blocking antibody (25 μg/mL anti-IL28RA). The media was analyzed at 20 hpi for secreted IFN-λ1 by ELISA. Data from one representative experiment of two independent experiments is presented as arithmetic mean ± SD of three or four technical replicates.
    Figure Legend Snippet: IFN response to inactivated HRV. HIEs (j3) were inoculated with replication-competent HRV (MOI of 10 FFU) or with an equivalent amount (in micrograms) of inactivated HRV particles. ( A ) Expression of type III IFN ( IFNL1 ), type I IFN ( IFNB1 ), and the ISG IFI44L was assessed at 6 hpi by RT-qPCR. Each data bar represents the geometric mean ± 95% CI of the geometric mean ( n = 4 technical replicates). Statistical analyses were performed by Mann–Whitney U test. ( B ) HIEs were infected as described in A and, after 1 h, HIEs were washed and resuspended in medium containing type III IFN receptor-blocking antibody (25 μg/mL anti-IL28RA). The media was analyzed at 20 hpi for secreted IFN-λ1 by ELISA. Data from one representative experiment of two independent experiments is presented as arithmetic mean ± SD of three or four technical replicates.

    Techniques Used: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Infection, Blocking Assay, Enzyme-linked Immunosorbent Assay

    20) Product Images from "Tamm-Horsfall Protein Regulates Circulating and Renal Cytokines by Affecting Glomerular Filtration Rate and Acting as a Urinary Cytokine Trap *"

    Article Title: Tamm-Horsfall Protein Regulates Circulating and Renal Cytokines by Affecting Glomerular Filtration Rate and Acting as a Urinary Cytokine Trap *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.348243

    Renal cytokine content. A–F , total protein extracts from groups of wild-type ( WT ) and THP KO mice were used to assess cytokine production using ELISA. The cytokine levels were expressed as a ratio to the total kidney protein input ( e.g. pg of cytokine/mg of total proteins). Note that all the six cytokines assayed without LPS challenge were higher in KO mice than in WT controls, although the increases for TNF-α and CXCL1 were not statistically significant.
    Figure Legend Snippet: Renal cytokine content. A–F , total protein extracts from groups of wild-type ( WT ) and THP KO mice were used to assess cytokine production using ELISA. The cytokine levels were expressed as a ratio to the total kidney protein input ( e.g. pg of cytokine/mg of total proteins). Note that all the six cytokines assayed without LPS challenge were higher in KO mice than in WT controls, although the increases for TNF-α and CXCL1 were not statistically significant.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    In vivo binding of renal cytokines to THP. Top panels , co-immunoprecipitation of THP-bound cytokines followed by cytokine ELISA. Total kidney protein extracts from wild-type ( WT ) and THP KO mice (2 representative mice per genotype shown) were subject to IP using a rabbit anti-THP antibody. The IP products were loaded into microtiter wells pre-coated with antibodies against IFN-γ ( A ), IL1α ( B ), TNF-α ( C ), or IL13 ( D ), to capture THP-bound cytokines. This was followed by conventional ELISA (see “Experimental Procedures” for details). Starting materials for IP contained the same amount of total kidney proteins. Lower panels , ELISA quantification of renal cytokine input ( e.g. THP-bound and -unbound cytokines), expressed as per milligram of total kidney proteins. Note that, whereas THP KO mice had higher renal cytokines ( lower panels , filled bars ), little could be precipitated with anti-THP antibody ( upper panels , filled bars ). In contrast, in wild-type mice, each of the four tested cytokines could be precipitated by the anti-THP antibody ( upper panels , open bars ) strongly suggesting an in vivo THP-cytokine interaction (see text for details).
    Figure Legend Snippet: In vivo binding of renal cytokines to THP. Top panels , co-immunoprecipitation of THP-bound cytokines followed by cytokine ELISA. Total kidney protein extracts from wild-type ( WT ) and THP KO mice (2 representative mice per genotype shown) were subject to IP using a rabbit anti-THP antibody. The IP products were loaded into microtiter wells pre-coated with antibodies against IFN-γ ( A ), IL1α ( B ), TNF-α ( C ), or IL13 ( D ), to capture THP-bound cytokines. This was followed by conventional ELISA (see “Experimental Procedures” for details). Starting materials for IP contained the same amount of total kidney proteins. Lower panels , ELISA quantification of renal cytokine input ( e.g. THP-bound and -unbound cytokines), expressed as per milligram of total kidney proteins. Note that, whereas THP KO mice had higher renal cytokines ( lower panels , filled bars ), little could be precipitated with anti-THP antibody ( upper panels , filled bars ). In contrast, in wild-type mice, each of the four tested cytokines could be precipitated by the anti-THP antibody ( upper panels , open bars ) strongly suggesting an in vivo THP-cytokine interaction (see text for details).

    Techniques Used: In Vivo, Binding Assay, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Mouse Assay

    Circulating cytokines in response to LPS treatment. Wild-type ( WT , filled circle ; n = 8) and THP knock-out ( KO , filled square ) mice ( n = 8) were injected intraperitoneally with LPS, and their blood was obtained at 1.5, 3, 6, 12, and 24 h after injection and processed for ELISA. Note that although serum cytokine levels increased in both WT and KO mice in response to LPS, KO mice had significantly higher levels of cytokines, except IL13, at various time points than WT mice ( asterisks denote the time points where statistical significance was present; see text for details). IL13 levels were also higher in KO mice than in WT mice at all time points, but did not reach statistical significance.
    Figure Legend Snippet: Circulating cytokines in response to LPS treatment. Wild-type ( WT , filled circle ; n = 8) and THP knock-out ( KO , filled square ) mice ( n = 8) were injected intraperitoneally with LPS, and their blood was obtained at 1.5, 3, 6, 12, and 24 h after injection and processed for ELISA. Note that although serum cytokine levels increased in both WT and KO mice in response to LPS, KO mice had significantly higher levels of cytokines, except IL13, at various time points than WT mice ( asterisks denote the time points where statistical significance was present; see text for details). IL13 levels were also higher in KO mice than in WT mice at all time points, but did not reach statistical significance.

    Techniques Used: Knock-Out, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

    21) Product Images from "Cytosolic Extract of Human Adipose Stem Cells Reverses the Amyloid Beta-Induced Mitochondrial Apoptosis via P53/Foxo3a Pathway"

    Article Title: Cytosolic Extract of Human Adipose Stem Cells Reverses the Amyloid Beta-Induced Mitochondrial Apoptosis via P53/Foxo3a Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0168859

    The hASC extract reduces amyloid beta level, p53 and foxo3a protein levels in AD in vitro neurons. (A) AD in vitro cells were treated with hASC extract for 48h and Aβ42 and Aβ40 were determined by ELISA. Graph shows the reduction of the Aβ40, Aβ42 and ratio of Aβ42/Aβ40 by hASC extract treatment. (B) WT and AD in vitro cells were differentiated for 3 days and treated with 100 μg/ml hASC extract for 48h. Immunoblotting showed expression levels of Bax, cleaved caspase-3, and Bcl2 proteins after treatment with the hASC extract. Quantified graph showed the reduction of Bax, Bcl2 and cleaved caspase-3 protein expressions by hASC extract treatment. A representative experiment is shown. (C) WT and AD in vitro cells differentiated for 3 days. Cells were treated with or without 100 μg/ml hASC extract and equal amount of cell lysate were subjected in immunoblotting for p53, foxo3a and β-actin. (D) WT and AD in vitro cells were differentiated for 3 days and treated with hASC extract for 48h. Cells were subjected in immunocytochemistry for foxo3a and DAPI. Images were captured by confocal microscope. (E) Quantified graph showed the changes of foxo3a intensity both in total (right panel) and in the nucleus (left panel). ( n = 3 each). Error bars represent S.E.M. Scale bar = 10 μm
    Figure Legend Snippet: The hASC extract reduces amyloid beta level, p53 and foxo3a protein levels in AD in vitro neurons. (A) AD in vitro cells were treated with hASC extract for 48h and Aβ42 and Aβ40 were determined by ELISA. Graph shows the reduction of the Aβ40, Aβ42 and ratio of Aβ42/Aβ40 by hASC extract treatment. (B) WT and AD in vitro cells were differentiated for 3 days and treated with 100 μg/ml hASC extract for 48h. Immunoblotting showed expression levels of Bax, cleaved caspase-3, and Bcl2 proteins after treatment with the hASC extract. Quantified graph showed the reduction of Bax, Bcl2 and cleaved caspase-3 protein expressions by hASC extract treatment. A representative experiment is shown. (C) WT and AD in vitro cells differentiated for 3 days. Cells were treated with or without 100 μg/ml hASC extract and equal amount of cell lysate were subjected in immunoblotting for p53, foxo3a and β-actin. (D) WT and AD in vitro cells were differentiated for 3 days and treated with hASC extract for 48h. Cells were subjected in immunocytochemistry for foxo3a and DAPI. Images were captured by confocal microscope. (E) Quantified graph showed the changes of foxo3a intensity both in total (right panel) and in the nucleus (left panel). ( n = 3 each). Error bars represent S.E.M. Scale bar = 10 μm

    Techniques Used: In Vitro, Enzyme-linked Immunosorbent Assay, Expressing, Immunocytochemistry, Microscopy

    22) Product Images from "Culture supernatant of adipose stem cells can ameliorate allergic airway inflammation via recruitment of CD4+CD25+Foxp3 T cells"

    Article Title: Culture supernatant of adipose stem cells can ameliorate allergic airway inflammation via recruitment of CD4+CD25+Foxp3 T cells

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-016-0462-5

    Protein analysis of con sup and ASC sup. a IL-10 and TGF-β expression level in ASC sup and con sup were measured by ELISA. b Both concentrated culture medium. 30 μg was loaded and the proteins were separated using SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane and incubated with antibody specific for TGF-β. ( *** p
    Figure Legend Snippet: Protein analysis of con sup and ASC sup. a IL-10 and TGF-β expression level in ASC sup and con sup were measured by ELISA. b Both concentrated culture medium. 30 μg was loaded and the proteins were separated using SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane and incubated with antibody specific for TGF-β. ( *** p

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, SDS Page, Incubation

    Treating with ASC sup regulated cytokine expression levels in OVA-induced BALF and LLNs. Cytokine concentrations in BALF( a ) and in the culture medium of CD3-stimulated lymphocytes isolated from LLNs( b ). For activation of lymphocytes from LLN, the wells were incubated with 1 μg/ml of anti-CD3 antibody for 16 hours at 4 °C. The lymphocytes (4 × 10 5 cells per well) were introduced to the well and incubated for 3 days. After activation, the levels of several cytokines were measured in the supernatant using ELISA kits; ELISA assays were conducted in accordance with the manufacturer’s instructions. ( PBS; BALF or culture supernatant of LLN from PBS-treated mice, OVA; BALF or culture supernatant of LLN from OVA-Alum-induced airway inflammation mice, con sup + OVA; BALF or culture supernatant of LLN from OVA-Alum-induced airway inflammation mice with con sup treatment, ASC sup + OVA; BALF or culture supernatant of LLN from OVA-Alum-induced airway inflammation mice with ASC sup treatment. * p
    Figure Legend Snippet: Treating with ASC sup regulated cytokine expression levels in OVA-induced BALF and LLNs. Cytokine concentrations in BALF( a ) and in the culture medium of CD3-stimulated lymphocytes isolated from LLNs( b ). For activation of lymphocytes from LLN, the wells were incubated with 1 μg/ml of anti-CD3 antibody for 16 hours at 4 °C. The lymphocytes (4 × 10 5 cells per well) were introduced to the well and incubated for 3 days. After activation, the levels of several cytokines were measured in the supernatant using ELISA kits; ELISA assays were conducted in accordance with the manufacturer’s instructions. ( PBS; BALF or culture supernatant of LLN from PBS-treated mice, OVA; BALF or culture supernatant of LLN from OVA-Alum-induced airway inflammation mice, con sup + OVA; BALF or culture supernatant of LLN from OVA-Alum-induced airway inflammation mice with con sup treatment, ASC sup + OVA; BALF or culture supernatant of LLN from OVA-Alum-induced airway inflammation mice with ASC sup treatment. * p

    Techniques Used: Expressing, Isolation, Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Mouse Assay

    23) Product Images from "Peptides of Presenilin-1 Bind the Amyloid Precursor Protein Ectodomain and Offer a Novel and Specific Therapeutic Approach to Reduce ß-Amyloid in Alzheimer’s Disease"

    Article Title: Peptides of Presenilin-1 Bind the Amyloid Precursor Protein Ectodomain and Offer a Novel and Specific Therapeutic Approach to Reduce ß-Amyloid in Alzheimer’s Disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0122451

    Effects Of Peptides P1 Through P10 On Aβ40 and 42 Production In Vitro . A and B. Peptides P1(residues 1–80 of PS-1), scrambled P1 (SP1), P2 (residues 1–40 of PS-1) and P3 (residues 41–80 of PS-1) (0–5 μM) were added to cultures of IMR-32 cells for 24 h and the culture media were analysed for Aβ40 (A) and Aβ42 (B) by ELISA. Aβ levels in pg/ml were expressed as a percentage of levels observed in the absence of added peptide. Values represent group means of 3–4 independent experiments per peptide ±s.e.m. Only P3 produced a clear dose-dependent decrease in both Aβ40 and -42, shown in red. C and D. Overlapping peptides from the P3 region (P4–P10) (0–5 μM) were next tested in IMR-32 cultures as in A and B above for their effect on Aβ40 (C) and -42 (D) production. A family of peptides, P6, P7 and P8 and an independent peptide, P4, produced substantial dose-dependent reduction in Aβ40 and 42, shown in red.
    Figure Legend Snippet: Effects Of Peptides P1 Through P10 On Aβ40 and 42 Production In Vitro . A and B. Peptides P1(residues 1–80 of PS-1), scrambled P1 (SP1), P2 (residues 1–40 of PS-1) and P3 (residues 41–80 of PS-1) (0–5 μM) were added to cultures of IMR-32 cells for 24 h and the culture media were analysed for Aβ40 (A) and Aβ42 (B) by ELISA. Aβ levels in pg/ml were expressed as a percentage of levels observed in the absence of added peptide. Values represent group means of 3–4 independent experiments per peptide ±s.e.m. Only P3 produced a clear dose-dependent decrease in both Aβ40 and -42, shown in red. C and D. Overlapping peptides from the P3 region (P4–P10) (0–5 μM) were next tested in IMR-32 cultures as in A and B above for their effect on Aβ40 (C) and -42 (D) production. A family of peptides, P6, P7 and P8 and an independent peptide, P4, produced substantial dose-dependent reduction in Aβ40 and 42, shown in red.

    Techniques Used: In Vitro, Enzyme-linked Immunosorbent Assay, Produced

    24) Product Images from "Factor VIIa induces biased cytoprotective signal in mice through the cleavage of PAR1 at canonical Arg41 site"

    Article Title: Factor VIIa induces biased cytoprotective signal in mice through the cleavage of PAR1 at canonical Arg41 site

    Journal: Arteriosclerosis, thrombosis, and vascular biology

    doi: 10.1161/ATVBAHA.120.314244

    FVIIa-mediated suppression of LPS-induced inflammatory cytokine elaboration in the lung tissues requires the R41 but not the R46 cleavage site of PAR1. Wild-type C57 BL/6J (WT), PAR1-R41Q, or PAR1-R46Q mice were administered with saline, human FVIIa (250 μg/kg body weight) or human APC (250 μg/kg body weight) i.v. via the tail vein. After 1 h, the mice receiving the saline were left either unchallenged (Con) or administered with LPS (5 mg/kg; LPS), i.p. The same dose of LPS was administered in parallel to mice injected with FVIIa (FVIIa, LPS) or APC (APC, LPS). Six hours following LPS administration, the mice were anesthetized using ketamine/xylazine and perfused with saline to remove blood in vascular beds. Lung tissue was collected and homogenized in the RIPA buffer containing protease inhibitors. Tissue extracts were centrifuged at 10,000 × g at 4°C to remove tissue debris. The supernatants were used to measure the cytokines by ELISA. The cytokines measured were TNFα (A, D) , IL-1ß (B, E) , and CCL2 (C, F) . Note: In this and all other Figures, the abbreviations “R41Q” and “R46Q” indicate studies using mice homozygous for the indicated mutation, i.e., mice that were QQ41 or QQ46 in PAR1. Data are the mean ± SEM of three independent experiments, a total of 6–10 animals in a group. One-way ANOVA, followed by Tukey’s multiple comparison test, was used to determine statistically significant differences. *, p
    Figure Legend Snippet: FVIIa-mediated suppression of LPS-induced inflammatory cytokine elaboration in the lung tissues requires the R41 but not the R46 cleavage site of PAR1. Wild-type C57 BL/6J (WT), PAR1-R41Q, or PAR1-R46Q mice were administered with saline, human FVIIa (250 μg/kg body weight) or human APC (250 μg/kg body weight) i.v. via the tail vein. After 1 h, the mice receiving the saline were left either unchallenged (Con) or administered with LPS (5 mg/kg; LPS), i.p. The same dose of LPS was administered in parallel to mice injected with FVIIa (FVIIa, LPS) or APC (APC, LPS). Six hours following LPS administration, the mice were anesthetized using ketamine/xylazine and perfused with saline to remove blood in vascular beds. Lung tissue was collected and homogenized in the RIPA buffer containing protease inhibitors. Tissue extracts were centrifuged at 10,000 × g at 4°C to remove tissue debris. The supernatants were used to measure the cytokines by ELISA. The cytokines measured were TNFα (A, D) , IL-1ß (B, E) , and CCL2 (C, F) . Note: In this and all other Figures, the abbreviations “R41Q” and “R46Q” indicate studies using mice homozygous for the indicated mutation, i.e., mice that were QQ41 or QQ46 in PAR1. Data are the mean ± SEM of three independent experiments, a total of 6–10 animals in a group. One-way ANOVA, followed by Tukey’s multiple comparison test, was used to determine statistically significant differences. *, p

    Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Mutagenesis

    25) Product Images from "HMGB1 Increases IL-1β Production in Vascular Smooth Muscle Cells via NLRP3 Inflammasome"

    Article Title: HMGB1 Increases IL-1β Production in Vascular Smooth Muscle Cells via NLRP3 Inflammasome

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2018.00313

    Effects of HMGB1 on IL-1β expression and its release from VSMCs. VSMCs were treated with HMGB1 (100 ng/ml) for 0–24 h, and were also treated with HMGB1 (0–500 ng/ml) for 6 h. (A,B) The mRNA levels of IL-1β were determined by RT-PCR. GAPDH was used as a control. Data are expressed as means ± SEMs of duplicates pooled from 4 independent experiments. (C,D) The protein levels of active IL-1β were determined by Western blot. β-Actin expression served as an internal control. Data are expressed as means ± SEMs of duplicates pooled from 4 independent experiments. (E,F) VSMCs were treated with HMGB1 (100 ng/ml) for 0–48 h, and were also treated with HMGB1 (0–500 ng/ml) for 24 h. The levels of IL-1β in the culture media were quantified by ELISA. Data are expressed as means ± SEMs of triplicates pooled from 4 independent experiments. * P
    Figure Legend Snippet: Effects of HMGB1 on IL-1β expression and its release from VSMCs. VSMCs were treated with HMGB1 (100 ng/ml) for 0–24 h, and were also treated with HMGB1 (0–500 ng/ml) for 6 h. (A,B) The mRNA levels of IL-1β were determined by RT-PCR. GAPDH was used as a control. Data are expressed as means ± SEMs of duplicates pooled from 4 independent experiments. (C,D) The protein levels of active IL-1β were determined by Western blot. β-Actin expression served as an internal control. Data are expressed as means ± SEMs of duplicates pooled from 4 independent experiments. (E,F) VSMCs were treated with HMGB1 (100 ng/ml) for 0–48 h, and were also treated with HMGB1 (0–500 ng/ml) for 24 h. The levels of IL-1β in the culture media were quantified by ELISA. Data are expressed as means ± SEMs of triplicates pooled from 4 independent experiments. * P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

    Functional role of HMGB1 receptors on the production of IL-1β in HMGB1-stimulated VSMCs. (A) VSMCs were transfected with siRNA (200 nM) for TLR2, TLR4, and RAGE, and then incubated with HMGB1 for 24 h. The levels of IL-1β in the culture media were quantified by ELISA. Data are expressed as means ± SEMs of triplicates pooled from 4 independent experiments. ** P
    Figure Legend Snippet: Functional role of HMGB1 receptors on the production of IL-1β in HMGB1-stimulated VSMCs. (A) VSMCs were transfected with siRNA (200 nM) for TLR2, TLR4, and RAGE, and then incubated with HMGB1 for 24 h. The levels of IL-1β in the culture media were quantified by ELISA. Data are expressed as means ± SEMs of triplicates pooled from 4 independent experiments. ** P

    Techniques Used: Functional Assay, Transfection, Incubation, Enzyme-linked Immunosorbent Assay

    26) Product Images from "Post-Exposure Immunization by Capsid-Modified AdC7 Vector Expressing Pseudomonas aeruginosa OprF Clears P. aeruginosa Respiratory Infection"

    Article Title: Post-Exposure Immunization by Capsid-Modified AdC7 Vector Expressing Pseudomonas aeruginosa OprF Clears P. aeruginosa Respiratory Infection

    Journal: Vaccine

    doi: 10.1016/j.vaccine.2017.10.078

    Cellular immune responses after the immunization with AdC7OprF.RGD in mice with established P. aeruginosa respiratory infection C57BL/6 mice were immunized intranasally with either 1×10 10 particle units of AdC7OprF.RGD, AdC7Null or PBS 4 days after the administration of P. aeruginosa embedded in agar beads. Two weeks after the immunization, the splenocytes were collected, cultured and stimulated with OprF protein for 48 h. A. IFN-γ and B. IL-4 levels were measured in the culture supernatants by ELISA. Data are presented as mean ± SEM of n=8 mice per group. * and ** denote significance of p
    Figure Legend Snippet: Cellular immune responses after the immunization with AdC7OprF.RGD in mice with established P. aeruginosa respiratory infection C57BL/6 mice were immunized intranasally with either 1×10 10 particle units of AdC7OprF.RGD, AdC7Null or PBS 4 days after the administration of P. aeruginosa embedded in agar beads. Two weeks after the immunization, the splenocytes were collected, cultured and stimulated with OprF protein for 48 h. A. IFN-γ and B. IL-4 levels were measured in the culture supernatants by ELISA. Data are presented as mean ± SEM of n=8 mice per group. * and ** denote significance of p

    Techniques Used: Mouse Assay, Infection, Cell Culture, Enzyme-linked Immunosorbent Assay

    27) Product Images from "B7-H1/CD80 interaction is required for the induction and maintenance of peripheral T-cell tolerance"

    Article Title: B7-H1/CD80 interaction is required for the induction and maintenance of peripheral T-cell tolerance

    Journal: Blood

    doi: 10.1182/blood-2010-01-265975

    Prevention and restoration of oral tolerance by blockade of B7-H1/CD80 interaction . B6 mice were given drinking water supplemented with OVA protein (○ or ●) or without OVA (□) from day 0 to day 7. On day 14, the mice were immunized subcutaneously with OVA protein emulsified in CFA. The mice were also treated intraperitoneally with 150 μg of 43H12 (●) or control rat IgG (○) on days 0, 4, 8, and 12 (A) or on days 14 and 17 (B). On day 21, draining LN cells were harvested from the mice and cultured with the indicated doses of OVA protein. After 48 hours, production of IFN-γ, IL-2, and IL-4 in culture supernatant was measured by ELISA. IL-17 level was measured 24, 48, and 72 hours after culture with 25 μg/mL OVA protein. Proliferative activity was assessed by an incorporation of 3 H-thymidine. All experiments were repeated at least 3 times. Representative data are shown as mean ± SD of triplicate wells in each group.
    Figure Legend Snippet: Prevention and restoration of oral tolerance by blockade of B7-H1/CD80 interaction . B6 mice were given drinking water supplemented with OVA protein (○ or ●) or without OVA (□) from day 0 to day 7. On day 14, the mice were immunized subcutaneously with OVA protein emulsified in CFA. The mice were also treated intraperitoneally with 150 μg of 43H12 (●) or control rat IgG (○) on days 0, 4, 8, and 12 (A) or on days 14 and 17 (B). On day 21, draining LN cells were harvested from the mice and cultured with the indicated doses of OVA protein. After 48 hours, production of IFN-γ, IL-2, and IL-4 in culture supernatant was measured by ELISA. IL-17 level was measured 24, 48, and 72 hours after culture with 25 μg/mL OVA protein. Proliferative activity was assessed by an incorporation of 3 H-thymidine. All experiments were repeated at least 3 times. Representative data are shown as mean ± SD of triplicate wells in each group.

    Techniques Used: Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Activity Assay

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    Thermo Fisher human mouse tgf ãŽâ²1 elisa ready set go kit
    Number of brain MNCs and their CNS antigen-specific autoreactive phenotype 14 d after MCAo. A , Number of MNCs infiltrating ipsilateral and contralateral hemisphere of the brain in vehicle and SNS/HPA blockade group (three individual experiments; vehicle, N = 15; SNS/HPA block, N = 20). B–D , Elispot experiments. B , Higher number of total brain MNCs responded to pMOG 35–55 stimulation with IFN-γ production (Th1 cells) per equal number of brain MNCs in SNS/HPA block group compared with the vehicle group. C , A similar effect was observed in ipsilateral hemisphere MNCs when only the SNS axis of SIDS was blocked with propranolol (two experiments; vehicle group, N = 5–8; SNS block, N = 6 or 7). D , Number of splenocytes reacting to pMOG 35–55 stimulation with IFN-γ, IL-4, or IL-17 secretion did not differ between the groups. E , F , <t>ELISA</t> experiments. <t>TGF-β1,</t> IL-10, IL-4, IL-17, and IFN-γ secretion (pg/ml) by pMOG 35–55 -stimulated brain MNCs, ipsilateral hemisphere ( E ) and by pMOG 35–55 -stimulated splenocytes ( F ).
    Human Mouse Tgf ãŽâ²1 Elisa Ready Set Go Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ifn α
    Evaluating the replication kinetics of, and level of interferon activation by, WT MHV and DUBmut in cell culture. (A) Replication kinetics of WT and DUBmut virus in DBT cells. (B) IFN-α11 mRNA levels in WT- and DUBmut-infected BMDMs were assessed at indicated time points by reverse transcription-quantitative PCR (qRT-PCR). (C) <t>IFN-α</t> protein levels in the supernatants of infected BMDMs were evaluated at the times indicated. (D) Comparison of IFN-α11 mRNA levels in B6 versus MDA5 −/− BMDMs at 12 h postinfection (h p.i.). (E) Assessment of levels of viral nucleocapsid (N) mRNA by qRT-PCR. (F) Replication kinetics of WT and DUBmut virus in BMDM cells. Data are representative of at least two independent experiments and are presented as means ± SD. Data in panels B and C were statistically analyzed using unpaired t tests. *, P
    Ifn α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher human tim 3
    Biochemical characterization of interactions between CEACAM1 and <t>TIM-3</t> a , hTIM-3 does not co-immunoprecipitate (co-IP) with ITGA5 despite interactions with hCEACAM1. HEK293T cells transfected with Flag–ITGA5 and HA–TIM-3 (ITGA5Tw) or Flag–CEACAM1 and HA–TIM-3 (CwTw). Immunoprecipitation with anti-HA antibody and immunoblotted (IB) with anti-Flag antibody are shown. Input represents anti-Flag immunoblot of lysates. b, Co-immunoprecipitation of human TIM-3 and CEACAM1 from activated primary human T cells after N -glycanase treatment of lystates followed by immunoprecipitation with anti-human TIM-3 antibodies (2E2, 2E12 or 3F9) or IgG as control and immunoblotted with anti-human CEACAM1 antibody (5F4). Protein lystates from HeLa-CEACAM1 transfectants treated with N -glycanase followed by immunoprecipitation with 5F4 and the immune complex used as positive control (pos). c , mTIM-3 interacts with mCEACAM1 in mouse T cells. Splenocytes from Ceacam1 4S Tg Ceacam1 −/− and Ceacam1-4L Tg Ceacam1 −/− mice cultured with anti-CD3 (1 μg ml −1 ) or anti-CD3 (1 μg ml −1 ) and anti-CD28 (1 μg ml −1 ) or medium for 96 h. Cell lysates immunoprecipitated with anti-mCEACAM1 antibody (cc1) or with mIgG and IB with 5D12 (anti-mTIM-3 antibody) are shown. Locations of mTIM-3 protein variants are indicated. CHO, carbohydrate. d , Immunoprecipitation and immunoblot as in a with tunicamycin treated, wild-type HA–hTIM-3 and Flag–hCEACAM1 co-transfected HEK293T cells. Arrowhead denotes core CEACAM1 protein. e , Potential hCEACAM1-interacting residues on hTIM-3 highlighted in blue. f , HEK293 T cells transiently co-transfected with Flag–hCEACAM1 and HA–hTIM-3 mutants. Immunoblotting of anti-HA were used to analyse hTIM-3 expression in HEK293T transfectants. Except for Pro50Ala mutation displaying enhanced overall protein expression, all other mutations in the IgV domain of hTIM-3 are equally detected by anti-HA antibody. g , Quantification of association of hTIM-3 mutants associated with wild-type hCEACAM1 shown in summing all experiments performed. Association between wild-type hCEACAM1 and hTIM-3 core protein are depicted as reference (set as 1, n = 3, mean ± s.e.m. shown, unpaired Student’s t -test). h , Immunoprecipitation with anti-Flag (hCEACAM1) and immunoblot with anti-HA (hTIM-3) or anti-Flag of wild-type hCEACAM1 and mutant hTIM-3 proteins are shown. i , Quantification of h as performed in g. j , HEK293T cells co-transfected with Flag–hCEACAM1 wild-type and HA– hTIM-3 mutants and immunoprecipitation/immunblot as in h revealing no effects of Cys52Ala or Cys63Ala mutations in hTIM-3 in affecting association with hCEACAM1 in contrast to Cys109Ala mutation of hTIM-3 that disrupts interactions with hCEACAM1. k , Potential hTIM-3-interacting-residues around the FG–CC′ cleft of hCEACAM1 highlighted in red. l , HEK293T cells transiently co-transfected with Flag–hCEACAM1 mutants and wild-type HA–hTIM-3. Immunoblot with anti-Flag antibody was used to analyse hCEACAM1 expression in HEK293T co-transfectants. All hCEACAM1 mutations in IgV domain equally detected. m . n–p , Analysis of Gly47Ala mutation of hCEACAM1 in hTIM-3 co-transfected HEK293T cells by immunoprecipitation with anti-HA (hTIM-3) and immunoblot with anti-Flag (hCEACAM1) to detect association ( n ), IB with anti-Flag to confirm similarity of hCEACAM1 transfection ( o ) and quantification of associated hCEACAM1 of n as shown in m. q–s , Analysis of hCEACAM1 mutants Asn42Ala and Arg43Ala association with hTIM-3 ( q ), similarity of transfections ( r ) and quantification of q as in n–p . Representative of four ( d, h ), three ( f, g, i, l–s ), two ( a–c ) and one ( j ) independent experiments. * P
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    Thermo Fisher il 12p70
    Rv2882c induced BMDM activation. (A) BMDMs (1 × 10 5 /well) were stimulated with 100 ng/mL LPS or 1, 5, or 10 μg/mL Rv2882c for 24 h. (A) Quantities of TNF-α, MCP-1, IL-6, IL-10, and <t>IL-12p70</t> in the culture supernatant were determined by ELISA. All data are expressed as the mean values ± SD ( n = 3). Significance levels (* p
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    Number of brain MNCs and their CNS antigen-specific autoreactive phenotype 14 d after MCAo. A , Number of MNCs infiltrating ipsilateral and contralateral hemisphere of the brain in vehicle and SNS/HPA blockade group (three individual experiments; vehicle, N = 15; SNS/HPA block, N = 20). B–D , Elispot experiments. B , Higher number of total brain MNCs responded to pMOG 35–55 stimulation with IFN-γ production (Th1 cells) per equal number of brain MNCs in SNS/HPA block group compared with the vehicle group. C , A similar effect was observed in ipsilateral hemisphere MNCs when only the SNS axis of SIDS was blocked with propranolol (two experiments; vehicle group, N = 5–8; SNS block, N = 6 or 7). D , Number of splenocytes reacting to pMOG 35–55 stimulation with IFN-γ, IL-4, or IL-17 secretion did not differ between the groups. E , F , ELISA experiments. TGF-β1, IL-10, IL-4, IL-17, and IFN-γ secretion (pg/ml) by pMOG 35–55 -stimulated brain MNCs, ipsilateral hemisphere ( E ) and by pMOG 35–55 -stimulated splenocytes ( F ).

    Journal: The Journal of Neuroscience

    Article Title: Blocking Stroke-Induced Immunodeficiency Increases CNS Antigen-Specific Autoreactivity But Does Not Worsen Functional Outcome after Experimental Stroke

    doi: 10.1523/JNEUROSCI.1532-14.2015

    Figure Lengend Snippet: Number of brain MNCs and their CNS antigen-specific autoreactive phenotype 14 d after MCAo. A , Number of MNCs infiltrating ipsilateral and contralateral hemisphere of the brain in vehicle and SNS/HPA blockade group (three individual experiments; vehicle, N = 15; SNS/HPA block, N = 20). B–D , Elispot experiments. B , Higher number of total brain MNCs responded to pMOG 35–55 stimulation with IFN-γ production (Th1 cells) per equal number of brain MNCs in SNS/HPA block group compared with the vehicle group. C , A similar effect was observed in ipsilateral hemisphere MNCs when only the SNS axis of SIDS was blocked with propranolol (two experiments; vehicle group, N = 5–8; SNS block, N = 6 or 7). D , Number of splenocytes reacting to pMOG 35–55 stimulation with IFN-γ, IL-4, or IL-17 secretion did not differ between the groups. E , F , ELISA experiments. TGF-β1, IL-10, IL-4, IL-17, and IFN-γ secretion (pg/ml) by pMOG 35–55 -stimulated brain MNCs, ipsilateral hemisphere ( E ) and by pMOG 35–55 -stimulated splenocytes ( F ).

    Article Snippet: Mouse multiplex kit (Millipore) and Human/Mouse TGF-β1 ELISA Ready-SET-Go kit (Affymetrix) were used according to the manufacturer's recommendations.

    Techniques: Blocking Assay, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay

    Evaluating the replication kinetics of, and level of interferon activation by, WT MHV and DUBmut in cell culture. (A) Replication kinetics of WT and DUBmut virus in DBT cells. (B) IFN-α11 mRNA levels in WT- and DUBmut-infected BMDMs were assessed at indicated time points by reverse transcription-quantitative PCR (qRT-PCR). (C) IFN-α protein levels in the supernatants of infected BMDMs were evaluated at the times indicated. (D) Comparison of IFN-α11 mRNA levels in B6 versus MDA5 −/− BMDMs at 12 h postinfection (h p.i.). (E) Assessment of levels of viral nucleocapsid (N) mRNA by qRT-PCR. (F) Replication kinetics of WT and DUBmut virus in BMDM cells. Data are representative of at least two independent experiments and are presented as means ± SD. Data in panels B and C were statistically analyzed using unpaired t tests. *, P

    Journal: Journal of Virology

    Article Title: Structure-Guided Mutagenesis Alters Deubiquitinating Activity and Attenuates Pathogenesis of a Murine Coronavirus

    doi: 10.1128/JVI.01734-19

    Figure Lengend Snippet: Evaluating the replication kinetics of, and level of interferon activation by, WT MHV and DUBmut in cell culture. (A) Replication kinetics of WT and DUBmut virus in DBT cells. (B) IFN-α11 mRNA levels in WT- and DUBmut-infected BMDMs were assessed at indicated time points by reverse transcription-quantitative PCR (qRT-PCR). (C) IFN-α protein levels in the supernatants of infected BMDMs were evaluated at the times indicated. (D) Comparison of IFN-α11 mRNA levels in B6 versus MDA5 −/− BMDMs at 12 h postinfection (h p.i.). (E) Assessment of levels of viral nucleocapsid (N) mRNA by qRT-PCR. (F) Replication kinetics of WT and DUBmut virus in BMDM cells. Data are representative of at least two independent experiments and are presented as means ± SD. Data in panels B and C were statistically analyzed using unpaired t tests. *, P

    Article Snippet: The secreted amount of IFN-α in culture supernatants was assayed using a mouse IFN-α ELISA kit (catalog no. BMS6027; eBioscience) according to the manufacturer’s instructions.

    Techniques: Activation Assay, Cell Culture, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Biochemical characterization of interactions between CEACAM1 and TIM-3 a , hTIM-3 does not co-immunoprecipitate (co-IP) with ITGA5 despite interactions with hCEACAM1. HEK293T cells transfected with Flag–ITGA5 and HA–TIM-3 (ITGA5Tw) or Flag–CEACAM1 and HA–TIM-3 (CwTw). Immunoprecipitation with anti-HA antibody and immunoblotted (IB) with anti-Flag antibody are shown. Input represents anti-Flag immunoblot of lysates. b, Co-immunoprecipitation of human TIM-3 and CEACAM1 from activated primary human T cells after N -glycanase treatment of lystates followed by immunoprecipitation with anti-human TIM-3 antibodies (2E2, 2E12 or 3F9) or IgG as control and immunoblotted with anti-human CEACAM1 antibody (5F4). Protein lystates from HeLa-CEACAM1 transfectants treated with N -glycanase followed by immunoprecipitation with 5F4 and the immune complex used as positive control (pos). c , mTIM-3 interacts with mCEACAM1 in mouse T cells. Splenocytes from Ceacam1 4S Tg Ceacam1 −/− and Ceacam1-4L Tg Ceacam1 −/− mice cultured with anti-CD3 (1 μg ml −1 ) or anti-CD3 (1 μg ml −1 ) and anti-CD28 (1 μg ml −1 ) or medium for 96 h. Cell lysates immunoprecipitated with anti-mCEACAM1 antibody (cc1) or with mIgG and IB with 5D12 (anti-mTIM-3 antibody) are shown. Locations of mTIM-3 protein variants are indicated. CHO, carbohydrate. d , Immunoprecipitation and immunoblot as in a with tunicamycin treated, wild-type HA–hTIM-3 and Flag–hCEACAM1 co-transfected HEK293T cells. Arrowhead denotes core CEACAM1 protein. e , Potential hCEACAM1-interacting residues on hTIM-3 highlighted in blue. f , HEK293 T cells transiently co-transfected with Flag–hCEACAM1 and HA–hTIM-3 mutants. Immunoblotting of anti-HA were used to analyse hTIM-3 expression in HEK293T transfectants. Except for Pro50Ala mutation displaying enhanced overall protein expression, all other mutations in the IgV domain of hTIM-3 are equally detected by anti-HA antibody. g , Quantification of association of hTIM-3 mutants associated with wild-type hCEACAM1 shown in summing all experiments performed. Association between wild-type hCEACAM1 and hTIM-3 core protein are depicted as reference (set as 1, n = 3, mean ± s.e.m. shown, unpaired Student’s t -test). h , Immunoprecipitation with anti-Flag (hCEACAM1) and immunoblot with anti-HA (hTIM-3) or anti-Flag of wild-type hCEACAM1 and mutant hTIM-3 proteins are shown. i , Quantification of h as performed in g. j , HEK293T cells co-transfected with Flag–hCEACAM1 wild-type and HA– hTIM-3 mutants and immunoprecipitation/immunblot as in h revealing no effects of Cys52Ala or Cys63Ala mutations in hTIM-3 in affecting association with hCEACAM1 in contrast to Cys109Ala mutation of hTIM-3 that disrupts interactions with hCEACAM1. k , Potential hTIM-3-interacting-residues around the FG–CC′ cleft of hCEACAM1 highlighted in red. l , HEK293T cells transiently co-transfected with Flag–hCEACAM1 mutants and wild-type HA–hTIM-3. Immunoblot with anti-Flag antibody was used to analyse hCEACAM1 expression in HEK293T co-transfectants. All hCEACAM1 mutations in IgV domain equally detected. m . n–p , Analysis of Gly47Ala mutation of hCEACAM1 in hTIM-3 co-transfected HEK293T cells by immunoprecipitation with anti-HA (hTIM-3) and immunoblot with anti-Flag (hCEACAM1) to detect association ( n ), IB with anti-Flag to confirm similarity of hCEACAM1 transfection ( o ) and quantification of associated hCEACAM1 of n as shown in m. q–s , Analysis of hCEACAM1 mutants Asn42Ala and Arg43Ala association with hTIM-3 ( q ), similarity of transfections ( r ) and quantification of q as in n–p . Representative of four ( d, h ), three ( f, g, i, l–s ), two ( a–c ) and one ( j ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: Biochemical characterization of interactions between CEACAM1 and TIM-3 a , hTIM-3 does not co-immunoprecipitate (co-IP) with ITGA5 despite interactions with hCEACAM1. HEK293T cells transfected with Flag–ITGA5 and HA–TIM-3 (ITGA5Tw) or Flag–CEACAM1 and HA–TIM-3 (CwTw). Immunoprecipitation with anti-HA antibody and immunoblotted (IB) with anti-Flag antibody are shown. Input represents anti-Flag immunoblot of lysates. b, Co-immunoprecipitation of human TIM-3 and CEACAM1 from activated primary human T cells after N -glycanase treatment of lystates followed by immunoprecipitation with anti-human TIM-3 antibodies (2E2, 2E12 or 3F9) or IgG as control and immunoblotted with anti-human CEACAM1 antibody (5F4). Protein lystates from HeLa-CEACAM1 transfectants treated with N -glycanase followed by immunoprecipitation with 5F4 and the immune complex used as positive control (pos). c , mTIM-3 interacts with mCEACAM1 in mouse T cells. Splenocytes from Ceacam1 4S Tg Ceacam1 −/− and Ceacam1-4L Tg Ceacam1 −/− mice cultured with anti-CD3 (1 μg ml −1 ) or anti-CD3 (1 μg ml −1 ) and anti-CD28 (1 μg ml −1 ) or medium for 96 h. Cell lysates immunoprecipitated with anti-mCEACAM1 antibody (cc1) or with mIgG and IB with 5D12 (anti-mTIM-3 antibody) are shown. Locations of mTIM-3 protein variants are indicated. CHO, carbohydrate. d , Immunoprecipitation and immunoblot as in a with tunicamycin treated, wild-type HA–hTIM-3 and Flag–hCEACAM1 co-transfected HEK293T cells. Arrowhead denotes core CEACAM1 protein. e , Potential hCEACAM1-interacting residues on hTIM-3 highlighted in blue. f , HEK293 T cells transiently co-transfected with Flag–hCEACAM1 and HA–hTIM-3 mutants. Immunoblotting of anti-HA were used to analyse hTIM-3 expression in HEK293T transfectants. Except for Pro50Ala mutation displaying enhanced overall protein expression, all other mutations in the IgV domain of hTIM-3 are equally detected by anti-HA antibody. g , Quantification of association of hTIM-3 mutants associated with wild-type hCEACAM1 shown in summing all experiments performed. Association between wild-type hCEACAM1 and hTIM-3 core protein are depicted as reference (set as 1, n = 3, mean ± s.e.m. shown, unpaired Student’s t -test). h , Immunoprecipitation with anti-Flag (hCEACAM1) and immunoblot with anti-HA (hTIM-3) or anti-Flag of wild-type hCEACAM1 and mutant hTIM-3 proteins are shown. i , Quantification of h as performed in g. j , HEK293T cells co-transfected with Flag–hCEACAM1 wild-type and HA– hTIM-3 mutants and immunoprecipitation/immunblot as in h revealing no effects of Cys52Ala or Cys63Ala mutations in hTIM-3 in affecting association with hCEACAM1 in contrast to Cys109Ala mutation of hTIM-3 that disrupts interactions with hCEACAM1. k , Potential hTIM-3-interacting-residues around the FG–CC′ cleft of hCEACAM1 highlighted in red. l , HEK293T cells transiently co-transfected with Flag–hCEACAM1 mutants and wild-type HA–hTIM-3. Immunoblot with anti-Flag antibody was used to analyse hCEACAM1 expression in HEK293T co-transfectants. All hCEACAM1 mutations in IgV domain equally detected. m . n–p , Analysis of Gly47Ala mutation of hCEACAM1 in hTIM-3 co-transfected HEK293T cells by immunoprecipitation with anti-HA (hTIM-3) and immunoblot with anti-Flag (hCEACAM1) to detect association ( n ), IB with anti-Flag to confirm similarity of hCEACAM1 transfection ( o ) and quantification of associated hCEACAM1 of n as shown in m. q–s , Analysis of hCEACAM1 mutants Asn42Ala and Arg43Ala association with hTIM-3 ( q ), similarity of transfections ( r ) and quantification of q as in n–p . Representative of four ( d, h ), three ( f, g, i, l–s ), two ( a–c ) and one ( j ) independent experiments. * P

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Positive Control, Mouse Assay, Cell Culture, Expressing, Mutagenesis

    Structural similarities between CEACAM1 and TIM-3 IgV-like N-terminal domains and biochemical association a–c , Interaction between TIM-3–Ig fusion protein and membrane protein of 60 kDa after deglycosylation derived from surface-biotinylated TK-1 cells. TIM-3–Ig fusion proteins and human IgG-precipitated proteins were deglycosylated by PNGase F and separated by SDS–PAGE. TIM-3–Ig-binding membrane proteins detected by immunoblot. A 60-kDa membrane protein (red circles) and 32-kDa protein consistent with galectin-9 (black circles) are found specifically associated with soluble (s) TIM-3–Ig fusion protein (a, lane 5) and full-length (f) TIM-3–Ig proteins ( b , lane 5), but not with the pre-clear controls (lanes 3 and 4) or human IgG (lanes 2 and 6). c , sTIM-3–Ig and full-length (fl) TIM-3–Ig interacting proteins were de-glycosylated by PNGase F and separated by SDS–PAGE. Proteins detected by silver staining. A band of 60kDa (red circle) isspecifically associated with sTIM-3–Ig proteins (lane 2), but not with human IgG (lane 5), or TIM-1–Ig or TIM-4–Ig (lanes 3, 4, 6–10). d , Superimposition of previously described IgV-like domains of mCEACAM1 and mTIM-3 demonstrate structural similarity with a score of 2.42 by the structural alignment and root mean square deviation (r.m.s.d.) calculated by Pymol. e , Sequence alignment of the IgV-like domains of mCEACAM1 and mTIM-3 on the basis of the secondary structure alignment in d. f , Sequence alignments of IgV domain sequences of CEACAM1 and overall mTIM and hTIM family members. α helices (orange) and β strands (blue) denoted as underlined segments in hCEACAM1 and mTIM-3. β strands labelled with upper- and lower-case letters for hCEACAM1 and mTIM-3, respectively. Conserved residues are shaded red. Mutated residues are shaded violet for hCEACAM1, and green for hTIM-3. Asterisk (*) indicates positions having a single, fully conserved residue; a dagger (†) indicates conservation between groups of weakly similar residues; a double-dagger (‡) indicates conservation between groups of strongly similar residues. g , Computational modelling as defined by energy calculations (score) relative to r.m.s. values of docking models to define potential cis and trans and amino acids involved. h , CEACAM1 expression on mouse fibroblast 3T3 cells used to identify a galectin-9-independent ligand. i , CEACAM1 expression on mouse TK-1 cells as in a–c . Representative of three ( a–c, h, i ) independent experiments.

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: Structural similarities between CEACAM1 and TIM-3 IgV-like N-terminal domains and biochemical association a–c , Interaction between TIM-3–Ig fusion protein and membrane protein of 60 kDa after deglycosylation derived from surface-biotinylated TK-1 cells. TIM-3–Ig fusion proteins and human IgG-precipitated proteins were deglycosylated by PNGase F and separated by SDS–PAGE. TIM-3–Ig-binding membrane proteins detected by immunoblot. A 60-kDa membrane protein (red circles) and 32-kDa protein consistent with galectin-9 (black circles) are found specifically associated with soluble (s) TIM-3–Ig fusion protein (a, lane 5) and full-length (f) TIM-3–Ig proteins ( b , lane 5), but not with the pre-clear controls (lanes 3 and 4) or human IgG (lanes 2 and 6). c , sTIM-3–Ig and full-length (fl) TIM-3–Ig interacting proteins were de-glycosylated by PNGase F and separated by SDS–PAGE. Proteins detected by silver staining. A band of 60kDa (red circle) isspecifically associated with sTIM-3–Ig proteins (lane 2), but not with human IgG (lane 5), or TIM-1–Ig or TIM-4–Ig (lanes 3, 4, 6–10). d , Superimposition of previously described IgV-like domains of mCEACAM1 and mTIM-3 demonstrate structural similarity with a score of 2.42 by the structural alignment and root mean square deviation (r.m.s.d.) calculated by Pymol. e , Sequence alignment of the IgV-like domains of mCEACAM1 and mTIM-3 on the basis of the secondary structure alignment in d. f , Sequence alignments of IgV domain sequences of CEACAM1 and overall mTIM and hTIM family members. α helices (orange) and β strands (blue) denoted as underlined segments in hCEACAM1 and mTIM-3. β strands labelled with upper- and lower-case letters for hCEACAM1 and mTIM-3, respectively. Conserved residues are shaded red. Mutated residues are shaded violet for hCEACAM1, and green for hTIM-3. Asterisk (*) indicates positions having a single, fully conserved residue; a dagger (†) indicates conservation between groups of weakly similar residues; a double-dagger (‡) indicates conservation between groups of strongly similar residues. g , Computational modelling as defined by energy calculations (score) relative to r.m.s. values of docking models to define potential cis and trans and amino acids involved. h , CEACAM1 expression on mouse fibroblast 3T3 cells used to identify a galectin-9-independent ligand. i , CEACAM1 expression on mouse TK-1 cells as in a–c . Representative of three ( a–c, h, i ) independent experiments.

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Derivative Assay, SDS Page, Binding Assay, Silver Staining, Sequencing, Expressing

    CEACAM1 is essential for TIM-3 mediated T cell tolerance a , Schematic diagram of OVA antigen-specific tolerance induction model. b , Schematic diagram of OVA immunization. c , Tracking in vivo antigen-specific T-cell responses of CFSE-labelled OT-II transgenic Rag2 −/− T cells in total lymphocyte gate of mesenteric lymph nodes, peripheral lymph node or spleen of wild-type or Ceacam1 −/− recipients after gating on CFSE-positive cells and staining for CEACAM1 in PBS and OVA 323–339 immunized mice. Hyper-responsiveness of OT-II transgenic Rag2 −/− T cells in Ceacam1 −/− mice was not due to decreased regulatory T-cell induction (data not shown) or increased initial parking on the basis of cell numbers shown. d , TIM-3 expression on CEACAM1-positive and -negative CFSE + cells as in c. e , Schematic diagram of SEB-induced T-cell tolerance model. f , mCEACAM1 and mTIM-3 expression on CD4 + Vβ8 + T cells after SEB tolerance induction. g , hCEACAM1 and hTIM-3 expression on activated primary human T cells defined by staining with indicated antibodies. h , CEACAM1 expression on TIM-3-silenced primary human T cells after re-activation by flow cytometry. Relative TIM-3, CEACAM1 or CD4 expression on T cells expressing control shRNA ( lacZ control, red) or three independent shRNAs directed at TIM3 (overlay, blue). shRNA target sequences shown. i – l , CEACAM1 and TIM-3 expression and functional consequences on T cells in HIV infection. CD4 + IFN-γ + T cells are decreased among CEACAM1 + TIM-3 + CD4 + T cells in HIV infection in response to Gag peptides ( i ). Although proportions of CEACAM1 + TIM-3 + CD8 + T cells are similar in HIV-infected and -uninfected subjects ( j ), CEACAM1 + TIM-3 + CD8 + T cells express little IFN-γ after stimulation with HIV Gag peptides or SEB relative to TIM-3 + CEACAM1 − CD8 + T cells ( k, l ). C, hCEACAM1; T, hTIM-3 ( n = 4 per group, mean ± s.e.m.). m – o , In situ proximity ligation analysis (PLA) of CEACAM1 and TIM-3. m , HEK293T cells transiently co-transfected with Flag–hCEACAM1 or HA–hTIM-3. Cells stained with DAPI (left), anti-tubulin (middle), anti-HA (rabbit) and anti-Flag (mouse) (middle right) or merged (right). Several examples of a positive PLA signal (middle right and right panels: red fluorescent dots) indicative of a maximum distance of 30–40 nm between hCEACAM1 and hTIM-3. n , Negative control, co-expression of Flag–PLK1 (protein kinase I) and HA–TIM-3 failed to generate fluorescent dots (that is, PLA negative). Cells stained with DAPI, anti-tubulin, anti-HA/anti-Flag or merged as in m. o , Negative control, co-expression of HA–ADAP (adhesion and degranulation promoting adaptor protein) failed to show a signal (that is, PLA negative) with staining as in m. p, q , CEACAM1 and TIM-3 colocalization at immunological synapse of primary human CD4 and CD8 T cells. Confocal microscopy of hTIM-3 + hCEACAM1 + primary CD4 + and CD8 + T cells forming conjugates with SEB-loaded B cells. DIC, differential interference contrast. Blue denotes B cell; red denotes CD3; purple denotes CEACAM1; green denotes TIM-3. White indicates colocalization between CEACAM1 and TIM-3 ( p ). Average Pearson correlation coefficients for CD4 + and CD8 + T cells were 0.543 and 0.566, respectively, representing strong co-localization ( q ). Data are mean ± s.e.m. and representative of five ( f, g ), four ( p, q ), three ( c, d, m–o ) and two ( h ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: CEACAM1 is essential for TIM-3 mediated T cell tolerance a , Schematic diagram of OVA antigen-specific tolerance induction model. b , Schematic diagram of OVA immunization. c , Tracking in vivo antigen-specific T-cell responses of CFSE-labelled OT-II transgenic Rag2 −/− T cells in total lymphocyte gate of mesenteric lymph nodes, peripheral lymph node or spleen of wild-type or Ceacam1 −/− recipients after gating on CFSE-positive cells and staining for CEACAM1 in PBS and OVA 323–339 immunized mice. Hyper-responsiveness of OT-II transgenic Rag2 −/− T cells in Ceacam1 −/− mice was not due to decreased regulatory T-cell induction (data not shown) or increased initial parking on the basis of cell numbers shown. d , TIM-3 expression on CEACAM1-positive and -negative CFSE + cells as in c. e , Schematic diagram of SEB-induced T-cell tolerance model. f , mCEACAM1 and mTIM-3 expression on CD4 + Vβ8 + T cells after SEB tolerance induction. g , hCEACAM1 and hTIM-3 expression on activated primary human T cells defined by staining with indicated antibodies. h , CEACAM1 expression on TIM-3-silenced primary human T cells after re-activation by flow cytometry. Relative TIM-3, CEACAM1 or CD4 expression on T cells expressing control shRNA ( lacZ control, red) or three independent shRNAs directed at TIM3 (overlay, blue). shRNA target sequences shown. i – l , CEACAM1 and TIM-3 expression and functional consequences on T cells in HIV infection. CD4 + IFN-γ + T cells are decreased among CEACAM1 + TIM-3 + CD4 + T cells in HIV infection in response to Gag peptides ( i ). Although proportions of CEACAM1 + TIM-3 + CD8 + T cells are similar in HIV-infected and -uninfected subjects ( j ), CEACAM1 + TIM-3 + CD8 + T cells express little IFN-γ after stimulation with HIV Gag peptides or SEB relative to TIM-3 + CEACAM1 − CD8 + T cells ( k, l ). C, hCEACAM1; T, hTIM-3 ( n = 4 per group, mean ± s.e.m.). m – o , In situ proximity ligation analysis (PLA) of CEACAM1 and TIM-3. m , HEK293T cells transiently co-transfected with Flag–hCEACAM1 or HA–hTIM-3. Cells stained with DAPI (left), anti-tubulin (middle), anti-HA (rabbit) and anti-Flag (mouse) (middle right) or merged (right). Several examples of a positive PLA signal (middle right and right panels: red fluorescent dots) indicative of a maximum distance of 30–40 nm between hCEACAM1 and hTIM-3. n , Negative control, co-expression of Flag–PLK1 (protein kinase I) and HA–TIM-3 failed to generate fluorescent dots (that is, PLA negative). Cells stained with DAPI, anti-tubulin, anti-HA/anti-Flag or merged as in m. o , Negative control, co-expression of HA–ADAP (adhesion and degranulation promoting adaptor protein) failed to show a signal (that is, PLA negative) with staining as in m. p, q , CEACAM1 and TIM-3 colocalization at immunological synapse of primary human CD4 and CD8 T cells. Confocal microscopy of hTIM-3 + hCEACAM1 + primary CD4 + and CD8 + T cells forming conjugates with SEB-loaded B cells. DIC, differential interference contrast. Blue denotes B cell; red denotes CD3; purple denotes CEACAM1; green denotes TIM-3. White indicates colocalization between CEACAM1 and TIM-3 ( p ). Average Pearson correlation coefficients for CD4 + and CD8 + T cells were 0.543 and 0.566, respectively, representing strong co-localization ( q ). Data are mean ± s.e.m. and representative of five ( f, g ), four ( p, q ), three ( c, d, m–o ) and two ( h ) independent experiments. * P

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: In Vivo, Transgenic Assay, Staining, Mouse Assay, Expressing, Activation Assay, Flow Cytometry, Cytometry, shRNA, Functional Assay, Infection, In Situ, Ligation, Proximity Ligation Assay, Transfection, Negative Control, Negative Staining, Confocal Microscopy

    CEACAM1 determines TIM-3 expression and function a , HEK293T cells transiently co-transfected with Flag–hCEACAM1 and wild-type or mutants of HA–hTIM-3. Flow cytometry detecting HA–hTIM-3 (detected with anti-HA) and Flag–hCEACAM1 (detected with 5F4) proteins at cell surface (top), Golgi apparatus (middle) or endoplasmic reticulum (bottom) using monensin and brefeldin A, respectively. b , Cellular distribution of wild-type or mutant hTIM-3 when co-expressed with wild-type hCEACAM1. Total counts of hTIM-3 at surface, Golgi apparatus and endoplasmic reticulum summed up to 100%. Depicted as percentage of hTIM-3. c , HEK293T cells transiently co-transfected with wild-type HA–hTIM-3 (detected with 2E2) and wild-type or mutant Flag–hCEACAM1 (detected with anti-Flag). Flow cytometry analyses as in a. d , Cellular distribution of c , as in b . Depicted as percentage of hTIM-3. e , Immunoblot for wild-type or Thr101Ile variant of hTIM-3 showing maturation status in presence of wild-type or mutated (Gln44Leu) hCEACAM1. f , Normal association of Thr101Ile variant of hTIM-3 with hCEACAM1. g , Analysis of CD4 + Vβ8 + T cells after SEB tolerance induction from experimental mice of indicated genotypes. h , Galectin-9 induction of apoptosis. Annexin V + propidium iodide staining of T H 1 cells polarized from Tim3 Tg or Tim3 Tg Ceacam1 −/− mice after treatment with galectin-9 (2 μg ml −1 ) for 8 h. Note decreased apoptosis in Tim3 Tg Ceacam1 −/− T cells. i , Schematic diagram of protocol used for protein pull-down using in-column IgV domain of GST–hTIM-3 incubated with hCEACAM1 protein derived from transfected HEK293T cells as in . j , GST or GST–hTIM-3 staining of hCEACAM1-4L–transfected Jurkat T cells. k , Wild-type CD4 + T cells stimulated with anti-CD3 and/or anti-CD28 in the presence or absence of mCEACAM1 NFc, or IgG1-Fc as control, and cells analysed for secretion of IFN-γ and IL-2. l, m , Characterization of tolerance in SEB model. Tim3 Tg (l) and Tim3 Tg Ceacam1 −/− ( m . Lymph node cells collected after SEB treatment and re-stimulated with soluble anti-CD3 at indicated doses and IL-2 measured by ELISA after 72 h. Note tolerance in Tim3 Tg but not Tim3 Tg Ceacam1 −/− mice. n = 3 per group. n , Anti-mTIM-3 blockade with 2C12 antibody of mCEACAM1 NFc or control IgG-Fc staining of CD4 + T cells from indicated genotypes expressed as levels relative to Ceacam1 −/− mice. o, p , Analysis of mTIM-3 cytoplasmic tail function in transmitting mCEACAM1-induced signals. Activated mouse CD4 + T cells from wild-type ( o ) or Ceacam1 −/− ( p ) mice were retrovirally transduced, sorted and stimulated with anti-CD3 with either human IgG-Fc (IgG, control) or mCEACAM1 N-terminal domain as NFc and TNF-α secretion assessed by ELISA after 72 h. Note ability of CEACAM1 N-terminal domain to transduce a signal associated with inhibition of TNF-α secretion in wild-type but not Ceacam1 −/− T cells. n = 3 per group. Data are mean ± s.e.m. and represent three ( f, g, k–p ) and two ( a–e, h, j ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: CEACAM1 determines TIM-3 expression and function a , HEK293T cells transiently co-transfected with Flag–hCEACAM1 and wild-type or mutants of HA–hTIM-3. Flow cytometry detecting HA–hTIM-3 (detected with anti-HA) and Flag–hCEACAM1 (detected with 5F4) proteins at cell surface (top), Golgi apparatus (middle) or endoplasmic reticulum (bottom) using monensin and brefeldin A, respectively. b , Cellular distribution of wild-type or mutant hTIM-3 when co-expressed with wild-type hCEACAM1. Total counts of hTIM-3 at surface, Golgi apparatus and endoplasmic reticulum summed up to 100%. Depicted as percentage of hTIM-3. c , HEK293T cells transiently co-transfected with wild-type HA–hTIM-3 (detected with 2E2) and wild-type or mutant Flag–hCEACAM1 (detected with anti-Flag). Flow cytometry analyses as in a. d , Cellular distribution of c , as in b . Depicted as percentage of hTIM-3. e , Immunoblot for wild-type or Thr101Ile variant of hTIM-3 showing maturation status in presence of wild-type or mutated (Gln44Leu) hCEACAM1. f , Normal association of Thr101Ile variant of hTIM-3 with hCEACAM1. g , Analysis of CD4 + Vβ8 + T cells after SEB tolerance induction from experimental mice of indicated genotypes. h , Galectin-9 induction of apoptosis. Annexin V + propidium iodide staining of T H 1 cells polarized from Tim3 Tg or Tim3 Tg Ceacam1 −/− mice after treatment with galectin-9 (2 μg ml −1 ) for 8 h. Note decreased apoptosis in Tim3 Tg Ceacam1 −/− T cells. i , Schematic diagram of protocol used for protein pull-down using in-column IgV domain of GST–hTIM-3 incubated with hCEACAM1 protein derived from transfected HEK293T cells as in . j , GST or GST–hTIM-3 staining of hCEACAM1-4L–transfected Jurkat T cells. k , Wild-type CD4 + T cells stimulated with anti-CD3 and/or anti-CD28 in the presence or absence of mCEACAM1 NFc, or IgG1-Fc as control, and cells analysed for secretion of IFN-γ and IL-2. l, m , Characterization of tolerance in SEB model. Tim3 Tg (l) and Tim3 Tg Ceacam1 −/− ( m . Lymph node cells collected after SEB treatment and re-stimulated with soluble anti-CD3 at indicated doses and IL-2 measured by ELISA after 72 h. Note tolerance in Tim3 Tg but not Tim3 Tg Ceacam1 −/− mice. n = 3 per group. n , Anti-mTIM-3 blockade with 2C12 antibody of mCEACAM1 NFc or control IgG-Fc staining of CD4 + T cells from indicated genotypes expressed as levels relative to Ceacam1 −/− mice. o, p , Analysis of mTIM-3 cytoplasmic tail function in transmitting mCEACAM1-induced signals. Activated mouse CD4 + T cells from wild-type ( o ) or Ceacam1 −/− ( p ) mice were retrovirally transduced, sorted and stimulated with anti-CD3 with either human IgG-Fc (IgG, control) or mCEACAM1 N-terminal domain as NFc and TNF-α secretion assessed by ELISA after 72 h. Note ability of CEACAM1 N-terminal domain to transduce a signal associated with inhibition of TNF-α secretion in wild-type but not Ceacam1 −/− T cells. n = 3 per group. Data are mean ± s.e.m. and represent three ( f, g, k–p ) and two ( a–e, h, j ) independent experiments. * P

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Expressing, Transfection, Flow Cytometry, Cytometry, Mutagenesis, Variant Assay, Mouse Assay, Staining, Incubation, Derivative Assay, Enzyme-linked Immunosorbent Assay, Transduction, Inhibition

    Blockade of CEACAM1 and TIM-3 or genetic loss of CEACAM1 increases anti-tumour immunity a , Schematic presentation of antibody blockade protocol described in . b , Schematic presentation of antibody blockade protocol referred to in panel c. c , Prevention of CT26 tumour growth with indicated combinations of antibodies as in ( b ) ( n = 5 per group, post-hoc Dunnett’s correction followed by Friedman test). d , Schematic of schedule used for therapeutic antibody administration as described in e. e , Synergy of CEACAM1 and programmed death-ligand1 (PD-L1) blockade in a therapeutic protocol as described in d was performed in wild-type BALB/c mice that received a subcutaneous inoculation of CT26 tumour cells. Mean tumour size ( n = 5 per group, with linear regression analysis). Note synergistic increase in anti-tumour effect when CEACAM1 and PD-L1 co-blockade was performed. f , TILs were analysed for the relative proportion of CD4 + ( n = 4, unpaired Student’s t -test with Mann–Whitney U correction). g , Percentages of CD8 + T cells from spleen show that antibody treatments have no effects on total CD8 + T cell numbers ( n = 7/8, unpaired two-tailed t -test). h , Negative correlation of the numbers of AH1 tet + CD8 + (Pearson’s correlation coefficient, r = 0.9560, P = 0.044). i , Representative flow cytometry for tumour-specific (AH1-tetramer, tet + ) CD8 + T cells in draining lymph nodes of mice from the indicated genotypes. Data are mean ± s.e.m. and represent three ( f–i ), two ( e ) and one ( c ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: Blockade of CEACAM1 and TIM-3 or genetic loss of CEACAM1 increases anti-tumour immunity a , Schematic presentation of antibody blockade protocol described in . b , Schematic presentation of antibody blockade protocol referred to in panel c. c , Prevention of CT26 tumour growth with indicated combinations of antibodies as in ( b ) ( n = 5 per group, post-hoc Dunnett’s correction followed by Friedman test). d , Schematic of schedule used for therapeutic antibody administration as described in e. e , Synergy of CEACAM1 and programmed death-ligand1 (PD-L1) blockade in a therapeutic protocol as described in d was performed in wild-type BALB/c mice that received a subcutaneous inoculation of CT26 tumour cells. Mean tumour size ( n = 5 per group, with linear regression analysis). Note synergistic increase in anti-tumour effect when CEACAM1 and PD-L1 co-blockade was performed. f , TILs were analysed for the relative proportion of CD4 + ( n = 4, unpaired Student’s t -test with Mann–Whitney U correction). g , Percentages of CD8 + T cells from spleen show that antibody treatments have no effects on total CD8 + T cell numbers ( n = 7/8, unpaired two-tailed t -test). h , Negative correlation of the numbers of AH1 tet + CD8 + (Pearson’s correlation coefficient, r = 0.9560, P = 0.044). i , Representative flow cytometry for tumour-specific (AH1-tetramer, tet + ) CD8 + T cells in draining lymph nodes of mice from the indicated genotypes. Data are mean ± s.e.m. and represent three ( f–i ), two ( e ) and one ( c ) independent experiments. * P

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Mouse Assay, MANN-WHITNEY, Two Tailed Test, Flow Cytometry, Cytometry

    CEACAM1 and TIM-3 heterodimerize and serve as heterophilic ligands a, b , Co-immunoprecipitation (IP) and immunoblot (IB) of wild-type hCEACAM1 and hTIM-3 in co-transfected HEK293T cells, c, d , Co-immunoprecipitation and immunoblot of wild-type hCEACAM1 and hTIM-3 mutants ( c ) or wild-type hTIM-3 and hCEACAM1 mutants ( d ) as in a and b. e , Human CEACAM1 (IgV)-TIM-3 (IgV) heterodimer structure, f, g , 2 F o — F c maps contoured at 0.9σ showing electron densities, h, i , Autoradiogram of anti-haemagglutinin (HA) (hTIM-3) immunoprecipitate from metabolic-labelled ( h ) and pulse-chase metabolic-labelled ( i ) co-transfected HEK293T cells. CHO, carbohydrate; core T, non-glycosylated hTIM-3; Cw, wild-type hCEACAM1; EndoH, endoglycosidaseH; H2-MA, HA-tagged influenza virus A M2 protein; T, hTIM-3 (Thr101Ile); Tw, wild-type hTIM-3. hTIM-3 isoforms noted. j , Quantification of densities in i ( n = 3 per group). k , Immunoblot for mTIM-3 from PBS-treated (−) or SEB-treated (+) CD4 + T cells. Labelling as in h and i. 1 , mTIM-3 expression after SEB tolerance induction, m , Column-bound glutathione S -transferase (GST)-hTIM-3 IgV-domain pull-down of hCEACAM1 detected by immunoblot. GST 2 , GST-hTIM-3 dimer. Ft, flow through, n , Suppression of mouse CD4 + T-cell proliferation by mCEACAM1 N-terminal domain-Fc fusion protein (NFc). o , Immunoprecipitation of mTIM-3 and immunoblot for BAT3 or mTIM-3 from lysates of CD4 + T cells. p, q , Proliferation of CD4 + T cells from wild-type ( p ) and CeaCAM1 −/− ( q ) mice transduced with wild-type mTIM-3 (Tw), mTIM-3 Δ252–281 (Tmut) or vector exposed to anti-CD3 and either NFc or IgG1-Fc (IgG1). Data are mean ± s.e.m. and represent five ( a, b ), four ( c, d ), three ( h-j, l, n, p, q ) and two ( k, m, o ) independent experiments. NS, not significant; * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: CEACAM1 and TIM-3 heterodimerize and serve as heterophilic ligands a, b , Co-immunoprecipitation (IP) and immunoblot (IB) of wild-type hCEACAM1 and hTIM-3 in co-transfected HEK293T cells, c, d , Co-immunoprecipitation and immunoblot of wild-type hCEACAM1 and hTIM-3 mutants ( c ) or wild-type hTIM-3 and hCEACAM1 mutants ( d ) as in a and b. e , Human CEACAM1 (IgV)-TIM-3 (IgV) heterodimer structure, f, g , 2 F o — F c maps contoured at 0.9σ showing electron densities, h, i , Autoradiogram of anti-haemagglutinin (HA) (hTIM-3) immunoprecipitate from metabolic-labelled ( h ) and pulse-chase metabolic-labelled ( i ) co-transfected HEK293T cells. CHO, carbohydrate; core T, non-glycosylated hTIM-3; Cw, wild-type hCEACAM1; EndoH, endoglycosidaseH; H2-MA, HA-tagged influenza virus A M2 protein; T, hTIM-3 (Thr101Ile); Tw, wild-type hTIM-3. hTIM-3 isoforms noted. j , Quantification of densities in i ( n = 3 per group). k , Immunoblot for mTIM-3 from PBS-treated (−) or SEB-treated (+) CD4 + T cells. Labelling as in h and i. 1 , mTIM-3 expression after SEB tolerance induction, m , Column-bound glutathione S -transferase (GST)-hTIM-3 IgV-domain pull-down of hCEACAM1 detected by immunoblot. GST 2 , GST-hTIM-3 dimer. Ft, flow through, n , Suppression of mouse CD4 + T-cell proliferation by mCEACAM1 N-terminal domain-Fc fusion protein (NFc). o , Immunoprecipitation of mTIM-3 and immunoblot for BAT3 or mTIM-3 from lysates of CD4 + T cells. p, q , Proliferation of CD4 + T cells from wild-type ( p ) and CeaCAM1 −/− ( q ) mice transduced with wild-type mTIM-3 (Tw), mTIM-3 Δ252–281 (Tmut) or vector exposed to anti-CD3 and either NFc or IgG1-Fc (IgG1). Data are mean ± s.e.m. and represent five ( a, b ), four ( c, d ), three ( h-j, l, n, p, q ) and two ( k, m, o ) independent experiments. NS, not significant; * P

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Immunoprecipitation, Transfection, Pulse Chase, Expressing, Flow Cytometry, Mouse Assay, Transduction, Plasmid Preparation

    TIM-3 regulation of mucosa-associated inflammation requires CEACAM1 a , mCEACAM1 and mTIM-3 expression on colonic lamina propria CD4 + T cells, b , Intracellular cytokines in cells described in a. c, d , mTIM-3 ( c ) and intracellular TNF-α (d) expression in lamina propria CD4 + T cells from indicated donors, e , Body weights relative to weights on day 14 of groups in c and d . Five mice expired (†). f , Score of surviving mice of groups in e. g , Body weights of genotypes as in e. h , Score of groups described in g. i, j , Nanostring ( i ) and quantitative PCR ( j ) of lamina propria mononuclear cells. Actb , β-actin gene. All data are mean ± s.e.m. and represent six ( a ), four ( b ) and three ( c–j ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: TIM-3 regulation of mucosa-associated inflammation requires CEACAM1 a , mCEACAM1 and mTIM-3 expression on colonic lamina propria CD4 + T cells, b , Intracellular cytokines in cells described in a. c, d , mTIM-3 ( c ) and intracellular TNF-α (d) expression in lamina propria CD4 + T cells from indicated donors, e , Body weights relative to weights on day 14 of groups in c and d . Five mice expired (†). f , Score of surviving mice of groups in e. g , Body weights of genotypes as in e. h , Score of groups described in g. i, j , Nanostring ( i ) and quantitative PCR ( j ) of lamina propria mononuclear cells. Actb , β-actin gene. All data are mean ± s.e.m. and represent six ( a ), four ( b ) and three ( c–j ) independent experiments. * P

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    CEACAM1 determines TIM-3 regulation of anti-tumour immune responses a , Survival curves in AOM/2.5% DSS model. b–d , Assessment of polyp numbers ( b ), polyp size ( c ) and cancer grades ( d ) in AOM/1.5% DSS model, e , Staining of CD8 + T cells associated with CT26 tumours. f , Intracellular cytokine expression in TIL subsets after anti-CD3 stimulation. g , Prevention of CT26 tumour growth in wild-type mice ( n = 5 per group). mAb, monoclonal antibody. h–k , Analysis of TILs for relative proportion of CD8 + ( h ) and CD4 + ( i ) T cells, IFN-γ + CD8 + T cells ( j ) and tumour-specific (AH1-tetramer, tet + ) CD8 + T cells in draining lymph nodes (dLN) ( k ) in groups described in g. l–n , Growth of CT26 cells (1), AH1 tet + CD8 + T cells in dLN ( m ) and TIM-3 expression on TILs ( n ) in wild-type and CeaCAM1 −/− mice. Data are mean ± s.e.m. and represent four ( e ), three ( g–k ) and two ( a–d, f, l–n ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: CEACAM1 determines TIM-3 regulation of anti-tumour immune responses a , Survival curves in AOM/2.5% DSS model. b–d , Assessment of polyp numbers ( b ), polyp size ( c ) and cancer grades ( d ) in AOM/1.5% DSS model, e , Staining of CD8 + T cells associated with CT26 tumours. f , Intracellular cytokine expression in TIL subsets after anti-CD3 stimulation. g , Prevention of CT26 tumour growth in wild-type mice ( n = 5 per group). mAb, monoclonal antibody. h–k , Analysis of TILs for relative proportion of CD8 + ( h ) and CD4 + ( i ) T cells, IFN-γ + CD8 + T cells ( j ) and tumour-specific (AH1-tetramer, tet + ) CD8 + T cells in draining lymph nodes (dLN) ( k ) in groups described in g. l–n , Growth of CT26 cells (1), AH1 tet + CD8 + T cells in dLN ( m ) and TIM-3 expression on TILs ( n ) in wild-type and CeaCAM1 −/− mice. Data are mean ± s.e.m. and represent four ( e ), three ( g–k ) and two ( a–d, f, l–n ) independent experiments. * P

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Staining, Expressing, Mouse Assay

    CEACAM1 and TIM-3 cooperatively regulate inflammation and anti-tumour immunity a , Representative haematoxylin and eosin staining of groups described in . Scale bar, 50 μm. b , Flow cytometry for intracellular cytokine assessment of TNF-α expression from infiltrating CD4 + T cells from inflamed colonic lamina propria of Ceacam1 −/− Rag2 −/− recipients, 6weeks after transfer with naive CD4 + CD44 lo CD62L high T cells from indicated genotypes. c , Anorectal prolapse of indicated genotypes. d . Scale bar, 50 μm. e , RNA expression defined by nanostring of lamina propria mononuclear cells in indicated groups (mean of n = 3 per group). f , Schematic overview of protocol for AOM/DSS colitis-associated cancer model. g , Representative haematoxylin and eosin staining of colon from wild-type mice in AOM/1.5% DSS model. Scale bar, 50 μm; h , Representative photograph of distal colons of wild-type mice ( n = 3 per group, anorectal junction at left end) in AOM/1.5% DSS model. Vertical arrows show the sites for dissection of the polyps (black) and the vicinity of the polyps (red). i , Representative flow cytometry analyses on infiltrating lymphocytes of invading distal colonic polyps or from the vicinity of the polyps or from mesenteric lymph nodes for CD4 + and CD8 + T cells and expression of CEACAM1 and TIM-3orPD-1 and TIM-3. Note that vicinity of polyps exhibit highest numbers of T cells with an exhausted phenotype. j , Summary of flow cytometry on infiltrating lymphocytes from invading distal colonic polyps or from vicinity of polyps and from mesenteric lymph nodes for CD4 + and CD8 + T cells expressing CEACAM1 and TIM-3 or PD-1 and TIM-3 ( n = 3, median shown). k , Representative pathology in AOM/1.5% DSS model. Scale bar, 60 μm. HGD, high grade dysplasia. Representative of three independent experiments ( a–e, g–k ). Fig. 3e

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: CEACAM1 and TIM-3 cooperatively regulate inflammation and anti-tumour immunity a , Representative haematoxylin and eosin staining of groups described in . Scale bar, 50 μm. b , Flow cytometry for intracellular cytokine assessment of TNF-α expression from infiltrating CD4 + T cells from inflamed colonic lamina propria of Ceacam1 −/− Rag2 −/− recipients, 6weeks after transfer with naive CD4 + CD44 lo CD62L high T cells from indicated genotypes. c , Anorectal prolapse of indicated genotypes. d . Scale bar, 50 μm. e , RNA expression defined by nanostring of lamina propria mononuclear cells in indicated groups (mean of n = 3 per group). f , Schematic overview of protocol for AOM/DSS colitis-associated cancer model. g , Representative haematoxylin and eosin staining of colon from wild-type mice in AOM/1.5% DSS model. Scale bar, 50 μm; h , Representative photograph of distal colons of wild-type mice ( n = 3 per group, anorectal junction at left end) in AOM/1.5% DSS model. Vertical arrows show the sites for dissection of the polyps (black) and the vicinity of the polyps (red). i , Representative flow cytometry analyses on infiltrating lymphocytes of invading distal colonic polyps or from the vicinity of the polyps or from mesenteric lymph nodes for CD4 + and CD8 + T cells and expression of CEACAM1 and TIM-3orPD-1 and TIM-3. Note that vicinity of polyps exhibit highest numbers of T cells with an exhausted phenotype. j , Summary of flow cytometry on infiltrating lymphocytes from invading distal colonic polyps or from vicinity of polyps and from mesenteric lymph nodes for CD4 + and CD8 + T cells expressing CEACAM1 and TIM-3 or PD-1 and TIM-3 ( n = 3, median shown). k , Representative pathology in AOM/1.5% DSS model. Scale bar, 60 μm. HGD, high grade dysplasia. Representative of three independent experiments ( a–e, g–k ). Fig. 3e

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Staining, Flow Cytometry, Cytometry, Expressing, RNA Expression, Mouse Assay, Dissection

    TIM-3 and CEACAM1 are co-expressed on T cells during induction of tolerance a, b , Tolerance induction in indicated mice. Median c.p.m., counts per minute. c, d , Responses of CFSE-labelled transgenic OT-II Rag2 −/− T cells in mesenteric lymph nodes (MLN), peripheral lymph node (LN) or spleen of wild-type (WT) or Ceacam1 −/− recipients to PBS ( n = 3 per group) or OVA ( n = 5 per group) for proliferation ( c ) and CEACAM1 or TIM-3 ( d ) expression. ND, not detectable. e , hCEACAM1 and hTIM-3 expression in co-transfected HEK293T cells. Percentage and mean fluorescence intensity (MFI) of hTIM-3 indicated. BFA, brefeldin A; ER, endoplasmic reticulum. f , hCEACAM1 and hTIM-3 expression on activated primary CD4 + human T cells. g, h , CEACAM1 + TIM-3 + CD4 + T cells ( g ) and intracellular cytokine staining for IFN-γ in CD4 + T cells after SEB stimulation ( h ) in HIV infection. C, CEACAM1; T, TIM-3 ( n = 4 per group). i , In situ proximity ligation assay of hCEACAM1 and hTIM-3 co-transfected HEK293T as in e . DAPI, 4′,6-diamidino-2-phenylindole. All data are mean ± s.e.m. and represent five ( e, f ), three ( c, d, i ) and two ( a, b ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: TIM-3 and CEACAM1 are co-expressed on T cells during induction of tolerance a, b , Tolerance induction in indicated mice. Median c.p.m., counts per minute. c, d , Responses of CFSE-labelled transgenic OT-II Rag2 −/− T cells in mesenteric lymph nodes (MLN), peripheral lymph node (LN) or spleen of wild-type (WT) or Ceacam1 −/− recipients to PBS ( n = 3 per group) or OVA ( n = 5 per group) for proliferation ( c ) and CEACAM1 or TIM-3 ( d ) expression. ND, not detectable. e , hCEACAM1 and hTIM-3 expression in co-transfected HEK293T cells. Percentage and mean fluorescence intensity (MFI) of hTIM-3 indicated. BFA, brefeldin A; ER, endoplasmic reticulum. f , hCEACAM1 and hTIM-3 expression on activated primary CD4 + human T cells. g, h , CEACAM1 + TIM-3 + CD4 + T cells ( g ) and intracellular cytokine staining for IFN-γ in CD4 + T cells after SEB stimulation ( h ) in HIV infection. C, CEACAM1; T, TIM-3 ( n = 4 per group). i , In situ proximity ligation assay of hCEACAM1 and hTIM-3 co-transfected HEK293T as in e . DAPI, 4′,6-diamidino-2-phenylindole. All data are mean ± s.e.m. and represent five ( e, f ), three ( c, d, i ) and two ( a, b ) independent experiments. * P

    Article Snippet: Previously described vectors containing the human CEACAM1–3L variant and human TIM-3 in the pDisplay vector (Invitrogen) were used as the template for all mutations.

    Techniques: Mouse Assay, Transgenic Assay, Expressing, Transfection, Fluorescence, Staining, Infection, In Situ, Proximity Ligation Assay

    Rv2882c induced BMDM activation. (A) BMDMs (1 × 10 5 /well) were stimulated with 100 ng/mL LPS or 1, 5, or 10 μg/mL Rv2882c for 24 h. (A) Quantities of TNF-α, MCP-1, IL-6, IL-10, and IL-12p70 in the culture supernatant were determined by ELISA. All data are expressed as the mean values ± SD ( n = 3). Significance levels (* p

    Journal: PLoS ONE

    Article Title: Mycobacterium tuberculosis Rv2882c Protein Induces Activation of Macrophages through TLR4 and Exhibits Vaccine Potential

    doi: 10.1371/journal.pone.0164458

    Figure Lengend Snippet: Rv2882c induced BMDM activation. (A) BMDMs (1 × 10 5 /well) were stimulated with 100 ng/mL LPS or 1, 5, or 10 μg/mL Rv2882c for 24 h. (A) Quantities of TNF-α, MCP-1, IL-6, IL-10, and IL-12p70 in the culture supernatant were determined by ELISA. All data are expressed as the mean values ± SD ( n = 3). Significance levels (* p

    Article Snippet: Mouse TNF-α, MCP-1, IL-6, IL-10, IL-12p70, IFN-γ, and IL-2 ELISA kits were obtained from eBioscience.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay