anti mouse il 6 neutralizing antibody  (R&D Systems)

Journal: Alzheimer's & Dementia

Article Title: Endothelial NAD + depletion drives vascular senescence and neuroinflammation via mtDNA‐cGAS/STING‐CD38 signaling in Alzheimer's disease

doi: 10.1002/alz.71423

Figure Lengend Snippet: NAD + supplementation suppresses cGAS/STING pathway activation in cerebral endothelial cells of APP/PS1 mice. (A) Heatmap of differentially expressed key cGAS/STING pathway‐related genes (such as Cgas , Sting1 , Irf3 ) identified by RNA‐seq of cerebral vessel‐enriched fractions from APPtg and APPtg + NR mice ( n = 3 per group). (B, C) Representative western blot image (B) and densitometric quantification of cGAS, STING, phospho‐TBK1 Ser172 (p‐TBK1), and phospho‐IRF3 Ser396 (p‐IRF3) in cerebral vessel‐enriched fractions from APPwt, APPtg, and APPtg + NR mice (C; n = 6 per group). (D–G) Representative immunofluorescence images of hippocampus and cortex from APPtg and APPtg + NR mice showing CD31 (green) co‐stained with STING (D, red) or cGAS (F, red); quantification of STING (E) and cGAS (G) fluorescence intensity within CD31 + cerebral vessels were shown ( n = 5 or 6 mice per group); nuclei were counterstained with DAPI (blue). (H) qPCR analysis of SASP genes ( Il6 , Tnf , Il1b , Cxcl10 , Cxcl2 ) in cerebral vessel‐enriched fractions from APPtg and APPtg + NR mice ( n = 5 per group). (I) ELISA quantification of IL‐6, TNF‐α, and IL‐1β in the culture supernatants of bEnd.3 endothelial cells treated with vehicle control, NR, Aβ, or Aβ + NR ( n = 6 per group). (J) SA‐β‐galactosidase staining of bEnd.3 endothelial cells transfected with control siRNA (si‐Ctrl), Cgas siRNA (si‐ Cgas ), or Sting1 siRNA (si‐ Sting ) followed by Aβ stimulation or vehicle control; representative images show SA‐β‐gal + cells indicated by white arrows, with enlarged insets provided; the percentage of SA‐β‐gal + cells were quantified ( n = 5 per group). Data are presented as mean ± SEM. Statistical analyses were performed using one‐way ANOVA followed by Tukey's multiple comparisons test (C, E, G, I, J) or unpaired two‐tailed Student's t ‐test (H). p ‐values are indicated in the figure.

Article Snippet: For IL‐6 pathway analysis, BV‐2 microglia were incubated with 10 ng/ml anti‐mouse IL‐6 neutralizing antibody (α‐IL‐6; R&D systems, #MAB406) or anti‐mouse IL‐6Rα blocking antibody (α‐IL‐6R; R&D systems, #AF1830) in CM‐containing medium from bEnd.3 cultures.

Techniques: Activation Assay, RNA Sequencing, Western Blot, Immunofluorescence, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Control, Transfection, Two Tailed Test

Journal: Alzheimer's & Dementia

Article Title: Endothelial NAD + depletion drives vascular senescence and neuroinflammation via mtDNA‐cGAS/STING‐CD38 signaling in Alzheimer's disease

doi: 10.1002/alz.71423

Figure Lengend Snippet: NAD + supplementation suppresses cGAS/STING activation by enhancing mitochondrial function and preventing cytosolic mtDNA leakage. (A) Quantification of mitochondrial membrane potential using JC‐1 staining in bEnd.3 endothelial cells treated with Aβ, Aβ + NR, or control conditions; representative images are shown in Figure ( n = 5 per group). (B, C) Flow cytometric analysis of intracellular ROS levels in bEnd.3 cells under indicated treatments ( n = 4 per group). (D) qPCR quantification of cytosolic mitochondrial DNA (mtDNA; D‐loop , Non‐Numt , Cox1 ) and nuclear DNA (nDNA; Tert , B2m ) in cerebral vessel‐enriched fractions isolated from APPwt, APPwt + NR, APPtg, and APPtg + NR mice ( n ≥5 per group). (E, F) Representative immunofluorescence images (E) and quantification (F) of co‐localization of CD31 (green) and oxidative DNA damage marker 8‐OHdG (red) in hippocampal and cortex of APPtg and APPtg + NR mice; nuclei were counterstained with DAPI (blue) ( n ≥5 mice per group). (G) Quantification of cytosolic mtDNA and nDNA levels in bEnd.3 cells transfected with siRNA targeting control (si‐Ctrl), Cgas (si‐ Cgas ), or Sting1 (si‐ Sting ) followed by Aβ treatment ( n = 4 per group). (H) Quantification of cytosolic mtDNA and nDNA levels in bEnd.3 cells treated with Aβ, Aβ + mtDNA depletion (ddC), or Aβ + ddC + NR ( n = 4 per group). (I) Relative mRNA expression of SASP‐related cytokines (IL‐6, TNF‐α, IL‐1β, CXCL10, CXCL2) under the same treatment conditions as in (H) ( n = 4 per group). (J, K) Western blot analysis (J) and quantification (K) of cGAS/STING pathway components (cGAS, STING, p‐TBK1, p‐IRF3) and tight junction proteins (ZO‐1, Occludin) in bEnd.3 cells under treatments with Aβ, Aβ + ddC, and Aβ + ddC + NR ( n = 4 per group). Data are presented as mean ± SEM. Statistical significance was assessed using one‐way ANOVA followed by Tukey's multiple comparisons test. P ‐values are indicated in the figure.

Article Snippet: For IL‐6 pathway analysis, BV‐2 microglia were incubated with 10 ng/ml anti‐mouse IL‐6 neutralizing antibody (α‐IL‐6; R&D systems, #MAB406) or anti‐mouse IL‐6Rα blocking antibody (α‐IL‐6R; R&D systems, #AF1830) in CM‐containing medium from bEnd.3 cultures.

Techniques: Activation Assay, Membrane, Staining, Control, Isolation, Immunofluorescence, Marker, Transfection, Expressing, Western Blot

Journal: Alzheimer's & Dementia

Article Title: Endothelial NAD + depletion drives vascular senescence and neuroinflammation via mtDNA‐cGAS/STING‐CD38 signaling in Alzheimer's disease

doi: 10.1002/alz.71423

Figure Lengend Snippet: NAD + supplementation disrupts IL‐6‐mediated endothelial‐microglial inflammatory crosstalk in AD. (A) Representative immunofluorescence images showing co‐staining of microglial marker Iba1 (red) and endothelial marker CD31 (green) in the cortex and hippocampus of APP/PS1 mice; white arrows indicate perivascular microglia closely associated with cerebral vessels. (B) Quantification of the proportion of perivascular microglia relative to total microglia ( n ≥ 5 per group). (C) Triple immunofluorescence staining of Iba1 (red), CD31 (green), and IL‐6R (gray) to visualize IL‐6R expression in perivascular microglia; yellow arrows indicate IL‐6R‐positive perivascular microglia. (D) Quantification of IL‐6R fluorescence intensity in vessel‐associated microglia ( n ≥5 per group). (E–F) Western blot analysis (E) and densitometric quantification (F) of IL‐6R, JAK1, and phosphorylation levels of STAT3 and NF‐κB p65 in microglia stimulated with conditioned media from bEnd.3 cells treated with vehicle (Con), NR, Aβ, or Aβ + NR ( n = 6 per group). (G–H) Western blot analysis (G) and quantification (H) of IL‐6R, JAK1, and p‐STAT3/p‐NF‐κB p65 in microglia co‐treated with Aβ‐challenged endothelial conditioned medium and isotype IgG, IL‐6‐neutralizing antibody (α‐IL‐6), or IL‐6R‐neutralizing antibody (α‐IL‐6R) ( n = 4 per group). Data are presented as mean ± SEM. Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test. P ‐values are indicated in the figure.

Article Snippet: For IL‐6 pathway analysis, BV‐2 microglia were incubated with 10 ng/ml anti‐mouse IL‐6 neutralizing antibody (α‐IL‐6; R&D systems, #MAB406) or anti‐mouse IL‐6Rα blocking antibody (α‐IL‐6R; R&D systems, #AF1830) in CM‐containing medium from bEnd.3 cultures.

Techniques: Immunofluorescence, Staining, Marker, Expressing, Fluorescence, Western Blot, Phospho-proteomics

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Combined IL6 and CCR2 blockade potentiates antitumor activity of NK cells in HPV-negative head and neck cancer

doi: 10.1186/s13046-024-03002-1

Figure Lengend Snippet: Gene expression and GSEA analysis of IL6 in TCGA HNSCC cohorts. A Differential analysis of IL6 expression between HPV − and HPV + HNSCC cohorts using TCGA dataset. B DEGs stratified by IL6 high and low expression in HPV − HNSCC cohort. C Expression correlation analysis between IL6 and the top nine upregulated DEGs identified in the IL6 high-expression group. D Pathway enrichment analysis (IL6 high expression vs IL6 low expression) revealing the ‘natural killer cell mediated cytotoxicity’ pathway in TCGA HPV − HNSCC cohort. E Correlation between the expression of IL6 and the other 4 DEGs upregulated in HPV − HNSCC cells vs HPV + HNSCC cells. F Correlation analysis of the gene expression between IL6 and CCR2 in TCGA HPV − HNSCC cohorts

Article Snippet: Ten days after cell inoculation, tumor-bearing mice were randomized into two groups to receive murine anti-IL6 neutralizing antibody (αIL6, clone MP5-20F3, BioXCell) and IgG2 isotype.

Techniques: Gene Expression, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Combined IL6 and CCR2 blockade potentiates antitumor activity of NK cells in HPV-negative head and neck cancer

doi: 10.1186/s13046-024-03002-1

Figure Lengend Snippet: Blocking IL6 signaling induces the remission of HPV − tumors in orthotopic tumor mouse models. A IL6 concentration in the peripheral serum of C57BL/6 mice bearing MOC2 or TC-1 tumors. B Schematic showing the timeline of experimental procedures. Male C57BL/6 mice at 6–8 weeks of age received intramucosal injection of 1.0 × 10 6 MOC2 or TC-1 cells. When tumors were established (10 days after cell inoculation), mice were randomized to receive the treatment of IgG2 isotype or αIL6 (100 µg/mouse) ( n = 5/group). C , D MOC2 tumor growth curve and weight in each treatment group. E , F TC-1 tumor growth curve and weight in each treatment group. In ( C ) and ( E ), representative tumors from C57BL/6 mice with indicated treatment are shown in the upper panel. * p < 0.05; ns: not significant

Article Snippet: Ten days after cell inoculation, tumor-bearing mice were randomized into two groups to receive murine anti-IL6 neutralizing antibody (αIL6, clone MP5-20F3, BioXCell) and IgG2 isotype.

Techniques: Blocking Assay, Concentration Assay, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Combined IL6 and CCR2 blockade potentiates antitumor activity of NK cells in HPV-negative head and neck cancer

doi: 10.1186/s13046-024-03002-1

Figure Lengend Snippet: Blocking IL6 signaling enhances the antitumor activity of NK cells in HPV − tumors. A , B Percent of CD8 + T cells and CD161 + NK cells in MOC2 tumors treated with isotype or αIL6. C Percent of Ki67 + NK cells in MOC2 tumors treated with isotype or αIL6. D , E Percent of CD8 + T cells and CD161 + NK cells in TC-1 tumors treated with isotype or αIL6. F Percent of Ki67 + NK cells in TC-1 tumors treated with isotype or αIL6. G Immunofluorescence staining of anti-CD161 antibody in MOC2 and TC-1 tumors treated with isotype or αIL6. Representative images and quantitative data are shown in the left and right panels. In ( A , B , D , E ), T cells and NK cells were gated from CD3 + and CD3 − population, respectively. * p < 0.05; ** p < 0.01; ns: not significant

Article Snippet: Ten days after cell inoculation, tumor-bearing mice were randomized into two groups to receive murine anti-IL6 neutralizing antibody (αIL6, clone MP5-20F3, BioXCell) and IgG2 isotype.

Techniques: Blocking Assay, Activity Assay, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Combined IL6 and CCR2 blockade potentiates antitumor activity of NK cells in HPV-negative head and neck cancer

doi: 10.1186/s13046-024-03002-1

Figure Lengend Snippet: αIL6 and RS504393 promote NK cell antitumor activity and repress MOC2 tumors more effectively when combined. A , F Schematic showing the timeline of experimental procedures. Male C57BL/6 mice at 6–8 weeks of age received intramucosal injection of 1.0 × 10 6 MOC2 or TC-1 cells. When tumors were established (10 days after cell inoculation), mice were randomized to receive the treatment of IgG2 isotype, αIL6 (100 µg/mouse), RS504393 (6 mg/kg) or the combination of αIL6 and RS504393 ( n = 5/group). B , C MOC2 tumor growth curve and weight in each treatment group. Representative tumors from C57BL/6 mice with indicated treatment are shown in the upper panel of ( B ). D , I Percent of CD161 + NK cells in MOC2 ( D ) or TC-1 ( I ) tumors treated with αIL6 and RS504393 alone or in combination. Representative images and quantitative data are shown in the left and right panels. E , K Percent of Ki67 + NK cells in MOC2 ( E ) or TC-1 ( K ) tumors treated with αIL6 and RS504393 alone or in combination. Representative images and quantitative data are shown in the left and right panels. G , H TC-1 tumor growth curve and weight in each treatment group. Representative tumors from C57BL/6 mice with indicated treatment are shown in the upper panel of ( G ). * p < 0.05; ** p < 0.01; ns: not significant

Article Snippet: Ten days after cell inoculation, tumor-bearing mice were randomized into two groups to receive murine anti-IL6 neutralizing antibody (αIL6, clone MP5-20F3, BioXCell) and IgG2 isotype.

Techniques: Activity Assay, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Combined IL6 and CCR2 blockade potentiates antitumor activity of NK cells in HPV-negative head and neck cancer

doi: 10.1186/s13046-024-03002-1

Figure Lengend Snippet: Depletion of NK cells attenuates the antitumor activity of the αIL6 and RS504393 combination in orthotopic mouse models. A Schematic showing the timeline of experimental procedures. Male C57BL/6 mice at 6–8 weeks of age received intramucosal injection of either 1.0 × 10 6 MOC2 or MOC1 cells. Tumor-bearing mice were randomized to receive the combination treatment of the αIL6 (100 µg/mouse) and RS504393 (6 mg/kg), in the presence of αASGM1 or rabbit polyclonal IgG isotype ( n = 10/group). B , C Individual and average MOC2 tumor growth curves. D , E Individual and average MOC1 tumor growth curves. F , G MOC2 and MOC1 tumor weight in each treatment group. * p < 0.05; ** p < 0.01; ns: not significant

Article Snippet: Ten days after cell inoculation, tumor-bearing mice were randomized into two groups to receive murine anti-IL6 neutralizing antibody (αIL6, clone MP5-20F3, BioXCell) and IgG2 isotype.

Techniques: Activity Assay, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Combined IL6 and CCR2 blockade potentiates antitumor activity of NK cells in HPV-negative head and neck cancer

doi: 10.1186/s13046-024-03002-1

Figure Lengend Snippet: A proposed model for this study. A In the microenvironment of HPV − HNSCC, IL6/IL6R and CCL2/CCR2 signaling exert a significant influence on the ability of HPV − tumor cells to evade immune attacks by NK cells. B Simultaneous blockade of IL6/IL6R and CCL2/CCR2 with αIL6 and RS504393 is effective in enhancing the antitumor activity of NK cells in HPV − HNSCC

Article Snippet: Ten days after cell inoculation, tumor-bearing mice were randomized into two groups to receive murine anti-IL6 neutralizing antibody (αIL6, clone MP5-20F3, BioXCell) and IgG2 isotype.

Techniques: Activity Assay

Journal: Cell Chemical Biology

Article Title: Therapeutic potency of compound RMY-205 for pulmonary fibrosis induced by SARS-CoV-2 nucleocapsid protein

doi: 10.1016/j.chembiol.2023.02.004

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-IL-6 for neutralization , Proteintech , Cat# 69001-1-Ig; RRID: AB_2918847.

Techniques: Neutralization, Recombinant, Enzyme-linked Immunosorbent Assay, shRNA