human il 6  (R&D Systems)

 
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    Name:
    Recombinant Human IL 6 Protein CF
    Description:
    The Recombinant Human IL 6 Protein from R D Systems is derived from E coli The Recombinant Human IL 6 Protein has been validated for the following applications Bioactivity
    Catalog Number:
    206-il-001mg/cf
    Price:
    3200
    Applications:
    Bioactivity
    Purity:
    >97%, by SDS-PAGE under reducing conditions and visualized by silver stain.
    Conjugate:
    Unconjugated
    Size:
    1 mg
    Category:
    Proteins and Enzymes
    Source:
    E. coli-derived Recombinant Human IL-6 Protein
    Buy from Supplier


    Structured Review

    R&D Systems human il 6
    Recombinant Human IL 6 Protein CF
    The Recombinant Human IL 6 Protein from R D Systems is derived from E coli The Recombinant Human IL 6 Protein has been validated for the following applications Bioactivity
    https://www.bioz.com/result/human il 6/product/R&D Systems
    Average 99 stars, based on 221 article reviews
    Price from $9.99 to $1999.99
    human il 6 - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "TGF-β2 downregulates osteogenesis under inflammatory conditions in dental follicle stem cells"

    Article Title: TGF-β2 downregulates osteogenesis under inflammatory conditions in dental follicle stem cells

    Journal: International Journal of Oral Science

    doi: 10.1038/s41368-018-0028-8

    Downregulation of the osteogenic differentiation of DFSCs after exposure to P.g.- derived LPS. a MTT assays were performed to determine cell viability after LPS treatment. LPS at 100 ng · mL −1 had no effect on cell viability. b Real-time PCR showed that the pro-inflammatory cytokines IL-6 and IL-8 were secreted after treatment of cells with 100 ng · mL −1 LPS treatment in conditional medium. c IL-6 and IL-8 were also expressed during the osteogenic differentiation of cells treated with 100 ng · mL −1 LPS. d , e Calcium deposition during osteogenesis was inhibited by 100 ng · mL −1 LPS treatment. The dissolved mineral content of the medium was decreased approximately 4.5-fold compared to the control without LPS treatment. f Osteocalcin gene expression was significantly inhibited. g Comparisons of TGF-β1 and TGF-β2 gene expression by RT-PCR were performed after differentiating osteogenic tissue in the presence of 100 ng · mL −1 LPS for 2 weeks. During osteogenesis, TGF-β1 expression increased significantly, whereas TGF-β2 showed decreased expression. During LPS treatment, TGF-β1 and TGF-β2 expression changed in an inverse manner. LPS triggered higher TGF-β2 expression during osteogenesis. The data are presented as the mean ± SD. * P
    Figure Legend Snippet: Downregulation of the osteogenic differentiation of DFSCs after exposure to P.g.- derived LPS. a MTT assays were performed to determine cell viability after LPS treatment. LPS at 100 ng · mL −1 had no effect on cell viability. b Real-time PCR showed that the pro-inflammatory cytokines IL-6 and IL-8 were secreted after treatment of cells with 100 ng · mL −1 LPS treatment in conditional medium. c IL-6 and IL-8 were also expressed during the osteogenic differentiation of cells treated with 100 ng · mL −1 LPS. d , e Calcium deposition during osteogenesis was inhibited by 100 ng · mL −1 LPS treatment. The dissolved mineral content of the medium was decreased approximately 4.5-fold compared to the control without LPS treatment. f Osteocalcin gene expression was significantly inhibited. g Comparisons of TGF-β1 and TGF-β2 gene expression by RT-PCR were performed after differentiating osteogenic tissue in the presence of 100 ng · mL −1 LPS for 2 weeks. During osteogenesis, TGF-β1 expression increased significantly, whereas TGF-β2 showed decreased expression. During LPS treatment, TGF-β1 and TGF-β2 expression changed in an inverse manner. LPS triggered higher TGF-β2 expression during osteogenesis. The data are presented as the mean ± SD. * P

    Techniques Used: Derivative Assay, MTT Assay, Real-time Polymerase Chain Reaction, Expressing, Reverse Transcription Polymerase Chain Reaction

    Inhibition of TGF-β2 overcomes the downregulation of bone formation caused by LPS. a , b On day 28, alizarin red S solution was used to stain calcium deposits in cultures treated with TGF-β2 inhibitor and LPS. The dissolved mineral content of the medium decreased after LPS treatment. However, treatment with 1 μg · mL −1 TGF-β2 inhibitor neutralised the TGF-β2 secreted by LPS treatment. Interestingly, inhibition of TGF-β2 increased the osteogenic differentiation of DFSCs. c The results of ALPase activity measurements also supported the conclusion that inhibition of TGF-β2 increased the early stage of osteogenesis in DFSCs. d When the TGF-β2 secreted during LPS-induced inflammation was neutralised, the levels of the pro-inflammatory cytokines IL-6 and IL-8 decreased. In contrast, osteocalcin (OCN) and type 1 collagen (Col1) expression increased after treatment of the cells with TGF-β2 inhibitor during LPS-induced inflammation. e Treatment with 100 ng · mL −1 LPS for 30 min activated smad2/3 signalling. f DFSCs activated with LPS for 30 min were treated with 0.5 μg · mL −1 TGF-β2 inhibitor for 7 days during osteogenic differentiation. In the presence of a TGF-β2 inhibitor, Runx2 expression was overcome. The data are presented as the mean ± SD. * P
    Figure Legend Snippet: Inhibition of TGF-β2 overcomes the downregulation of bone formation caused by LPS. a , b On day 28, alizarin red S solution was used to stain calcium deposits in cultures treated with TGF-β2 inhibitor and LPS. The dissolved mineral content of the medium decreased after LPS treatment. However, treatment with 1 μg · mL −1 TGF-β2 inhibitor neutralised the TGF-β2 secreted by LPS treatment. Interestingly, inhibition of TGF-β2 increased the osteogenic differentiation of DFSCs. c The results of ALPase activity measurements also supported the conclusion that inhibition of TGF-β2 increased the early stage of osteogenesis in DFSCs. d When the TGF-β2 secreted during LPS-induced inflammation was neutralised, the levels of the pro-inflammatory cytokines IL-6 and IL-8 decreased. In contrast, osteocalcin (OCN) and type 1 collagen (Col1) expression increased after treatment of the cells with TGF-β2 inhibitor during LPS-induced inflammation. e Treatment with 100 ng · mL −1 LPS for 30 min activated smad2/3 signalling. f DFSCs activated with LPS for 30 min were treated with 0.5 μg · mL −1 TGF-β2 inhibitor for 7 days during osteogenic differentiation. In the presence of a TGF-β2 inhibitor, Runx2 expression was overcome. The data are presented as the mean ± SD. * P

    Techniques Used: Inhibition, Staining, Activity Assay, Expressing

    Porphyromonas gingivalis ( P.g .)-derived LPS-induced inflammation mimics inflammatory conditions in DFSCs. a Cultured DFSCs were treated with various concentrations of LPS (10, 100 and 1000 ng · mL −1 ) and allowed to secrete nitric oxide (NO) for 24 and 48 h. b The levels of the pro-inflammatory cytokines IL-6 and IL-8 were increased by treatment with 1000 ng · mL −1 LPS for 48 h. However, there was no significant difference in the gene expression of TGF-β1 and TGF-β2 in cells maintained in conditional medium with LPS treatment. c , d The protein levels of IL-6 and IL-8 were also increased after LPS treatment. The data are presented as the mean ± SD. * P
    Figure Legend Snippet: Porphyromonas gingivalis ( P.g .)-derived LPS-induced inflammation mimics inflammatory conditions in DFSCs. a Cultured DFSCs were treated with various concentrations of LPS (10, 100 and 1000 ng · mL −1 ) and allowed to secrete nitric oxide (NO) for 24 and 48 h. b The levels of the pro-inflammatory cytokines IL-6 and IL-8 were increased by treatment with 1000 ng · mL −1 LPS for 48 h. However, there was no significant difference in the gene expression of TGF-β1 and TGF-β2 in cells maintained in conditional medium with LPS treatment. c , d The protein levels of IL-6 and IL-8 were also increased after LPS treatment. The data are presented as the mean ± SD. * P

    Techniques Used: Derivative Assay, Cell Culture, Expressing

    Gene profiling by RT-PCR. a The levels of the pro-inflammatory cytokines IL-6, IL-8 and IL-1β in DFSCs isolated from inflamed tissue were measured. b From the set of proteins with Mascot scores greater than 30 selected from LC-MS/MS analysis of normal and inflamed DFSCs, proteins related to osteogenic differentiation were selected. The level of expression of the genes encoding these proteins in normal and inflamed DFSCs during osteogenesis was determined by RT-PCR using specific primers. Differences in TGF-β2 gene expression in normal and inflamed DFSCs during osteogenesis were detected
    Figure Legend Snippet: Gene profiling by RT-PCR. a The levels of the pro-inflammatory cytokines IL-6, IL-8 and IL-1β in DFSCs isolated from inflamed tissue were measured. b From the set of proteins with Mascot scores greater than 30 selected from LC-MS/MS analysis of normal and inflamed DFSCs, proteins related to osteogenic differentiation were selected. The level of expression of the genes encoding these proteins in normal and inflamed DFSCs during osteogenesis was determined by RT-PCR using specific primers. Differences in TGF-β2 gene expression in normal and inflamed DFSCs during osteogenesis were detected

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Expressing

    2) Product Images from "Mesenchymal stem cell-derived inflammatory fibroblasts promote monocyte transition into myeloid fibroblasts via an IL-6-dependent mechanism in the aging mouse heart"

    Article Title: Mesenchymal stem cell-derived inflammatory fibroblasts promote monocyte transition into myeloid fibroblasts via an IL-6-dependent mechanism in the aging mouse heart

    Journal: The FASEB Journal

    doi: 10.1096/fj.14-268136

    The expression of IL-6 in cardiac mesenchymal fibroblasts is affected by aging. A ) Expression of IL-6 in quiescent fibroblasts derived from young and old MSCs were analyzed by quantitative PCR and by ELISA, 24 h after the cell cycle was synchronized. B ) Double immunofluorescence labeling of IL-6 (red) and DDR2 (green) in 3- and 30-mo-old mouse hearts. Double positive cells are visualized in yellow/orange color. Middle panel shows magnified image of the section marked by white squares. Bottom panel shows morphometric analysis of IL-6 + DDR2 + cells in heart sections. Nuclei were stained with DAPI. Scale bar, 20 μm. P
    Figure Legend Snippet: The expression of IL-6 in cardiac mesenchymal fibroblasts is affected by aging. A ) Expression of IL-6 in quiescent fibroblasts derived from young and old MSCs were analyzed by quantitative PCR and by ELISA, 24 h after the cell cycle was synchronized. B ) Double immunofluorescence labeling of IL-6 (red) and DDR2 (green) in 3- and 30-mo-old mouse hearts. Double positive cells are visualized in yellow/orange color. Middle panel shows magnified image of the section marked by white squares. Bottom panel shows morphometric analysis of IL-6 + DDR2 + cells in heart sections. Nuclei were stained with DAPI. Scale bar, 20 μm. P

    Techniques Used: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Labeling, Staining

    The FTase-ERK pathway contributes to IL-6 expression in cardiac fibroblasts. Quiescent fibroblasts derived from the old MSCs were treated with 100 nM FTI-277 (FTI) or 2 μ M PD 0325901 (PD) for 24 h. A–C ) IL-6 mRNA level ( A ), IL-6 secretion ( B ), and cytoplasmic IL-6 ( C ) are shown. To visualize cytoplasmic IL-6 cells were treated with 10 ng/ml brefeldin A for 6 h. Scale bar, 50 μm. P
    Figure Legend Snippet: The FTase-ERK pathway contributes to IL-6 expression in cardiac fibroblasts. Quiescent fibroblasts derived from the old MSCs were treated with 100 nM FTI-277 (FTI) or 2 μ M PD 0325901 (PD) for 24 h. A–C ) IL-6 mRNA level ( A ), IL-6 secretion ( B ), and cytoplasmic IL-6 ( C ) are shown. To visualize cytoplasmic IL-6 cells were treated with 10 ng/ml brefeldin A for 6 h. Scale bar, 50 μm. P

    Techniques Used: Expressing, Derivative Assay

    Differential response to recombinant IL-6 in fibroblasts derived from young and old MSCs. Quiescent fibroblasts were treated with one dose of recombinant IL-6 (50 ng/ml) and then cells were harvested 24 and 48 h later. A ) Response of fibroblast derived from the young MSCs. B ) Responses from fibroblasts derived from the old MSCs. P
    Figure Legend Snippet: Differential response to recombinant IL-6 in fibroblasts derived from young and old MSCs. Quiescent fibroblasts were treated with one dose of recombinant IL-6 (50 ng/ml) and then cells were harvested 24 and 48 h later. A ) Response of fibroblast derived from the young MSCs. B ) Responses from fibroblasts derived from the old MSCs. P

    Techniques Used: Recombinant, Derivative Assay

    RasGrf1 expression controls IL-6 transcription in fibroblasts derived from the old MSCs. A ). Silencing RasGrf1 by siRNA in fibroblasts derived from old MSCs reduced RasGrf1 mRNA (left panel) and protein (right panel) levels. Fibroblasts were transfected with scrambled siRNA (labeled: control) or siRNA targeting RasGrf1 (labeled: siRNA). RNA and protein analysis was carried 48 h posttransfection. B and C ) RasGrf1 silencing caused a decrease of IL-6 transcription ( B ) and reduced levels of secreted IL-6 ( C ). D) Cells transfected with scrambled siRNA or RasGrf1 siRNA were treated with 10 ng/ml brefeldin A for 6 h (starting 42 h posttransfection). Cytoplasmic IL-6 was visualized by immunofluorescence labeling. Scale bar, 50 μm. P
    Figure Legend Snippet: RasGrf1 expression controls IL-6 transcription in fibroblasts derived from the old MSCs. A ). Silencing RasGrf1 by siRNA in fibroblasts derived from old MSCs reduced RasGrf1 mRNA (left panel) and protein (right panel) levels. Fibroblasts were transfected with scrambled siRNA (labeled: control) or siRNA targeting RasGrf1 (labeled: siRNA). RNA and protein analysis was carried 48 h posttransfection. B and C ) RasGrf1 silencing caused a decrease of IL-6 transcription ( B ) and reduced levels of secreted IL-6 ( C ). D) Cells transfected with scrambled siRNA or RasGrf1 siRNA were treated with 10 ng/ml brefeldin A for 6 h (starting 42 h posttransfection). Cytoplasmic IL-6 was visualized by immunofluorescence labeling. Scale bar, 50 μm. P

    Techniques Used: Expressing, Derivative Assay, Transfection, Labeling, Immunofluorescence

    Role of elevated RasGrf1 expression in amplified IL-6 levels in fibroblasts derived from the old MSCs. Increased expression of RasGrf1 promotes exchange of GDP into GTP on Ras and renders its activation. Ras activation is also controlled by FTase activity since use of the FTase inhibitor (FTI-277) reduces IL-6 expression. Downstream of Ras and FTase, ERK activation controls IL-6 expression; use of PD 0325901 (ERK inhibitor) down-regulates IL-6 levels. Finally, IL-6 that is secreted activates its own transcription in a feed-forward loop.
    Figure Legend Snippet: Role of elevated RasGrf1 expression in amplified IL-6 levels in fibroblasts derived from the old MSCs. Increased expression of RasGrf1 promotes exchange of GDP into GTP on Ras and renders its activation. Ras activation is also controlled by FTase activity since use of the FTase inhibitor (FTI-277) reduces IL-6 expression. Downstream of Ras and FTase, ERK activation controls IL-6 expression; use of PD 0325901 (ERK inhibitor) down-regulates IL-6 levels. Finally, IL-6 that is secreted activates its own transcription in a feed-forward loop.

    Techniques Used: Expressing, Amplification, Derivative Assay, Activation Assay, Activity Assay

    IL-6 facilitates MCP-1-driven monocyte to myeloid fibroblast maturation. For experiments ( A–C ) PBMCs were added on top of a confluent endothelial cell monolayer seeded on an insert. Medium below the insert was supplemented as indicated. The following reagents were used: IL-6 (10 ng/ml), MCP-1 (650 ng/ml), IL-6R (200 ng/ml), soluble gp130 (300 ng/ml), sc-144 hydrochloride (5 μM), PD 0325901 (1 μM), Jak inhibitor (2 μM). noRX, no supplementation; sc-144, sc-144 hydrochloride; PD, PD 0325901; Jaki, Jak inhibitor. D ) Fibroblasts derived from young and old MSCs secrete the same level of soluble IL-6R. P
    Figure Legend Snippet: IL-6 facilitates MCP-1-driven monocyte to myeloid fibroblast maturation. For experiments ( A–C ) PBMCs were added on top of a confluent endothelial cell monolayer seeded on an insert. Medium below the insert was supplemented as indicated. The following reagents were used: IL-6 (10 ng/ml), MCP-1 (650 ng/ml), IL-6R (200 ng/ml), soluble gp130 (300 ng/ml), sc-144 hydrochloride (5 μM), PD 0325901 (1 μM), Jak inhibitor (2 μM). noRX, no supplementation; sc-144, sc-144 hydrochloride; PD, PD 0325901; Jaki, Jak inhibitor. D ) Fibroblasts derived from young and old MSCs secrete the same level of soluble IL-6R. P

    Techniques Used: Derivative Assay

    3) Product Images from "Treatment with anti-IL-6 receptor antibody prevented increase in serum hepcidin levels and improved anemia in mice inoculated with IL-6–producing lung carcinoma cells"

    Article Title: Treatment with anti-IL-6 receptor antibody prevented increase in serum hepcidin levels and improved anemia in mice inoculated with IL-6–producing lung carcinoma cells

    Journal: BMC Cancer

    doi: 10.1186/s12885-016-2305-2

    Effects of MR16-1 on iron indices and pro-inflammatory cytokines in the LC-06-JCK mouse model. a Murine hepcidin levels, b murine ferritin levels, c murine TNF-α levels, d murine IL-1β levels, and e murine IL-6 levels were measured in mice treated for 5 weeks. Results are the mean + SD for 19–20 mice in each group. * P
    Figure Legend Snippet: Effects of MR16-1 on iron indices and pro-inflammatory cytokines in the LC-06-JCK mouse model. a Murine hepcidin levels, b murine ferritin levels, c murine TNF-α levels, d murine IL-1β levels, and e murine IL-6 levels were measured in mice treated for 5 weeks. Results are the mean + SD for 19–20 mice in each group. * P

    Techniques Used: Mouse Assay

    Changes in the parameters during the development of anemia in the LC-06-JCK mouse model. a Human IL-6 levels, b Hb levels, c HCT levels, and d MCV values were measured in mice treated for 0, 2, and 4 weeks. Open squares, NTB group; open circles, TB group; closed circles, MR16-1 group. Results are the mean + SD for 8 mice in each group. * P
    Figure Legend Snippet: Changes in the parameters during the development of anemia in the LC-06-JCK mouse model. a Human IL-6 levels, b Hb levels, c HCT levels, and d MCV values were measured in mice treated for 0, 2, and 4 weeks. Open squares, NTB group; open circles, TB group; closed circles, MR16-1 group. Results are the mean + SD for 8 mice in each group. * P

    Techniques Used: Mouse Assay

    4) Product Images from "A Dual Target-directed Agent against Interleukin-6 Receptor and Tumor Necrosis Factor α ameliorates experimental arthritis"

    Article Title: A Dual Target-directed Agent against Interleukin-6 Receptor and Tumor Necrosis Factor α ameliorates experimental arthritis

    Journal: Scientific Reports

    doi: 10.1038/srep20150

    The inhibitory effects of DTA(A7/sTNFR2) on the production of inflammatory mediators and proliferation, and migration of RA-FLS. The inhibitory effects of DTA(A7/sTNFR2) on TNFα-induced GM-CSF and VEGF-C expression ( a ) or IL-6-induced MMP3 and MMP13 expression ( b ) in RA-FLS. RA-FLS were incubated with or without human TNFα ( a ) or IL-6 and sIL-6R ( b ) for 24 h. RA-FLS was also treated with 100 μg/mL DTA(A7/sTNFR2) or PBS (as a negative control) during the incubation. The mRNA expression of GM-CSF and VEGF-C or MMP3 and MMP13 was analyzed by realtime RT-PCR in triplicate. All values were normalized to that of GAPDH (Mean ± SEM). (c) The inhibitory effects of DTA(A7/sTNFR2) on GM-CSF and MMP3 expression induced by both of TNFα and IL-6 in RA-FLS. RA-FLS was incubated with or without TNFα, IL-6 and sIL-6R for 24h. RA-FLS was also treated with or without 100 μg/mL DTA(A7/sTNFR2) or PBS (as a negative control) during the incubation. The mRNA expression of GM-CSF and MMP3 was analyzed by realtime RT-PCR in triplicate. All values were normalized to GAPDH (Mean ± SEM). ( d ) Anti-proliferative effects of DTA(A7/TNFR2) on RA-FLS. RA-FLS were incubated with or without human TNFα, IL-6, and soluble IL-6R(sIL-6R) for 72 h. 100 μg/mL of sTNFR2, A7, DTA(A7/sTNFR2), or IgG was added during the incubation. Cell proliferation was assessed using CCK8 assay. Proliferation rates were normalized to the negative control (n.c.) value. Values are means ± SEM. ( e , left) Representative results of the scratch assay to determine the anti-migration effects of DTA(A7/sTNFR2) on RA-FLS. After wounds were formed with sterile pipette tips, cells (with the exception of n.c.) were incubated with IgG, sTNFR2, A7, or DTA(A7/sTNFR2) in the presence of TNFα, IL-6, and IL-6R for 16 h. Black lines indicate the boundaries of the wounds. ( e , right) Migration activities. Migration activities were calculated as follows: migration activity = 1—(wounded area at 16 h)/(wounded area at 0 h). The values were normalized to n.c. Mean ± SEM is presented. All results are representative of at least three independent experiments. P -values were obtained from Student’s t -test (*p
    Figure Legend Snippet: The inhibitory effects of DTA(A7/sTNFR2) on the production of inflammatory mediators and proliferation, and migration of RA-FLS. The inhibitory effects of DTA(A7/sTNFR2) on TNFα-induced GM-CSF and VEGF-C expression ( a ) or IL-6-induced MMP3 and MMP13 expression ( b ) in RA-FLS. RA-FLS were incubated with or without human TNFα ( a ) or IL-6 and sIL-6R ( b ) for 24 h. RA-FLS was also treated with 100 μg/mL DTA(A7/sTNFR2) or PBS (as a negative control) during the incubation. The mRNA expression of GM-CSF and VEGF-C or MMP3 and MMP13 was analyzed by realtime RT-PCR in triplicate. All values were normalized to that of GAPDH (Mean ± SEM). (c) The inhibitory effects of DTA(A7/sTNFR2) on GM-CSF and MMP3 expression induced by both of TNFα and IL-6 in RA-FLS. RA-FLS was incubated with or without TNFα, IL-6 and sIL-6R for 24h. RA-FLS was also treated with or without 100 μg/mL DTA(A7/sTNFR2) or PBS (as a negative control) during the incubation. The mRNA expression of GM-CSF and MMP3 was analyzed by realtime RT-PCR in triplicate. All values were normalized to GAPDH (Mean ± SEM). ( d ) Anti-proliferative effects of DTA(A7/TNFR2) on RA-FLS. RA-FLS were incubated with or without human TNFα, IL-6, and soluble IL-6R(sIL-6R) for 72 h. 100 μg/mL of sTNFR2, A7, DTA(A7/sTNFR2), or IgG was added during the incubation. Cell proliferation was assessed using CCK8 assay. Proliferation rates were normalized to the negative control (n.c.) value. Values are means ± SEM. ( e , left) Representative results of the scratch assay to determine the anti-migration effects of DTA(A7/sTNFR2) on RA-FLS. After wounds were formed with sterile pipette tips, cells (with the exception of n.c.) were incubated with IgG, sTNFR2, A7, or DTA(A7/sTNFR2) in the presence of TNFα, IL-6, and IL-6R for 16 h. Black lines indicate the boundaries of the wounds. ( e , right) Migration activities. Migration activities were calculated as follows: migration activity = 1—(wounded area at 16 h)/(wounded area at 0 h). The values were normalized to n.c. Mean ± SEM is presented. All results are representative of at least three independent experiments. P -values were obtained from Student’s t -test (*p

    Techniques Used: Migration, Expressing, Incubation, Negative Control, Reverse Transcription Polymerase Chain Reaction, CCK-8 Assay, Wound Healing Assay, Transferring, Activity Assay

    5) Product Images from "Immunization against an IL-6 peptide induces anti-IL-6 antibodies and modulates the Delayed-Type Hypersensitivity reaction in cynomolgus monkeys"

    Article Title: Immunization against an IL-6 peptide induces anti-IL-6 antibodies and modulates the Delayed-Type Hypersensitivity reaction in cynomolgus monkeys

    Journal: Scientific Reports

    doi: 10.1038/srep19549

    Kinetics of the anti-IL-6 antibody production in the cynomolgus monkeys immunized against the hIS200 peptide immunogen. The control groups did not exhibit any antibodies against IL-6 detected by ELISA and are thus not shown.
    Figure Legend Snippet: Kinetics of the anti-IL-6 antibody production in the cynomolgus monkeys immunized against the hIS200 peptide immunogen. The control groups did not exhibit any antibodies against IL-6 detected by ELISA and are thus not shown.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Neutralizing capacity of the antibodies purified from sera of the immunized monkeys at d-8, d59, d90, d105 and d120. The four hIS200-immunized monkeys (in black) exhibit a significant IL-6 neutralization capacity higher than the background, while the neutralizing capacity of the control monkeys (in grey) is always below background value (threshold of 20% marked by the dotted line). Only one control group (KLH) is shown for clarity of the figure (the values of the other control group are also all below background value).
    Figure Legend Snippet: Neutralizing capacity of the antibodies purified from sera of the immunized monkeys at d-8, d59, d90, d105 and d120. The four hIS200-immunized monkeys (in black) exhibit a significant IL-6 neutralization capacity higher than the background, while the neutralizing capacity of the control monkeys (in grey) is always below background value (threshold of 20% marked by the dotted line). Only one control group (KLH) is shown for clarity of the figure (the values of the other control group are also all below background value).

    Techniques Used: Purification, Neutralization

    ( a ) IL-6 levels in the serum of monkeys were measured by ELISA. Data are presented as mean ± SD for each group at each time point. The mean level is always slightly higher in the hIS200-immunized group at each timepoint. ( b ) Analysis by the Mann-Whitney test shows that the mean IL-6 concentration over time is significantly higher in hIS200-immunized monkeys compared to the two control groups.
    Figure Legend Snippet: ( a ) IL-6 levels in the serum of monkeys were measured by ELISA. Data are presented as mean ± SD for each group at each time point. The mean level is always slightly higher in the hIS200-immunized group at each timepoint. ( b ) Analysis by the Mann-Whitney test shows that the mean IL-6 concentration over time is significantly higher in hIS200-immunized monkeys compared to the two control groups.

    Techniques Used: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Concentration Assay

    6) Product Images from "Disruption of a self-amplifying catecholamine loop reduces cytokine release syndrome"

    Article Title: Disruption of a self-amplifying catecholamine loop reduces cytokine release syndrome

    Journal: Nature

    doi: 10.1038/s41586-018-0774-y

    MTR and ANP prevent cytokine release in syngeneic Eμ-ALL model without compromising anti-tumour efficacy. a , b , Circulating catecholamines (left to right, n = 3, 4, 3, 4, 4, 4, 3, 4, 3, 4, 4, 4 per column/graph) ( a ) and murine cytokines IL-6 ( n = 3 per column), KC ( n = 3, 3, 3, 4, 3, 3, 4, 4, 3 per column), IL-1α ( n = 3, 3, 3, 4, 3, 3, 4, 3, 3 per column) and G-CSF ( n = 3, 3, 3, 4, 4, 3, 4, 3, 3 per column) ( b ), assessed at 24 and 72 h after mCART19 injection. Data are presented as means ± s.d. with individual data points shown, analysed by two-tailed t -test. c , BLI was performed before and 10 days after mCART19 cell injection, with or without ANP and MTR pre-treatment ( n = 5 animals per group). BLI radiance was used to quantify the tumour burden during the treatment course (right). d , Percentage survival of Eμ-ALL-mice after mCART19 cell transfer ( n = 8 mice per group). Survival differences were analysed by two-sided log-rank test.
    Figure Legend Snippet: MTR and ANP prevent cytokine release in syngeneic Eμ-ALL model without compromising anti-tumour efficacy. a , b , Circulating catecholamines (left to right, n = 3, 4, 3, 4, 4, 4, 3, 4, 3, 4, 4, 4 per column/graph) ( a ) and murine cytokines IL-6 ( n = 3 per column), KC ( n = 3, 3, 3, 4, 3, 3, 4, 4, 3 per column), IL-1α ( n = 3, 3, 3, 4, 3, 3, 4, 3, 3 per column) and G-CSF ( n = 3, 3, 3, 4, 4, 3, 4, 3, 3 per column) ( b ), assessed at 24 and 72 h after mCART19 injection. Data are presented as means ± s.d. with individual data points shown, analysed by two-tailed t -test. c , BLI was performed before and 10 days after mCART19 cell injection, with or without ANP and MTR pre-treatment ( n = 5 animals per group). BLI radiance was used to quantify the tumour burden during the treatment course (right). d , Percentage survival of Eμ-ALL-mice after mCART19 cell transfer ( n = 8 mice per group). Survival differences were analysed by two-sided log-rank test.

    Techniques Used: Aqueous Normal-phase Chromatography, Injection, Two Tailed Test, Mouse Assay

    Catecholamines modulate the cytokine release in macrophages in vitro. a , b , Human U937 macrophage-like cells were pre-treated with ANP or MTR for 10 min, then stimulated with LPS at 1 μg ml −1 and/or adrenaline at 15 ng ml −1 . Culture supernatants were analysed for catecholamines ( n = 3 per column) ( a ) as well as the indicated cytokines ( n = 3 per column) ( b ). c , TH expression of baseline and LPS-stimulated Th +/+ or Th ΔLysM macrophages ( n = 3 per group), analysed by RT–PCR; results are normalized to ubiquitin C ( UBC ) expression. d , e , Supernatants of collected peritoneal macrophages from Th +/+ or Th ΔLysM mice, stimulated with LPS at 50 μg ml −1 , adrenaline 15 μg ml −1 or both for 24 h, were analysed for levels of adrenaline ( n = 3), noradrenaline ( n = 3) ( d ) and cytokines IL-6 ( n = 3), KC ( n = 3), MIP-2 ( n = 3) and TNF ( n = 3) ( e ). All data are presented as mean ± s.d. with individual data points shown, analysed by two-tailed t -test.
    Figure Legend Snippet: Catecholamines modulate the cytokine release in macrophages in vitro. a , b , Human U937 macrophage-like cells were pre-treated with ANP or MTR for 10 min, then stimulated with LPS at 1 μg ml −1 and/or adrenaline at 15 ng ml −1 . Culture supernatants were analysed for catecholamines ( n = 3 per column) ( a ) as well as the indicated cytokines ( n = 3 per column) ( b ). c , TH expression of baseline and LPS-stimulated Th +/+ or Th ΔLysM macrophages ( n = 3 per group), analysed by RT–PCR; results are normalized to ubiquitin C ( UBC ) expression. d , e , Supernatants of collected peritoneal macrophages from Th +/+ or Th ΔLysM mice, stimulated with LPS at 50 μg ml −1 , adrenaline 15 μg ml −1 or both for 24 h, were analysed for levels of adrenaline ( n = 3), noradrenaline ( n = 3) ( d ) and cytokines IL-6 ( n = 3), KC ( n = 3), MIP-2 ( n = 3) and TNF ( n = 3) ( e ). All data are presented as mean ± s.d. with individual data points shown, analysed by two-tailed t -test.

    Techniques Used: In Vitro, Aqueous Normal-phase Chromatography, Expressing, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Two Tailed Test

    Inhibition of catecholamine synthesis with MTR does not impair the therapeutic response of hCART19. a , Serial bioluminescence imaging (BLI) of Raji-bearing NSGS mice (low tumour burden)at day 6 and 19 after treatment with 1.5 × 10 7 hCART19, with or without MTR ( n = 10 mice per group) compared to control (UT-T), with or without MTR ( n = 5 mice per group). BLI counts were used to quantify the tumour burden during the treatment course (right). Statistical differences were evaluated by one-tailed t -test. b , Corresponding Kaplan–Meier curve of Raji-bearing NSGS mice with low tumour burden, treated with 1.5 × 10 7 hCART19, with or without MTR pre-treatment ( n = 10 mice per group) in comparison to control (UT-T), with or without MTR ( n ). c , d , Levels of plasma adrenaline ( n = 3, 3, 4 per column) and noradrenaline ( n = 3, 4, 7) ( c ) and Hs IFN-γ ( n = 4), Hs TNF ( n = 4, 3, 3), and mouse cytokines Mm IL-6 ( n = 3) and Mm KC ( n = 3) ( d ), assessed 72 h after hCART19 treatment. Data are presented as mean ± s.d. with individual data points shown, analysed by two-tailed t -test. e , Scheme showing how inhibition of the catecholamine pathway may reduce CRS. TLR, toll-like receptor.
    Figure Legend Snippet: Inhibition of catecholamine synthesis with MTR does not impair the therapeutic response of hCART19. a , Serial bioluminescence imaging (BLI) of Raji-bearing NSGS mice (low tumour burden)at day 6 and 19 after treatment with 1.5 × 10 7 hCART19, with or without MTR ( n = 10 mice per group) compared to control (UT-T), with or without MTR ( n = 5 mice per group). BLI counts were used to quantify the tumour burden during the treatment course (right). Statistical differences were evaluated by one-tailed t -test. b , Corresponding Kaplan–Meier curve of Raji-bearing NSGS mice with low tumour burden, treated with 1.5 × 10 7 hCART19, with or without MTR pre-treatment ( n = 10 mice per group) in comparison to control (UT-T), with or without MTR ( n ). c , d , Levels of plasma adrenaline ( n = 3, 3, 4 per column) and noradrenaline ( n = 3, 4, 7) ( c ) and Hs IFN-γ ( n = 4), Hs TNF ( n = 4, 3, 3), and mouse cytokines Mm IL-6 ( n = 3) and Mm KC ( n = 3) ( d ), assessed 72 h after hCART19 treatment. Data are presented as mean ± s.d. with individual data points shown, analysed by two-tailed t -test. e , Scheme showing how inhibition of the catecholamine pathway may reduce CRS. TLR, toll-like receptor.

    Techniques Used: Inhibition, Imaging, Mouse Assay, One-tailed Test, Two Tailed Test

    Modulation of catecholamine synthesis by MTR dose-dependently determines survival and cytokine release. a , Survival of BALB/c mice stimulated with a lethal dose of LPS and treated with the indicated dose of MTR: MTR 20 mg kg −1 ( n = 5 mice per group); MTR 30 mg kg −1 ( n = 10 mice), MTR 40 mg kg −1 ( n = 12) compared to LPS ( n = 10 mice). Survival differences were analysed by two-sided log-rank test. b , c , Levels of plasma catecholamines ( n = 4 per column) ( b ) and IL-6 ( n = 4 per column), KC (left to right, n = 4, 4, 3 per column), IFN-γ ( n = 4) and TNF ( n = 4, 4, 3) ( c ) at different MTR doses measured 24 h after LPS injection. d , e , 24-h-time course of circulating adrenaline ( n = 5, 5, 5, 4, 5, 4, 5, 5), noradrenaline ( n = 5) and dopamine ( n = 5) ( d ) and corresponding levels of IL-6 ( n = 4), KC ( n = 7, 7, 7, 6, 5, 4, 5, 5), IFN-γ ( n = 6, 6, 6, 8, 4, 8, 6, 4) and TNF ( n = 6, 6, 6, 6, 6, 4, 7, 7) ( e ) in LPS-treated mice receiving MTR 40 mg kg −1 . Data are presented as mean ± s.d. with individual data points shown, analysed by two-tailed t -test ( b – e ).
    Figure Legend Snippet: Modulation of catecholamine synthesis by MTR dose-dependently determines survival and cytokine release. a , Survival of BALB/c mice stimulated with a lethal dose of LPS and treated with the indicated dose of MTR: MTR 20 mg kg −1 ( n = 5 mice per group); MTR 30 mg kg −1 ( n = 10 mice), MTR 40 mg kg −1 ( n = 12) compared to LPS ( n = 10 mice). Survival differences were analysed by two-sided log-rank test. b , c , Levels of plasma catecholamines ( n = 4 per column) ( b ) and IL-6 ( n = 4 per column), KC (left to right, n = 4, 4, 3 per column), IFN-γ ( n = 4) and TNF ( n = 4, 4, 3) ( c ) at different MTR doses measured 24 h after LPS injection. d , e , 24-h-time course of circulating adrenaline ( n = 5, 5, 5, 4, 5, 4, 5, 5), noradrenaline ( n = 5) and dopamine ( n = 5) ( d ) and corresponding levels of IL-6 ( n = 4), KC ( n = 7, 7, 7, 6, 5, 4, 5, 5), IFN-γ ( n = 6, 6, 6, 8, 4, 8, 6, 4) and TNF ( n = 6, 6, 6, 6, 6, 4, 7, 7) ( e ) in LPS-treated mice receiving MTR 40 mg kg −1 . Data are presented as mean ± s.d. with individual data points shown, analysed by two-tailed t -test ( b – e ).

    Techniques Used: Mouse Assay, Injection, Two Tailed Test

    Adrenaline enhances the inflammatory response. a , Survival of BALB/c mice implanted with the indicated catecholamine pump and stimulated with a sublethal dose of LPS ( n = 14 mice per group) compared to LPS alone ( n = 19 mice). Survival differences were analysed by Gehan–Breslow–Wilcoxon test. b , Survival of BALB/c mice with indicated catecholamine pump without LPS stimulation ( n = 5 mice per group). c , d , 24 h plasma levels of adrenaline (left to right, n = 3, 4, 3, 3, 3, 4, 4, 3 per column), noradrenaline ( n = 3, 3, 3, 3, 3, 4, 4, 3) and dopamine ( n = 3, 3, 3, 3, 3, 4, 4, 3) ( c ) as well as levels of IL-6 ( n = 4 per column), TNF ( n = 5 per column) and KC ( n = 4 per column) ( d ) in mice receiving the indicated treatments. e , Dopamine concentration in LPS- and adrenaline-treated peritoneal macrophages pre-incubated with ANP or MTR ( n = 3 per column), measured after 24 h. f , g , Levels of catecholamines ( n = 3 independent samples per column) ( f ) and several cytokines ( n = 3 independent samples per column) ( g ) in adrenaline (15 ng ml −1 )-treated peritoneal macrophages pre-incubated with ANP or MTR and measured after 24 h. Data are presented as mean ± s.d. with individual data points shown, analysed by two-tailed t -test ( c – g ).
    Figure Legend Snippet: Adrenaline enhances the inflammatory response. a , Survival of BALB/c mice implanted with the indicated catecholamine pump and stimulated with a sublethal dose of LPS ( n = 14 mice per group) compared to LPS alone ( n = 19 mice). Survival differences were analysed by Gehan–Breslow–Wilcoxon test. b , Survival of BALB/c mice with indicated catecholamine pump without LPS stimulation ( n = 5 mice per group). c , d , 24 h plasma levels of adrenaline (left to right, n = 3, 4, 3, 3, 3, 4, 4, 3 per column), noradrenaline ( n = 3, 3, 3, 3, 3, 4, 4, 3) and dopamine ( n = 3, 3, 3, 3, 3, 4, 4, 3) ( c ) as well as levels of IL-6 ( n = 4 per column), TNF ( n = 5 per column) and KC ( n = 4 per column) ( d ) in mice receiving the indicated treatments. e , Dopamine concentration in LPS- and adrenaline-treated peritoneal macrophages pre-incubated with ANP or MTR ( n = 3 per column), measured after 24 h. f , g , Levels of catecholamines ( n = 3 independent samples per column) ( f ) and several cytokines ( n = 3 independent samples per column) ( g ) in adrenaline (15 ng ml −1 )-treated peritoneal macrophages pre-incubated with ANP or MTR and measured after 24 h. Data are presented as mean ± s.d. with individual data points shown, analysed by two-tailed t -test ( c – g ).

    Techniques Used: Mouse Assay, Concentration Assay, Incubation, Aqueous Normal-phase Chromatography, Two Tailed Test

    Catecholamine production in myeloid cells is essential for cytokine release. a , Peritoneal macrophages were pre-incubated with ANP or MTR for 10 min and then stimulated with LPS (50 μg ml −1 ) or a combination of LPS and adrenaline (15 ng ml −1 ) in vitro. Shown are the levels of adrenaline (left to right, n = 3, 3, 3, 6, 6, 6, 3, 3, 3 per column) and noradrenaline ( n = 3) in the supernatant after 24 h. b , Corresponding cytokines from macrophage culture supernatants: IL-6 ( n = 3, 3, 3, 4, 4, 4, 3, 3, 3), MIP-2 ( n = 4, 4, 4, 4, 5, 5, 4, 3, 3), KC ( n = 3, 3, 3, 5, 5, 5, 3, 3, 3) and TNF ( n = 3, 3, 3, 3, 5, 6, 4, 3, 3). c , Survival of Th +/+ and Th ΔLysM mice treated with LPS and analysed with two-sided log-rank test ( n = 12; 6 male, 6 female). d , e , Plasma levels of adrenaline ( n = 4, 4, 7, 6) and noradrenaline ( n = 3, 3, 7, 6) ( d ) and indicated cytokines ( n = 3, 3, 4, 3) ( e ) at baseline and 24 h after LPS treatment in Th +/+ or Th ΔLysM mice. Data are presented as mean ± s.d. with individual data points shown, analysed by two-tailed t -test ( a , b , d , e ).
    Figure Legend Snippet: Catecholamine production in myeloid cells is essential for cytokine release. a , Peritoneal macrophages were pre-incubated with ANP or MTR for 10 min and then stimulated with LPS (50 μg ml −1 ) or a combination of LPS and adrenaline (15 ng ml −1 ) in vitro. Shown are the levels of adrenaline (left to right, n = 3, 3, 3, 6, 6, 6, 3, 3, 3 per column) and noradrenaline ( n = 3) in the supernatant after 24 h. b , Corresponding cytokines from macrophage culture supernatants: IL-6 ( n = 3, 3, 3, 4, 4, 4, 3, 3, 3), MIP-2 ( n = 4, 4, 4, 4, 5, 5, 4, 3, 3), KC ( n = 3, 3, 3, 5, 5, 5, 3, 3, 3) and TNF ( n = 3, 3, 3, 3, 5, 6, 4, 3, 3). c , Survival of Th +/+ and Th ΔLysM mice treated with LPS and analysed with two-sided log-rank test ( n = 12; 6 male, 6 female). d , e , Plasma levels of adrenaline ( n = 4, 4, 7, 6) and noradrenaline ( n = 3, 3, 7, 6) ( d ) and indicated cytokines ( n = 3, 3, 4, 3) ( e ) at baseline and 24 h after LPS treatment in Th +/+ or Th ΔLysM mice. Data are presented as mean ± s.d. with individual data points shown, analysed by two-tailed t -test ( a , b , d , e ).

    Techniques Used: Incubation, Aqueous Normal-phase Chromatography, In Vitro, Mouse Assay, Two Tailed Test

    MTR and ANP prevent cytokine release in Raji/hCART19 mouse model. a , Bioluminescent images (BLI) of Raji-bearing NSGS mice with high tumour burden. At day 0, tumour engraftment was quantified by BLI and mice were assigned to the treatment groups (untreated, MTR, hCART19, hCART19+MTR, n = 5 mice per group; UT-T, n = 4). b , c , Levels of dopamine (left to right, n = 3, 3, 3, 4, 4, 4, 3, 4, 4, 4 per column) ( b ) and indicated cytokines ( n = 4) ( c ) measured in mice (with high tumour burden) 24 and 72 h after hCART19 and UT-T injection. d , BLI of Raji-bearing NSGS mice with low tumour burden. At day 0, mice were randomly assigned based on tumour burden to receive hCART19, with or without MTR ( n = 10 mice per group) or UT-T, with or without MTR ( n = 5 mice per group). e , Levels of Hs IL-2 ( n = 4, 4, 3) and Mm MIP-2 ( n = 3, 3, 4) assessed 72 h after hCART19 injection in mice with low tumour burden. f , g , NSGS mice were injected with hCART19 4 days after Raji implantation and treated with ANP delivered via subcutaneously implanted osmotic pumps. Levels of circulating catecholamines ( n = 4 per column) ( f ) and Mm IL-6, Mm KC and Mm MIP-2 ( n = 4, 4, 3, 4) as well as Hs IL-2 ( n = 4) ( g ) were assessed 24 h after hCART19 administration. h , Survival of Raji cell-bearing NSGS mice treated with hCART19 and ANP ( n = 5 per group); analysed by two-sided log-rank test. i , Level of circulating hCART19 10 days after treatment, determined by C q from RT–PCR and analysed in triplicates ( n = 4 per group). Data are presented as mean ± s.d. with individual data points shown, analysed by two-tailed t -test ( b , c , e – i ).
    Figure Legend Snippet: MTR and ANP prevent cytokine release in Raji/hCART19 mouse model. a , Bioluminescent images (BLI) of Raji-bearing NSGS mice with high tumour burden. At day 0, tumour engraftment was quantified by BLI and mice were assigned to the treatment groups (untreated, MTR, hCART19, hCART19+MTR, n = 5 mice per group; UT-T, n = 4). b , c , Levels of dopamine (left to right, n = 3, 3, 3, 4, 4, 4, 3, 4, 4, 4 per column) ( b ) and indicated cytokines ( n = 4) ( c ) measured in mice (with high tumour burden) 24 and 72 h after hCART19 and UT-T injection. d , BLI of Raji-bearing NSGS mice with low tumour burden. At day 0, mice were randomly assigned based on tumour burden to receive hCART19, with or without MTR ( n = 10 mice per group) or UT-T, with or without MTR ( n = 5 mice per group). e , Levels of Hs IL-2 ( n = 4, 4, 3) and Mm MIP-2 ( n = 3, 3, 4) assessed 72 h after hCART19 injection in mice with low tumour burden. f , g , NSGS mice were injected with hCART19 4 days after Raji implantation and treated with ANP delivered via subcutaneously implanted osmotic pumps. Levels of circulating catecholamines ( n = 4 per column) ( f ) and Mm IL-6, Mm KC and Mm MIP-2 ( n = 4, 4, 3, 4) as well as Hs IL-2 ( n = 4) ( g ) were assessed 24 h after hCART19 administration. h , Survival of Raji cell-bearing NSGS mice treated with hCART19 and ANP ( n = 5 per group); analysed by two-sided log-rank test. i , Level of circulating hCART19 10 days after treatment, determined by C q from RT–PCR and analysed in triplicates ( n = 4 per group). Data are presented as mean ± s.d. with individual data points shown, analysed by two-tailed t -test ( b , c , e – i ).

    Techniques Used: Aqueous Normal-phase Chromatography, Mouse Assay, Injection, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

    7) Product Images from "Stromal-derived interleukin 6 drives epithelial-to-mesenchymal transition and therapy resistance in esophageal adenocarcinoma"

    Article Title: Stromal-derived interleukin 6 drives epithelial-to-mesenchymal transition and therapy resistance in esophageal adenocarcinoma

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

    doi: 10.1073/pnas.1820459116

    Stromal CAF-secreted IL-6 drives therapy resistance. ( A – C ) Cell viability assays were performed on primary 007B cells incubated for 168 h in the following culture conditions: unconditioned medium without chemotherapeutics (untreated, untr), unconditioned medium with chemotherapeutics (control), conditioned medium with chemotherapeutics, medium supplemented with the indicated cytokines and chemotherapeutics (colored bars), or 081RF supernatant with or without neutralizing antibodies for the indicated cytokines and chemotherapeutics. Graphs show means ± SEM of data normalized to t = 0, n = 3. P values were by one-way ANOVA and compared with the control or 081RF (–) sup only condition. ( D – F ) As for A – C , using 031M cells. ( G ) Human IL-6 was measured by ELISA in 3 d-incubated supernatant of the indicated cultures (5 d for 243RF culture) and media not incubated on cells. ( H ) Mouse IL-6 was measured by ELISA as for G in supernatants from indicated (co)cultures. ( I ) 293T, 007B, and 031M cells were stimulated for 20 min with medium containing 081RF supernatant incubated for 3 d, diluted 1 in 4. Recombinant IL-6 was used as a positive control, and IL-6–neutralizing antibody was used as a negative control for IL-6–induced STAT3 phosphorylation. Following exposure, cells were lysed and processed for Western blot analysis for the indicated antigens. * P
    Figure Legend Snippet: Stromal CAF-secreted IL-6 drives therapy resistance. ( A – C ) Cell viability assays were performed on primary 007B cells incubated for 168 h in the following culture conditions: unconditioned medium without chemotherapeutics (untreated, untr), unconditioned medium with chemotherapeutics (control), conditioned medium with chemotherapeutics, medium supplemented with the indicated cytokines and chemotherapeutics (colored bars), or 081RF supernatant with or without neutralizing antibodies for the indicated cytokines and chemotherapeutics. Graphs show means ± SEM of data normalized to t = 0, n = 3. P values were by one-way ANOVA and compared with the control or 081RF (–) sup only condition. ( D – F ) As for A – C , using 031M cells. ( G ) Human IL-6 was measured by ELISA in 3 d-incubated supernatant of the indicated cultures (5 d for 243RF culture) and media not incubated on cells. ( H ) Mouse IL-6 was measured by ELISA as for G in supernatants from indicated (co)cultures. ( I ) 293T, 007B, and 031M cells were stimulated for 20 min with medium containing 081RF supernatant incubated for 3 d, diluted 1 in 4. Recombinant IL-6 was used as a positive control, and IL-6–neutralizing antibody was used as a negative control for IL-6–induced STAT3 phosphorylation. Following exposure, cells were lysed and processed for Western blot analysis for the indicated antigens. * P

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay, Recombinant, Positive Control, Negative Control, Western Blot

    CAF-secreted IL-6 induces epithelial-to-mesenchymal transition. ( A ) The 007B and 031M cultures were exposed to the following conditions for 14 d: unconditioned medium (untreated), 081RF supernatant (1 in 4 diluted), recombinant IL-6, and 081RF + IL-6–neutralizing antibody. Morphology was assessed by phase-contrast microscopy. (Scale bar: 200 μm.) ( B ) As for A using 007B and 031M organoid cultures. Dashed lines indicate the migratory front of cells migrating out of the organoid. Arrows indicate the edge of the Matrigel cushion. ( C ) Transwell migration assays on 007B and 031M cells cultured for 14 d in the conditions as for A before the assay. In the transwell assays, 1% FCS was used as a chemoattractant. Migration shown is corrected for no-attractant controls (medium without FCS), n = 3. P values were determined by two-way ANOVA and Tukey’s multiple comparisons correction, one-phase exponential curves were fitted, and the lines of matching color indicate the SD. ( D ) Limiting dilution assays were performed using 007B and 031M cells after incubation for 14 d in the indicated conditions. Cells were sorted into 96-well plates. Bar graphs show means ± SEM, n = 3. * P
    Figure Legend Snippet: CAF-secreted IL-6 induces epithelial-to-mesenchymal transition. ( A ) The 007B and 031M cultures were exposed to the following conditions for 14 d: unconditioned medium (untreated), 081RF supernatant (1 in 4 diluted), recombinant IL-6, and 081RF + IL-6–neutralizing antibody. Morphology was assessed by phase-contrast microscopy. (Scale bar: 200 μm.) ( B ) As for A using 007B and 031M organoid cultures. Dashed lines indicate the migratory front of cells migrating out of the organoid. Arrows indicate the edge of the Matrigel cushion. ( C ) Transwell migration assays on 007B and 031M cells cultured for 14 d in the conditions as for A before the assay. In the transwell assays, 1% FCS was used as a chemoattractant. Migration shown is corrected for no-attractant controls (medium without FCS), n = 3. P values were determined by two-way ANOVA and Tukey’s multiple comparisons correction, one-phase exponential curves were fitted, and the lines of matching color indicate the SD. ( D ) Limiting dilution assays were performed using 007B and 031M cells after incubation for 14 d in the indicated conditions. Cells were sorted into 96-well plates. Bar graphs show means ± SEM, n = 3. * P

    Techniques Used: Recombinant, Microscopy, Migration, Cell Culture, Incubation

    Stroma-derived IL-6 confers resistance to radiochemotherapy in EAC patients. ( A ) Clonogenic assays were performed on 007B cells after receiving one dose of the indicated cytostatic agents and seven doses of 1-Gy radiation. Cells were cultured in the following conditions for 10 d before the assay: control, 081RF supernatant, 081RF supernatant + IL-6–neutralizing antibody (500 ng/mL), or recombinant IL-6 (2 ng/mL). ( B ) As for A , using 031M cells. ( C ) The 007B and 031M organoid cultures were exposed to conditions as for A and B . The culture conditions were maintained throughout the assay, and morphology was monitored by phase-contrast microscopy. Shown are passage 1 (4 wk after treatment) and passage 2 (10 d after passage 1). (Scale bar: 200 μm.) ( D ) Blood was drawn and processed for serum storage from pretreatment EAC patients seen at the Academic Medical Center (AMC) ( n = 80). All patients then received the neoadjuvant CROSS regimen, and Mandard score was determined by a pathologist. IL-6 serum levels of pretreated EAC patients were measured using ELISA. ( E ) The same serum samples as for D were used to measure ADAM12. Correlation of serum IL-6 and ADAM12 levels was determined on all samples, including those with blank measurements. The log-scale plot excludes blanks. ( F ) As for D , showing ADAM12 serum levels. Graphs show means ± SD. Significance was tested by the Mann–Whitney U test. ( G ) Indicated CAF lines were treated with IL-6 (10 ng/mL), IL-6–neutralizing antibody (1 µg/mL), or TGF-β (5 ng/mL) for 3 d. Supernatant was harvested after an additional 7 d, and ADAM12 levels were analyzed by ELISA. ** P
    Figure Legend Snippet: Stroma-derived IL-6 confers resistance to radiochemotherapy in EAC patients. ( A ) Clonogenic assays were performed on 007B cells after receiving one dose of the indicated cytostatic agents and seven doses of 1-Gy radiation. Cells were cultured in the following conditions for 10 d before the assay: control, 081RF supernatant, 081RF supernatant + IL-6–neutralizing antibody (500 ng/mL), or recombinant IL-6 (2 ng/mL). ( B ) As for A , using 031M cells. ( C ) The 007B and 031M organoid cultures were exposed to conditions as for A and B . The culture conditions were maintained throughout the assay, and morphology was monitored by phase-contrast microscopy. Shown are passage 1 (4 wk after treatment) and passage 2 (10 d after passage 1). (Scale bar: 200 μm.) ( D ) Blood was drawn and processed for serum storage from pretreatment EAC patients seen at the Academic Medical Center (AMC) ( n = 80). All patients then received the neoadjuvant CROSS regimen, and Mandard score was determined by a pathologist. IL-6 serum levels of pretreated EAC patients were measured using ELISA. ( E ) The same serum samples as for D were used to measure ADAM12. Correlation of serum IL-6 and ADAM12 levels was determined on all samples, including those with blank measurements. The log-scale plot excludes blanks. ( F ) As for D , showing ADAM12 serum levels. Graphs show means ± SD. Significance was tested by the Mann–Whitney U test. ( G ) Indicated CAF lines were treated with IL-6 (10 ng/mL), IL-6–neutralizing antibody (1 µg/mL), or TGF-β (5 ng/mL) for 3 d. Supernatant was harvested after an additional 7 d, and ADAM12 levels were analyzed by ELISA. ** P

    Techniques Used: Derivative Assay, Cell Culture, Recombinant, Microscopy, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    8) Product Images from "Role of the microenvironment in mantle cell lymphoma: IL-6 is an important survival factor for the tumor cells"

    Article Title: Role of the microenvironment in mantle cell lymphoma: IL-6 is an important survival factor for the tumor cells

    Journal: Blood

    doi: 10.1182/blood-2012-04-424630

    Role of STAT3 activation in IL-6–mediated protection of MCL cells. Western blot showing STAT3 phosphorylation (A) in Mino and Granta 519 cells examined at 15, 30, 60, and 120 minutes in cultures without or with addition of IL-6 (0.5 ng/mL) and IL-6–neutralizing antibodies (a-IL6; 50 μg/mL), (B) in Mino and Granta 519 cells that were pretreated with 25μM of STAT3 inhibitor V or JAK2 inhibitor, respectively, for 2 hours, before culturing with IL-6 (0.5 ng/mL) for 30 minutes, or (C) in Mino cells that were pretreated with 25μM of PI3K, mTOR, or MEK inhibitors, respectively, for 2 hours before culturing with IL-6 (0.5 ng/mL) for 30 minutes. Also shown are percentages of apoptotic (D) Mino and Granta 519 cells in cultures without or with addition of BTZ (10nM), IL-6 (0.5 ng/mL), or STAT3 inhibitor V (25μM); or (E) Mino cells in cultures without or with addition of IL-6 (0.5 ng/mL), BTZ (10nM), or inhibitors (25μM) to Jak2, STAT3, PI3K, MEK, or mTOR, respectively, for 48 hours. The P value in the graph shows comparison as indicated
    Figure Legend Snippet: Role of STAT3 activation in IL-6–mediated protection of MCL cells. Western blot showing STAT3 phosphorylation (A) in Mino and Granta 519 cells examined at 15, 30, 60, and 120 minutes in cultures without or with addition of IL-6 (0.5 ng/mL) and IL-6–neutralizing antibodies (a-IL6; 50 μg/mL), (B) in Mino and Granta 519 cells that were pretreated with 25μM of STAT3 inhibitor V or JAK2 inhibitor, respectively, for 2 hours, before culturing with IL-6 (0.5 ng/mL) for 30 minutes, or (C) in Mino cells that were pretreated with 25μM of PI3K, mTOR, or MEK inhibitors, respectively, for 2 hours before culturing with IL-6 (0.5 ng/mL) for 30 minutes. Also shown are percentages of apoptotic (D) Mino and Granta 519 cells in cultures without or with addition of BTZ (10nM), IL-6 (0.5 ng/mL), or STAT3 inhibitor V (25μM); or (E) Mino cells in cultures without or with addition of IL-6 (0.5 ng/mL), BTZ (10nM), or inhibitors (25μM) to Jak2, STAT3, PI3K, MEK, or mTOR, respectively, for 48 hours. The P value in the graph shows comparison as indicated

    Techniques Used: Activation Assay, Western Blot

    Autocrine and exogenous IL-6 protects MCL cells from apoptosis. (A) Percentages of apoptotic cells in 48-hour cultures of primary MCL cells of 6 patients (PT1-PT6) in medium with or without addition of IL-6–neutralizing antibodies (α-IL-6; 50 μg/mL) and/or gp130-blocking antibodies (α-gp130; 5 μg/mL) or control IgG1. (B) Proliferation (CPM) of IL-6 + MCL SP53 and Granta 519 cells in a 7-day culture in medium in the presence or absence of α-IL-6 (50 μg/mL) and α-gp130 (5 μg/mL) antibodies or control IgG1. (C) Percentages of apoptotic Mino cells in 48-hour cultures in the presence of BTZ (10nM), atiprimod (ATI; 2μM), or cytarabine (Ara-c; 2μM). Mino cells were precultured with rIL-6 (0.5 ng/mL) for 2 hours before the drugs were added. Percentages of apoptotic cells induced by (D) BTZ (10nM) or (E) serum starvation. MCL cell cultures were first added with rIL-6 (50-5000 pg/mL) and 2 hours later were treated with BTZ or serum starvation. (F) Pooled data from 3 patients (PT1-PT3) showing that rIL-6 protected both MCL cell lines and primary MCL cells against BTZ-induced apoptosis. The presence of 50 μg/mL of α-IL-6 and/or 5 μg/mL of α-gp130 antibodies abrogated the protection of rIL-6 against BTZ-induced apoptosis. Results of 3 independent experiments are shown. The P values in the graphs show comparison as indicated. * P
    Figure Legend Snippet: Autocrine and exogenous IL-6 protects MCL cells from apoptosis. (A) Percentages of apoptotic cells in 48-hour cultures of primary MCL cells of 6 patients (PT1-PT6) in medium with or without addition of IL-6–neutralizing antibodies (α-IL-6; 50 μg/mL) and/or gp130-blocking antibodies (α-gp130; 5 μg/mL) or control IgG1. (B) Proliferation (CPM) of IL-6 + MCL SP53 and Granta 519 cells in a 7-day culture in medium in the presence or absence of α-IL-6 (50 μg/mL) and α-gp130 (5 μg/mL) antibodies or control IgG1. (C) Percentages of apoptotic Mino cells in 48-hour cultures in the presence of BTZ (10nM), atiprimod (ATI; 2μM), or cytarabine (Ara-c; 2μM). Mino cells were precultured with rIL-6 (0.5 ng/mL) for 2 hours before the drugs were added. Percentages of apoptotic cells induced by (D) BTZ (10nM) or (E) serum starvation. MCL cell cultures were first added with rIL-6 (50-5000 pg/mL) and 2 hours later were treated with BTZ or serum starvation. (F) Pooled data from 3 patients (PT1-PT3) showing that rIL-6 protected both MCL cell lines and primary MCL cells against BTZ-induced apoptosis. The presence of 50 μg/mL of α-IL-6 and/or 5 μg/mL of α-gp130 antibodies abrogated the protection of rIL-6 against BTZ-induced apoptosis. Results of 3 independent experiments are shown. The P values in the graphs show comparison as indicated. * P

    Techniques Used: Blocking Assay, Acetylene Reduction Assay

    Paracrine or exogenous IL-6 protects MCL cells against chemotherapy drug–induced apoptosis. (A) Viability of freshly isolated primary MCL cells from 3 patients (PT1-PT3) cocultured for 24 hours with PBMCs from 2 blood donors (PBMC1 and PBMC2) in Transwell inserts in the presence of BTZ (10nM) with the addition of IL-6–neutralizing (α-IL-6; 50 μg/mL) and gp130-blocking (α-gp130; 5 μg/mL) antibodies or control IgG1. After the culture, PBMCs in Transwell inserts were removed and MCL cells in the culture wells were subjected to MTS assay (* P
    Figure Legend Snippet: Paracrine or exogenous IL-6 protects MCL cells against chemotherapy drug–induced apoptosis. (A) Viability of freshly isolated primary MCL cells from 3 patients (PT1-PT3) cocultured for 24 hours with PBMCs from 2 blood donors (PBMC1 and PBMC2) in Transwell inserts in the presence of BTZ (10nM) with the addition of IL-6–neutralizing (α-IL-6; 50 μg/mL) and gp130-blocking (α-gp130; 5 μg/mL) antibodies or control IgG1. After the culture, PBMCs in Transwell inserts were removed and MCL cells in the culture wells were subjected to MTS assay (* P

    Techniques Used: Isolation, Blocking Assay, MTS Assay

    Autocrine or paracrine gp80 protects MCL cells against apoptosis. Percentages of apoptotic: (A) Mino cells in cultures with or without addition of IL-6 (0.5 ng/mL), gp80-neutralizing antibodies (α-gp80; 10 μg/mL), or control IgG1 for 2 hours before BTZ (10nM) was added to the culture for additional 48 hours; (B) gp80-knockdown (KD), wild-type (WT), or empty vector-transfected Mino cells (Empty vector) in cultures with or without addition of IL-6 (0.5 ng/mL) or Minosup for 2 hours before BTZ (10nM) was added to the culture for additional 48 hours; or (C) SP53 cells in cultures with or without addition of IL-6 (0.5 ng/mL), Minosup, gp80-neutralizing antibodies (α-gp80; 10 μg/mL) or control IgG1 for 2 hours before BTZ (10nM) was added to the culture for additional 48 hours. (D) ELISA showing the levels of gp80 secreted by conditional, gp80-expressing stable SP53 cells induced by doxycycline (DOX) or vehicle. (E) Percentage of apoptotic gp80-transfected (S80), empty vector-transfected (Svec), or parental SP53 cells, pretreated with PBS or DOX overnight, in cultures with addition of gp80-neutralizing antibodies (α-gp80; 10 μg/mL) or control IgG1, and/or BTZ (10nM) for 48 hours. Results of 3 independent experiments are shown. The P values in the graphs show comparison as indicated.
    Figure Legend Snippet: Autocrine or paracrine gp80 protects MCL cells against apoptosis. Percentages of apoptotic: (A) Mino cells in cultures with or without addition of IL-6 (0.5 ng/mL), gp80-neutralizing antibodies (α-gp80; 10 μg/mL), or control IgG1 for 2 hours before BTZ (10nM) was added to the culture for additional 48 hours; (B) gp80-knockdown (KD), wild-type (WT), or empty vector-transfected Mino cells (Empty vector) in cultures with or without addition of IL-6 (0.5 ng/mL) or Minosup for 2 hours before BTZ (10nM) was added to the culture for additional 48 hours; or (C) SP53 cells in cultures with or without addition of IL-6 (0.5 ng/mL), Minosup, gp80-neutralizing antibodies (α-gp80; 10 μg/mL) or control IgG1 for 2 hours before BTZ (10nM) was added to the culture for additional 48 hours. (D) ELISA showing the levels of gp80 secreted by conditional, gp80-expressing stable SP53 cells induced by doxycycline (DOX) or vehicle. (E) Percentage of apoptotic gp80-transfected (S80), empty vector-transfected (Svec), or parental SP53 cells, pretreated with PBS or DOX overnight, in cultures with addition of gp80-neutralizing antibodies (α-gp80; 10 μg/mL) or control IgG1, and/or BTZ (10nM) for 48 hours. Results of 3 independent experiments are shown. The P values in the graphs show comparison as indicated.

    Techniques Used: Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Expressing

    In vivo effect of IL-6 on MCL growth and survival. (A) Tumor burdens in SCID mice (5 per group) injected with parental-, gp80-overexpressing SP53 cells (S80)–, and empty vector-transfected SP53 (Svec) cells on week 4 after tumor inoculation. (B) ELISA showing the levels of secreted human gp80 in the sera of Svec or S80 SP53-inoculated SCID mice and in culture media of dissected cells from the subcutaneous tumor mass. Mouse serum was collected when tumor mass reached 15 mm in diameter. (C) IHC staining showing the expression of gp80 (top) or phosphorylated (p) STAT3 (bottom) by S80 and Svec tumors in mice. Phosphorylated STAT3 was intensively stained in the nuclei of the tumor cells. (D) Tumor burdens and (E) survival of parental-, Svec-, and S80-bearing SCID mice (10 per group) after BTZ treatment.
    Figure Legend Snippet: In vivo effect of IL-6 on MCL growth and survival. (A) Tumor burdens in SCID mice (5 per group) injected with parental-, gp80-overexpressing SP53 cells (S80)–, and empty vector-transfected SP53 (Svec) cells on week 4 after tumor inoculation. (B) ELISA showing the levels of secreted human gp80 in the sera of Svec or S80 SP53-inoculated SCID mice and in culture media of dissected cells from the subcutaneous tumor mass. Mouse serum was collected when tumor mass reached 15 mm in diameter. (C) IHC staining showing the expression of gp80 (top) or phosphorylated (p) STAT3 (bottom) by S80 and Svec tumors in mice. Phosphorylated STAT3 was intensively stained in the nuclei of the tumor cells. (D) Tumor burdens and (E) survival of parental-, Svec-, and S80-bearing SCID mice (10 per group) after BTZ treatment.

    Techniques Used: In Vivo, Mouse Assay, Injection, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Expressing

    Expression of IL-6 and its receptors by MCL. ELISA detected the levels of (A) IL-6 or (B) gp80 in culture media of MCL cell lines and primary MCL cells from 7 patients (PT1-PT7), (C) IL-6, and (D) gp80 in culture media of MCL cells after serum starvation or treatment with a low dose (2nM) of BTZ for 12 hours, and (E) IL-6 and gp80 in culture media of PBMCs from 4 blood donors (PBMC1-PBMC4). Also shown is flow cytometry analysis for the expression of gp130 on (F) MCL cell lines and (G) primary MCL cells from 4 patients (PT1-PT4).
    Figure Legend Snippet: Expression of IL-6 and its receptors by MCL. ELISA detected the levels of (A) IL-6 or (B) gp80 in culture media of MCL cell lines and primary MCL cells from 7 patients (PT1-PT7), (C) IL-6, and (D) gp80 in culture media of MCL cells after serum starvation or treatment with a low dose (2nM) of BTZ for 12 hours, and (E) IL-6 and gp80 in culture media of PBMCs from 4 blood donors (PBMC1-PBMC4). Also shown is flow cytometry analysis for the expression of gp130 on (F) MCL cell lines and (G) primary MCL cells from 4 patients (PT1-PT4).

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    9) Product Images from "Intraperitoneal cancer-immune microenvironment promotes peritoneal dissemination of gastric cancer"

    Article Title: Intraperitoneal cancer-immune microenvironment promotes peritoneal dissemination of gastric cancer

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2019.1671760

    Clinical correlation between IL-6 and TAMs in the peritoneal cavity. (a) and (b) The levels of IL-6 and CD163+ macrophages were evaluated in stage I and stage IV patients. (c) and (d) The levels of IL-6 and CD163+ macrophages were compared between cytology-positive only and peritoneal dissemination-positive patients.A value of IL-6 less than detectable sensitivity was defined as one. A value greater than 4 pg/ml, which is a serum reference value, was defined as positive. The values represent means ± SD. (* p
    Figure Legend Snippet: Clinical correlation between IL-6 and TAMs in the peritoneal cavity. (a) and (b) The levels of IL-6 and CD163+ macrophages were evaluated in stage I and stage IV patients. (c) and (d) The levels of IL-6 and CD163+ macrophages were compared between cytology-positive only and peritoneal dissemination-positive patients.A value of IL-6 less than detectable sensitivity was defined as one. A value greater than 4 pg/ml, which is a serum reference value, was defined as positive. The values represent means ± SD. (* p

    Techniques Used:

    IL-6 potentiates migration and invasion of GC and induces phosphorylation of STAT3. (a) Migration and invasion assay of GCIY cells treated with the medium containing IL-6 (0–100 ng/ml). Migrated and invaded cells were counted by staining with 0.5% crystal violet. The values represent means ± S. D. (* p
    Figure Legend Snippet: IL-6 potentiates migration and invasion of GC and induces phosphorylation of STAT3. (a) Migration and invasion assay of GCIY cells treated with the medium containing IL-6 (0–100 ng/ml). Migrated and invaded cells were counted by staining with 0.5% crystal violet. The values represent means ± S. D. (* p

    Techniques Used: Migration, Invasion Assay, Staining

    IL-6 is elevated with the co-culture of TDMs and GC cells. (a) Cytokine and chemokine arrays of supernatants from transwell co-culture TDMs and GCIY cells. The values are normalized by TDMs mono-culture supernatants. (b) IL-6 concentration in TDMs/GCIY transwell co-culture supernatants were quantified by ELISA (n = 3). As a control, each mono-culture supernatant of GCIY or TDMs was analyzed. The values represent means ± SD. (* p
    Figure Legend Snippet: IL-6 is elevated with the co-culture of TDMs and GC cells. (a) Cytokine and chemokine arrays of supernatants from transwell co-culture TDMs and GCIY cells. The values are normalized by TDMs mono-culture supernatants. (b) IL-6 concentration in TDMs/GCIY transwell co-culture supernatants were quantified by ELISA (n = 3). As a control, each mono-culture supernatant of GCIY or TDMs was analyzed. The values represent means ± SD. (* p

    Techniques Used: Co-Culture Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

    10) Product Images from "High density lipoprotein mediates anti-inflammatory transcriptional reprogramming of macrophages via the transcriptional repressor ATF3"

    Article Title: High density lipoprotein mediates anti-inflammatory transcriptional reprogramming of macrophages via the transcriptional repressor ATF3

    Journal: Nature immunology

    doi: 10.1038/ni.2784

    ATF3 is active following induction by HDL. a – c , ATF3 ChIP sequencing of BMDMs pre-treated with HDL (2 mg/ml) for 6 h followed by stimulation with CpG (100 nM) for 4 h. Distribution of ATF3 binding within the genome (upper) and promoter regions (lower panel) ( a ) and global ATF3 peak localization relative to transcription start site (TSS); normalized average of tags per peak per bp from −1kb to +1kb ( b ). Genomic loci of Il6 , Il12p40 and Tnf with ChIP-Seq signals for ATF3 binding under the stimulation conditions, red bars indicate significant peaks ( c ). d , e qPCR analysis of ATF3, IL-6, IL-12p40 or Ch25h mRNA expression in livers from Apoe -deficient mice fed on a high fat diet diet and injected intravenously (i.v.) with PBS or HDL (100 mg/kg) ( n= 5) . a – c , At least three biological replicates per condition were generated. d , e Data are shown as the mean ±S.E.M, PBS versus HDL **p
    Figure Legend Snippet: ATF3 is active following induction by HDL. a – c , ATF3 ChIP sequencing of BMDMs pre-treated with HDL (2 mg/ml) for 6 h followed by stimulation with CpG (100 nM) for 4 h. Distribution of ATF3 binding within the genome (upper) and promoter regions (lower panel) ( a ) and global ATF3 peak localization relative to transcription start site (TSS); normalized average of tags per peak per bp from −1kb to +1kb ( b ). Genomic loci of Il6 , Il12p40 and Tnf with ChIP-Seq signals for ATF3 binding under the stimulation conditions, red bars indicate significant peaks ( c ). d , e qPCR analysis of ATF3, IL-6, IL-12p40 or Ch25h mRNA expression in livers from Apoe -deficient mice fed on a high fat diet diet and injected intravenously (i.v.) with PBS or HDL (100 mg/kg) ( n= 5) . a – c , At least three biological replicates per condition were generated. d , e Data are shown as the mean ±S.E.M, PBS versus HDL **p

    Techniques Used: Chromatin Immunoprecipitation, Sequencing, Binding Assay, Real-time Polymerase Chain Reaction, Expressing, Mouse Assay, Injection, Generated

    HDL inhibits TLR-induced pro-inflammatory cytokine transcription. a , Bodipy-labeled LPS, Alexa 647-labeled CpG 1826 or HDL were run over an S200 size exclusion column separately ( a , upper panel) or together ( a , lower panel) and absorbance profiles examined. b , LPS (200 ng/ml), CpG (100 nM) or P3C (50 ng/ml) were incubated with HDL (2 mg/ml), and BMDMs stimulated for 30 min. Whole cell lysates were analyzed for p38 phosphorylation (p-p38) relative to total β-actin. c , d BMDMs were pre-treated with HDL (2 mg/ml) for 6 h before stimulation with CpG (100 nM) for indicated times: total cellular cholesterol ( c ) and phosphorylation of p38, JNK, NF-κB p65 and IκBα degradation ( d ). e , f BMDMs were pre-treated with HDL as before, and stimulated with CpG (100 nM) for 30 min: subcellular localization of NF-κB p65 (β-tubulin: cytoplasmic loading control; poly ADP ribose polymerase (PARP): nuclear loading control) ( e ) and NF-κB binding to a target probe as analysed by EMSA ( f ). g , h BMDMs were pre-treated with HDL as before: CpG-induced phospho-NF-κB p65 and intracellular IL-6 and IL-1β were measured in whole cell extracts, ( g ); secreted IL-6 measured by ELISA ( h ). mRNA expression of BMDMs pre-treated with HDL as before, and stimulated with 100 nM CpG for 4 h ( i ). j , Liver mRNA profile 1 h after C57BL/6 mice were injected with CpG following 6 h HDL pre-treatment ( n =8). a , Data is representative of at least three independent experiments. b , Representative immunoblot and densitometric analysis combined from three independent experiments (mean ±S.E.M, each ligand; no HDL versus HDL incubation ***p
    Figure Legend Snippet: HDL inhibits TLR-induced pro-inflammatory cytokine transcription. a , Bodipy-labeled LPS, Alexa 647-labeled CpG 1826 or HDL were run over an S200 size exclusion column separately ( a , upper panel) or together ( a , lower panel) and absorbance profiles examined. b , LPS (200 ng/ml), CpG (100 nM) or P3C (50 ng/ml) were incubated with HDL (2 mg/ml), and BMDMs stimulated for 30 min. Whole cell lysates were analyzed for p38 phosphorylation (p-p38) relative to total β-actin. c , d BMDMs were pre-treated with HDL (2 mg/ml) for 6 h before stimulation with CpG (100 nM) for indicated times: total cellular cholesterol ( c ) and phosphorylation of p38, JNK, NF-κB p65 and IκBα degradation ( d ). e , f BMDMs were pre-treated with HDL as before, and stimulated with CpG (100 nM) for 30 min: subcellular localization of NF-κB p65 (β-tubulin: cytoplasmic loading control; poly ADP ribose polymerase (PARP): nuclear loading control) ( e ) and NF-κB binding to a target probe as analysed by EMSA ( f ). g , h BMDMs were pre-treated with HDL as before: CpG-induced phospho-NF-κB p65 and intracellular IL-6 and IL-1β were measured in whole cell extracts, ( g ); secreted IL-6 measured by ELISA ( h ). mRNA expression of BMDMs pre-treated with HDL as before, and stimulated with 100 nM CpG for 4 h ( i ). j , Liver mRNA profile 1 h after C57BL/6 mice were injected with CpG following 6 h HDL pre-treatment ( n =8). a , Data is representative of at least three independent experiments. b , Representative immunoblot and densitometric analysis combined from three independent experiments (mean ±S.E.M, each ligand; no HDL versus HDL incubation ***p

    Techniques Used: Labeling, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Mouse Assay, Injection

    Microarray analysis identifies ATF3 as a candidate gene for the anti-inflammatory function of HDL. a , Immortalized-BMDMs were pre-treated with HDL (2 mg/ml) for indicated times, then either washed twice in serum-free DMEM, or left unwashed, before overnight stimulation with CpG (100 nM). TNF secretion was measured by ELISA and normalized to CpG treatment alone. b , BMDMs were pre-treated with HDL (2 mg/ml) for 12 h, then either washed twice in serum-free DMEM, or left unwashed, before stimulation with CpG (100 nM) for indicated times. IL-6 was measured in culture supernatants and normalized to CpG treatment alone. c – f , Microarray analysis of BMDMs pre-treated for 6 h with HDL (2 mg/ml) then stimulated with CpG (100 nM) for 4 h. c , Venn diagram shows genes with differential expression (vs control) and the overlap in these genes (Fold Change limit 1.8, False discovery rate p
    Figure Legend Snippet: Microarray analysis identifies ATF3 as a candidate gene for the anti-inflammatory function of HDL. a , Immortalized-BMDMs were pre-treated with HDL (2 mg/ml) for indicated times, then either washed twice in serum-free DMEM, or left unwashed, before overnight stimulation with CpG (100 nM). TNF secretion was measured by ELISA and normalized to CpG treatment alone. b , BMDMs were pre-treated with HDL (2 mg/ml) for 12 h, then either washed twice in serum-free DMEM, or left unwashed, before stimulation with CpG (100 nM) for indicated times. IL-6 was measured in culture supernatants and normalized to CpG treatment alone. c – f , Microarray analysis of BMDMs pre-treated for 6 h with HDL (2 mg/ml) then stimulated with CpG (100 nM) for 4 h. c , Venn diagram shows genes with differential expression (vs control) and the overlap in these genes (Fold Change limit 1.8, False discovery rate p

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

    HDL inhibits TLR-induced cytokine production from macrophages in vivo and in vitro. ( a – d ) Mice were injected intraperitoneally (i.p.) with 2 mg HDL for 6 h before i.p. injection of TLR ligand (CpG: 20 μg, P3C 2 μg) and D-galactosamine (10 mg). Serum cytokines were measured after 1 h and serum alanine aminotransferase (ALT) and liver histology after 10 h . a C3H/HeJ mice injected with native HDL and CpG: ALT release ( n =12) and serum cytokines (CpG n= 28, native HDL+CpG n = 27) were measured. b , c C57BL/6 mice injected with HDL and CpG: ALT release ( n =9); serum cytokines (CpG n =18, HDL+CpG n =19) ( b ), and liver histology by hemotoxylin and eosin staining shows hepatic cell death. ( c ). d , C57BL/6 injected with HDL and P3C: ALT release ( n =15) and serum cytokines ( n =15) were measured. e , ELISA of BMDMs treated with 2 mg/ml HDL for 6 h followed by overnight stimulation with the TLR4 ligand, LPS (100 ng/ml); TLR9 ligand, CpG (100 nM); TLR7/8 ligand, R848 (5 ng/ml); or TLR2/1 ligand P3C (50 ng/ml). f , Cell viability was measured with CellTitre- Blue® reagent. g , BMDMs were pre-treated with recombinant or native HDL (2 mg/ml) for 6 h and stimulated overnight with CpG (100 nM) or P3C (50 ng/ml) and IL-6 cytokine secretion measured by ELISA. a , b , d Data are presented as mean values ±S.E.M, CpG versus HDLs+CpG *p
    Figure Legend Snippet: HDL inhibits TLR-induced cytokine production from macrophages in vivo and in vitro. ( a – d ) Mice were injected intraperitoneally (i.p.) with 2 mg HDL for 6 h before i.p. injection of TLR ligand (CpG: 20 μg, P3C 2 μg) and D-galactosamine (10 mg). Serum cytokines were measured after 1 h and serum alanine aminotransferase (ALT) and liver histology after 10 h . a C3H/HeJ mice injected with native HDL and CpG: ALT release ( n =12) and serum cytokines (CpG n= 28, native HDL+CpG n = 27) were measured. b , c C57BL/6 mice injected with HDL and CpG: ALT release ( n =9); serum cytokines (CpG n =18, HDL+CpG n =19) ( b ), and liver histology by hemotoxylin and eosin staining shows hepatic cell death. ( c ). d , C57BL/6 injected with HDL and P3C: ALT release ( n =15) and serum cytokines ( n =15) were measured. e , ELISA of BMDMs treated with 2 mg/ml HDL for 6 h followed by overnight stimulation with the TLR4 ligand, LPS (100 ng/ml); TLR9 ligand, CpG (100 nM); TLR7/8 ligand, R848 (5 ng/ml); or TLR2/1 ligand P3C (50 ng/ml). f , Cell viability was measured with CellTitre- Blue® reagent. g , BMDMs were pre-treated with recombinant or native HDL (2 mg/ml) for 6 h and stimulated overnight with CpG (100 nM) or P3C (50 ng/ml) and IL-6 cytokine secretion measured by ELISA. a , b , d Data are presented as mean values ±S.E.M, CpG versus HDLs+CpG *p

    Techniques Used: In Vivo, In Vitro, Mouse Assay, Injection, Staining, Enzyme-linked Immunosorbent Assay, Recombinant

    11) Product Images from "Effects of prostaglandin lipid mediators on agonist-induced lung endothelial permeability and inflammation"

    Article Title: Effects of prostaglandin lipid mediators on agonist-induced lung endothelial permeability and inflammation

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00519.2016

    Effects of PGA 2 , PGE 2 , PGD 2 , PGF 2α , PGJ 2 , and PGI 2 on EC barrier dysfunction caused by IL-6. A : pulmonary EC pretreated with indicated PG (0.5 µM, first arrow) were stimulated with a combination of IL-6 (20 ng/ml) and IL-6 soluble receptor (SR, 40 ng/ml) as shown by second arrow. Changes in TER were monitored over 15-h time period. B : analysis of EC permeability using XPert assay. Bar graph depicts permeability changes after 6 h of IL-6/SR treatment; n = 5. * P
    Figure Legend Snippet: Effects of PGA 2 , PGE 2 , PGD 2 , PGF 2α , PGJ 2 , and PGI 2 on EC barrier dysfunction caused by IL-6. A : pulmonary EC pretreated with indicated PG (0.5 µM, first arrow) were stimulated with a combination of IL-6 (20 ng/ml) and IL-6 soluble receptor (SR, 40 ng/ml) as shown by second arrow. Changes in TER were monitored over 15-h time period. B : analysis of EC permeability using XPert assay. Bar graph depicts permeability changes after 6 h of IL-6/SR treatment; n = 5. * P

    Techniques Used: Permeability

    12) Product Images from "Elevated levels of adrenomedullin in eutopic endometrium and plasma from women with endometriosis"

    Article Title: Elevated levels of adrenomedullin in eutopic endometrium and plasma from women with endometriosis

    Journal: Fertility and sterility

    doi: 10.1016/j.fertnstert.2018.02.004

    IL-6 activation of STAT3 upregulates Adm expression in Ishikawa cells. A, Western blot analysis of pSTAT3 expression in Isihawa cells following a 15 minute treatment of varying doses of IL-6. B, qRT-PCR analysis of Adm expression in Ishikawa cells after a 15 minute treatment of 100 ng/mL IL-6. ***p
    Figure Legend Snippet: IL-6 activation of STAT3 upregulates Adm expression in Ishikawa cells. A, Western blot analysis of pSTAT3 expression in Isihawa cells following a 15 minute treatment of varying doses of IL-6. B, qRT-PCR analysis of Adm expression in Ishikawa cells after a 15 minute treatment of 100 ng/mL IL-6. ***p

    Techniques Used: Activation Assay, Expressing, Western Blot, Quantitative RT-PCR

    13) Product Images from "Combined treatment with antioxidants and immunosuppressants on cytokine release by human peripheral blood mononuclear cells - chemically injured keratocyte reaction"

    Article Title: Combined treatment with antioxidants and immunosuppressants on cytokine release by human peripheral blood mononuclear cells - chemically injured keratocyte reaction

    Journal: Molecular Vision

    doi:

    IL-6 levels measured by ELISA. IL-6 levels decreased in treatment with dexamethasone and anti-oxidants (Top left). The combination of immunosuppressants and antioxidants are more effective to suppress the production of IL-6 .
    Figure Legend Snippet: IL-6 levels measured by ELISA. IL-6 levels decreased in treatment with dexamethasone and anti-oxidants (Top left). The combination of immunosuppressants and antioxidants are more effective to suppress the production of IL-6 .

    Techniques Used: Enzyme-linked Immunosorbent Assay

    14) Product Images from "Increased Expression of Chondroitin Sulfotransferases following AngII may Contribute to Pathophysiology Underlying Covid-19 Respiratory Failure: Impact may be Exacerbated by Decline in Arylsulfatase B Activity"

    Article Title: Increased Expression of Chondroitin Sulfotransferases following AngII may Contribute to Pathophysiology Underlying Covid-19 Respiratory Failure: Impact may be Exacerbated by Decline in Arylsulfatase B Activity

    Journal: bioRxiv

    doi: 10.1101/2020.06.25.171975

    Schematic of Overall Pathway The overall pathway indicates that transcriptional events arise following the stimulation of the ACE2 receptor, leading to increased expression of CHST11 and CHST15. The increased sulfotransferase expression, attributable either to AngII or to spike protein receptor binding, leads to increased production of C4S and CSE. Accumulation of these sulfated glycosaminoglycans causes decline in oxygenation, leading to decline in ARSB activity, and the further accumulation of C4S and CSE and expression of IL-6, and increasing respiratory insufficiency and cytokine storm.
    Figure Legend Snippet: Schematic of Overall Pathway The overall pathway indicates that transcriptional events arise following the stimulation of the ACE2 receptor, leading to increased expression of CHST11 and CHST15. The increased sulfotransferase expression, attributable either to AngII or to spike protein receptor binding, leads to increased production of C4S and CSE. Accumulation of these sulfated glycosaminoglycans causes decline in oxygenation, leading to decline in ARSB activity, and the further accumulation of C4S and CSE and expression of IL-6, and increasing respiratory insufficiency and cytokine storm.

    Techniques Used: Expressing, Binding Assay, Activity Assay

    Inverse relationship between Interleukin-6 and Arylsulfatase B A.Plasma Interleukin (IL)-6 levels were markedly increased in cystic fibrosis (n=16) compared to the normal controls (n=11) (65.5 ± 6.3 pg/ml vs. 5.1 ± 0.7 pg/ml; p
    Figure Legend Snippet: Inverse relationship between Interleukin-6 and Arylsulfatase B A.Plasma Interleukin (IL)-6 levels were markedly increased in cystic fibrosis (n=16) compared to the normal controls (n=11) (65.5 ± 6.3 pg/ml vs. 5.1 ± 0.7 pg/ml; p

    Techniques Used:

    15) Product Images from "TBK1-associated Protein in Endolysosomes (TAPE)/CC2D1A Is a Key Regulator Linking RIG-I-like Receptors to Antiviral Immunity *"

    Article Title: TBK1-associated Protein in Endolysosomes (TAPE)/CC2D1A Is a Key Regulator Linking RIG-I-like Receptors to Antiviral Immunity *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.C112.394346

    Tape deficiency impairs IFN-β production upon RLR ligand stimulation. A and C , Tape +/+ and Tape −/− MEFs transfected with IFN-β-Luc were treated with poly(I-C) (0.2 μg/ml) ( A ) or 5′-ppp dsRNA (0.5 μg/ml) ( C ) transfection to analyze the IFN-β promoter activity. t Poly(I-C) , poly(I-C) transfection. B and D , likewise, supernatants from treated MEFs were measured by ELISA for IFN-β ( left ) or IL-6 ( right ) production. E , supernatants from Tape +/+ and Tape −/− fetal liver macrophages with poly(I-C) transfection were measured by ELISA for RANTES ( left ) or IL-6 ( right ) production.
    Figure Legend Snippet: Tape deficiency impairs IFN-β production upon RLR ligand stimulation. A and C , Tape +/+ and Tape −/− MEFs transfected with IFN-β-Luc were treated with poly(I-C) (0.2 μg/ml) ( A ) or 5′-ppp dsRNA (0.5 μg/ml) ( C ) transfection to analyze the IFN-β promoter activity. t Poly(I-C) , poly(I-C) transfection. B and D , likewise, supernatants from treated MEFs were measured by ELISA for IFN-β ( left ) or IL-6 ( right ) production. E , supernatants from Tape +/+ and Tape −/− fetal liver macrophages with poly(I-C) transfection were measured by ELISA for RANTES ( left ) or IL-6 ( right ) production.

    Techniques Used: Transfection, Activity Assay, Enzyme-linked Immunosorbent Assay

    Roles of TAPE in antiviral immune responses. A , 293 cells treated with the indicated siRNAs were transfected with IFN-β-Luc, together with RIG-I, mTAPE, or hTAPE as indicated, and then infected with IAV (A/WSN/33) (4 MOI) to analyze the IFN-β promoter activity. Ctrl , control. B , A549 cells treated with the indicated siRNAs together with NS1 siRNA were infected with IAV (3 MOI) for 24 h. NS1 siRNA was used to knockdown IAV NS1 protein, which is known to antagonize IFN-β induction. Supernatants were measured by ELISA for IFN-β ( left ) or IL-6 ( right ) production. C , supernatants from Tape +/+ and Tape −/− MEFs infected with IAV (A/WSN/33) (3 MOI) were measured for RANTES production by ELISA ( left ). Ifnb mRNA expression levels in MEFs infected with IAV (PR8 strain) (3 MOI for 10 h) were monitored by quantitative RT-PCR. D , 293 cells treated with the indicated siRNAs were transfected with RIG-I or a control vector and then infected with VSV (0.001 MOI). Viral titers in supernatants were measured by plaque assays. E , 293 cells treated with the indicated siRNAs were transfected with IFN-β-Luc, together with MDA5, and then infected with EMCV (2 MOI) to analyze the IFN-β promoter activity.
    Figure Legend Snippet: Roles of TAPE in antiviral immune responses. A , 293 cells treated with the indicated siRNAs were transfected with IFN-β-Luc, together with RIG-I, mTAPE, or hTAPE as indicated, and then infected with IAV (A/WSN/33) (4 MOI) to analyze the IFN-β promoter activity. Ctrl , control. B , A549 cells treated with the indicated siRNAs together with NS1 siRNA were infected with IAV (3 MOI) for 24 h. NS1 siRNA was used to knockdown IAV NS1 protein, which is known to antagonize IFN-β induction. Supernatants were measured by ELISA for IFN-β ( left ) or IL-6 ( right ) production. C , supernatants from Tape +/+ and Tape −/− MEFs infected with IAV (A/WSN/33) (3 MOI) were measured for RANTES production by ELISA ( left ). Ifnb mRNA expression levels in MEFs infected with IAV (PR8 strain) (3 MOI for 10 h) were monitored by quantitative RT-PCR. D , 293 cells treated with the indicated siRNAs were transfected with RIG-I or a control vector and then infected with VSV (0.001 MOI). Viral titers in supernatants were measured by plaque assays. E , 293 cells treated with the indicated siRNAs were transfected with IFN-β-Luc, together with MDA5, and then infected with EMCV (2 MOI) to analyze the IFN-β promoter activity.

    Techniques Used: Transfection, Infection, Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Plasmid Preparation

    16) Product Images from "Endotoxin-Induced Systemic Inflammation Activates Microglia: [11C]PBR28 Positron Emission Tomography in Nonhuman Primates"

    Article Title: Endotoxin-Induced Systemic Inflammation Activates Microglia: [11C]PBR28 Positron Emission Tomography in Nonhuman Primates

    Journal: NeuroImage

    doi: 10.1016/j.neuroimage.2012.06.055

    The physiologic and immune response to LPS administration. LPS was administered at 0h (arrows), and each data point pre- and post-LPS administration represents data (mean and standard deviation) from all baboons measured at a given time: n = 6 for the pre-LPS through 3h period, n = 3 for the 4h through 6h period, and n = 2 for the 22h through 25h period. (A) Heart rate increased after LPS administration and stayed elevated until the following day. (B) Systolic and diastolic blood pressure increased slightly after LPS administration and subsequently decreased. Systolic blood pressure normalized after approximately 4 hours, while diastolic blood pressure remained lower until the following day. (C) Rectal temperature increased approximately 4 hours after LPS administration and remained elevated until the following day. (D) Levels of inflammatory cytokines (TNFα, IL-1β, IL-6, and IL-8) increased after LPS administration. IL-6 levels were measured with both electrochemiluminenscence and immunsorbent assays (see Methods for details). Abbreviations: ELISA, enzyme-linked immunosorbent assay; IL, interleukin; LPS, lipopolysaccharide; TNF, tumor necrosis factor.
    Figure Legend Snippet: The physiologic and immune response to LPS administration. LPS was administered at 0h (arrows), and each data point pre- and post-LPS administration represents data (mean and standard deviation) from all baboons measured at a given time: n = 6 for the pre-LPS through 3h period, n = 3 for the 4h through 6h period, and n = 2 for the 22h through 25h period. (A) Heart rate increased after LPS administration and stayed elevated until the following day. (B) Systolic and diastolic blood pressure increased slightly after LPS administration and subsequently decreased. Systolic blood pressure normalized after approximately 4 hours, while diastolic blood pressure remained lower until the following day. (C) Rectal temperature increased approximately 4 hours after LPS administration and remained elevated until the following day. (D) Levels of inflammatory cytokines (TNFα, IL-1β, IL-6, and IL-8) increased after LPS administration. IL-6 levels were measured with both electrochemiluminenscence and immunsorbent assays (see Methods for details). Abbreviations: ELISA, enzyme-linked immunosorbent assay; IL, interleukin; LPS, lipopolysaccharide; TNF, tumor necrosis factor.

    Techniques Used: Standard Deviation, Enzyme-linked Immunosorbent Assay

    17) Product Images from "Cultured Human Airway Epithelial Cells (Calu-3): A Model of Human Respiratory Function, Structure, and Inflammatory Responses"

    Article Title: Cultured Human Airway Epithelial Cells (Calu-3): A Model of Human Respiratory Function, Structure, and Inflammatory Responses

    Journal: Critical Care Research and Practice

    doi: 10.1155/2010/394578

    proinflammatory cytokines in ASF from normoxia- or hyperoxia-exposed Calu-3 monolayers with or without +20 cmH 2 O. Both IL-6 (a) and IL-8 (b) increased in pressure-exposed groups over time ( P
    Figure Legend Snippet: proinflammatory cytokines in ASF from normoxia- or hyperoxia-exposed Calu-3 monolayers with or without +20 cmH 2 O. Both IL-6 (a) and IL-8 (b) increased in pressure-exposed groups over time ( P

    Techniques Used:

    18) Product Images from "Activation of Protein Tyrosine Phosphatase Non-Receptor Type 2 by Spermidine Exerts Anti-Inflammatory Effects in Human THP-1 Monocytes and in a Mouse Model of Acute Colitis"

    Article Title: Activation of Protein Tyrosine Phosphatase Non-Receptor Type 2 by Spermidine Exerts Anti-Inflammatory Effects in Human THP-1 Monocytes and in a Mouse Model of Acute Colitis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073703

    Cytokine signaling in interferon-gamma (IFN-γ)- and/or spermidine-treated THP-1 cells. The graphs show ( a ) mRNA levels of intracellular cell adhesion molecule (ICAM)-1, ( b ) secretion of interleukin (IL)-6, and ( c ) secretion of monocyte chemoattractant protein (MCP)-1 in THP-1 cells treated with IFN-γ (1000 U/ml) and/or spermidine (100 µM) for 24 h (n = 3). Data are expressed as fold induction with respect to untreated cells. Significant differences compared to untreated cells (** = p
    Figure Legend Snippet: Cytokine signaling in interferon-gamma (IFN-γ)- and/or spermidine-treated THP-1 cells. The graphs show ( a ) mRNA levels of intracellular cell adhesion molecule (ICAM)-1, ( b ) secretion of interleukin (IL)-6, and ( c ) secretion of monocyte chemoattractant protein (MCP)-1 in THP-1 cells treated with IFN-γ (1000 U/ml) and/or spermidine (100 µM) for 24 h (n = 3). Data are expressed as fold induction with respect to untreated cells. Significant differences compared to untreated cells (** = p

    Techniques Used:

    Effect of protein tyrosine phosphatase non-receptor type 2 (PTPN2) knockdown on cytokine signaling in human monocytic THP-1 cells treated with interferon-gamma (IFN-γ) and/or spermidine. THP-1 cells were transfected with either nonspecific small interfering RNA (siRNA) or ptpn2 siRNA. The graphs show secretion levels of ( a ) monocyte chemoattractant protein (MCP)-1, and ( b ) interleukin (IL)-6 in THP-1 cells treated with IFN-γ (1000 U/ml) and/or spermidine (100 µM) for 24 h (n = 3). Data are expressed as fold induction respect to untreated cells transfected with non-specific siRNA. Significant differences compared to untreated (*** = p
    Figure Legend Snippet: Effect of protein tyrosine phosphatase non-receptor type 2 (PTPN2) knockdown on cytokine signaling in human monocytic THP-1 cells treated with interferon-gamma (IFN-γ) and/or spermidine. THP-1 cells were transfected with either nonspecific small interfering RNA (siRNA) or ptpn2 siRNA. The graphs show secretion levels of ( a ) monocyte chemoattractant protein (MCP)-1, and ( b ) interleukin (IL)-6 in THP-1 cells treated with IFN-γ (1000 U/ml) and/or spermidine (100 µM) for 24 h (n = 3). Data are expressed as fold induction respect to untreated cells transfected with non-specific siRNA. Significant differences compared to untreated (*** = p

    Techniques Used: Transfection, Small Interfering RNA

    19) Product Images from "STING signaling remodels the tumor microenvironment by antagonizing myeloid-derived suppressor cell expansion"

    Article Title: STING signaling remodels the tumor microenvironment by antagonizing myeloid-derived suppressor cell expansion

    Journal: Cell Death and Differentiation

    doi: 10.1038/s41418-019-0302-0

    STING inhibits the differentiation of NPC-induced MDSCs. a Protein levels of STING and other signaling proteins in the NPC cell lines CNE2, C666, and TW03 or the normal NP cell line NP69. b Representative flow cytometry plot (left) and quantification (right) of MDSC differentiation assays are shown as the CD33 + CD11b + cells in the HLA-DR − gate induced by medium, NPC-empty vector (EV) or NPC-STING cells co-cultured with CD33 + cells isolated from healthy PBMCs. The percentage of MDSCs was decreased in STING-transfected TW03- and CNE2-mediated MDSC induction. c Representative plot (left) and quantification (right) are shown as the CD33 + CD11b + cells in the HLA-DR − gate induced by medium, NPC-shControl (shCtrl) or NPC-shSTING in different combinations. The percentage of MDSCs was increased in shSTING-transfected TW03- and CNE2-mediated MDSC induction. d Histogram of flow cytometry staining for the p-STAT3 and PD-L1 level in NPC-induced MDSCs mediated by CNE2-EV and CNE2-STING cells. e Representative histogram of p-STAT3 and PD-L1 expression in NPC-induced MDSCs mediated by CNE2-shCtrl and CNE2-shSTING-02 cells. f IL-6 and GM-CSF production were determined by ELISA in the cultural supernatants of NPC-EV, NPC-STING, NPC-shCtrl, and NPC-shSTING-02 cells. All of the experiments were performed at least three times, and quantification data are plotted as the means ± SEM. Statistical analyses were conducted with an unpaired Student’s t -test; * p
    Figure Legend Snippet: STING inhibits the differentiation of NPC-induced MDSCs. a Protein levels of STING and other signaling proteins in the NPC cell lines CNE2, C666, and TW03 or the normal NP cell line NP69. b Representative flow cytometry plot (left) and quantification (right) of MDSC differentiation assays are shown as the CD33 + CD11b + cells in the HLA-DR − gate induced by medium, NPC-empty vector (EV) or NPC-STING cells co-cultured with CD33 + cells isolated from healthy PBMCs. The percentage of MDSCs was decreased in STING-transfected TW03- and CNE2-mediated MDSC induction. c Representative plot (left) and quantification (right) are shown as the CD33 + CD11b + cells in the HLA-DR − gate induced by medium, NPC-shControl (shCtrl) or NPC-shSTING in different combinations. The percentage of MDSCs was increased in shSTING-transfected TW03- and CNE2-mediated MDSC induction. d Histogram of flow cytometry staining for the p-STAT3 and PD-L1 level in NPC-induced MDSCs mediated by CNE2-EV and CNE2-STING cells. e Representative histogram of p-STAT3 and PD-L1 expression in NPC-induced MDSCs mediated by CNE2-shCtrl and CNE2-shSTING-02 cells. f IL-6 and GM-CSF production were determined by ELISA in the cultural supernatants of NPC-EV, NPC-STING, NPC-shCtrl, and NPC-shSTING-02 cells. All of the experiments were performed at least three times, and quantification data are plotted as the means ± SEM. Statistical analyses were conducted with an unpaired Student’s t -test; * p

    Techniques Used: Flow Cytometry, Plasmid Preparation, Cell Culture, Isolation, Transfection, Staining, Expressing, Enzyme-linked Immunosorbent Assay

    STING downregulates STAT3 signaling during NPC-derived MDSC differentiation. a CNE2 cells were transfected with a Myc-tagged-empty vector (Myc-EV) and a Myc-tagged-STING (Myc-STING) expression vector for at least 24 h, followed by treatment with IL-6 (20 ng/ml) for 30 min. The STAT3 pY705, STAT3 pS727, total STAT3, Myc, and β-actin levels were detected by immunoblot assay. b Immunoblot analysis of the indicated CNE2 cells treated with IL-6 (20 ng/ml) for 30 min before collecting of the lysates. c CNE2 cells were transfected with a STAT3-targeted gene promoter-driven luciferase reporter (STAT3-luc) and TK-Renilla luciferase (TK-luc), together with expression plasmids encoding Myc-EV or Myc-STING, for at least 24 h before treatment with or without IL-6 stimulation for 30 min. Luciferase assays (top) were performed to determine the relative STAT3 luciferase expression (fold), and an immunoblot assay (bottom) was used to detect STING expression. STAT3 (WT) and STAT3 (Y705F) mutants were used as positive and negative controls for STAT3 transcriptional activity, respectively. d Control or STING-knockdown CNE2 cells were transfected with STAT3-luc and TK-luc expression vectors, followed by IL-6 stimulation for 30 min. After 24 h, luciferase assays (top) and an immunoblot assay (bottom) were performed to determine STAT3 activity and STING expression. e ELISA assay of IL-6 and GM-CSF production in the culture supernatants of shCtrl NPC cells or of shSTING-02 NPC cells treated with cryptotanshinone for 48 h. f Representative image (top) and quantification (bottom) of MDSC differentiation assays in which CD33 + cells were co-cultured with NPC-shCtrl or cryptotanshinone-treated shSTING-02 NPC cells for 48 h. CD33 + cells in medium alone were included as a control. All experiments were performed at least three times, and quantification data are plotted as the mean ± SEM. Statistics was conducted with an unpaired Student’s t -test; * p
    Figure Legend Snippet: STING downregulates STAT3 signaling during NPC-derived MDSC differentiation. a CNE2 cells were transfected with a Myc-tagged-empty vector (Myc-EV) and a Myc-tagged-STING (Myc-STING) expression vector for at least 24 h, followed by treatment with IL-6 (20 ng/ml) for 30 min. The STAT3 pY705, STAT3 pS727, total STAT3, Myc, and β-actin levels were detected by immunoblot assay. b Immunoblot analysis of the indicated CNE2 cells treated with IL-6 (20 ng/ml) for 30 min before collecting of the lysates. c CNE2 cells were transfected with a STAT3-targeted gene promoter-driven luciferase reporter (STAT3-luc) and TK-Renilla luciferase (TK-luc), together with expression plasmids encoding Myc-EV or Myc-STING, for at least 24 h before treatment with or without IL-6 stimulation for 30 min. Luciferase assays (top) were performed to determine the relative STAT3 luciferase expression (fold), and an immunoblot assay (bottom) was used to detect STING expression. STAT3 (WT) and STAT3 (Y705F) mutants were used as positive and negative controls for STAT3 transcriptional activity, respectively. d Control or STING-knockdown CNE2 cells were transfected with STAT3-luc and TK-luc expression vectors, followed by IL-6 stimulation for 30 min. After 24 h, luciferase assays (top) and an immunoblot assay (bottom) were performed to determine STAT3 activity and STING expression. e ELISA assay of IL-6 and GM-CSF production in the culture supernatants of shCtrl NPC cells or of shSTING-02 NPC cells treated with cryptotanshinone for 48 h. f Representative image (top) and quantification (bottom) of MDSC differentiation assays in which CD33 + cells were co-cultured with NPC-shCtrl or cryptotanshinone-treated shSTING-02 NPC cells for 48 h. CD33 + cells in medium alone were included as a control. All experiments were performed at least three times, and quantification data are plotted as the mean ± SEM. Statistics was conducted with an unpaired Student’s t -test; * p

    Techniques Used: Derivative Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

    STING-mediated Type I IFN signaling inhibits NPC-induced MDSC differentiation. a Top: schematic diagram of the STING wild-type (WT) and STING S358A mutant (SA). Bottom: the lysates of NPC cells stably expressing Flag-tagged EV, STING-WT, and STING-SA were subjected to immunoblotting with anti-Flag antibody. b Luciferase assay of the empty vector (EV), STING-WT, and STING-SA NPC cells transfected with IFNβ-luc (top) and ISRE-luc (bottom), together with TK-luc. c The lysates of NPC cells stably expressing Flag-tagged EV, STING-WT, and STING-SA were subjected to immunoblotting with the indicated antibodies. d ELISA assay of IL-6 and GM-CSF levels in the culture supernatants of cells stably expressing EV, STING WT, and STING SA. e Quantification analysis of the percentage of MDSCs induced by NPC cells stably expressing EV, STING WT, and STING SA. f Wild type (WT) and TBK1-KO TW03 cells were transfected with Flag-tagged EV and STING for 48 h. The cell lysates were immunoblotted with the indicated antibodies. g ELISA assay and h MDSC differentiation assay of the supernatants from WT and TBK1-KO TW03 cells transfected with Flag-tagged EV and STING for 48 h. All experiments were performed at least three times, and the quantification data are plotted as the mean ± SEM. Statistical analyses were conducted with an unpaired Student’s t -test; * p
    Figure Legend Snippet: STING-mediated Type I IFN signaling inhibits NPC-induced MDSC differentiation. a Top: schematic diagram of the STING wild-type (WT) and STING S358A mutant (SA). Bottom: the lysates of NPC cells stably expressing Flag-tagged EV, STING-WT, and STING-SA were subjected to immunoblotting with anti-Flag antibody. b Luciferase assay of the empty vector (EV), STING-WT, and STING-SA NPC cells transfected with IFNβ-luc (top) and ISRE-luc (bottom), together with TK-luc. c The lysates of NPC cells stably expressing Flag-tagged EV, STING-WT, and STING-SA were subjected to immunoblotting with the indicated antibodies. d ELISA assay of IL-6 and GM-CSF levels in the culture supernatants of cells stably expressing EV, STING WT, and STING SA. e Quantification analysis of the percentage of MDSCs induced by NPC cells stably expressing EV, STING WT, and STING SA. f Wild type (WT) and TBK1-KO TW03 cells were transfected with Flag-tagged EV and STING for 48 h. The cell lysates were immunoblotted with the indicated antibodies. g ELISA assay and h MDSC differentiation assay of the supernatants from WT and TBK1-KO TW03 cells transfected with Flag-tagged EV and STING for 48 h. All experiments were performed at least three times, and the quantification data are plotted as the mean ± SEM. Statistical analyses were conducted with an unpaired Student’s t -test; * p

    Techniques Used: Mutagenesis, Stable Transfection, Expressing, Luciferase, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Differentiation Assay

    20) Product Images from "Damage-associated molecular patterns in intensive care unit patients with acute liver injuries"

    Article Title: Damage-associated molecular patterns in intensive care unit patients with acute liver injuries

    Journal: Medicine

    doi: 10.1097/MD.0000000000012780

    3.2. Association of extracellular histones, HMGB1, sTM, and IL-6 with ALI
    Figure Legend Snippet: 3.2. Association of extracellular histones, HMGB1, sTM, and IL-6 with ALI

    Techniques Used:

    3.2. Association of extracellular histones, HMGB1, sTM, and IL-6 with ALI
    Figure Legend Snippet: 3.2. Association of extracellular histones, HMGB1, sTM, and IL-6 with ALI

    Techniques Used:

    21) Product Images from "Death-Associated Protein Kinase Controls STAT3 Activity in Intestinal Epithelial Cells"

    Article Title: Death-Associated Protein Kinase Controls STAT3 Activity in Intestinal Epithelial Cells

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2012.11.026

    DAPK attenuates TNF-induced IL-6 secretion and STAT3 Y705 phosphorylation in HCEC cells. A – C : HCEC cells were treated with 0.66 ng/mL TNF for 24 and 48 hours in the presence (DAPK- or nonspecific siRNA) or absence of siRNA. A : DAPK knockdown and STAT3 Y705 phosphorylation was assessed by Western blotting of whole cell lysates. B : IL-6 mRNA expression was analyzed by real-time reverse transcription-PCR. Two similar experiments were performed. * P
    Figure Legend Snippet: DAPK attenuates TNF-induced IL-6 secretion and STAT3 Y705 phosphorylation in HCEC cells. A – C : HCEC cells were treated with 0.66 ng/mL TNF for 24 and 48 hours in the presence (DAPK- or nonspecific siRNA) or absence of siRNA. A : DAPK knockdown and STAT3 Y705 phosphorylation was assessed by Western blotting of whole cell lysates. B : IL-6 mRNA expression was analyzed by real-time reverse transcription-PCR. Two similar experiments were performed. * P

    Techniques Used: Western Blot, Expressing, Polymerase Chain Reaction

    A : DAPK and pSTAT3 Y705 expression pattern in DALM. Representative images of DAPK and pSTAT3 Y705 staining in DALM tissue sections ( n = 11) depicting heterogeneous pattern of expression are shown. Areas with high DAPK–high pSTAT3 Y705 ( filled arrowheads ); high DAPK–low pSTAT3 Y705 ( open arrowheads ); low DAPK–high pSTAT3 Y705 ( asterisks ); and low DAPK–low pSTAT3 Y705 ( arrows ) expression are indicated. Note the serial sections for DAPK and pSTAT3 Y705 allowing direct comparison of expression in the same region of interest. TNF-induced functions in normal human colon epithelial cells: HCEC cells were stimulated with 0.66 ng/mL of TNF for various time points (1, 6, 24, 48, or 72 hours). DAPK ( B ) and STAT3 ( C ) expression/activation were assessed by immunoblotting using the corresponding antibodies. Representative Western blots of five independent experiments are shown. IL-6 ( D ) and IL-8 ( E ) secretion was measured in cell culture supernatants by ELISA. Data were obtained from more than five independent experiments performed in duplicate or triplicate. * P
    Figure Legend Snippet: A : DAPK and pSTAT3 Y705 expression pattern in DALM. Representative images of DAPK and pSTAT3 Y705 staining in DALM tissue sections ( n = 11) depicting heterogeneous pattern of expression are shown. Areas with high DAPK–high pSTAT3 Y705 ( filled arrowheads ); high DAPK–low pSTAT3 Y705 ( open arrowheads ); low DAPK–high pSTAT3 Y705 ( asterisks ); and low DAPK–low pSTAT3 Y705 ( arrows ) expression are indicated. Note the serial sections for DAPK and pSTAT3 Y705 allowing direct comparison of expression in the same region of interest. TNF-induced functions in normal human colon epithelial cells: HCEC cells were stimulated with 0.66 ng/mL of TNF for various time points (1, 6, 24, 48, or 72 hours). DAPK ( B ) and STAT3 ( C ) expression/activation were assessed by immunoblotting using the corresponding antibodies. Representative Western blots of five independent experiments are shown. IL-6 ( D ) and IL-8 ( E ) secretion was measured in cell culture supernatants by ELISA. Data were obtained from more than five independent experiments performed in duplicate or triplicate. * P

    Techniques Used: Expressing, Staining, Activation Assay, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

    22) Product Images from "AMP-activated protein kinase reduces inflammatory responses and cellular senescence in pulmonary emphysema"

    Article Title: AMP-activated protein kinase reduces inflammatory responses and cellular senescence in pulmonary emphysema

    Journal: Oncotarget

    doi: 10.18632/oncotarget.15116

    Effect of AMPK on CSE-induced release of pro-inflammatory mediators in human lung epithelial cells Both BEAS-2B and SAECs were treated with AICAR (1 mM) or Compound C (5 μM) for 24 h in the presence or absence of CSE (0.25% and 0.5%) treatment. Culture supernatants were collected for the measurement of IL-6 and IL-8 by ELISA. Data are expressed as the mean ± SEM. N = 4–5. * P
    Figure Legend Snippet: Effect of AMPK on CSE-induced release of pro-inflammatory mediators in human lung epithelial cells Both BEAS-2B and SAECs were treated with AICAR (1 mM) or Compound C (5 μM) for 24 h in the presence or absence of CSE (0.25% and 0.5%) treatment. Culture supernatants were collected for the measurement of IL-6 and IL-8 by ELISA. Data are expressed as the mean ± SEM. N = 4–5. * P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    23) Product Images from "Carbocisteine attenuates TNF-α-induced inflammation in human alveolar epithelial cells in vitro through suppressing NF-κB and ERK1/2 MAPK signaling pathways"

    Article Title: Carbocisteine attenuates TNF-α-induced inflammation in human alveolar epithelial cells in vitro through suppressing NF-κB and ERK1/2 MAPK signaling pathways

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2015.150

    Effects of carbocisteine on TNF-α-induced IL-6 and IL-8 release in A549 cells. Control group: same amount of vehicle (PBS); TNF-α group: 10 ng/mL; Carbocisteine+TNF-α group: pre-treated with 10, 100, or 1000 μmol/L carbocisteine for 24 h, then exposure to 10 ng/mL TNF-α for 24 h; TNF-α+Carbocisteine group: pre-treated with 10 ng/mL TNF-α for 24 h, then exposure to 10, 100, or 1000 μmol/L carbocisteine for 24 h. Mean±SD. n =6. * P
    Figure Legend Snippet: Effects of carbocisteine on TNF-α-induced IL-6 and IL-8 release in A549 cells. Control group: same amount of vehicle (PBS); TNF-α group: 10 ng/mL; Carbocisteine+TNF-α group: pre-treated with 10, 100, or 1000 μmol/L carbocisteine for 24 h, then exposure to 10 ng/mL TNF-α for 24 h; TNF-α+Carbocisteine group: pre-treated with 10 ng/mL TNF-α for 24 h, then exposure to 10, 100, or 1000 μmol/L carbocisteine for 24 h. Mean±SD. n =6. * P

    Techniques Used:

    Effects of carbocisteine on the TNF-α-induced expression of the IL-6, IL-8, TNF-α, MCP-1 and MIP-1β genes in A549 cells. Control group: same amount of vehicle (PBS); TNF-α group: 10 ng/mL; Carbocisteine+TNF-α group: pre-treated with 10, 100, and 1000 μmol/L carbocisteine for 24 h, then exposure to 10 ng/mL TNF-α for 24 h; TNF-α+Carbocisteine group: pre-treated with 10 ng/mL TNF-α for 24 h, then exposure to 10, 100, and 1000 μmol/L carbocisteine for 24 h. (A) IL-6, (B) IL-8, (C) TNF-α, (D) MCP-1, (E) MIP-1β. Mean±SD. n =3. * P
    Figure Legend Snippet: Effects of carbocisteine on the TNF-α-induced expression of the IL-6, IL-8, TNF-α, MCP-1 and MIP-1β genes in A549 cells. Control group: same amount of vehicle (PBS); TNF-α group: 10 ng/mL; Carbocisteine+TNF-α group: pre-treated with 10, 100, and 1000 μmol/L carbocisteine for 24 h, then exposure to 10 ng/mL TNF-α for 24 h; TNF-α+Carbocisteine group: pre-treated with 10 ng/mL TNF-α for 24 h, then exposure to 10, 100, and 1000 μmol/L carbocisteine for 24 h. (A) IL-6, (B) IL-8, (C) TNF-α, (D) MCP-1, (E) MIP-1β. Mean±SD. n =3. * P

    Techniques Used: Expressing

    24) Product Images from "Cysteamine suppresses human peripheral blood mononuclear cells - human corneal endothelial cell reaction via reactive oxygen species reduction"

    Article Title: Cysteamine suppresses human peripheral blood mononuclear cells - human corneal endothelial cell reaction via reactive oxygen species reduction

    Journal: Molecular Vision

    doi:

    IL-6 levels measured by ELISA. The IL-6 levels decreased with an increase in the CYS concentration (p=0.003; Kruskal–Wallis test). PHA-stimulated PBMCs served as the positive control and unstimulated PBMCs served as the negative control. *: statistically significant by the Mann–Whitney U test.
    Figure Legend Snippet: IL-6 levels measured by ELISA. The IL-6 levels decreased with an increase in the CYS concentration (p=0.003; Kruskal–Wallis test). PHA-stimulated PBMCs served as the positive control and unstimulated PBMCs served as the negative control. *: statistically significant by the Mann–Whitney U test.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay, Positive Control, Negative Control, MANN-WHITNEY

    25) Product Images from "A 2,3-diphenylpyrido[1,2-a] pyrimidin-4-one derivative inhibits specific angiogenic factors induced by TNF-α"

    Article Title: A 2,3-diphenylpyrido[1,2-a] pyrimidin-4-one derivative inhibits specific angiogenic factors induced by TNF-α

    Journal: Saudi Pharmaceutical Journal : SPJ

    doi: 10.1016/j.jsps.2019.09.014

    DB103 inhibited IL-6, MCP-1 and Ang-2 but not IL-8 cell release. ECs were incubated for 24 h with vehicle or with 10 ng/mL of TNF-α in the continued presence of DB103 or apigenin. MCP-1 ( A ), IL-6 ( B ), Ang-2 ( C ) and IL-8 ( D ) expression were measured on cell supernatant by ELISA assay. Each bar represents the mean of three independent experiments, each performed in six replicates. ANOVA with Bonferroni’s post hoc comparison. # P
    Figure Legend Snippet: DB103 inhibited IL-6, MCP-1 and Ang-2 but not IL-8 cell release. ECs were incubated for 24 h with vehicle or with 10 ng/mL of TNF-α in the continued presence of DB103 or apigenin. MCP-1 ( A ), IL-6 ( B ), Ang-2 ( C ) and IL-8 ( D ) expression were measured on cell supernatant by ELISA assay. Each bar represents the mean of three independent experiments, each performed in six replicates. ANOVA with Bonferroni’s post hoc comparison. # P

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

    26) Product Images from "HBV replication is significantly reduced by IL-6"

    Article Title: HBV replication is significantly reduced by IL-6

    Journal: Journal of Biomedical Science

    doi: 10.1186/1423-0127-16-41

    IL-6 does not disrupt the HBV nucleocapsids . To assess the IL-6 effect on the stability of HBV nucleocapsids, confluent cells were labeled with [ 35 S] methionine-cysteine protein labeling mix for 6 h and then chased in the absence or presence of IL-6 (20 ng/ml or 40 ng/ml) for 24 h or 48 h. Cell lysates were harvested and the nucleocapsids were separated from free core protein by centrifugation through a Centricon-100 filter with a retention cutoff of 100 kDa. Total core protein and nucleocapsids were then immunoprecipitated using an anti-core antibody and separated by SDS-polyacrylamide gel electrophoresis. The labeled core protein was visualized by autoradiography. The signals were quantified by densitometry analysis and expressed as percentage of the respective control cells to indicate the IL-6 effect on capsid stability. Results shown are representative of three independent experiments.
    Figure Legend Snippet: IL-6 does not disrupt the HBV nucleocapsids . To assess the IL-6 effect on the stability of HBV nucleocapsids, confluent cells were labeled with [ 35 S] methionine-cysteine protein labeling mix for 6 h and then chased in the absence or presence of IL-6 (20 ng/ml or 40 ng/ml) for 24 h or 48 h. Cell lysates were harvested and the nucleocapsids were separated from free core protein by centrifugation through a Centricon-100 filter with a retention cutoff of 100 kDa. Total core protein and nucleocapsids were then immunoprecipitated using an anti-core antibody and separated by SDS-polyacrylamide gel electrophoresis. The labeled core protein was visualized by autoradiography. The signals were quantified by densitometry analysis and expressed as percentage of the respective control cells to indicate the IL-6 effect on capsid stability. Results shown are representative of three independent experiments.

    Techniques Used: Labeling, Centrifugation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Autoradiography

    IL-6 decreases the formation of HBV cccDNA . Confluent cells (day0) were treated with or without 20 ng/ml of IL-6 for 4 days (day4) or 6 days (day6) and subjected to Hirt extraction to prepare HBV cccDNA. The Hirt extract was thermally denatured at 85°C for 5 min, digested with EcoRI and analyzed by Southern blotting using an HBV-specific probe. Bands corresponding to HBV cccDNA and HBV single-stranded DNA (SS) are indicated. The signals were quantified by densitometry analysis and expressed as percentage of the control cells to indicate the IL-6 effect on the accumulation of HBV cccDNA.
    Figure Legend Snippet: IL-6 decreases the formation of HBV cccDNA . Confluent cells (day0) were treated with or without 20 ng/ml of IL-6 for 4 days (day4) or 6 days (day6) and subjected to Hirt extraction to prepare HBV cccDNA. The Hirt extract was thermally denatured at 85°C for 5 min, digested with EcoRI and analyzed by Southern blotting using an HBV-specific probe. Bands corresponding to HBV cccDNA and HBV single-stranded DNA (SS) are indicated. The signals were quantified by densitometry analysis and expressed as percentage of the control cells to indicate the IL-6 effect on the accumulation of HBV cccDNA.

    Techniques Used: Southern Blot

    IL-6 suppresses HBV replication in a time-dependent manner . Confluent cells (day0) were treated with or without 20 ng/ml of IL-6 for 2 days (day2), 4 days (day4) or 6 days (day6). For the analysis of HBV replicative intermediates, equal amounts of total DNA were subjected to HindIII digestion and separated by electrophoresis. The integrated viral genome and viral replicative intermediates were then examined by Southern blot analysis using an HBV-specific probe. Bands corresponding to the integrated genome, the relaxed circular double-stranded DNA (RC) and single-stranded DNA (SS) are indicated individually. The intensity of the integrated HBV genome in each lane reflected the amounts of total DNA and was served as the internal control for sample loading. The signals were quantified by densitometry analysis and expressed as percentage of the respective control cells to indicate the inhibitory effect of IL-6.
    Figure Legend Snippet: IL-6 suppresses HBV replication in a time-dependent manner . Confluent cells (day0) were treated with or without 20 ng/ml of IL-6 for 2 days (day2), 4 days (day4) or 6 days (day6). For the analysis of HBV replicative intermediates, equal amounts of total DNA were subjected to HindIII digestion and separated by electrophoresis. The integrated viral genome and viral replicative intermediates were then examined by Southern blot analysis using an HBV-specific probe. Bands corresponding to the integrated genome, the relaxed circular double-stranded DNA (RC) and single-stranded DNA (SS) are indicated individually. The intensity of the integrated HBV genome in each lane reflected the amounts of total DNA and was served as the internal control for sample loading. The signals were quantified by densitometry analysis and expressed as percentage of the respective control cells to indicate the inhibitory effect of IL-6.

    Techniques Used: Electrophoresis, Southern Blot

    IL-6 decreases the levels of HBV transcripts, core protein, and genome-containing nucleocapsids . Confluent cells (day0) were treated with or without 20 ng/ml of IL-6 for 2 days (day2), 4 days (day4) or 6 days (day6). For RNA analysis (panel for RNA), total RNAs were extracted and equal amounts of samples were subjected to Northern blot analysis to reveal the expression profile of the HBV transcripts, namely those of 3.5-kb and 2.4-kb/2.1-kb respectively. The expression level of 18S rRNA was used as an internal control for sample loading. For Western blot analysis (panel for Proteins), cell lysates were harvested and equal amounts of samples were separated by SDS-polyacrylamide gel electrophoresis. The total core protein was subsequently detected by immunoblot analysis with antibodies against core protein. The expression level of β-actin was used as an internal control for sample loading. For particle blot analysis, equal amounts of cell lysates were assayed for viral capsid by native agarose gel electrophoresis. HBV genome-containing nucleocapsids were then detected by Southern blot analysis of the disrupted nucleocapsids using an HBV-specific probe (panel for Genome-containing nucleocapsids). The signals including 3.5-kb RNA, core proteins and genome-containing nucleocapsids were quantified by densitometry analysis and expressed as the average percentage of respective control cells from three independent experiments to indicate the inhibitory effect of IL-6. Results shown are representative of three independent experiments. *: The reduction in levels of HBV genome-containing nucleocapsids was compared with that of core proteins and found to be highly significant (P
    Figure Legend Snippet: IL-6 decreases the levels of HBV transcripts, core protein, and genome-containing nucleocapsids . Confluent cells (day0) were treated with or without 20 ng/ml of IL-6 for 2 days (day2), 4 days (day4) or 6 days (day6). For RNA analysis (panel for RNA), total RNAs were extracted and equal amounts of samples were subjected to Northern blot analysis to reveal the expression profile of the HBV transcripts, namely those of 3.5-kb and 2.4-kb/2.1-kb respectively. The expression level of 18S rRNA was used as an internal control for sample loading. For Western blot analysis (panel for Proteins), cell lysates were harvested and equal amounts of samples were separated by SDS-polyacrylamide gel electrophoresis. The total core protein was subsequently detected by immunoblot analysis with antibodies against core protein. The expression level of β-actin was used as an internal control for sample loading. For particle blot analysis, equal amounts of cell lysates were assayed for viral capsid by native agarose gel electrophoresis. HBV genome-containing nucleocapsids were then detected by Southern blot analysis of the disrupted nucleocapsids using an HBV-specific probe (panel for Genome-containing nucleocapsids). The signals including 3.5-kb RNA, core proteins and genome-containing nucleocapsids were quantified by densitometry analysis and expressed as the average percentage of respective control cells from three independent experiments to indicate the inhibitory effect of IL-6. Results shown are representative of three independent experiments. *: The reduction in levels of HBV genome-containing nucleocapsids was compared with that of core proteins and found to be highly significant (P

    Techniques Used: Northern Blot, Expressing, Western Blot, Polyacrylamide Gel Electrophoresis, Agarose Gel Electrophoresis, Southern Blot

    IL-6 does not suppress the levels of secreted HBeAg and HBsAg . Confluent cells were treated for 6 days with various doses of IL-6 (0, 5, 10, 20, 40 ng/ml). (A) The level of extracellular viral genome-containing nucleocapsids in the culture medium was measured by real-time PCR method and normalized with total cell number. (B and C) The levels of secreted HBeAg and HBsAg in the culture medium were measured by ELISA. Results shown are representative of three independent experiments.
    Figure Legend Snippet: IL-6 does not suppress the levels of secreted HBeAg and HBsAg . Confluent cells were treated for 6 days with various doses of IL-6 (0, 5, 10, 20, 40 ng/ml). (A) The level of extracellular viral genome-containing nucleocapsids in the culture medium was measured by real-time PCR method and normalized with total cell number. (B and C) The levels of secreted HBeAg and HBsAg in the culture medium were measured by ELISA. Results shown are representative of three independent experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    The antiviral effect of IL-6 on HBV replication is not mediated through induction of IFNs . (A) Confluent 1.3ES2 cells were treated with or without 20 ng/ml of IL-6 for 2 day (day2) or 4 days (day4). Total RNA of each sample was extracted for the detection of IFN-α2b, IFN-β, IFN-γ and β-actin by RT-PCR analysis. Cells were transfected with 20 μg poly (I:C) for 6 h and served as the positive control for the induction of IFN-α2b and IFN-β simultaneously. Plasmid containing IFN-γ gene was served as the positive control for the detection of IFN-γ. (B) Confluent 1.3ES2 cells were treated with or without 20 ng/ml of IL-6 for 1 day (day1), 3 days (day3) or 6 days (day6). Total RNA of each sample was extracted for the detection of GBP-1 (target gene for IFN-γ), MxA (target gene for type I IFN) and GAPDH by RT-PCR analysis. Cells were treated with IFN-β or IFN-γ for 18 h and served as the positive control for the induction of GBP-1 and MxA genes. (C) Confluent 1.3ES2 cells were treated with various concentration of IL-6 or IFN-β for 4 days in the absence or presence of anti-IFN-β polyclonal antibody (400 U/ml). The level of HBV genome-containing nucleocapsids was measured as mentioned above. The expression level of β-actin was used as an internal control for sample loading.
    Figure Legend Snippet: The antiviral effect of IL-6 on HBV replication is not mediated through induction of IFNs . (A) Confluent 1.3ES2 cells were treated with or without 20 ng/ml of IL-6 for 2 day (day2) or 4 days (day4). Total RNA of each sample was extracted for the detection of IFN-α2b, IFN-β, IFN-γ and β-actin by RT-PCR analysis. Cells were transfected with 20 μg poly (I:C) for 6 h and served as the positive control for the induction of IFN-α2b and IFN-β simultaneously. Plasmid containing IFN-γ gene was served as the positive control for the detection of IFN-γ. (B) Confluent 1.3ES2 cells were treated with or without 20 ng/ml of IL-6 for 1 day (day1), 3 days (day3) or 6 days (day6). Total RNA of each sample was extracted for the detection of GBP-1 (target gene for IFN-γ), MxA (target gene for type I IFN) and GAPDH by RT-PCR analysis. Cells were treated with IFN-β or IFN-γ for 18 h and served as the positive control for the induction of GBP-1 and MxA genes. (C) Confluent 1.3ES2 cells were treated with various concentration of IL-6 or IFN-β for 4 days in the absence or presence of anti-IFN-β polyclonal antibody (400 U/ml). The level of HBV genome-containing nucleocapsids was measured as mentioned above. The expression level of β-actin was used as an internal control for sample loading.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Transfection, Positive Control, Plasmid Preparation, Concentration Assay, Expressing

    IL-6 suppresses HBV replication in a dose-dependent manner . (A) 1.3ES2 cells were treated 6 days after plating (when the cells had reached confluence) with various doses of IL-6 (0, 5, 10, 20, 40 ng/ml). The cell lysates were harvested after 4 days treatment with IL-6 and equal amounts of sample were analyzed by particle blot analysis using native agarose gel electrophoresis. HBV genome-containing nucleocapsids were detected by Southern blot analysis of the disrupted nucleocapsids, using an HBV-specific probe. Expression of β-actin was used as an internal control for sample loading. The signals were quantified by densitometry analysis and expressed as percentage of the control cells to indicate the inhibitory effect of IL-6. (B) Effect of IL-6 on growth curve. The cells were treated with IL-6, as described above. The cell number was determined by the trypan blue exclusion method.
    Figure Legend Snippet: IL-6 suppresses HBV replication in a dose-dependent manner . (A) 1.3ES2 cells were treated 6 days after plating (when the cells had reached confluence) with various doses of IL-6 (0, 5, 10, 20, 40 ng/ml). The cell lysates were harvested after 4 days treatment with IL-6 and equal amounts of sample were analyzed by particle blot analysis using native agarose gel electrophoresis. HBV genome-containing nucleocapsids were detected by Southern blot analysis of the disrupted nucleocapsids, using an HBV-specific probe. Expression of β-actin was used as an internal control for sample loading. The signals were quantified by densitometry analysis and expressed as percentage of the control cells to indicate the inhibitory effect of IL-6. (B) Effect of IL-6 on growth curve. The cells were treated with IL-6, as described above. The cell number was determined by the trypan blue exclusion method.

    Techniques Used: Agarose Gel Electrophoresis, Southern Blot, Expressing

    27) Product Images from "The Transcription Factor Twist1 Limits T Helper 17 and T Follicular Helper Cell Development by Repressing the Gene Encoding the Interleukin-6 Receptor α Chain *"

    Article Title: The Transcription Factor Twist1 Limits T Helper 17 and T Follicular Helper Cell Development by Repressing the Gene Encoding the Interleukin-6 Receptor α Chain *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.497248

    Twist1 binds to Tfh cell-associated genes. A–C , naïve WT CD4 + T cells were activated with or without IL-6 for 2 days. Cells were harvested daily to analyze STAT3 binding to the Twist1 promoter ( A ) or Twist1 binding to the indicated promoters
    Figure Legend Snippet: Twist1 binds to Tfh cell-associated genes. A–C , naïve WT CD4 + T cells were activated with or without IL-6 for 2 days. Cells were harvested daily to analyze STAT3 binding to the Twist1 promoter ( A ) or Twist1 binding to the indicated promoters

    Techniques Used: Binding Assay

    Twist1 impairs IL-6-STAT3 signaling in Th17 cells. A–D , naïve CD4 + T cells were isolated from WT and Twist1 fl/fl CD4 -Cre mice and differentiated under Th17 polarizing conditions. The levels of phospho-STAT3 (pSTAT3) and phospho-STAT5 (pSTAT5)
    Figure Legend Snippet: Twist1 impairs IL-6-STAT3 signaling in Th17 cells. A–D , naïve CD4 + T cells were isolated from WT and Twist1 fl/fl CD4 -Cre mice and differentiated under Th17 polarizing conditions. The levels of phospho-STAT3 (pSTAT3) and phospho-STAT5 (pSTAT5)

    Techniques Used: Isolation, Mouse Assay

    28) Product Images from "Proteomic Profiling of the Amniotic Fluid to Detect Inflammation, Infection, and Neonatal Sepsis"

    Article Title: Proteomic Profiling of the Amniotic Fluid to Detect Inflammation, Infection, and Neonatal Sepsis

    Journal: PLoS Medicine

    doi: 10.1371/journal.pmed.0040018

    Proteomic Profiling of AF Versus Results of Other Diagnostic Tests of Intra-amniotic Inflammation and/or Infection (A) AF median WBC count, (B) ELISA for the IL-6 (C) and MMP-8 levels, and (D) glucose concentration in the same samples of AF ( n = 169) varied with the degree of inflammation. Several degrees of inflammation were established using SELDI-TOF mass spectrometry (MR = 0 indicates “no” inflammation; MR = 2 indicates “minimal” inflammation; and MR = 3–4 indicates “severe” inflammation). Statistical comparisons were performed using Kruskal-Wallis ANOVA.
    Figure Legend Snippet: Proteomic Profiling of AF Versus Results of Other Diagnostic Tests of Intra-amniotic Inflammation and/or Infection (A) AF median WBC count, (B) ELISA for the IL-6 (C) and MMP-8 levels, and (D) glucose concentration in the same samples of AF ( n = 169) varied with the degree of inflammation. Several degrees of inflammation were established using SELDI-TOF mass spectrometry (MR = 0 indicates “no” inflammation; MR = 2 indicates “minimal” inflammation; and MR = 3–4 indicates “severe” inflammation). Statistical comparisons were performed using Kruskal-Wallis ANOVA.

    Techniques Used: Diagnostic Assay, Infection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Mass Spectrometry

    29) Product Images from "Differential interleukin-6/Stat3 signaling as a function of cellular context mediates Ras-induced transformation"

    Article Title: Differential interleukin-6/Stat3 signaling as a function of cellular context mediates Ras-induced transformation

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/bcr2725

    IL-6 is required for tumorigenesis of Ras transformed MCF10A cells . (a) Ras transformed MCF10A cells expressing control (C) or IL-6 shRNA (IL-6sh) were stimulated with TNF-α and analyzed for levels of IL-6 and normalized to actin by RT-PCR. (b) Extracts from MCF10A-Ras (RasC) and MCF10AIL6Sh (RasIL6sh) were analyzed for Stat3, pStat3, Ras and Tubulin levels. (c) MCF10A-Ras (RasC) and MCF10AIL6Sh (RasIL6Sh) cells were plated in six-well dishes and cell numbers were determined daily for seven days. Additionally, RasC cells were treated with IL-6 (10 ng/ml) (Ras+IL6) daily and cell numbers were determined. Each data point represents the mean value from triplicate wells. (d) MCF10A-Ras (C) and MCF10AIL6Sh (IL6Sh) cells were plated into Boyden chambers and cell migration was determined in the absence or presence of IL-6 (C+IL6, S3Sh+IL-6) with crystal violet staining after 24 hrs. Results are expressed as cells/field (mean ± SD of triplicates from three independent experiments). (e) Tumor growth in nude mice using MCF10A-Ras cells expressing control (C) or IL-6 shRNA (IL-6sh) was determined (mean ± SD from five independent injections).
    Figure Legend Snippet: IL-6 is required for tumorigenesis of Ras transformed MCF10A cells . (a) Ras transformed MCF10A cells expressing control (C) or IL-6 shRNA (IL-6sh) were stimulated with TNF-α and analyzed for levels of IL-6 and normalized to actin by RT-PCR. (b) Extracts from MCF10A-Ras (RasC) and MCF10AIL6Sh (RasIL6sh) were analyzed for Stat3, pStat3, Ras and Tubulin levels. (c) MCF10A-Ras (RasC) and MCF10AIL6Sh (RasIL6Sh) cells were plated in six-well dishes and cell numbers were determined daily for seven days. Additionally, RasC cells were treated with IL-6 (10 ng/ml) (Ras+IL6) daily and cell numbers were determined. Each data point represents the mean value from triplicate wells. (d) MCF10A-Ras (C) and MCF10AIL6Sh (IL6Sh) cells were plated into Boyden chambers and cell migration was determined in the absence or presence of IL-6 (C+IL6, S3Sh+IL-6) with crystal violet staining after 24 hrs. Results are expressed as cells/field (mean ± SD of triplicates from three independent experiments). (e) Tumor growth in nude mice using MCF10A-Ras cells expressing control (C) or IL-6 shRNA (IL-6sh) was determined (mean ± SD from five independent injections).

    Techniques Used: Transformation Assay, Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction, Migration, Staining, Mouse Assay

    Ras expressing mammary tumors exhibit high levels of activated Stat3 and IL-6 . (a) Paraffin embedded tumors from MCF10A-Ras cells expressing control (Ras-C) or Stat3shRNA (RasS3sh) were examined by hematoxyline and eosin (H E), pStat3 and IL-6 by immunohistochemistry. Extracts from MCF10A-Ras expressing control (C) or Stat3 shRNA (S3sh) tumors were analyzed for pStat3 and Tubulin by Western blot analysis. (b) Paraffin embedded tumors from MMTV-promoter driven K-Ras (MMTV-K-Ras) were analyzed by hematoxyline and eosin stain (H E), for pStat3 by immunohistochemistry and for IL-6 by immunofluorescence. Extracts from normal mammary tissue (N) or MMTV-K-Ras (K-Ras) were analyzed for pStat3, Stat3 and Tubulin.
    Figure Legend Snippet: Ras expressing mammary tumors exhibit high levels of activated Stat3 and IL-6 . (a) Paraffin embedded tumors from MCF10A-Ras cells expressing control (Ras-C) or Stat3shRNA (RasS3sh) were examined by hematoxyline and eosin (H E), pStat3 and IL-6 by immunohistochemistry. Extracts from MCF10A-Ras expressing control (C) or Stat3 shRNA (S3sh) tumors were analyzed for pStat3 and Tubulin by Western blot analysis. (b) Paraffin embedded tumors from MMTV-promoter driven K-Ras (MMTV-K-Ras) were analyzed by hematoxyline and eosin stain (H E), for pStat3 by immunohistochemistry and for IL-6 by immunofluorescence. Extracts from normal mammary tissue (N) or MMTV-K-Ras (K-Ras) were analyzed for pStat3, Stat3 and Tubulin.

    Techniques Used: Expressing, Immunohistochemistry, shRNA, Western Blot, Staining, Immunofluorescence

    Exogenous IL-6 enhances pStat3 levels, binding activity and cell migration in MCF10A-Ras cells . (a) Extracts from MCF10A-Ras cells (C) and MCF10A-Ras treated with IL-6 (10ng/ml) for one hour (IL-6) were analyzed by Western blot analysis for pStat3, Stat3 and Tubulin (b) Human IL-6 mRNA levels from MCF10A-Ras cells (C) and MCF10A-Ras cells treated with IL-6 for four hours (IL-6) were determined by RT-PCR and normalized to β-Actin. (c) EMSA was performed with nuclear extracts from the cell lines described in a. Stat3 DNA binding was supershifted with anti-Stat3 antibody (IL6+Ab). (d) MCF10A-Ras cells (C) or Stat3 shRNA (S3sh) were plated into Boyden chambers and cell migration was determined in the absence or presence of IL-6 (C+IL6, S3Sh+IL-6) with crystal violet staining after 24 hrs. Results are expressed as cells/field (mean ± SD of triplicates from three independent experiments).
    Figure Legend Snippet: Exogenous IL-6 enhances pStat3 levels, binding activity and cell migration in MCF10A-Ras cells . (a) Extracts from MCF10A-Ras cells (C) and MCF10A-Ras treated with IL-6 (10ng/ml) for one hour (IL-6) were analyzed by Western blot analysis for pStat3, Stat3 and Tubulin (b) Human IL-6 mRNA levels from MCF10A-Ras cells (C) and MCF10A-Ras cells treated with IL-6 for four hours (IL-6) were determined by RT-PCR and normalized to β-Actin. (c) EMSA was performed with nuclear extracts from the cell lines described in a. Stat3 DNA binding was supershifted with anti-Stat3 antibody (IL6+Ab). (d) MCF10A-Ras cells (C) or Stat3 shRNA (S3sh) were plated into Boyden chambers and cell migration was determined in the absence or presence of IL-6 (C+IL6, S3Sh+IL-6) with crystal violet staining after 24 hrs. Results are expressed as cells/field (mean ± SD of triplicates from three independent experiments).

    Techniques Used: Binding Assay, Activity Assay, Migration, Western Blot, Reverse Transcription Polymerase Chain Reaction, shRNA, Staining

    3-D morphogenic assays of MCF10A-Ras cells display IL-6/Jak/pStat3 signaling which regulate E-Cadherin levels . (a) MCF10A cells were grown in Matrigel as were MCF10A-Ras cells which were stained for pStat3 and Dapi by immunofluorescence treated with DMSO control (Ras-C), P6 (a pan-Jak inhibitor) or a blocking antibody to IL-6 (α-IL-6). (b) MCF10A-Ras cells expressing control (HRas-C) or Stat3 shRNA (S3sh) were grown in Matrigel and stained with Dapi and E-Cadherin (D/E-Cad) by immunofluorescence. (c) MCF10A-Ras cells were grown in matrigel and treated with DMSO (HRas-C), P6 or a blocking antibody to IL-6 (α-IL-6) and stained for Dapi and E-Cadherin (D/E-Cad).
    Figure Legend Snippet: 3-D morphogenic assays of MCF10A-Ras cells display IL-6/Jak/pStat3 signaling which regulate E-Cadherin levels . (a) MCF10A cells were grown in Matrigel as were MCF10A-Ras cells which were stained for pStat3 and Dapi by immunofluorescence treated with DMSO control (Ras-C), P6 (a pan-Jak inhibitor) or a blocking antibody to IL-6 (α-IL-6). (b) MCF10A-Ras cells expressing control (HRas-C) or Stat3 shRNA (S3sh) were grown in Matrigel and stained with Dapi and E-Cadherin (D/E-Cad) by immunofluorescence. (c) MCF10A-Ras cells were grown in matrigel and treated with DMSO (HRas-C), P6 or a blocking antibody to IL-6 (α-IL-6) and stained for Dapi and E-Cadherin (D/E-Cad).

    Techniques Used: Staining, Immunofluorescence, Blocking Assay, Expressing, shRNA

    Growth of MCF10A-Ras cells in two- versus three-dimensions alters the expression levels of pStat3, IL-6 and E-Cadherin . (a) Tumors arising from immunocompromised mice injected with MCF10A-Ras cells were cultured and passaged four times on tissue culture plates. Extracts were isolated from an MCF10A-Ras primary tumor (Tu) and cultured cells following serial passage (P1, P2, P3 and P4) and analyzed for pStat3, Stat3, E-Cadherin and Tubulin by Western blot. Quantitative evaluation of pStat3/Stat3 (grey bars) and pStat3/Tubulin (black bars) immunoblots by densitometry. Results are representative of three individual experiments. Statistical significance is indicated with asterisks (*, P
    Figure Legend Snippet: Growth of MCF10A-Ras cells in two- versus three-dimensions alters the expression levels of pStat3, IL-6 and E-Cadherin . (a) Tumors arising from immunocompromised mice injected with MCF10A-Ras cells were cultured and passaged four times on tissue culture plates. Extracts were isolated from an MCF10A-Ras primary tumor (Tu) and cultured cells following serial passage (P1, P2, P3 and P4) and analyzed for pStat3, Stat3, E-Cadherin and Tubulin by Western blot. Quantitative evaluation of pStat3/Stat3 (grey bars) and pStat3/Tubulin (black bars) immunoblots by densitometry. Results are representative of three individual experiments. Statistical significance is indicated with asterisks (*, P

    Techniques Used: Expressing, Mouse Assay, Injection, Cell Culture, Isolation, Western Blot

    30) Product Images from "Tumour YAP1 and PTEN expression correlates with tumour‐associated myeloid suppressor cell expansion and reduced survival in colorectal cancer"

    Article Title: Tumour YAP1 and PTEN expression correlates with tumour‐associated myeloid suppressor cell expansion and reduced survival in colorectal cancer

    Journal: Immunology

    doi: 10.1111/imm.12949

    Blockage of Yes‐associated protein 1 (YAP1) or phosphatase and tensin homologue (PTEN) alters granulocyte–macrophage colony‐stimulating factor (GM‐CSF) secretion and is associated with the activation of P‐AKT, P‐p65 and COX‐2. (a) ELISA array for the levels of GM‐CSF, interleukin‐6 (IL‐6) and IL‐1 β in the supernatants of LS174T and SW620 cell cultures with or without the presence of small interfering RNA (siRNA) ‐PTEN, siRNA‐YAP1 or siRNA‐PTEN +siRNA‐YAP1. The data are derived from three independent experiments. (b) Western blot analysis showed that P‐AKT, P‐p65, and COX‐2 expression, but not NLRP3 and IL‐1β expression, was increased with the blockage of YAP1 or PTEN. Representative data are shown; the experiment was repeated five times. (c) The signalling pathway of YAP1 and PTEN regulates tumour‐associated myeloid‐derived suppressor cell (MDSC) expansion in CRC.
    Figure Legend Snippet: Blockage of Yes‐associated protein 1 (YAP1) or phosphatase and tensin homologue (PTEN) alters granulocyte–macrophage colony‐stimulating factor (GM‐CSF) secretion and is associated with the activation of P‐AKT, P‐p65 and COX‐2. (a) ELISA array for the levels of GM‐CSF, interleukin‐6 (IL‐6) and IL‐1 β in the supernatants of LS174T and SW620 cell cultures with or without the presence of small interfering RNA (siRNA) ‐PTEN, siRNA‐YAP1 or siRNA‐PTEN +siRNA‐YAP1. The data are derived from three independent experiments. (b) Western blot analysis showed that P‐AKT, P‐p65, and COX‐2 expression, but not NLRP3 and IL‐1β expression, was increased with the blockage of YAP1 or PTEN. Representative data are shown; the experiment was repeated five times. (c) The signalling pathway of YAP1 and PTEN regulates tumour‐associated myeloid‐derived suppressor cell (MDSC) expansion in CRC.

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Small Interfering RNA, Derivative Assay, Western Blot, Expressing

    31) Product Images from "CD28 Autonomous Signaling Orchestrates IL-22 Expression and IL-22-Regulated Epithelial Barrier Functions in Human T Lymphocytes"

    Article Title: CD28 Autonomous Signaling Orchestrates IL-22 Expression and IL-22-Regulated Epithelial Barrier Functions in Human T Lymphocytes

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.590964

    IL-22 regulation by CD28 autonomous stimulation in human CD4 + T cells. (A) CD4 + T cells were stimulated for indicated times with 2 μg ml −1 isotype control (Ig) or anti-CD28.2 or anti-CD3 (UCHT1) or anti-CD3 plus anti-CD28.2 Abs. IL-22 mRNA expression was measured by real-time PCR and values, normalized to GAPDH, expressed as fold induction (F.I.) over the basal level of cells stimulated with isotype control Ig. Data show the mean ± SEM of one out of three HD subjects. Statistical significance was calculated by Student t test. (B) IL-22 mRNA levels of CD4 + T cells from HD (n = 7) stimulated for 0, 6, or 24 h with crosslinked anti-CD28.2 Abs. IL-22 mRNA levels were measured by real-time PCR and values, normalized to GAPDH, expressed as arbitrary units (AU). Data show the mean ± SEM and statistical significance was calculated by Mann-Whitney test. Mean values: 0 h = 8.5, 6 h = 329.6, 24 h = 1336. (C) IL-22 mRNA levels (AU) of CD4 + T cells from HD (n = 24) stimulated for 24 h with isotype control Ig or anti-CD28.2 Abs. Data show the mean ± SEM and statistical significance was calculated by Mann-Whitney test. Mean values: Ig = 38.1; CD28 = 496.7. (D) CD4 + T cells from HD (n = 6) were stimulated for 0, 24 or 48 h with isotype control or crosslinked anti-CD28.2 Abs. IL-22 levels in culture supernatant were measured by ELISA. Lines represent median values and statistical significance was calculated by Student t test. Median values: 0 h = 17.7 pg ml −1 , 24 h = 1840 pg ml −1 , 48 h = 2329 pg ml −1 . (E) CD4 + T cells from HD (n = 12) were stimulated for 48 h with isotype control Ig or anti-CD28.2 Abs. IL-22 levels in culture supernatant were measured by ELISA. Lines represent median values and statistical significance was calculated by Student t test. Median values: Ig = 14.4 pg ml −1 , CD28 = 545 pg ml −1 . (F) CD4 + T cells from HD (n = 8) were stimulated for 24 h with isotype control Ig or anti-CD28.2 Abs. IL-6 levels in culture supernatant were measured by ELISA. Lines represent median values and statistical significance was calculated by Student t test. Median values: Ig = 0 pg ml −1 , CD28 = 99.3 pg ml −1 . (G) CD4 + T cells from HD (n = 3) were stimulated for 24 h with isotype control Ig in the presence or absence of 50 ng ml −1 recombinant IL-6 (rIL-6). IL-22 mRNA levels were measured by real-time PCR and values, normalized to GAPDH, expressed as arbitrary units (AU). Data show the mean ± SEM and statistical significance was calculated by Student t test. (H, I) IL-22 mRNA expression (H) and secretion (I) in CD4 + T cells from HD stimulated for 24 h (H) or 48 (I) with isotype control Ig or anti-CD28.2 Abs in the presence of 10 μg ml −1 isotype control or neutralizing anti-IL-6 Abs. IL-22 mRNA levels (H) were measured by real-time PCR and values (n = 7), normalized to GAPDH, expressed as fold induction (F.I.) over the basal level of cells stimulated with isotype control Ig. Data show the mean ± SEM and statistical significance was calculated by Wilcoxon test. Mean values: CD28 = 201.3, CD28 anti-IL-6 = 25.7. IL-22 levels (I) in culture supernatants were measured by ELISA (n = 6). Data show the mean ± SEM and statistical significance was calculated by Student t test. Mean values: Ig = 0 pg ml −1 , CD28 = 1123 pg ml −1 , CD28 anti-IL-6 = 0 pg ml −1 . (*) p
    Figure Legend Snippet: IL-22 regulation by CD28 autonomous stimulation in human CD4 + T cells. (A) CD4 + T cells were stimulated for indicated times with 2 μg ml −1 isotype control (Ig) or anti-CD28.2 or anti-CD3 (UCHT1) or anti-CD3 plus anti-CD28.2 Abs. IL-22 mRNA expression was measured by real-time PCR and values, normalized to GAPDH, expressed as fold induction (F.I.) over the basal level of cells stimulated with isotype control Ig. Data show the mean ± SEM of one out of three HD subjects. Statistical significance was calculated by Student t test. (B) IL-22 mRNA levels of CD4 + T cells from HD (n = 7) stimulated for 0, 6, or 24 h with crosslinked anti-CD28.2 Abs. IL-22 mRNA levels were measured by real-time PCR and values, normalized to GAPDH, expressed as arbitrary units (AU). Data show the mean ± SEM and statistical significance was calculated by Mann-Whitney test. Mean values: 0 h = 8.5, 6 h = 329.6, 24 h = 1336. (C) IL-22 mRNA levels (AU) of CD4 + T cells from HD (n = 24) stimulated for 24 h with isotype control Ig or anti-CD28.2 Abs. Data show the mean ± SEM and statistical significance was calculated by Mann-Whitney test. Mean values: Ig = 38.1; CD28 = 496.7. (D) CD4 + T cells from HD (n = 6) were stimulated for 0, 24 or 48 h with isotype control or crosslinked anti-CD28.2 Abs. IL-22 levels in culture supernatant were measured by ELISA. Lines represent median values and statistical significance was calculated by Student t test. Median values: 0 h = 17.7 pg ml −1 , 24 h = 1840 pg ml −1 , 48 h = 2329 pg ml −1 . (E) CD4 + T cells from HD (n = 12) were stimulated for 48 h with isotype control Ig or anti-CD28.2 Abs. IL-22 levels in culture supernatant were measured by ELISA. Lines represent median values and statistical significance was calculated by Student t test. Median values: Ig = 14.4 pg ml −1 , CD28 = 545 pg ml −1 . (F) CD4 + T cells from HD (n = 8) were stimulated for 24 h with isotype control Ig or anti-CD28.2 Abs. IL-6 levels in culture supernatant were measured by ELISA. Lines represent median values and statistical significance was calculated by Student t test. Median values: Ig = 0 pg ml −1 , CD28 = 99.3 pg ml −1 . (G) CD4 + T cells from HD (n = 3) were stimulated for 24 h with isotype control Ig in the presence or absence of 50 ng ml −1 recombinant IL-6 (rIL-6). IL-22 mRNA levels were measured by real-time PCR and values, normalized to GAPDH, expressed as arbitrary units (AU). Data show the mean ± SEM and statistical significance was calculated by Student t test. (H, I) IL-22 mRNA expression (H) and secretion (I) in CD4 + T cells from HD stimulated for 24 h (H) or 48 (I) with isotype control Ig or anti-CD28.2 Abs in the presence of 10 μg ml −1 isotype control or neutralizing anti-IL-6 Abs. IL-22 mRNA levels (H) were measured by real-time PCR and values (n = 7), normalized to GAPDH, expressed as fold induction (F.I.) over the basal level of cells stimulated with isotype control Ig. Data show the mean ± SEM and statistical significance was calculated by Wilcoxon test. Mean values: CD28 = 201.3, CD28 anti-IL-6 = 25.7. IL-22 levels (I) in culture supernatants were measured by ELISA (n = 6). Data show the mean ± SEM and statistical significance was calculated by Student t test. Mean values: Ig = 0 pg ml −1 , CD28 = 1123 pg ml −1 , CD28 anti-IL-6 = 0 pg ml −1 . (*) p

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

    CD28-mediated IL-22 production by CD4 + T cells induces the up-regulation of MUC1 and MMP9 in CACO-2 epithelial cells. (A–C) CACO-2 cells were cultured in triplicates in 24 trans-well plates with medium alone or CD4 + T cells stimulated for the indicated times with isotype control Ig or crosslinked anti-CD28.2 or anti-CD3 (UCHT1) or anti-CD3 plus anti-CD28.2 Abs. MMP9 (A) , MUC1 (B) and S100A9 (C) mRNA levels in CACO-2 cells were measured by real-time PCR and values, normalized to GAPDH, expressed as fold inductions (F.I.) over the basal level of CACO-2 cultured with medium alone. Data show the mean ± SEM of one out of three HD. Statistical significance was calculated by Student t test. (D, E) MMP9 (D) and MUC1 (E) mRNA levels in CACO-2 cells cultured with medium alone or with CD4 + T cells from HD (n = 8) stimulated for 48 h with isotype control Ig or cross-linked anti-CD28.2 Abs. Values, normalized to GAPDH, were expressed as fold inductions (F.I.) over the basal level of CACO-2 cultured with medium alone. Data show the mean ± SEM and statistical significance was calculated by Student t test. Mean values: MMP9, T Ig = 62.4, T CD28 = 773.4; MUC1, T Ig = 11.5, T CD28 = 131.1. (F) Anti- MUC1 or anti- GAPDH western blotting of CACO-2 cells cultured with medium alone (Med) or co-cultured with CD4 + T cells stimulated for 48 h with isotype control Ig or cross-linked anti-CD28.2 Abs. MUC1 fold inductions (F.I.) were quantified by densitometric analysis and normalized to GAPDH levels. Bars represent mean F.I. ± SEM of five HD. (G) CACO-2 cells were cultured with medium alone or co-cultured with CD4 + T cells from HD (n = 5) stimulated for 48 h with isotype control Ig or anti-CD28.2 Abs in the presence of 10 μg ml −1 isotype control or neutralizing anti-IL-22 Abs. MMP9 and MUC1 mRNA levels were analyzed by real time PCR and, after normalization to GAPDH, fold inductions (F.I.) were calculated over the basal level of CACO-2 cultured with medium alone. Values of CACO-2 cells co-cultured with CD4 + T cells treated with DMSO and stimulated with anti-CD28 Abs were assumed as 100%. Data express the mean ± SEM. Statistical significance was calculated by Student t test. (H) CACO-2 cells were cultured with medium alone or co-cultured with CD4 + T cells from HD (n = 3) stimulated for 48 h with isotype control Ig or anti-CD28.2 Abs in the presence of 10 μg ml −1 isotype control or neutralizing anti-IL-6 Abs. MUC1 mRNA levels were analyzed by real time PCR and, after normalization to GAPDH, fold inductions (F.I.) were calculated over the basal level of CACO-2 cultured with medium alone. Data express the mean ± SEM. Statistical significance was calculated by Student t test. Mean values: T Ig = 1.3, T CD28 = 37.8, T CD28 anti-IL-6 = 7.2. (*) p
    Figure Legend Snippet: CD28-mediated IL-22 production by CD4 + T cells induces the up-regulation of MUC1 and MMP9 in CACO-2 epithelial cells. (A–C) CACO-2 cells were cultured in triplicates in 24 trans-well plates with medium alone or CD4 + T cells stimulated for the indicated times with isotype control Ig or crosslinked anti-CD28.2 or anti-CD3 (UCHT1) or anti-CD3 plus anti-CD28.2 Abs. MMP9 (A) , MUC1 (B) and S100A9 (C) mRNA levels in CACO-2 cells were measured by real-time PCR and values, normalized to GAPDH, expressed as fold inductions (F.I.) over the basal level of CACO-2 cultured with medium alone. Data show the mean ± SEM of one out of three HD. Statistical significance was calculated by Student t test. (D, E) MMP9 (D) and MUC1 (E) mRNA levels in CACO-2 cells cultured with medium alone or with CD4 + T cells from HD (n = 8) stimulated for 48 h with isotype control Ig or cross-linked anti-CD28.2 Abs. Values, normalized to GAPDH, were expressed as fold inductions (F.I.) over the basal level of CACO-2 cultured with medium alone. Data show the mean ± SEM and statistical significance was calculated by Student t test. Mean values: MMP9, T Ig = 62.4, T CD28 = 773.4; MUC1, T Ig = 11.5, T CD28 = 131.1. (F) Anti- MUC1 or anti- GAPDH western blotting of CACO-2 cells cultured with medium alone (Med) or co-cultured with CD4 + T cells stimulated for 48 h with isotype control Ig or cross-linked anti-CD28.2 Abs. MUC1 fold inductions (F.I.) were quantified by densitometric analysis and normalized to GAPDH levels. Bars represent mean F.I. ± SEM of five HD. (G) CACO-2 cells were cultured with medium alone or co-cultured with CD4 + T cells from HD (n = 5) stimulated for 48 h with isotype control Ig or anti-CD28.2 Abs in the presence of 10 μg ml −1 isotype control or neutralizing anti-IL-22 Abs. MMP9 and MUC1 mRNA levels were analyzed by real time PCR and, after normalization to GAPDH, fold inductions (F.I.) were calculated over the basal level of CACO-2 cultured with medium alone. Values of CACO-2 cells co-cultured with CD4 + T cells treated with DMSO and stimulated with anti-CD28 Abs were assumed as 100%. Data express the mean ± SEM. Statistical significance was calculated by Student t test. (H) CACO-2 cells were cultured with medium alone or co-cultured with CD4 + T cells from HD (n = 3) stimulated for 48 h with isotype control Ig or anti-CD28.2 Abs in the presence of 10 μg ml −1 isotype control or neutralizing anti-IL-6 Abs. MUC1 mRNA levels were analyzed by real time PCR and, after normalization to GAPDH, fold inductions (F.I.) were calculated over the basal level of CACO-2 cultured with medium alone. Data express the mean ± SEM. Statistical significance was calculated by Student t test. Mean values: T Ig = 1.3, T CD28 = 37.8, T CD28 anti-IL-6 = 7.2. (*) p

    Techniques Used: Cell Culture, Real-time Polymerase Chain Reaction, Western Blot

    CD28-associated class 1A PI3K regulates IL-22 production and IL-22–mediated epithelial cell functions. (A) CACO-2 cells were cultured for 48 h with DMSO, as vehicle control, or 10 μM AS605240. Cell death was analyzed by flow cytometry by quantifying the ability of cells to incorporate propidium iodide (PI). (B) The percentage of PI positive cells was calculated. Results express the mean ± SEM and statistical significance was calculated by Student t test. (C) MMP9 and MUC1 mRNA levels in CACO-2 cells cultured with medium alone or CD4 + T cells from HD (n = 3) treated with DMSO, as vehicle control, or 10 μM AS605240 and stimulated for 48 h with isotype control Ig or anti-CD28.2 Abs. mRNA levels were analyzed by real-time PCR and, after normalization to GAPDH, fold inductions (F.I.) were calculated over the basal level of CACO-2 cultured with medium alone. Values of CACO-2 cells co-cultured with CD4 + T cells treated with DMSO and stimulated with anti-CD28 Abs were assumed as 100%. Data express the mean ± SEM. Statistical significance was calculated by Student t test. (D) IL-22 levels in culture supernatant, measured by ELISA, of CD4 + T cells from HD (n = 7) treated with DMSO, as vehicle control, or 10 μM AS605240 and stimulated for 48 h with isotype control Ig or crosslinked anti-CD28.2 Abs. Data express mean ± SEM and statistical significance was calculated by Student t test. Mean values: DMSO Ig = 112.1, DMSO CD28 = 2897, AS605240 CD28 = 328.5. (E) IL-6 levels in culture supernatant, measured by ELISA, of CD4 + T cells from HD (n = 7) treated with DMSO, as vehicle control, or 10 μM AS605240 and stimulated for 24 h with isotype control Ig or crosslinked anti-CD28.2 Abs. Data express mean ± SEM and statistical significance was calculated by Student t test. Mean values: DMSO Ig = 0 pg ml −1 , DMSO CD28 = 184.7 pg ml −1 , AS605240 CD28 = 0 pg ml −1 . (F) IL-22 mRNA levels in CD4 + T cells from HD (n = 3) treated with DMSO, as vehicle control, or 10 μM AS605240 and stimulated for 24 h with isotype control Ig or anti-CD28.2 Abs. Fold inductions (F.I.) were calculated over the basal level of DMSO-treated T cells stimulated with isotype control Ig. Values of CD4 + T cells treated with DMSO and stimulated with anti-CD28 Abs were assumed as 100%. Data express the mean ± SEM and statistical significance was calculated by Student t test. (*) p
    Figure Legend Snippet: CD28-associated class 1A PI3K regulates IL-22 production and IL-22–mediated epithelial cell functions. (A) CACO-2 cells were cultured for 48 h with DMSO, as vehicle control, or 10 μM AS605240. Cell death was analyzed by flow cytometry by quantifying the ability of cells to incorporate propidium iodide (PI). (B) The percentage of PI positive cells was calculated. Results express the mean ± SEM and statistical significance was calculated by Student t test. (C) MMP9 and MUC1 mRNA levels in CACO-2 cells cultured with medium alone or CD4 + T cells from HD (n = 3) treated with DMSO, as vehicle control, or 10 μM AS605240 and stimulated for 48 h with isotype control Ig or anti-CD28.2 Abs. mRNA levels were analyzed by real-time PCR and, after normalization to GAPDH, fold inductions (F.I.) were calculated over the basal level of CACO-2 cultured with medium alone. Values of CACO-2 cells co-cultured with CD4 + T cells treated with DMSO and stimulated with anti-CD28 Abs were assumed as 100%. Data express the mean ± SEM. Statistical significance was calculated by Student t test. (D) IL-22 levels in culture supernatant, measured by ELISA, of CD4 + T cells from HD (n = 7) treated with DMSO, as vehicle control, or 10 μM AS605240 and stimulated for 48 h with isotype control Ig or crosslinked anti-CD28.2 Abs. Data express mean ± SEM and statistical significance was calculated by Student t test. Mean values: DMSO Ig = 112.1, DMSO CD28 = 2897, AS605240 CD28 = 328.5. (E) IL-6 levels in culture supernatant, measured by ELISA, of CD4 + T cells from HD (n = 7) treated with DMSO, as vehicle control, or 10 μM AS605240 and stimulated for 24 h with isotype control Ig or crosslinked anti-CD28.2 Abs. Data express mean ± SEM and statistical significance was calculated by Student t test. Mean values: DMSO Ig = 0 pg ml −1 , DMSO CD28 = 184.7 pg ml −1 , AS605240 CD28 = 0 pg ml −1 . (F) IL-22 mRNA levels in CD4 + T cells from HD (n = 3) treated with DMSO, as vehicle control, or 10 μM AS605240 and stimulated for 24 h with isotype control Ig or anti-CD28.2 Abs. Fold inductions (F.I.) were calculated over the basal level of DMSO-treated T cells stimulated with isotype control Ig. Values of CD4 + T cells treated with DMSO and stimulated with anti-CD28 Abs were assumed as 100%. Data express the mean ± SEM and statistical significance was calculated by Student t test. (*) p

    Techniques Used: Cell Culture, Flow Cytometry, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    32) Product Images from "A revised model for the secretion of tPA and cytokines from cultured endothelial cells"

    Article Title: A revised model for the secretion of tPA and cytokines from cultured endothelial cells

    Journal: Blood

    doi: 10.1182/blood-2010-03-276170

    The tPA-EGFP organelle is a short-lived compartment from which unstimulated secretion of tPA-EGFP and cytokines arises . (A-B) Representative examples of fixed cells expressing proregion-EGFP (Ai-ii; 24 hours after Nucleofection) or tPA-EGFP (Bi-ii; 6-7 hours after Nucleofection) and stained with a specific antibody to TGN46 (red in color images) in the absence of BFA (vehicle control; Ai and Bi) or after 1-hour treatment with 5μM BFA. Solid squares and line in panel Aiii represent the mean numbers of WPBs, expressed as a percentage of the total number at t = 3 minutes after addition of BFA, present in cells at the times indicated (n = 6 cells; BFA added at t = 0 minutes). Numbers of WPBs in vehicle-treated control cells at 3, 15, and 60 minute time points, expressed in the same way, are shown in open squares with dashed line (n = 4 cells). (Biii) Similar data for tPA-EGFP-containing puncta. In this case, the numbers of tPA-EGFP-containing granules in BFA-treated cells are plotted individually because of differences in the precise timing of measurements between experiments (n = 9 cells). The solid black line indicates a single exponential decline fitted to the pooled data for all cells. Superimposed on the plot (red circles and red broken line) is the mean decrease in unstimulated secretion of tPA-EGFP in cells treated with BFA. These data are expressed as a percentage of control cells (vehicle-treated) at the times indicated (n = 4 independent experiments each carried out in duplicate; BFA added at t = 0 minutes). Panels Aiv represent ELISA data for secreted EGFP from cells expressing proregion-EGFP. Cells exposed to BFA or vehicle control (1 hour) were stimulated with histamine or vehicle control (10 minutes) as indicated. Data shown are an individual experiment carried out in triplicate and are representative of 3 separate experiments. Panel Biv represents the mean decrease in unstimulated secretion of IL-6 (□), GRO-α (■), and MCP-1 (●) from IL-1β–treated cells exposed to BFA at t = 0. Data are normalized to that of vehicle control-treated cells at the times indicated and represent data pooled from 3 or 4 independent experiments each carried out in duplicate. For comparison, the data for unstimulated secretion of tPA-EGFP in BFA treated-cells plotted in panel Biii are included in red.
    Figure Legend Snippet: The tPA-EGFP organelle is a short-lived compartment from which unstimulated secretion of tPA-EGFP and cytokines arises . (A-B) Representative examples of fixed cells expressing proregion-EGFP (Ai-ii; 24 hours after Nucleofection) or tPA-EGFP (Bi-ii; 6-7 hours after Nucleofection) and stained with a specific antibody to TGN46 (red in color images) in the absence of BFA (vehicle control; Ai and Bi) or after 1-hour treatment with 5μM BFA. Solid squares and line in panel Aiii represent the mean numbers of WPBs, expressed as a percentage of the total number at t = 3 minutes after addition of BFA, present in cells at the times indicated (n = 6 cells; BFA added at t = 0 minutes). Numbers of WPBs in vehicle-treated control cells at 3, 15, and 60 minute time points, expressed in the same way, are shown in open squares with dashed line (n = 4 cells). (Biii) Similar data for tPA-EGFP-containing puncta. In this case, the numbers of tPA-EGFP-containing granules in BFA-treated cells are plotted individually because of differences in the precise timing of measurements between experiments (n = 9 cells). The solid black line indicates a single exponential decline fitted to the pooled data for all cells. Superimposed on the plot (red circles and red broken line) is the mean decrease in unstimulated secretion of tPA-EGFP in cells treated with BFA. These data are expressed as a percentage of control cells (vehicle-treated) at the times indicated (n = 4 independent experiments each carried out in duplicate; BFA added at t = 0 minutes). Panels Aiv represent ELISA data for secreted EGFP from cells expressing proregion-EGFP. Cells exposed to BFA or vehicle control (1 hour) were stimulated with histamine or vehicle control (10 minutes) as indicated. Data shown are an individual experiment carried out in triplicate and are representative of 3 separate experiments. Panel Biv represents the mean decrease in unstimulated secretion of IL-6 (□), GRO-α (■), and MCP-1 (●) from IL-1β–treated cells exposed to BFA at t = 0. Data are normalized to that of vehicle control-treated cells at the times indicated and represent data pooled from 3 or 4 independent experiments each carried out in duplicate. For comparison, the data for unstimulated secretion of tPA-EGFP in BFA treated-cells plotted in panel Biii are included in red.

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

    BFA- and CHX-insensitive stimulated secretion and storage efficiency of tPA and cytokines in HUVECs . (A) Top panels: Histamine-stimulated secretion of tPA (i), IL-8 (ii), IL-6 (iii), MCP-1 (iv), and GRO-α (v) in the absence (BFA vehicle, 1 hour) or after BFA treatment (1 hour) as indicated. Cells were pretreated for 24 hours with either 3mM Na-butyrate (i) or 1 ng/mL rhIL-1β (ii-v) before experiments. Bottom panels: Histamine- or ionomycin-stimulated secretion of tPA and the cytokines in control (CHX vehicle for 24 hours) or CHX-treated (24 hours) cells as indicated. Na-butyrate or rhIL-1β was included in the media during CHX treatment. Data shown in each case are an individual experiment, carried out in triplicate, and are representative of 3 or 4 replicate experiments. * P
    Figure Legend Snippet: BFA- and CHX-insensitive stimulated secretion and storage efficiency of tPA and cytokines in HUVECs . (A) Top panels: Histamine-stimulated secretion of tPA (i), IL-8 (ii), IL-6 (iii), MCP-1 (iv), and GRO-α (v) in the absence (BFA vehicle, 1 hour) or after BFA treatment (1 hour) as indicated. Cells were pretreated for 24 hours with either 3mM Na-butyrate (i) or 1 ng/mL rhIL-1β (ii-v) before experiments. Bottom panels: Histamine- or ionomycin-stimulated secretion of tPA and the cytokines in control (CHX vehicle for 24 hours) or CHX-treated (24 hours) cells as indicated. Na-butyrate or rhIL-1β was included in the media during CHX treatment. Data shown in each case are an individual experiment, carried out in triplicate, and are representative of 3 or 4 replicate experiments. * P

    Techniques Used:

    IL-6 is present in the tPA-EGFP organelle and WPBs in IL-1β–treated cells . (A-B) Examples of single cells labeled for endogenous IL-6 (green in color-merged panels) and endogenous VWF immunoreactivity (red in color-merged panels). (C) Endogenous IL-6 immunoreactivity (red in color-merged image) in a cell expressing tPA-EGFP (EGFP fluorescence green in color-merged image). Regions indicated by the white boxes are shown in grayscale to the right of each image. (D) Endogenous IL-6 (red and top panel in grayscale images), GRO-α (green and middle panel in grayscale images), and VWF (blue and bottom panel in grayscale images) immunoreactivity in an IL-1β-pretreated cell.
    Figure Legend Snippet: IL-6 is present in the tPA-EGFP organelle and WPBs in IL-1β–treated cells . (A-B) Examples of single cells labeled for endogenous IL-6 (green in color-merged panels) and endogenous VWF immunoreactivity (red in color-merged panels). (C) Endogenous IL-6 immunoreactivity (red in color-merged image) in a cell expressing tPA-EGFP (EGFP fluorescence green in color-merged image). Regions indicated by the white boxes are shown in grayscale to the right of each image. (D) Endogenous IL-6 (red and top panel in grayscale images), GRO-α (green and middle panel in grayscale images), and VWF (blue and bottom panel in grayscale images) immunoreactivity in an IL-1β-pretreated cell.

    Techniques Used: Labeling, Expressing, Fluorescence

    33) Product Images from "Human Mesenchymal Stem Cell Secretome from Bone Marrow or Adipose-Derived Tissue Sources for Treatment of Hypoxia-Induced Pulmonary Epithelial Injury"

    Article Title: Human Mesenchymal Stem Cell Secretome from Bone Marrow or Adipose-Derived Tissue Sources for Treatment of Hypoxia-Induced Pulmonary Epithelial Injury

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19102996

    ( A ) Cell viability and release of pro-inflammatory ( B ) IL-6, ( C ) IL-8 and anti-inflammatory ( D ) IL-10 cytokines from A549 cells treated with recombinant glucose-regulated proteins (GRP75, GRP78 and GRP94) treatment during hypoxic (0.5% O 2 ) exposure for 24 h. ELISAs were used to measure secreted cytokines in the supernatant of A549 cells. Data presented as mean ± SD; n = 3 (* p
    Figure Legend Snippet: ( A ) Cell viability and release of pro-inflammatory ( B ) IL-6, ( C ) IL-8 and anti-inflammatory ( D ) IL-10 cytokines from A549 cells treated with recombinant glucose-regulated proteins (GRP75, GRP78 and GRP94) treatment during hypoxic (0.5% O 2 ) exposure for 24 h. ELISAs were used to measure secreted cytokines in the supernatant of A549 cells. Data presented as mean ± SD; n = 3 (* p

    Techniques Used: Recombinant

    The release of pro-inflammatory; ( A ) CINC-1/CXCL-1, ( B ) IL-1β, ( C ) IL-6, ( D ) TNF-α and anti-inflammatory cytokines; ( E ) IL-10 from primary rat AECs pre-treated with human BMSC/ADSC-CM during hypoxic (1.5% O 2 ) exposure for 24 h. ELISAs were used to measure secreted inflammatory cytokines in the supernatant of primary AECs. Data presented as mean ± SD; n = 3 (* p
    Figure Legend Snippet: The release of pro-inflammatory; ( A ) CINC-1/CXCL-1, ( B ) IL-1β, ( C ) IL-6, ( D ) TNF-α and anti-inflammatory cytokines; ( E ) IL-10 from primary rat AECs pre-treated with human BMSC/ADSC-CM during hypoxic (1.5% O 2 ) exposure for 24 h. ELISAs were used to measure secreted inflammatory cytokines in the supernatant of primary AECs. Data presented as mean ± SD; n = 3 (* p

    Techniques Used:

    34) Product Images from "Sodium thiosulfate attenuates acute lung injury in mice"

    Article Title: Sodium thiosulfate attenuates acute lung injury in mice

    Journal: Anesthesiology

    doi: 10.1097/ALN.0000000000000456

    Effects of sodium thiosulfate on lipopolysaccharide or tumor necrosis factor alpha-induced cytokine production in human umbilical vein endothelial cells and human lung microvascular endothelial cells Interleukin-6 (IL-6) levels in human umbilical vein endothelial cells (HUVEC; A), and human lung microvascular endothelial cells (HMVEC-L; B) culture medium incubated with lipopolysaccharide (LPS) or Tumor Necrosis Factor alpha (TNFα) for 20 h with or without varying concentrations of sodium thiosulfate (STS). *** p
    Figure Legend Snippet: Effects of sodium thiosulfate on lipopolysaccharide or tumor necrosis factor alpha-induced cytokine production in human umbilical vein endothelial cells and human lung microvascular endothelial cells Interleukin-6 (IL-6) levels in human umbilical vein endothelial cells (HUVEC; A), and human lung microvascular endothelial cells (HMVEC-L; B) culture medium incubated with lipopolysaccharide (LPS) or Tumor Necrosis Factor alpha (TNFα) for 20 h with or without varying concentrations of sodium thiosulfate (STS). *** p

    Techniques Used: Incubation

    Effects of sodium thiosulfate on lipopolysaccharide- or polymicrobial sepsis-induced lung injury (A) Total number of leukocytes and (B) polymorphonuclear neutrophils (PMNs) in bronchoalveolar lavage fluid (BALF). (C) Myeloperoxidase (MPO) activity and (D) total protein concentration in BALF. (E) Mouse lung wet dry ratio. (F, G). Inflammatory cytokine levels in BALF. (H) Interleukin-6 (IL-6) levels in the mouse lung subjected to cecal ligation and puncture (CLP). *** p
    Figure Legend Snippet: Effects of sodium thiosulfate on lipopolysaccharide- or polymicrobial sepsis-induced lung injury (A) Total number of leukocytes and (B) polymorphonuclear neutrophils (PMNs) in bronchoalveolar lavage fluid (BALF). (C) Myeloperoxidase (MPO) activity and (D) total protein concentration in BALF. (E) Mouse lung wet dry ratio. (F, G). Inflammatory cytokine levels in BALF. (H) Interleukin-6 (IL-6) levels in the mouse lung subjected to cecal ligation and puncture (CLP). *** p

    Techniques Used: Activity Assay, Protein Concentration, Ligation

    35) Product Images from "Tempol, an Intracellular Antioxidant, Inhibits Tissue Factor Expression, Attenuates Dendritic Cell Function, and Is Partially Protective in a Murine Model of Cerebral Malaria"

    Article Title: Tempol, an Intracellular Antioxidant, Inhibits Tissue Factor Expression, Attenuates Dendritic Cell Function, and Is Partially Protective in a Murine Model of Cerebral Malaria

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0087140

    Tempol inhibits transcription and functional expression of tissue factor in endothelial cells, and cytokine production. A, Inhibition of tissue factor (TF) activity. MVEC were incubated overnight with Tempol (0, 1, and 3 mM) followed by washing the wells and addition of lipopolysaccharide (LPS) (200 ng/mL). A mixture containing FX and FVIIa was then added to the cells, and FXa generation was estimated in the supernatant using chromogenic substrate (S2222), as described in Materials and Methods. B, Inhibition of TF generation. Cells were incubated overnight with Tempol (0, 1, and 3 mM) followed by washing of the wells and addition of LPS (200 ng/mL) for six hours. Wells were washed and cells were lysed with 0.1% Triton X-100. The supernatant was used to detect TF antigen by ELISA. C, Inhibition of TF transcription. Cells were incubated overnight with Tempol (0, 1, and 3 mM) followed by addition of LPS (200 ng/mL) for 2 h. Cells were washed and trypsinized for extraction of mRNA. Real-time PCR of the samples was evaluated as described in Materials and Methods. The figure shows a representative result from two independent experiments. D–F, Inhibition of cytokine generation. MVEC were incubated overnight with Tempol (0, 1, and 3 mM) followed by washing of the wells and addition of LPS (200 ng/mL). After six hours, the supernatant of the cells was collected and used for detection of D, IL-6, E, IL-8, and F, MCP-1 by ELISA (n = 8). *, P≤0.05 (analysis of variance, Bonferroni post-test).
    Figure Legend Snippet: Tempol inhibits transcription and functional expression of tissue factor in endothelial cells, and cytokine production. A, Inhibition of tissue factor (TF) activity. MVEC were incubated overnight with Tempol (0, 1, and 3 mM) followed by washing the wells and addition of lipopolysaccharide (LPS) (200 ng/mL). A mixture containing FX and FVIIa was then added to the cells, and FXa generation was estimated in the supernatant using chromogenic substrate (S2222), as described in Materials and Methods. B, Inhibition of TF generation. Cells were incubated overnight with Tempol (0, 1, and 3 mM) followed by washing of the wells and addition of LPS (200 ng/mL) for six hours. Wells were washed and cells were lysed with 0.1% Triton X-100. The supernatant was used to detect TF antigen by ELISA. C, Inhibition of TF transcription. Cells were incubated overnight with Tempol (0, 1, and 3 mM) followed by addition of LPS (200 ng/mL) for 2 h. Cells were washed and trypsinized for extraction of mRNA. Real-time PCR of the samples was evaluated as described in Materials and Methods. The figure shows a representative result from two independent experiments. D–F, Inhibition of cytokine generation. MVEC were incubated overnight with Tempol (0, 1, and 3 mM) followed by washing of the wells and addition of LPS (200 ng/mL). After six hours, the supernatant of the cells was collected and used for detection of D, IL-6, E, IL-8, and F, MCP-1 by ELISA (n = 8). *, P≤0.05 (analysis of variance, Bonferroni post-test).

    Techniques Used: Functional Assay, Expressing, Inhibition, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

    Tempol inhibits MCP-1 plasma levels of mice infected with Plasmodium berghei Anka. P. berghei Anka parasitized red blood cells (10 6 ) ( n = 10) were injected intraperitoneally (i.p.). A, Tempol (20 mg/kg), was started at day 1 or day 4 post infection (p.i.). At day 6 p.i., several parameters were determined including A, clinical score and B, parasitemia (determined by Giemsa-stained smears of tail blood). Serum was collected for determination of C, MCP-1, D, IFN-γ, E, TNF-α, F, IL-2, G, IL-6, and H, IL-10 by enzyme-linked immunosorbent assay as described in Materials and Methods. *, P≤0.05 (analysis of variance, Tukey post test). NS, non-significant. Ten mice were analyzed per group.
    Figure Legend Snippet: Tempol inhibits MCP-1 plasma levels of mice infected with Plasmodium berghei Anka. P. berghei Anka parasitized red blood cells (10 6 ) ( n = 10) were injected intraperitoneally (i.p.). A, Tempol (20 mg/kg), was started at day 1 or day 4 post infection (p.i.). At day 6 p.i., several parameters were determined including A, clinical score and B, parasitemia (determined by Giemsa-stained smears of tail blood). Serum was collected for determination of C, MCP-1, D, IFN-γ, E, TNF-α, F, IL-2, G, IL-6, and H, IL-10 by enzyme-linked immunosorbent assay as described in Materials and Methods. *, P≤0.05 (analysis of variance, Tukey post test). NS, non-significant. Ten mice were analyzed per group.

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

    Tempol inhibits dendritic cell (DC) functions. Bone marrow-derived DCs (10 6 cells/mL) were preincubated overnight with medium or Tempol at indicated concentrations and stimulated with lipopolysaccharide (LPS) (200 ng/mL) for 24 hours. Detection of A, TNF-α was evaluated in cell-free culture supernatants after six hours, and the levels of B, IL-6 and C, IL-12p70 were evaluated after 24 hours. D, Cells were stained with fluorochrome-labeled monoclonal antibodies to CD11c, CD40, CD80, CD86, and MHC class II and analyzed by flow cytometry. E, DCs incubated with medium or Tempol were pulsed with OVA (100 µg/mL) plus LPS (200 ng/mL) for four hours. After repeated washings, DCs were co-incubated with purified CD4 + cells from DO11.10 mice (DC:CD4 + ratio = 1:4) for 72 hours, and proliferation was measured as described in Materials and Methods. F, IL-2 and G , IFN-γ were evaluated in the culture supernatants from the proliferation assay. *, P≤0.05 versus control group ( –/– ); # , P≤0.05 versus LPS group ( +/− ) (analysis of variance).
    Figure Legend Snippet: Tempol inhibits dendritic cell (DC) functions. Bone marrow-derived DCs (10 6 cells/mL) were preincubated overnight with medium or Tempol at indicated concentrations and stimulated with lipopolysaccharide (LPS) (200 ng/mL) for 24 hours. Detection of A, TNF-α was evaluated in cell-free culture supernatants after six hours, and the levels of B, IL-6 and C, IL-12p70 were evaluated after 24 hours. D, Cells were stained with fluorochrome-labeled monoclonal antibodies to CD11c, CD40, CD80, CD86, and MHC class II and analyzed by flow cytometry. E, DCs incubated with medium or Tempol were pulsed with OVA (100 µg/mL) plus LPS (200 ng/mL) for four hours. After repeated washings, DCs were co-incubated with purified CD4 + cells from DO11.10 mice (DC:CD4 + ratio = 1:4) for 72 hours, and proliferation was measured as described in Materials and Methods. F, IL-2 and G , IFN-γ were evaluated in the culture supernatants from the proliferation assay. *, P≤0.05 versus control group ( –/– ); # , P≤0.05 versus LPS group ( +/− ) (analysis of variance).

    Techniques Used: Derivative Assay, Staining, Labeling, Flow Cytometry, Cytometry, Incubation, Purification, Mouse Assay, Proliferation Assay

    36) Product Images from "Interaction between Toll-Like Receptor 9-CpG Oligodeoxynucleotides and Hepatitis B Virus Virions Leads to Entry Inhibition in Hepatocytes and Reduction of Alpha Interferon Production by Plasmacytoid Dendritic Cells"

    Article Title: Interaction between Toll-Like Receptor 9-CpG Oligodeoxynucleotides and Hepatitis B Virus Virions Leads to Entry Inhibition in Hepatocytes and Reduction of Alpha Interferon Production by Plasmacytoid Dendritic Cells

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01741-17

    CpG ODN 2395 inhibits the establishment of HBV infection in dHepaRG cells. (A) dHepaRG cells were either mock infected or infected with HepG2.2.15 cell-derived (left panel) or HepAD38 cell-derived (right panel) HBV (100 vge/cell). CpG ODN 2395 was added (or not [- condition]) at 2 μM either during HBV inoculation (0/+1 condition; CpG is then washed away at the end of the inoculation period) or after 4 days of infection and until harvesting of supernatant and intracellular material (+4/+7 condition). HBeAg and HBsAg secretion was monitored by ELISA, and HBV DNA intracellular accumulation was monitored by qPCR. (B) Neutral red assay and monitoring of ApoB secretion were performed under the same conditions as those in panel A to check toxicity and impact of treatment on cell differentiation. (C) The secretion of IP-10 and IL-6 was monitored by ELISA 1 day after stimulation with CpG ODN 2395 of dHepaRG cells that were infected or not. Pam3CSK4 and poly(I·C)-HMW were used as positive controls for the production by dHepaRG cells of IP-10 and IL-6, respectively. Results are given as mean ± standard error of the mean for three independent assays using two different batches of HBV, and differences from the control condition were considered statistically significant when the P value was ≤0.05 (*), ≤0.01 (**), or ≤0.001 (***). ns, not significant.
    Figure Legend Snippet: CpG ODN 2395 inhibits the establishment of HBV infection in dHepaRG cells. (A) dHepaRG cells were either mock infected or infected with HepG2.2.15 cell-derived (left panel) or HepAD38 cell-derived (right panel) HBV (100 vge/cell). CpG ODN 2395 was added (or not [- condition]) at 2 μM either during HBV inoculation (0/+1 condition; CpG is then washed away at the end of the inoculation period) or after 4 days of infection and until harvesting of supernatant and intracellular material (+4/+7 condition). HBeAg and HBsAg secretion was monitored by ELISA, and HBV DNA intracellular accumulation was monitored by qPCR. (B) Neutral red assay and monitoring of ApoB secretion were performed under the same conditions as those in panel A to check toxicity and impact of treatment on cell differentiation. (C) The secretion of IP-10 and IL-6 was monitored by ELISA 1 day after stimulation with CpG ODN 2395 of dHepaRG cells that were infected or not. Pam3CSK4 and poly(I·C)-HMW were used as positive controls for the production by dHepaRG cells of IP-10 and IL-6, respectively. Results are given as mean ± standard error of the mean for three independent assays using two different batches of HBV, and differences from the control condition were considered statistically significant when the P value was ≤0.05 (*), ≤0.01 (**), or ≤0.001 (***). ns, not significant.

    Techniques Used: Infection, Derivative Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Neutral Red Assay, Cell Differentiation

    37) Product Images from "A Possible Inflammatory Role of Twist1 in Human White Adipocytes"

    Article Title: A Possible Inflammatory Role of Twist1 in Human White Adipocytes

    Journal: Diabetes

    doi: 10.2337/db09-0997

    ChIP in human in vitro–differentiated adipocytes. A : ChIP was performed on in vitro–differentiated adipocytes. ChIP assays were performed using antibodies specifically recognizing twist1, acetyl-histone H4 (positive control), and GLUT4 (negative control). Input DNA and immunoprecipitated DNA were quantified by PCR using specific primer sets recognizing human MCP-1 , IL-6 , and TNF- α promoter regions overlapping E-boxes. Regions complementary to the primers, transcriptional start sites (indicated by an arrow), and translational start sites in the promoters are shown to the right. Primer sequences and expected PCR products are listed in supplementary Table 3. B : Undifferentiated 3T3-L1 cells were transiently transfected with pIL6 together with either empty pcDNA3.1 or pTwist1. An internal control (cytomegalovirus-β-galactosidase–containing vector) was included in all transfections. Relative luciferase activity was normalized for β-galactosidase expression and expressed as fold over empty vector (pGL2-basic) + empty pcDNA3.1 or pGL2-basic + pTwist1. Samples were run in triplicate, and the results display mean ± SEM of four independent experiments. * P
    Figure Legend Snippet: ChIP in human in vitro–differentiated adipocytes. A : ChIP was performed on in vitro–differentiated adipocytes. ChIP assays were performed using antibodies specifically recognizing twist1, acetyl-histone H4 (positive control), and GLUT4 (negative control). Input DNA and immunoprecipitated DNA were quantified by PCR using specific primer sets recognizing human MCP-1 , IL-6 , and TNF- α promoter regions overlapping E-boxes. Regions complementary to the primers, transcriptional start sites (indicated by an arrow), and translational start sites in the promoters are shown to the right. Primer sequences and expected PCR products are listed in supplementary Table 3. B : Undifferentiated 3T3-L1 cells were transiently transfected with pIL6 together with either empty pcDNA3.1 or pTwist1. An internal control (cytomegalovirus-β-galactosidase–containing vector) was included in all transfections. Relative luciferase activity was normalized for β-galactosidase expression and expressed as fold over empty vector (pGL2-basic) + empty pcDNA3.1 or pGL2-basic + pTwist1. Samples were run in triplicate, and the results display mean ± SEM of four independent experiments. * P

    Techniques Used: Chromatin Immunoprecipitation, In Vitro, Positive Control, Negative Control, Immunoprecipitation, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Expressing

    Expression and release of IL-6, MCP-1, and adiponectin into conditioned media after siRNA treatment of cultured adipocytes. Human in vitro–differentiated adipocytes were treated with 50 nmol/l control siRNA or siRNA directed against twist1. Expression of the inflammatory genes IL-6 , TNF- α, and MCP-1 as well as adiponectin was measured after siRNA treatment ( A ). mRNA levels were normalized to the reference gene GAPDH . Release of IL-6 ( B ), MCP-1 ( C ), and adiponectin ( D ) was measured in conditioned media; n = 4 (IL-6) and n = 3 (MCP-1/adiponectin). Results shown are mean ± SEM and mRNA levels are shown as relative expression (fold change to control siRNA). ** P
    Figure Legend Snippet: Expression and release of IL-6, MCP-1, and adiponectin into conditioned media after siRNA treatment of cultured adipocytes. Human in vitro–differentiated adipocytes were treated with 50 nmol/l control siRNA or siRNA directed against twist1. Expression of the inflammatory genes IL-6 , TNF- α, and MCP-1 as well as adiponectin was measured after siRNA treatment ( A ). mRNA levels were normalized to the reference gene GAPDH . Release of IL-6 ( B ), MCP-1 ( C ), and adiponectin ( D ) was measured in conditioned media; n = 4 (IL-6) and n = 3 (MCP-1/adiponectin). Results shown are mean ± SEM and mRNA levels are shown as relative expression (fold change to control siRNA). ** P

    Techniques Used: Expressing, Cell Culture, In Vitro

    38) Product Images from "STAT3 Genotypic Variation and Cellular STAT3 Activation and Colon Leukocyte Recruitment in Pediatric Crohn Disease"

    Article Title: STAT3 Genotypic Variation and Cellular STAT3 Activation and Colon Leukocyte Recruitment in Pediatric Crohn Disease

    Journal: Journal of pediatric gastroenterology and nutrition

    doi: 10.1097/MPG.0b013e318246be78

    IL-6 Stimulated Nuclear Localization of STAT3 is Increased in EBL Carrying the STAT3 ”A” Risk Allele EBLs from IBD patients carrying the STAT3 “A” risk allele or disease controls carrying the “G” non-risk allele were examined under basal conditions and following IL-6 stimulation. ImageStream x (Amnis®) analysis demonstrated increased nuclear co-localization of pSTAT3 following IL-6 stimulation in the STAT3 “A” risk allele EBL (10.8 fold increase) compared to no-change in the non-risk “G” allele EBL.
    Figure Legend Snippet: IL-6 Stimulated Nuclear Localization of STAT3 is Increased in EBL Carrying the STAT3 ”A” Risk Allele EBLs from IBD patients carrying the STAT3 “A” risk allele or disease controls carrying the “G” non-risk allele were examined under basal conditions and following IL-6 stimulation. ImageStream x (Amnis®) analysis demonstrated increased nuclear co-localization of pSTAT3 following IL-6 stimulation in the STAT3 “A” risk allele EBL (10.8 fold increase) compared to no-change in the non-risk “G” allele EBL.

    Techniques Used:

    IL-6 Stimulated STAT3 Activation and Expression of STAT3 and SOCS3 are Increased in Immortalized B-cell lines from IBD Patients Carrying the STAT3 ”A” Risk Allele EBL nuclear protein abundance of (A) tyrosine phosphorylated STAT3 and (C) tyrosine phosphorylated STAT1before and after IL-6/IL-6R stimulation was measured by immunoblot analysis. Data are expressed as normalized chemoluminescent arbitrary units (AU) to loading control TFIIβ via LAS Image Reader and MultiGauge Software (Fujifilm®). Representative immunoblot for (B) pSTAT3 and (D) pSTAT1. EBL mRNA expression was measured by quantitative real time PCR for (E) STAT3 and (F) SOCS3 before and after stimulation with IL-6/ IL-6R. Data are expressed relative to glyceraldehyde phosphate dehydrogenase (GAPDH) as mean ± SEM. GG (n=6) and AA (n=6–9) : EBLs null or homozygous for STAT3 “A” risk allele. Differences were tested by A, C, and E) unpaired t-test with Welch's correction or F) unpaired t-test.
    Figure Legend Snippet: IL-6 Stimulated STAT3 Activation and Expression of STAT3 and SOCS3 are Increased in Immortalized B-cell lines from IBD Patients Carrying the STAT3 ”A” Risk Allele EBL nuclear protein abundance of (A) tyrosine phosphorylated STAT3 and (C) tyrosine phosphorylated STAT1before and after IL-6/IL-6R stimulation was measured by immunoblot analysis. Data are expressed as normalized chemoluminescent arbitrary units (AU) to loading control TFIIβ via LAS Image Reader and MultiGauge Software (Fujifilm®). Representative immunoblot for (B) pSTAT3 and (D) pSTAT1. EBL mRNA expression was measured by quantitative real time PCR for (E) STAT3 and (F) SOCS3 before and after stimulation with IL-6/ IL-6R. Data are expressed relative to glyceraldehyde phosphate dehydrogenase (GAPDH) as mean ± SEM. GG (n=6) and AA (n=6–9) : EBLs null or homozygous for STAT3 “A” risk allele. Differences were tested by A, C, and E) unpaired t-test with Welch's correction or F) unpaired t-test.

    Techniques Used: Activation Assay, Expressing, Software, Real-time Polymerase Chain Reaction

    Cytosolic protein abundance of STAT3, STAT1, IL-6 receptor, JAK2, GP130, nor SOCS3 varied by STAT ”A” risk allele EBL cytosolic protein abundance of (A) STAT3, (B) STAT1, (C) JAK2, (D) IL-6 receptor, (E) GP130, and (F) SOCS3 were measured by immunoblot analysis. Data are expressed as normalized chemoluminescent arbitrary units (AU) to loading control β-actin via LAS Image Reader and MultiGauge Software (Fujifilm®). Representative immunoblot for each are shown. GG (n=6) and AA (n=9) : EBLs null or homozygous for the STAT3 “A” risk allele. Differences were tested by unpaired t-test.
    Figure Legend Snippet: Cytosolic protein abundance of STAT3, STAT1, IL-6 receptor, JAK2, GP130, nor SOCS3 varied by STAT ”A” risk allele EBL cytosolic protein abundance of (A) STAT3, (B) STAT1, (C) JAK2, (D) IL-6 receptor, (E) GP130, and (F) SOCS3 were measured by immunoblot analysis. Data are expressed as normalized chemoluminescent arbitrary units (AU) to loading control β-actin via LAS Image Reader and MultiGauge Software (Fujifilm®). Representative immunoblot for each are shown. GG (n=6) and AA (n=9) : EBLs null or homozygous for the STAT3 “A” risk allele. Differences were tested by unpaired t-test.

    Techniques Used: Software

    Membrane Localization of the IL-6 receptor, GP130, and JAK2 are Increased in Immortalized B-cell lines from IBD Patients Carrying the STAT3 ”A” Risk Allele EBL Membrane protein abundance of (A) IL-6 receptor (C) GP130 and (E) JAK2 were measured by immunoblot analysis. Data are expressed as normalized chemoluminescent arbitrary units (AU) to loading control β-Tubulin via LAS Image Reader and MultiGauge Software (Fujifilm®). Representative immunoblot for (B) IL-6 receptor, (D) GP130, and (F) JAK2. GG (n=7) and AA (n=10) : EBLs null or homozygous for the STAT3 “A” risk allele. Differences were tested by unpaired t-test.
    Figure Legend Snippet: Membrane Localization of the IL-6 receptor, GP130, and JAK2 are Increased in Immortalized B-cell lines from IBD Patients Carrying the STAT3 ”A” Risk Allele EBL Membrane protein abundance of (A) IL-6 receptor (C) GP130 and (E) JAK2 were measured by immunoblot analysis. Data are expressed as normalized chemoluminescent arbitrary units (AU) to loading control β-Tubulin via LAS Image Reader and MultiGauge Software (Fujifilm®). Representative immunoblot for (B) IL-6 receptor, (D) GP130, and (F) JAK2. GG (n=7) and AA (n=10) : EBLs null or homozygous for the STAT3 “A” risk allele. Differences were tested by unpaired t-test.

    Techniques Used: Software

    39) Product Images from "Adenosine receptors in regulation of dendritic cell differentiation and function"

    Article Title: Adenosine receptors in regulation of dendritic cell differentiation and function

    Journal: Blood

    doi: 10.1182/blood-2008-02-136325

    Adenosine-differentiated CD1a low CD14 + DCs express VEGF, IL-8, IL-6, and IL-10, a combination of angiogenic, proinflammatory, and immune suppressive factors. (A,B) Immature DCs were generated from human monocytes in the presence of 30 μM NECA, and CD1a + and CD1a − CD14 + cells were separated by immunomagnetic technique. These cells, as well as freshly purified monocytes as control, were cultured under the same conditions with the addition of 20 ng/mL TNF-α. Cells were harvested after 6 hours for mRNA quantification; culture supernatants for measurements of secreted cytokines were collected from duplicate samples after 24 hours. Specific mRNA transcripts were quantified by real-time PCR, and concentrations of secreted cytokines were measured by ELISA. Average values from 3 different experiments are shown. Error bars denote SE.
    Figure Legend Snippet: Adenosine-differentiated CD1a low CD14 + DCs express VEGF, IL-8, IL-6, and IL-10, a combination of angiogenic, proinflammatory, and immune suppressive factors. (A,B) Immature DCs were generated from human monocytes in the presence of 30 μM NECA, and CD1a + and CD1a − CD14 + cells were separated by immunomagnetic technique. These cells, as well as freshly purified monocytes as control, were cultured under the same conditions with the addition of 20 ng/mL TNF-α. Cells were harvested after 6 hours for mRNA quantification; culture supernatants for measurements of secreted cytokines were collected from duplicate samples after 24 hours. Specific mRNA transcripts were quantified by real-time PCR, and concentrations of secreted cytokines were measured by ELISA. Average values from 3 different experiments are shown. Error bars denote SE.

    Techniques Used: Generated, Purification, Cell Culture, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    40) Product Images from "Selective Role of Vinculin in Contractile Mechanisms of Endothelial Permeability"

    Article Title: Selective Role of Vinculin in Contractile Mechanisms of Endothelial Permeability

    Journal: American Journal of Respiratory Cell and Molecular Biology

    doi: 10.1165/rcmb.2015-0328OC

    Effects of vinculin knockdown on thrombin– and IL-6–induced FA rearrangement, actin cytoskeletal remodeling, and endothelial permeability. Human pulmonary ECs were transfected with vinculin-specific (si-VNC) or nonspecific small interfering
    Figure Legend Snippet: Effects of vinculin knockdown on thrombin– and IL-6–induced FA rearrangement, actin cytoskeletal remodeling, and endothelial permeability. Human pulmonary ECs were transfected with vinculin-specific (si-VNC) or nonspecific small interfering

    Techniques Used: Permeability, Transfection

    Analysis of adherens junction (AJ) remodeling in response to IL-6. HPAECs were treated with IL-6 and SR (30 ng/ml and 60 ng/ml). ( A ) Levels of phosphorylated signal transducer and activator of transcription (STAT) and VE-cadherin in the cell lysates were
    Figure Legend Snippet: Analysis of adherens junction (AJ) remodeling in response to IL-6. HPAECs were treated with IL-6 and SR (30 ng/ml and 60 ng/ml). ( A ) Levels of phosphorylated signal transducer and activator of transcription (STAT) and VE-cadherin in the cell lysates were

    Techniques Used:

    Analysis of contractile signaling in response to IL-6. ( A ) IL-6–induced Rho activation was assessed by RhoGTP pulldown assay. Thrombin treatment (0.2 U/ml, 5 min) was used as a positive control. ( B ) Cytoskeletal remodeling in response to IL-6
    Figure Legend Snippet: Analysis of contractile signaling in response to IL-6. ( A ) IL-6–induced Rho activation was assessed by RhoGTP pulldown assay. Thrombin treatment (0.2 U/ml, 5 min) was used as a positive control. ( B ) Cytoskeletal remodeling in response to IL-6

    Techniques Used: Activation Assay, Positive Control

    Effects of thrombin and IL-6 on endothelial permeability and focal adhesion (FA) remodeling. ( A and B ) Human pulmonary artery endothelial cells (HPAECs) grown on glass coverslips ( A ) or in 96-well plates ( B ) with immobilized, biotinylated gelatin (0.25
    Figure Legend Snippet: Effects of thrombin and IL-6 on endothelial permeability and focal adhesion (FA) remodeling. ( A and B ) Human pulmonary artery endothelial cells (HPAECs) grown on glass coverslips ( A ) or in 96-well plates ( B ) with immobilized, biotinylated gelatin (0.25

    Techniques Used: Permeability

    Effect of vinculin knockdown on cell signaling induced by thrombin and IL-6. Human pulmonary ECs were transfected with vinculin-specific or nonspecific siRNA. ( A ) Thrombin-induced Rho activation was assessed by RhoGTP pulldown assay. Bar graphs depict
    Figure Legend Snippet: Effect of vinculin knockdown on cell signaling induced by thrombin and IL-6. Human pulmonary ECs were transfected with vinculin-specific or nonspecific siRNA. ( A ) Thrombin-induced Rho activation was assessed by RhoGTP pulldown assay. Bar graphs depict

    Techniques Used: Transfection, Activation Assay

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    Article Snippet: .. In Vivo Clearance 6 μg of rhIL-6 alone (R & D Systems) or rhIL-6 incubated with mAbs or BiS3Ab in a 1:1 epitope: paratope ratio in PBS (pH 7.4) was injected i.v. into mice in triplicate. .. Mouse plasma was collected 5, 15, 30, 60, 120, and 480 min after dosing, and the serum IL-6 levels were calculated using a Quantikine IL-6 assay kit (R & D Systems).

    Transfection:

    Article Title: Endogenous bone morphogenetic protein 2 plays a role in vascular smooth muscle cell calcification induced by interleukin 6 in vitro
    Article Snippet: .. After transfection, the cells of both groups were changed to regular medium culture and both were treated with rhIL-6 10 ng/mL. .. The cellular mRNA level of BMP2, BAP, OPG, and OPN were tested at 12 h and 48 h. To observe the effect of siRNA on calcification, 50 ng/mL rhIL-6 was used.

    Mouse Assay:

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    Article Snippet: .. In Vivo Clearance 6 μg of rhIL-6 alone (R & D Systems) or rhIL-6 incubated with mAbs or BiS3Ab in a 1:1 epitope: paratope ratio in PBS (pH 7.4) was injected i.v. into mice in triplicate. .. Mouse plasma was collected 5, 15, 30, 60, 120, and 480 min after dosing, and the serum IL-6 levels were calculated using a Quantikine IL-6 assay kit (R & D Systems).

    Enzyme-linked Immunosorbent Assay:

    Article Title: STING signaling remodels the tumor microenvironment by antagonizing myeloid-derived suppressor cell expansion
    Article Snippet: .. Enzyme-linked immunosorbent (ELISA) assayFor ELISA assays, human IL-6 (R & D Systems, Minneapolis, MN, USA), and GM-CSF ELISA kits (eBioscience, San Diego, CA, USA) were used. .. Briefly, a 96-well plate was coated with 100 µL of coating diluent at 4 °C overnight.

    Transgenic Assay:

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    Article Snippet: .. Transgenic expression of hIL-6 resulted in a 4-fold increase in hepcidin mRNA levels in transgenic larvae (7 days postfertilization; B). .. Markedly elevated hepcidin mRNA levels were also found in the livers of adult cmlc-IL-6 transgenic fish (supplemental Figure 2B).

    Incubation:

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    Article Snippet: .. After overnight incubation at 4°C and multiple washings, bound hIL-6 was detected with 2 μg/ml polyclonal anti–hIL-6 goat antibody and donkey anti–goat IgG HRP followed by chromogenic substrates. ..

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    Article Snippet: .. In Vivo Clearance 6 μg of rhIL-6 alone (R & D Systems) or rhIL-6 incubated with mAbs or BiS3Ab in a 1:1 epitope: paratope ratio in PBS (pH 7.4) was injected i.v. into mice in triplicate. .. Mouse plasma was collected 5, 15, 30, 60, 120, and 480 min after dosing, and the serum IL-6 levels were calculated using a Quantikine IL-6 assay kit (R & D Systems).

    other:

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    Article Snippet: Cells were treated with indicated doses of LPS or rhIL-6 for 24 h or 72 h, according to the experiment performed.

    Expressing:

    Article Title: Inhibition of bone morphogenetic protein signaling attenuates anemia associated with inflammation
    Article Snippet: .. Transgenic expression of hIL-6 resulted in a 4-fold increase in hepcidin mRNA levels in transgenic larvae (7 days postfertilization; B). .. Markedly elevated hepcidin mRNA levels were also found in the livers of adult cmlc-IL-6 transgenic fish (supplemental Figure 2B).

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    Article Snippet: .. The results of these experiments identified markedly increased secretion of the C-helix substitution variant and some increase in secretion upon substitution of the distal half of vIL-6 helix-D with that of hIL-6 (Fig. ). .. However, these sequences were unable to confer retention to hIL-6 when introduced into the human cytokine (Fig. ), indicating that the contribution of these regions to vIL-6 intracellular retention is context dependent.

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    OA cartilage explant culture with inhibition of synovial fluid IL-6 or addition of exogenous IL-6 . ( a, c ) GAG content and GAG release in OA cartilage explants from three donors cultured either in medium only, medium supplemented with 25% OA synovial fluid (SF) or medium with 25% OA synovial fluid in which IL-6 is blocked with an activity-inhibiting antibody (mean ± SD in mg/g); P = 0.06. ( b, d ) GAG content and GAG release in OA cartilage explants from eight donors cultured in the presence or absence of <t>rhIL-6</t> (50 ng/mL) with rhIL-6Rα (200 ng/mL).
    Rhil 6, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Correlation of serum Gd-IgA1 with ( a ) MCP-1 and ( b ) IL-6 from HMCs treated with IgA1 isolates from patients.

    Journal: Clinical Kidney Journal

    Article Title: Higher serum galactose-deficient immunoglobulin A1 concentration is associated with stronger mesangial cellular inflammatory response and more severe histologic findings in immunoglobulin A nephropathy

    doi: 10.1093/ckj/sfy068

    Figure Lengend Snippet: Correlation of serum Gd-IgA1 with ( a ) MCP-1 and ( b ) IL-6 from HMCs treated with IgA1 isolates from patients.

    Article Snippet: Cell culture supernatant was collected after 24 h stimulation and pro-inflammatory cytokines monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) were measured by ELISA using matched paired antibodies (R & D Systems, Minneapolis, MN, USA).

    Techniques:

    OA cartilage explant culture with inhibition of synovial fluid IL-6 or addition of exogenous IL-6 . ( a, c ) GAG content and GAG release in OA cartilage explants from three donors cultured either in medium only, medium supplemented with 25% OA synovial fluid (SF) or medium with 25% OA synovial fluid in which IL-6 is blocked with an activity-inhibiting antibody (mean ± SD in mg/g); P = 0.06. ( b, d ) GAG content and GAG release in OA cartilage explants from eight donors cultured in the presence or absence of rhIL-6 (50 ng/mL) with rhIL-6Rα (200 ng/mL).

    Journal: Arthritis Research & Therapy

    Article Title: Interleukin-6 is elevated in synovial fluid of patients with focal cartilage defects and stimulates cartilage matrix production in an in vitro regeneration model

    doi: 10.1186/ar4107

    Figure Lengend Snippet: OA cartilage explant culture with inhibition of synovial fluid IL-6 or addition of exogenous IL-6 . ( a, c ) GAG content and GAG release in OA cartilage explants from three donors cultured either in medium only, medium supplemented with 25% OA synovial fluid (SF) or medium with 25% OA synovial fluid in which IL-6 is blocked with an activity-inhibiting antibody (mean ± SD in mg/g); P = 0.06. ( b, d ) GAG content and GAG release in OA cartilage explants from eight donors cultured in the presence or absence of rhIL-6 (50 ng/mL) with rhIL-6Rα (200 ng/mL).

    Article Snippet: Endogenous IL-6 production was relatively low in healthy chondrocytes, so the possible effects of high concentrations of IL-6 were further investigated by the addition of 10 ng/mL rhIL-6 with 25 ng/mL rhIL-6Rα (R & D Systems, #206-IL, #227-SR) to both healthy (n = 3) and OA (n = 3) chondrocytes.

    Techniques: Inhibition, Cell Culture, Activity Assay

    Cartilage regeneration with addition of IL-6 . Cartilage regeneration cultures of three healthy (H) and three osteoarthritic (OA) donors with addition of rhIL-6 (10 ng/mL) and rhIL-6Rα (25 ng/mL). ( a-c ) GAG content, GAG release and DNA content of IL-6 supplemented samples depicted as percentage of control samples (mean ± SD); a = P = 0.009, b = P

    Journal: Arthritis Research & Therapy

    Article Title: Interleukin-6 is elevated in synovial fluid of patients with focal cartilage defects and stimulates cartilage matrix production in an in vitro regeneration model

    doi: 10.1186/ar4107

    Figure Lengend Snippet: Cartilage regeneration with addition of IL-6 . Cartilage regeneration cultures of three healthy (H) and three osteoarthritic (OA) donors with addition of rhIL-6 (10 ng/mL) and rhIL-6Rα (25 ng/mL). ( a-c ) GAG content, GAG release and DNA content of IL-6 supplemented samples depicted as percentage of control samples (mean ± SD); a = P = 0.009, b = P

    Article Snippet: Endogenous IL-6 production was relatively low in healthy chondrocytes, so the possible effects of high concentrations of IL-6 were further investigated by the addition of 10 ng/mL rhIL-6 with 25 ng/mL rhIL-6Rα (R & D Systems, #206-IL, #227-SR) to both healthy (n = 3) and OA (n = 3) chondrocytes.

    Techniques:

    M2-enhanced IL-6 and VM in glioma cells via PKC pathway ( A ) Glioma cells were incubated with THP-1-CM or M2-CM for indicated time, the phosphorylation of PKC (p-PKC, Pan) was determined by Western blotting. ( B , C ) Glioma cells were incubated with DMEM medium, M2-CM or M2-CM containing Bisindolylmaleimide I (PKC I) for 24 h, IL-6 transcription (B) and concentration (C) in CM were determined by qRT-PCR and ELISA respectively. ( D ) As previously treatment, the protein levels of VM markers were then detected by Western blotting. ( E ) Representative images and quantification of tubule formation assay in glioma cells incubated in M2-CM, M2-CM containing Bisindolylmaleimide I or M2-CM containing Bisindolylmaleimide I with rhIL-6 for 24 h (×100). Each bar represents the mean ± SEM ( n = 3). * P

    Journal: Oncotarget

    Article Title: M2-like tumor-associated macrophages drive vasculogenic mimicry through amplification of IL-6 expression in glioma cells

    doi: 10.18632/oncotarget.13661

    Figure Lengend Snippet: M2-enhanced IL-6 and VM in glioma cells via PKC pathway ( A ) Glioma cells were incubated with THP-1-CM or M2-CM for indicated time, the phosphorylation of PKC (p-PKC, Pan) was determined by Western blotting. ( B , C ) Glioma cells were incubated with DMEM medium, M2-CM or M2-CM containing Bisindolylmaleimide I (PKC I) for 24 h, IL-6 transcription (B) and concentration (C) in CM were determined by qRT-PCR and ELISA respectively. ( D ) As previously treatment, the protein levels of VM markers were then detected by Western blotting. ( E ) Representative images and quantification of tubule formation assay in glioma cells incubated in M2-CM, M2-CM containing Bisindolylmaleimide I or M2-CM containing Bisindolylmaleimide I with rhIL-6 for 24 h (×100). Each bar represents the mean ± SEM ( n = 3). * P

    Article Snippet: Reagents and antibodies Isotype-IgG, rhIL-4, rhIL-13, M-CSF, rhIL-6, anti-IL-6 and human IL-6 Quantikine ELISA were purchased from R & D Systems.

    Techniques: Incubation, Western Blot, Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Tube Formation Assay

    IL-6 upregulation is responsible for VM promotion in glioma cells ( A , B ) Glioma cells were treated with DMEM medium or M2-CM in the presence or absence of anti-IL-6 at 1 μg/ml, or isotype-matched IgG (IgG) control for 24 hours. Representative images (A) and quantification (B) of tubule formation assay on Matrigel matrix in vitro (×100). ( C ) As previously treatment, the protein levels of VM markers were then detected by Western blotting. ( D , E ) Glioma cells were treated with rhIL-6 in a concentration of 100 ng/mL or PBS control for 24 hours. Representative images (D) and quantification (E) of tubule formation assay on Matrigel matrix in vitro (×100). ( F ) VM markers protein of glioma cells, alone or after cultured with rhIL-6. Each bar represents the mean ± SEM ( n = 3). * P

    Journal: Oncotarget

    Article Title: M2-like tumor-associated macrophages drive vasculogenic mimicry through amplification of IL-6 expression in glioma cells

    doi: 10.18632/oncotarget.13661

    Figure Lengend Snippet: IL-6 upregulation is responsible for VM promotion in glioma cells ( A , B ) Glioma cells were treated with DMEM medium or M2-CM in the presence or absence of anti-IL-6 at 1 μg/ml, or isotype-matched IgG (IgG) control for 24 hours. Representative images (A) and quantification (B) of tubule formation assay on Matrigel matrix in vitro (×100). ( C ) As previously treatment, the protein levels of VM markers were then detected by Western blotting. ( D , E ) Glioma cells were treated with rhIL-6 in a concentration of 100 ng/mL or PBS control for 24 hours. Representative images (D) and quantification (E) of tubule formation assay on Matrigel matrix in vitro (×100). ( F ) VM markers protein of glioma cells, alone or after cultured with rhIL-6. Each bar represents the mean ± SEM ( n = 3). * P

    Article Snippet: Reagents and antibodies Isotype-IgG, rhIL-4, rhIL-13, M-CSF, rhIL-6, anti-IL-6 and human IL-6 Quantikine ELISA were purchased from R & D Systems.

    Techniques: Tube Formation Assay, In Vitro, Western Blot, Concentration Assay, Cell Culture

    Exogenous recombinant human IL-6, IL-8 and TGFβ1 induce reactivation of dormant breast cancer colonies but do not promote growth of growing colonies. a Cytokines induce regrowth of dormant colonies. One thousand MCF-7 cells were incubated in fibronectin-coated wells with FGF-2 for 6 days, followed by addition of recombinant human IL-6, IL-8 and TGFβ1 on day 6 and incubation for an additional 6 days, as described. Cells were stained with 0.1% crystal violet and growing and dormant colonies were counted on day 6 and 12. b Cytokines do not promote the growth of growing colonies. MCF-7 cells were incubated in fibronectin-coated wells with FGF-2 or recombinant human IL-6, IL-8 or TGFβ1, as described. Cells were stained with 0.1% crystal violet and growing and dormant colonies were counted on day 6. c Time lapse photography demonstrating regrowth potential of dormant clones. Photographs demonstrate growth expansion or degradation of both dormant and growing colonies. MCF cells were incubated on fibronectin-coated plates without and with FGF-2 for six days, followed by an additional 6 day incubation with fresh DMEM/10% FCS without or with rhIL-6, IL-8 and TGFβ1, as described. Time-lapse microscopic images of the same individual colonies in the same fields were obtained once daily for these six days. Size markers are 200 μM

    Journal: Cell Communication and Signaling : CCS

    Article Title: Reawakening of dormant estrogen-dependent human breast cancer cells by bone marrow stroma secretory senescence

    doi: 10.1186/s12964-018-0259-5

    Figure Lengend Snippet: Exogenous recombinant human IL-6, IL-8 and TGFβ1 induce reactivation of dormant breast cancer colonies but do not promote growth of growing colonies. a Cytokines induce regrowth of dormant colonies. One thousand MCF-7 cells were incubated in fibronectin-coated wells with FGF-2 for 6 days, followed by addition of recombinant human IL-6, IL-8 and TGFβ1 on day 6 and incubation for an additional 6 days, as described. Cells were stained with 0.1% crystal violet and growing and dormant colonies were counted on day 6 and 12. b Cytokines do not promote the growth of growing colonies. MCF-7 cells were incubated in fibronectin-coated wells with FGF-2 or recombinant human IL-6, IL-8 or TGFβ1, as described. Cells were stained with 0.1% crystal violet and growing and dormant colonies were counted on day 6. c Time lapse photography demonstrating regrowth potential of dormant clones. Photographs demonstrate growth expansion or degradation of both dormant and growing colonies. MCF cells were incubated on fibronectin-coated plates without and with FGF-2 for six days, followed by an additional 6 day incubation with fresh DMEM/10% FCS without or with rhIL-6, IL-8 and TGFβ1, as described. Time-lapse microscopic images of the same individual colonies in the same fields were obtained once daily for these six days. Size markers are 200 μM

    Article Snippet: Recombinant human (rh)FGF-2, rhIL-6, rhIL-8 and rhTGFβ1 and mouse blocking monoclonal antibody to CXCL1/KC (IL-8) and mouse immunoglobulin G (IgG) were purchased from R & D Systems (Minneapolis, MN).

    Techniques: Recombinant, Incubation, Staining, Clone Assay

    Dormant and reactivated dormant breast cancer colonies exhibit characteristics of an epithelial to mesenchymal transition. a Morphological changes from epithelial (left) to a spindle like (right) morphology were observed between growing MCF-7 colonies and reactivated dormant cells. Colonies were photographed at 40X magnification. b Western blots performed with lysates from MCF-7 cells incubated with or without FGF-2 in tissue culture for 6 days and dormant cells incubated with rhIL-6, IL-8 and TGFβ1 for an additional 6 days on fibronectin at clonogenic density. Blots were stained with antibodies to human E-cadherin, N-cadherin, SLUG, and ERα. Coomassie blue-stained gels were used as loading controls. Relative band intensity ratios to those of the corresponding loading control were determined

    Journal: Cell Communication and Signaling : CCS

    Article Title: Reawakening of dormant estrogen-dependent human breast cancer cells by bone marrow stroma secretory senescence

    doi: 10.1186/s12964-018-0259-5

    Figure Lengend Snippet: Dormant and reactivated dormant breast cancer colonies exhibit characteristics of an epithelial to mesenchymal transition. a Morphological changes from epithelial (left) to a spindle like (right) morphology were observed between growing MCF-7 colonies and reactivated dormant cells. Colonies were photographed at 40X magnification. b Western blots performed with lysates from MCF-7 cells incubated with or without FGF-2 in tissue culture for 6 days and dormant cells incubated with rhIL-6, IL-8 and TGFβ1 for an additional 6 days on fibronectin at clonogenic density. Blots were stained with antibodies to human E-cadherin, N-cadherin, SLUG, and ERα. Coomassie blue-stained gels were used as loading controls. Relative band intensity ratios to those of the corresponding loading control were determined

    Article Snippet: Recombinant human (rh)FGF-2, rhIL-6, rhIL-8 and rhTGFβ1 and mouse blocking monoclonal antibody to CXCL1/KC (IL-8) and mouse immunoglobulin G (IgG) were purchased from R & D Systems (Minneapolis, MN).

    Techniques: Western Blot, Incubation, Staining