Interleukin 6 (il 6)

Manufactured by Cell Signaling Technology

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IL-6 is a recombinant protein that functions as a cytokine. It is involved in the regulation of immune response and inflammation.

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Interleukin (IL)-6 (2 citations)

121 protocols using interleukin 6 (il 6)

ARPE-19 Cell Culture and IL-6 Treatment

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The human RPE cell line ARPE-19 was purchased from American Type Culture Collection (ATCC, Manassas, VA). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Life Technologies, Grand Island, NY) containing 10% fetal bovine serum (FBS; Gibco) at 37 °C in a humidified atmosphere containing 5% CO₂. The cell line was authenticated with short tandem repeat (STR) analysis. The STR results showed that the DNA of the cell line perfectly matched ARPE-19, and no cross contamination of human cells was detected (Appendix 1).
For IL-6 treatment, the cells were seeded in six- or 96-well plates and incubated with different concentrations of IL-6 (Cell Signaling, Danvers, MA). The pharmacological inhibitor of STAT3 signaling S3I-201 was purchased from Selleck Chemicals (Houston, TX), prepared in dimethyl sulfoxide (DMSO) as 10 mM stock solution, and added 60 min before treatment with IL-6.

Chen X., Yang W., Deng X., Ye S, & Xiao W. (2020). Interleukin-6 promotes proliferative vitreoretinopathy by inducing epithelial-mesenchymal transition via the JAK1/STAT3 signaling pathway. Molecular Vision, 26, 517-529.

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Optimizing IL-6 STAT3 Phosphorylation

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Human IL-6 was purchased from Cell Signaling (8904SC), and cells were treated with 100 ng/mL of IL-6 for 30 minutes based on an optimization time course in which 30 minutes had the strongest phosphorylation of STAT3 at this concentration.

Pinkham C., An S., Lundberg L., Bansal N., Benedict A., Narayanan A, & Kehn-Hall K. (2016). The Role of Signal Transducer and Activator of Transcription 3 in Rift Valley Fever Virus Infection. Virology, 496, 175-185.

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Proliferative Capability of BON1 Cells

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The proliferation capability of BON1 cells was firstly assessed by a MTS cell proliferation colorimetric assay kit (Biovision, Milpitas, CA, USA) according to the manufactures instructions. Briefly, BON1 cells (5 × 10³/well) were seeded in 96-well plate and incubated for 3 days with IL-6 (25 ng/mL, Cell Signaling) or CRP (20 µg/mL, R&D Systems). Twenty microliters of MTS reagent were added to each well. After 3 h of incubation, optical density was read at 490 nm and 650 nm using a SYNERGY/HTX multi-mode reader (BioTek Instruments).
Secondly, we used the BrdU Cell Proliferation ELISA Kit (Abcam), in order to assess the same mechanism, according to the manufactures instructions.

Schimmack S., Yang Y., Felix K., Herbst M., Li Y., Schenk M., Bergmann F., Hackert T, & Strobel O. (2019). C-reactive protein (CRP) promotes malignant properties in pancreatic neuroendocrine neoplasms. Endocrine Connections, 8(7), 1007-1019.

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Western Blotting Antibodies and Conditions

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Western blotting was performed, as described previously [49 (link)]. Antibodies used in this study are as follows: IL-6 (Cell Signaling Technology, Danvers, MA, USA, D3K2N), IL-6 (Immunoway, YT5348), IL-8 (Cell Signaling Technology, E5F5Q), H3 (Abcam, Cambridge, UK, ab1971), H3K9me2 (Cell Signaling Technology, D85B4), H3K9me3 (Millipore, Burlington, MA, USA, #07-523), H3K27me3 (Active Motif, Carlsbad, CA, USA, 61017), H3K27me3 (Millipore, #07-449), p-STING (Cell Signaling Technology, 19781), STING (Abways, Shanghai, China, CY7204), c-GAS (Cell Signaling Technology, 79978), c-GAS (Abcam, ab242363), β-actin (Sigma-Aldrich, St. Louis, MO, USA, A5228), CyclinA2 (Abcam, ab181591), CyclinA2 (Cell Signaling Technology, E6D1J), and Ki67 (GeneTex, Irvine, CA, USA, GTX16667). Secondary goat anti-mouse and goat anti-rabbit antibodies were obtained from ZSGB-BIO (Beijing, China).

Zhang N., Shang M., Li H., Wu L., Dong M., Huang B., Lu J, & Zhang Y. (2022). Dual Inhibition of H3K9me2 and H3K27me3 Promotes Tumor Cell Senescence without Triggering the Secretion of SASP. International Journal of Molecular Sciences, 23(7), 3911.

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Immunocytochemical Analysis of Inflammatory Cytokines

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The cultured peritoneal macrophages were fixed in 4% paraformaldehyde for 20 min and then washed with PBS three times. Following permeabilization with ice-cold 0.3% Triton X-100 for 10 min, the cells were blocked in 5% (m/v) bovine serum albumin (BSA, cat. no. A1993; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature and then incubated with primary rabbit anti-mouse antibodies as follows: TNF-α (diluted 1:200 with PBS/5% BSA; cat. no. BS6000; Bioworld Technology, Inc., St. Louis Park, MN, USA), IL-1β (diluted 1:50 with PBS/5% BSA; cat. no. 31202; Cell Signaling Technology, Inc., Danvers, MA, USA), IL-6 (diluted 1:200 with PBS/5% BSA; cat. no. 12912; Cell Signaling Technology, Inc.) overnight at 4°C. Subsequently, cells were washed with PBS three times, followed by incubation with corresponding secondary phycoerythrin-conjugated goat anti-rabbit IgG (1:200 diluted with PBS/5% BSA; cat. no. sc-3739; Santa Cruz Biotechnology, Inc. Dallas, TX, USA) for 1 h at room temperature. For nuclear staining, cells were covered with 10 µg/ml DAPI for 2 min at room temperature. The stained cells were visualized under a fluorescence microscope at ×400 magnification with green excitation light. Different groups of images were taken in the same software settings.

Wang M., Kong X., Xie Y., He C., Wang T, & Zhou H. (2019). Role of TLR-4 in anti-β2-glycoprotein I-induced activation of peritoneal macrophages and vascular endothelial cells in mice. Molecular Medicine Reports, 19(5), 4353-4363.

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Western Blot Analysis for Protein Expression

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Cells were lysed (30 min at 4 °C), cleared by centrifugation, and protein concentrations were determined using BCA protein assay (Pierce). 15–30 μg protein lysate were separated on a 4–12% Bis–Tris gel (Invitrogen) and blotted onto a Nitrocellulose Membrane using iBlot (Invitrogen). Membrane was blocked for 1 h in Odyssey Blocking Buffer (LiCor, Lincoln, NE USA) followed by incubation with primary antibodies. IRDye^® secondary antibodies (LiCor and Abcam, Cambridge, England) were used and signals were detected using the Odyssey Sa (LiCor). Following antibodies were used: MitoProfile^® Total OXPHOS Human WB Antibody Cocktail (abcam #ab110411), human CD40 (abcam #13545), IL10 (#ab133575), IL6 (Cell Signaling, #12153), β-actin (abcam), α-Tubulin (LiCor) and β-tubulin (Santa Cruz, Dallas, TX, USA and abcam) were used as loading controls. Original Blot for Figure 2 and for the cut bands shown in Figure 3B are presented in Appendix A (page 7).

Keuper M., Sachs S., Walheim E., Berti L., Raedle B., Tews D., Fischer-Posovszky P., Wabitsch M., Hrabě de Angelis M., Kastenmüller G., Tschöp M.H., Jastroch M., Staiger H, & Hofmann S.M. (2017). Activated macrophages control human adipocyte mitochondrial bioenergetics via secreted factors. Molecular Metabolism, 6(10), 1226-1239.

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Immunoblot Analysis of Colon Crypts

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For immunoblot analysis, isolated colon crypts or YAMC cells scraped off from the dish were lysed with NP40 lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% NP40, pH 8.0) supplied with protease inhibitor cocktail (Roche, USA) on ice for 20 min before 5 min 6000 rpm centrifugation at 4°C. Supernatants were collected for SDS-PAGE analysis. The primary antibodies were incubated overnight at 4°C.
Antibodies: GR/NR3C1 (Cell Signaling Technology, Cat.3660, 1:1000), REV-ERBα /NR1D1(Abcam, Cat. ab174309, 1:1000), NLRP3(Cell Signaling Technology, Cat.15101, 1:1000), IL1β(Abclonal, Cat.A16288, 1:1000), IL6 (Cell Signaling Technology, Cat.12912, 1:1000), CLDN1 (Thermo Fisher, Cat.37-4900, 1:10000), ORMDL3 (Abclonal, Cat.A14951, 1:2000), NPAS2 (Abclonal, Cat.A16930, 1:2000), PARD3 (Proteintech, Cat.11085-1-AP, 1:500), ACTB/β-actin (Abclonal, Cat.AC026, 1:10000), BMAL1 (Proteintech, Cat.14268-1-AP, 1:5000), CLOCK (Abclonal, Cat.A7265, 1:10000), Caspase-1 (Proteintech, Cat.22915-1-AP, 1:1000), IFIT3 (Abclonal, Cat.A3924, 1:1500), CDH1 (Abclonal, Cat.A11492, 1:1500), CDH3 (Abclonal, Cat.A14235, 1:1500).

Zheng G., Pang S., Wang J., Wang F., Wang Q., Yang L., Ji M., Xie D., Zhu S., Chen Y., Zhou Y., Higgins G.A., Wiley J.W., Hou X, & Lin R. (2023). Glucocorticoid receptor-mediated Nr1d1 chromatin circadian misalignment in stress-induced irritable bowel syndrome. iScience, 26(7), 107137.

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Inflammatory Pathway Protein Analysis

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Antibodies against phosphor-/total AMPK, phosphor-/total-ERK1/2, IL-6, IκBα, phosphor-/total-p38, phosphor-/total-p65, p53, and SIRT1 were from Cell Signaling Technology (Beverly, MA, USA). Anti-Ly-6B.2 monoclonal and anti-β-actin antibodies were purchased from Bio-Rad Laboratories Inc. (Hercules, CA, USA) and Sigma (Saint Louis, MO, USA), respectively. IRDye 680 goat anti-mouse and IRDye 800CW goat anti-rabbit secondary antibodies were purchased from Li-Cor Biosciences (Lincoln, NE). The Vectastain Elite ABC, and DAB peroxidase (HRP) substrate kits were purchased from Vector Laboratories Inc. (Burlingame, CA, USA). Trizol^® Reagent was purchased from Sigma (Saint Louis, MO, USA). DNase I and RNeasy Mini kit were purchased from Qiagen (Valencia, CA, USA), and iScript™ cDNA synthesis kit was purchased from Bio-Rad Laboratories Inc. (Hercules, CA, USA). Colitis grade DSS (MW = 36,000–50,000) was purchased from MP Biomedicals (Santa Ana, CA, USA).

Bibi S., Kang Y., Du M, & Zhu M.J. (2017). Maternal high fat diet consumption enhances offspring susceptibility to DSS-induced colitis in mice. Obesity (Silver Spring, Md.), 25(5), 901-908.

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Investigating Neuroinflammatory Mechanisms

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All chemicals, including anti-CT19 amyloid antibody and 10% neutral buffered formalin solution used in this study, were from Sigma Chemical (St. Louis, MO, USA). Specific antibodies to MAPKs, phospho-MAPKs, total tau, p-tau-T181, p-tau-S396, β-amyloid, TNF-α, MCP-1, GFAP, cleaved caspase-3, or IL-6 were purchased from Cell Signaling (Danver, MA, USA). Specific antibody to GSK-3β, p-GSK-3β-S9, CDK5, p-tau-T231, iNOS, 3-NT, CYP2E1, or NeuN was from Abcam (Cambridge, MA, USA). Antibody against p-CDK5-Tyr15, cleaved caspase-3, Bax, GAPDH, NeuN, GFAP, HSP90, HNF-1α, BNIP3, and secondary antibodies conjugated with horse radish peroxidase were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant HIV-1 Tat and gp120 (B.MN D11 strain) proteins were provided by the NIAID AIDS Reagent Program (National Institutes of Health, Bethesda, MD, USA). Neuro-2a cells were purchased from the ATCC (American Type Culture Cells (Manassas, VA). Other materials not described here were the highest grades available and/or the same, as recently described [21 (link), 22 (link)].

Cho Y.E., Lee M.H, & Song B.J. (2017). Neuronal Cell Death and Degeneration through Increased Nitroxidative Stress and Tau Phosphorylation in HIV-1 Transgenic Rats. PLoS ONE, 12(1), e0169945.

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Mitochondrial Protein Extraction and Analysis in Cerebral Infarction

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Total proteins of infarcted cortex were extracted using the protein extraction kit (BB-3101, Bestbio), and mitochondrial proteins of infarcted cortex were isolated using Tissue Mitochondria Isolation Kit (C3606, Beyotime). The concentration of proteins was measured using the bicinchoninic acid method, and loading buffer was added to obtain equal concentrations (3 μg/μL). Samples were subjected to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred on a 0.45 μm polyvinylidene difluoride membrane. After washing with tris-buffered saline with 0.1% Tween® 20, the membrane was sealed using a rapid sealing solution for 20 min and then incubated with primary antibodies at 4°C overnight, and the corresponding secondary antibodies (bs-0296G, Bioss) were incubated for 2 h at room temperature.
The following primary antibodies were used: LC3B (L7543, Sigma), p62 (18420-1-AP, Proteintech), mTOR (#2983, Cell Signaling Technology), p-mTOR (#5536, Cell Signaling Technology), IL-1β (16806-1-AP, Proteintech), IL-6 (#12153, Cell Signaling Technology), TNF-α (BS1857, Bioworld Technology) and β-actin (#3700, Cell Signaling Technology), COX IV (ab202554, Abcam), Drp1 (ab184247, Abcam), and Parkin (JF82-09, Novus).

Zhang Q.Q., Luo L., Liu M.X., Wang C.J., Wu Y, & Yu K.W. (2022). Enriched Environment-Induced Neuroprotection against Cerebral Ischemia-Reperfusion Injury Might Be Mediated via Enhancing Autophagy Flux and Mitophagy Flux. Mediators of Inflammation, 2022, 2396487.

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