Detroit 562 cells were purchased from ATCC and cultured in RPMI medium (Gibco) supplemented with 10% fetal bovine serum performance (Gibco), 100 U/ml penicillin and 100μg/ml streptomycin (Gibco), and 1 mM sodium pyruvate (Gibco). LPS from E.coli B4:111 (Sigma) at 1 μg/mL was used to stimulate the cells. Inhibition of NF-kB was done treating the cells with BAY 11–7082 (ChemCruz) at 100uM (stock solution at 50 mM in DMSO) for one hour prior and at 90uM (dilution 9/10 in the medium containing the ligand) during the LPS treatment (Fig. 4 b and c). RELA activation assay, RT-qPCR and ChIP-qPCR procedures are described in Additional file 11 .
Bay11 7082
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Germany
BAY11-7082 is an organic compound used as a research tool. It functions as an inhibitor of IκB kinase (IKK), thereby preventing the activation of the transcription factor NF-κB. This compound is commonly used in scientific research applications.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Parthenolide, by Santa Cruz Biotechnology (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Rpmi medium, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum performance, by Thermo Fisher Scientific (2 mentions)
Lps from e coli b4 111, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Mitomycin c mmc, by Merck Group (2 mentions)
U0126, by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Recombinant human il 1β, by Thermo Fisher Scientific (2 mentions)
Spectramax l microplate reader, by Molecular Devices (2 mentions)
One glo luciferase assay system, by Promega (2 mentions)
Fadu cell, by American Type Culture Collection (1 mentions)
Rottlerin, by Merck Group (1 mentions)
36 protocols using bay11 7082
1
NF-kB Inhibition Assay in Detroit 562 Cells
2
FaDu Cell Stimulation and NFKB Inhibition
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FaDu cells were purchased from ATCC and cultured in RPMI medium (Gibco) supplemented with 10% fetal bovine serum performance (Gibco), 100 U/ml penicillin and 100 ug/ml streptomycin (Gibco), and 1 mM sodium pyruvate (Gibco). LPS from E. coli B4:111 (Sigma) at 1 μg/mL was used to stimulate the cells. Inhibition of NFKB was achieved using BAY 11–7082 (ChemCruz) at 50 uM (stock solution at 50 mM in DMSO) for one hour prior and at 45 uM (dilution 9/10 in the medium containing the ligand) during the treatment. RELA activation as well as gene expression were performed as described in Supplementary Table S7 .
3
Ovarian Cancer Cell Line Maintenance and Characterization
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Epithelial OC cell lines (A2780, A2780_CR5, SKOV3, HEYC2, OV90, IOSE, IGROV, OVMUNA, OV90) were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA) as described previously [50 (link)]. Cisplatin-resistant A2780_CR5 was established by continuous exposure to increasing concentrations of cisplatin [50 (link)]. Cell lines were authenticated in 2012 by ATCC and tested for mycoplasma contamination (Manassas, VA). High-grade serous ovarian tumors (unpaired samples; chemo-naïve, stage 3–4 or recurrent, platinum resistant), and ovarian surface epithelium (OSE) were surgically collected with informed consent from all subjects (IRB approved protocol IUCRO-0280), snap-frozen, and stored in liquid nitrogen [51 (link)]. Cisplatin (CDDP) was purchased from Calbiochem (Billerica, MA), mitomycin C (MMC) was purchased from Sigma Chemical Co. (St. Louis MO), and H2O2 was purchased from EMD Millipore (Billerica, MA). NF-κB inhibitor Bay-11–7082 was purchased from Santa Cruz Biotech (Santa Cruz, CA). LZRS-HOTAIR was a gift from Dr. Howard Chang (Stanford University; Addgene plasmid # 26110). Full-length HOTAIR was cloned into pAV5S vector containing a 98-mer aptamer sequence and as a vector control, aptamer cloned into pAV5S was used to account for any possible RNA-dependent signaling effects [52 (link)].
4
Culturing Normal Human Astrocytes and U-87 Astrocytoma Cells
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Normal human astrocytes (NHA) isolated from the cerebrums of 5-month-old human fetuses were purchased from Cambrex (CC-2565, Walkersville, MD, USA), and cultured according to the manufacturer’s protocol. The astrocytoma human cell line U-87 was routinely grown in cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Rockville, MD, USA) containing 10% heat-inactivated fetal calf serum, 1% penicillin/streptomycin, and 2 mM L-glutamine (ICN Pharmaceuticals, CA, USA) at 37 °C in a humidified atmosphere of 5% CO2.
Bryostatin-1, prostratin, GF109203X, and rottlerin were purchased from Sigma (St. Louis, MO, USA). Pyrrolidine dithiocarbamate (PDTC), and BAY11-7082 were obtained from Santa Cruz Biotechnology (Santa Cruz CA, USA).
Bryostatin-1, prostratin, GF109203X, and rottlerin were purchased from Sigma (St. Louis, MO, USA). Pyrrolidine dithiocarbamate (PDTC), and BAY11-7082 were obtained from Santa Cruz Biotechnology (Santa Cruz CA, USA).
5
ILC Activation and NF-κB Inhibition
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Sorted PB or intestinal ILCs were cultured in Yssel’s-supplemented IMDM with 1% normal human serum and 10 U/ml IL-2 (PeproTech). In case of stimulation, cells were additionally treated with 50 ng/ml IL-1β (R&D Systems), 50 ng/ml IL-23 (R&D Systems), 50 ng/ml IL-18 (R&D Systems), 20 ng/ml TGF-β1 (R&D Systems), or IFN-γ (PeproTech) for 3−9 days.
To inhibit NF-κB signaling pathway BAY11-7082 (Santa Cruz Biotechnology) and BMS-345541 (Sigma-Aldrich) were used. Sorted ILCs were stimulated for 3 days with 10 U/ml IL-2 plus 50 ng/ml IL-1β in the presence or absence of the corresponding inhibitor. Different concentrations of the inhibitors were used: 0.5 μM and 1 μM of BAY11-7082, and 1, 2, and 4 μM of BMS-345541. Subsequently the cells were harvested and HLA-DR and co-stimulatory molecule expression were accessed by flow cytometry. Nonviable cells were excluded from the analysis based on the dead cell marker and CD45 staining.
To inhibit NF-κB signaling pathway BAY11-7082 (Santa Cruz Biotechnology) and BMS-345541 (Sigma-Aldrich) were used. Sorted ILCs were stimulated for 3 days with 10 U/ml IL-2 plus 50 ng/ml IL-1β in the presence or absence of the corresponding inhibitor. Different concentrations of the inhibitors were used: 0.5 μM and 1 μM of BAY11-7082, and 1, 2, and 4 μM of BMS-345541. Subsequently the cells were harvested and HLA-DR and co-stimulatory molecule expression were accessed by flow cytometry. Nonviable cells were excluded from the analysis based on the dead cell marker and CD45 staining.
6
Cytotoxicity Evaluation of Inflammatory Factors
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The human SFMSCs were seeded into 96-well plates at 10,000 cells/well and then treated with 10 ng/ml IL-1β, 10 ng/ml TNF-α, 10 ng/ml IL-8/IL-1β/IL-6/IL-10/TNF-α/IL-12p compound, or 10 μM NF-κB inhibitor (BAY11-7082; Cat. no. 19542-67-7; Santa Cruz Biotechnology, Inc.) for 12 h. We then measured the activity of LDH released from the cells using the cytotoxicity detection kit (LDH; Roche) following the manufacturer's instructions.
7
Histone Acetylation and Deacetylation Modulation
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Histone acetyltransferase inhibitor Garcinol (GAR), SIRT1 activator Resveratrol (RES), SIRT1 inhibitor Nicotinamide (NIC) and NF-κB inhibitor BAY 11-7082 (BAY) were obtained from Santa Cruz (Santa Cruz, CA). Antibodies against HMGB1 and β-actin were purchased from Cell Signaling Technology (Danvers, MA). Anti-acetyl lysine antibody (clone Kac-01) was purchased from PTM Biolab (Hangzhou, China). siRNAs were purchased from biotend (Shanghai, China). The bovine serum albumin (BSA) was purchased from Dingguo (Nanjing, China).
8
Regulatory Transcription Factors Modulation
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The HIF1α activator CoCl2 and the NF-κB inhibitor Bay 11-7082 were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The MEK inhibitors PD0325901 and U0126 as well as the HIF1α inhibitor Bay 87-2243 were purchased from SelleckChem (Munich, Germany). The PKCα activators PMA, bryostatin 1, and PDBu were purchased from Tocris Biosciences (Bristol, UK).
9
Cytokine-induced Inflammatory Responses
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The human SFMSCs were seeded into 10-mm plates and cultured until they reached 80% confluence. The cells were treated with cytokines and/or inhibitors. Inhibitors [BAY11-7082 (Cat. no. 19542-67-7; Santa Cruz Biotechnology, Inc.), anti-TNF-α (167348; Ebioscience, San Diego, CA, USA)] were added 1 h prior to cytokine treatment. The empty plate using the same culture medium, but without cells as the negative control. IL-6 and IL-8 expression in the culture medium was determined using a CBA kit (Human Inflammatory Cytokine kit; Cat. no. 551811; BD Biosciences). Double-stranded DNA was detected using Quant-iT PicoGreen dsDNA reagent (Invitrogen, Carlsbad, CA, USA) as an endogenous control.
10
Investigating Survivin and NF-κB Signaling
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The anti-Id1 antibody was purchased from Abcam; anti-survivin, and anti-NFκB/p65 antibodies were obtained from Cell Signaling Technology. BAY 11–7082 (the NFκB blocker), and YM155 (survivin blocker) were obtained from Santa Cruz Biotechnology.
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