Microglia from the hippocampus of rats' brain tissue were isolated and purified by primary isolation method. Cells were cultured in high glucose DMEM medium (11965092; ThermoFisherScientific) containing 1% penicillin-streptomycin (C0009; Beyotime) and 10% FBS (PS-FB5-SA; Peakserum) in a 37°C incubator with 5% CO2. The purified and cultured microglia were stimulated with the mixture of Aβ and IBO (referred to as Aβ microglia). The above cells were divided into 6 groups: KL overexpressing adenovirus transfected Aβ microglia (KL-OE); KL shRNA transfected Aβ microglia (KL-si); IKK-16 (a specific inhibitor of IKK) purchased from Selleck was added to Aβ microglia (IKK-16); KL overexpressing adenovirus was introduced into Aβ microglia, and then IKK-16 was added (KL-OE/IKK-16); empty vector was introduced into Aβ microglia (NC) and Aβ microglia (Control). After 48 h of adenovirus infection, IKK-16 (1 μM) or the same amount of DMSO was pretreated for 30 min, and then Aβ (0.55 μM) and IBO (0.6 μM) were mixed for 48 h, and the cells were collected.
Ikk 16
Manufactured by Selleck Chemicals
Sourced in United States, China
IKK-16 is a lab equipment product that functions as a precision temperature control system. It is designed to maintain consistent and accurate temperature levels within laboratory environments.
Automatically generated - may contain errors
Lab products found in correlation
Tpca 1, by Selleck Chemicals (4 mentions)
Tnf α, by Thermo Fisher Scientific (3 mentions)
Sb203580, by Selleck Chemicals (3 mentions)
Gdc 0941, by Selleck Chemicals (2 mentions)
Bms 345541, by Selleck Chemicals (2 mentions)
Jsh 23, by Selleck Chemicals (2 mentions)
Sp600125, by Selleck Chemicals (2 mentions)
Pf184, by Bio-Techne (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Mg132, by Selleck Chemicals (2 mentions)
Rabbit anti nf κb p65, by Cell Signaling Technology (2 mentions)
Hrp labeled goat anti rabbit igg, by Santa Cruz Biotechnology (2 mentions)
Hrp labeled goat anti mouse igg, by Santa Cruz Biotechnology (2 mentions)
Ros assay kit, by Beyotime (2 mentions)
1 6 hexanediol, by Merck Group (2 mentions)
Ciap1, by Cell Signaling Technology (2 mentions)
Ciap2, by Cell Signaling Technology (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
33 protocols using ikk 16
1
Microglia Isolation and Modulation
2
B-cell signaling inhibition assay
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For JQ1 and IKK-16 inhibition, the DT40 cells were suspended in 5 μM JQ1 (Selleckchem, USA) and 6 μM of IKK-16 (Selleckchem) 60 min before anti-IgM stimulation. The final concentration of DMSO was adjusted to 0.1% for JQ1, IKK-16 and control. For 1,6-hexanediol inhibition, 5% of 1,6-hexanediol (Sigma-Aldrich) was added after anti-IgM stimulation.
3
JQ1 and IKK-16 inhibition in DT40 cells
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For JQ1 and IKK-16 inhibition, the DT40 cells were suspended in 5 µM JQ1 (Selleckchem, USA) and 6 µM of IKK-16 (Selleckchem) 60 min before anti-IgM preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted December 6, 2021. ; https://doi.org/10.1101/2021.07.13.452147 doi: bioRxiv preprint stimulation. For 1,6-hexanediol inhibition, 5% of 1,6-hexanediol (Sigma-Aldrich) was added after anti-IgM stimulation.
The copyright holder for this this version posted December 6, 2021. ; https://doi.org/10.1101/2021.07.13.452147 doi: bioRxiv preprint stimulation. For 1,6-hexanediol inhibition, 5% of 1,6-hexanediol (Sigma-Aldrich) was added after anti-IgM stimulation.
4
Melanoma Cell Line Cultivation and Drug Preparation
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All melanoma cell lines were obtained from the Massachusetts General Hospital Cancer Center. C32, K2, MMACSF, SKMEL28, and WM115 cell lines were grown in DMEM/F12 (Invitrogen) supplemented with 5% fetal bovine serum (FBS) and 1% sodium pyruvate (Invitrogen). COLO858, LOXIMVI, MZ7MEL, RVH421, and WM1552C cell lines were grown in RMPI 1640 (VWR) supplemented with 5% fetal bovine serum (FBS) and 1% sodium pyruvate (Invitrogen). We added penicillin (50 U/ml) and streptomycin (50 μg/ml) to all growth media.
Chemical inhibitors from the following sources were dissolved in dimethyl sulfoxide (DMSO) as 10 mM stock solutions for in vitro studies: vemurafenib (PLX4032), PLX4720, SB590885, selumetinib (AZD6244) and AZ628 (all from MedChem Express), JNK-IN-8 (EMD Millipore), SP600125, doramapimod (BIRB796), and SB202190, GDC0941, tofacitinib (CP-690550), and IKK16 (all from Selleck Chemicals).
Chemical inhibitors from the following sources were dissolved in dimethyl sulfoxide (DMSO) as 10 mM stock solutions for in vitro studies: vemurafenib (PLX4032), PLX4720, SB590885, selumetinib (AZD6244) and AZ628 (all from MedChem Express), JNK-IN-8 (EMD Millipore), SP600125, doramapimod (BIRB796), and SB202190, GDC0941, tofacitinib (CP-690550), and IKK16 (all from Selleck Chemicals).
5
Apoptosis Induction and NF-κB Modulation
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Dulbecco’s Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute (RPMI)-1640 medium were purchased from Gibco (Shanghai, China). Fetal bovine serum (FBS) was obtained from Biological Industries (Cromwell, CT, USA). Acridine orange/ ethidium bromide (AO/EB) double fluorescence staining solution was obtained from Beyotime Institute of Biotechnology (Nanjing, China). Hoechst 33342 solution and cell counting kit-8 (CCK-8) were obtained from Dojindo Molecular Technologies, Inc. (Beijing, China). Pancaspase inhibitor (z-VAD-fmk) and NF-κB inhibitor (IKK 16) were bought from Selleckchem (Shanghai, China). Anti-Bcl-2, anti-Bax, anti-cytochrome c, anti-cleaved caspase-9 and anti-cleaved caspase-3 antibodies were acquired from Cell Signaling Technology (Shanghai, China). Anti-Ki-67, anti-PCNA, anti-E-cadherin, anti-N-cadherin, anti-Snail and anti-GAPDH antibodies were obtained from Abcam (Shanghai, China). Antibodies against NF-κB-p50, NF-κB-p65, IκB-α and p-IκB-α were obtained from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA).
6
Biochemical Assay for MALT1 Inhibitors
7
Ammonium Pyrrolidine Dithiocarbamate and IKK-16 Treatment
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The cells were allowed to grow for 24 h after seeding. Subsequently, they were treated with different concentrations of ammonium pyrrolidine dithiocarbamate (PDTC) (Sigma-Aldrich, Cat.# P 8765, Munich, Germany) or IKK-16 (Cat.# S2882, Selleckchem, Munich, Germany) (diluted in culture medium) for either 6 or 24 h. The reaction was stopped by removal of medium and addition of ice-cold phosphate-buffered saline. The fixation was performed with ice-cold methanol for proximity ligation assays. The procedure for the MTT assays is described in the MTT subsection.
8
TNFα Signaling Pathway Regulation
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Human recombinant TNFα protein was purchased from PeproTech (Rocky Hill, NJ, USA). Src-inhibitor saracatinib, JAK-inhibitor tofacitinib, mitogen-activated protein kinase-inhibitor SB203580, STAT3-inhibitor S3I-201, p65-inhibitor JSH-23, IKK-inhibitors TPCA-1 and IKK-16 were perchased from Selleckchem (Houston, TX, USA), JAK-inhibitor PDTC was perchased from Abcam (Cambridge, MA, USA). Antibody against HK2, phospho-IKKα/β (Ser176/180) and phospho-IKKε (Ser172) were purchased from Cell Signaling Techonology (Beverly, MA, USA), antibody against YAP was purchased from NOVUS Biologicals (Littleton, CO, USA), antibody against CD68 was pursed from Abcam, antibody against p65 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against β-actin, GFP, Myc, Flag, HA and TEAD4 were purchased from Sigma-Aldrich. Antibody against TNFR1, IKKα, IKKβ and IKKε were purchased from Abclonal Technology (Wuhan, China). Antibody against pan-phosphorylated serine/threonine was purchased from BD Biosciences (San Jose, CA, USA).
9
Signaling Inhibitors Modulate IL-6 Expression
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FLS (1 × 105/ml) (n = 5) were pretreated with signaling inhibitors (SP600125 (10 μM, inhibitor of JNK), SB203580 (25 μM, inhibitor of P38 MAPK), IKK-16 (10 μM, inhibitor of NF-κB), and FR180204 (10 μM, inhibitors of Erk1/2) (Selleck Chemicals, USA)) for 1 h and stimulated with IL-34 (50 ng/ml) for 24 h or 72 h at 37°C. The mRNA expression level of IL-6 was measured by RT-PCR, and IL-6 production was measured by ELISA.
10
NLRP3 Inflammasome Activation in HCASMCs
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All cells were kept at 5% CO2 and 37 °C. HCASMCs were treated with isolated NLRP3-YFP inflammasome particles (3:1 NLRP3-YFP particles/ cell) for 4 h (internalization, gene expression, migration, western blot) and 24 h (gene expression, extracellular matrix production). For mechanistic studies, NLRP3 inhibitor MCC950 (1 µM, Invivogen), caspase-1 inhibitor Ac-YVAD-cmk (25 µg/ml, Invivogen) and NFκB inhibitor IKK-16 (2 µM, Selleckchem) were added to the media 30 min before treatment. Endocytosis inhibitor Cytochalasin D (4 µM, Invitrogen) was added 30 min before treatment with NLRP3-YFP particles and was washed off 3 times by washing with culture medium.
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