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Ikk16

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IKK16 is a chemical compound that functions as a potent and selective inhibitor of the IKB kinase (IKK) enzyme complex. The IKK complex plays a central role in the activation of the NF-kB signaling pathway, which is involved in the regulation of various cellular processes, including inflammation and immune response.

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Lab products found in correlation

Lps eb ultrapure, by InvivoGen (2 mentions) Cycloheximide (chx), by Merck Group (2 mentions) Poly 1 c, by InvivoGen (2 mentions) Pf 3644022, by Bio-Techne (2 mentions) Tak1 inhibitor 5z 7 oxozeaenol, by Bio-Techne (2 mentions) Accuri flow cytometer, by BD (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Non essential amino acids (neaa), by MP Biomedicals (1 mentions) Mda mb 231, by Lonza (1 mentions) Dmem high glucose with l glutamine, by Lonza (1 mentions) 3 aminobenzamide 3 ab, by Merck Group (1 mentions) Chir99021, by Selleck Chemicals (1 mentions) Bix02189, by Selleck Chemicals (1 mentions) Sns 032, by Selleck Chemicals (1 mentions) Fr180204, by Bio-Techne (1 mentions) Tpca 1, by Bio-Techne (1 mentions) Ci 1040, by Selleck Chemicals (1 mentions) Danusertib, by Selleck Chemicals (1 mentions) Sb218078, by Bio-Techne (1 mentions)

10 protocols using ikk16

1

Macrophage Polarization by Glioblastoma Exosomes

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Macrophages were generated from primary human monocytes as previously described.28 (link) After differentiation, control macrophages (Mϕ Naive) were maintained in RPMI. GBex (25μg protein/2 × 106 cells/1 mL) were co-cultured with macrophages for 72 h. Macrophages were phenotyped by flow cytometry. In experiments with the NF-κB pathway inhibitor, macrophages were exposed to IKK16 (200 nmol, 2539, Tocris) for 30 min prior to the addition of GBex.
To study macrophages polarization, changes in expression of M1/M2 markers on macrophages were measured by flow cytometry after surface or intracellular staining with specific antibodies (Supplementary Table 1). Cell fluorescence was measured with an Accuri flow cytometer (BD Biosciences).
Azambuja J.H., Ludwig N., Yerneni S., Rao A., Braganhol E, & Whiteside T.L. (2020). Molecular profiles and immunomodulatory activities of glioblastoma-derived exosomes. Neuro-oncology Advances, 2(1), vdaa056.
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2

Modulation of Cell Death Pathways

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The Smac-mimetic Compound A, the caspase inhibitor IDN-6556 (Idun Pharmaceuticals) and the RIPK1 inhibitor Necrostatin were synthesized by TetraLogic Pharmaceuticals. The RIPK3 inhibitor GSK’872 was from Calbiochem. The TAK1 inhibitor (5Z)-7-Oxozeaenol, the IKK inhibitor IKK-16 and the MK2 inhibitor PF-3644022 were from Tocris Bioscience. Cycloheximide was from Sigma. Recombinant Fc-TNF was produced in house. Ultrapure LPS-EB and poly(I:C) were purchased from Invivogen.
Lalaoui N., Boyden S.E., Oda H., Wood G.M., Stone D.L., Chau D., Liu L., Stoffels M., Kratina T., Lawlor K.E., Zaal K.J., Hoffmann P.M., Etemadi N., Shield-Artin K., Biben C., Tsai W.L., Blake M.D., Kuehn H.S., Yang D., Anderton H., Silke N., Wachsmuth L., Zheng L., Moura N.S., Beck D.B., Gutierrez-Cruz G., Ombrello A.K., Pinto-Patarroyo G.P., Kueh A.J., Herold M.J., Hall C., Wang H., Chae J.J., Dmitrieva N.I., McKenzie M., Light A., Barham B.K., Jones A., Romeo T.M., Zhou Q., Aksentijevich I., Mullikin J.C., Gross A.J., Shum A.K., Hawkins E.D., Masters S.L., Lenardo M.J., Boehm M., Rosenzweig S.D., Pasparakis M., Voss A.K., Gadina M., Kastner D.L, & Silke J. (2019). Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease. Nature, 577(7788), 103-108.
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3

Modulation of Cell Death Pathways

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The Smac-mimetic Compound A, the caspase inhibitor IDN-6556 (Idun Pharmaceuticals) and the RIPK1 inhibitor Necrostatin were synthesized by TetraLogic Pharmaceuticals. The RIPK3 inhibitor GSK’872 was from Calbiochem. The TAK1 inhibitor (5Z)-7-Oxozeaenol, the IKK inhibitor IKK-16 and the MK2 inhibitor PF-3644022 were from Tocris Bioscience. Cycloheximide was from Sigma. Recombinant Fc-TNF was produced in house. Ultrapure LPS-EB and poly(I:C) were purchased from Invivogen.
Lalaoui N., Boyden S.E., Oda H., Wood G.M., Stone D.L., Chau D., Liu L., Stoffels M., Kratina T., Lawlor K.E., Zaal K.J., Hoffmann P.M., Etemadi N., Shield-Artin K., Biben C., Tsai W.L., Blake M.D., Kuehn H.S., Yang D., Anderton H., Silke N., Wachsmuth L., Zheng L., Moura N.S., Beck D.B., Gutierrez-Cruz G., Ombrello A.K., Pinto-Patarroyo G.P., Kueh A.J., Herold M.J., Hall C., Wang H., Chae J.J., Dmitrieva N.I., McKenzie M., Light A., Barham B.K., Jones A., Romeo T.M., Zhou Q., Aksentijevich I., Mullikin J.C., Gross A.J., Shum A.K., Hawkins E.D., Masters S.L., Lenardo M.J., Boehm M., Rosenzweig S.D., Pasparakis M., Voss A.K., Gadina M., Kastner D.L, & Silke J. (2019). Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease. Nature, 577(7788), 103-108.
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4

Breast Cancer Cell Culture Protocol

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MDA-MB-231 and MCF7 were purchased from the National Centre of Cell Culture (NCCS), Pune, Maharashtra, India. MDA-MB-231 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM high glucose with L-glutamine; Lonza) and MCF7 in Eagle’s Minimum Essential Medium (EMEM; Lonza supplemented with non-essential amino acids (NEAA) from MP biomedicals). All media were supplemented with heat-inactivated 10% fetal bovine serum (Gibco), 100 IU mg/mL penicillin/streptomycin (Gibco) at 37°C in a humidified atmosphere containing 5% CO2. ST08 was dissolved in DMSO such that all treatments had equal concentrations of dimethyl sulfoxide (DMSO) between 0.1–0.2%. Cisplatin(MP biomedicals, Santa Ana, California, USA) was dissolved in water. Phorbol ester (phorbol 12- myristate 13-acetate; PMA) and IKK-16 were purchased from Tocris Bioscience (Bristol, United Kingdom) and dissolved in DMSO. The structure of ST08 and molecular signatures of each cell line are tabulated in Table 1 (34 (link)).
Nirgude S., Desai S., Mahadeva R., Ravindran F, & Choudhary B. (2022). ST08 Altered NF-κB Pathway in Breast Cancer Cells In Vitro as Revealed by miRNA-mRNA Analysis and Enhanced the Effect of Cisplatin on Tumour Reduction in EAC Mouse Model. Frontiers in Oncology, 12, 835027.
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5

Estrogen Modulates Mesangial Cell Responses

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Mesangial cells [15 (link)] were maintained in DMEM with 10% fetal bovine serum (FBS). To determine the effect of estrogens, cells were cultured in phenol red free medium with charcoal/dextran treated FBS (Hyclone) for 72h. Cells were then treated with 17β-estradiol (E2 10−5 – 10−11 M) for 48h prior to TNFα stimulation. 10−7 and 10−5 M E2 were used to determine effects on TNFα stimulated VCAM-1 mRNA levels, with similar results. For PARP-1 inhibition, cells were pretreated with the inhibitors 3-aminobenzamide (3AB, Sigma, 0.01–1mM) or CEP 8983 (CEP, Cephalon/Teva, 1–10 μM) 30 min prior to TNFα stimulation. For NFκB inhibition, cells were pretreated with selective inhibitor of IκB kinase, IKK16 (Tocris Biosciences, 1μM) 30min prior to TNFα (1ng/ml) stimulation.
Jog N.R, & Caricchio R. (2015). 17β Estradiol Regulates Adhesion Molecule Expression in Mesangial Cells during Glomerulonephritis. Clinical immunology (Orlando, Fla.), 159(1), 13-22.
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6

Screening of Protein Kinase Inhibitors for TNBC

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The fifty-five protein kinase inhibitors (PKIs) were purchased from following sources: BML-275, FR 180204, IKK16, GW 843682X, NSC 109555, NU7441, PD407824, PF 573228, SB 218078, TCS PIM-1-1, TCS PIM-1-4a, and TPCA-1 from Tocris Biosciences (Bristol, UK); indirubin-3′-monoxime and Ro-31-8220 from Calbiochem (San Diego, CA, USA); A-769662, bosutinib, chelerythrine, CP690550, fasudil, gefitinib, imatinib, nilotinib, PKC412, roscovitine, SNS-314, and tozasertib from LC Laboratories (Woburn, MA, USA); AT7867, AT9283, AZD1152, AZD1480, BI 2536, BIX 02189, CHIR-99021, CI-1040, CYC116, danusertib, enzastaurin, GDC-0879, INCB018424, JNJ-7706621, KU-55933, LY2228820, MLN8237, PD-0325901, PF-4708671, PLX-4032, PLX-4720, SB216763, SNS-032, SP600125, VX-702, Y-27632, and ZM447439 from Selleck Chemicals (Houston, TX, USA); U0126 from Promega (Madison, WI, USA); TBCA from Millipore (Burlington, MA, USA).
All TNBC cells in this study were obtained from the American Type Culture Collection (Manassas, VA, USA). The cultured cells were monitor by trypan blue cell counting as described previously [58 (link)].
Yi Y.W., You K.S., Han S., Ha I.J., Park J.S., Lee S.G, & Seong Y.S. (2022). Inhibition of IκB Kinase Is a Potential Therapeutic Strategy to Circumvent Resistance to Epidermal Growth Factor Receptor Inhibition in Triple-Negative Breast Cancer Cells. Cancers, 14(21), 5215.
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7

Ischemia-Reperfusion Injury Amelioration

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Rats were randomly allocated into the following groups: (1) sham+vehicle (n=11); (2) IRI+vehicle (n=11); (3) IRI+IKK16 0.1 mg/kg (n=5); (4) IRI+IKK16 0.3 mg/kg (n=7); and (5) IRI+IKK16 1 mg/kg (n=9). Rats were administered vehicle (10% dimethyl sulfoxide) or N‐(4‐Pyrrolidin‐1‐yl‐piperidin‐1‐yl)‐[4‐(4‐benzo[b]thiophen‐2‐yl‐pyrimidin‐2‐ylamino)phenyl] carboxamide hydrochloride (IKK16) (a specific inhibitor of IKK) 24 hours after the onset of reperfusion via the tail vein at a volume of 1 mL/kg. IKK16 was purchased from Tocris Bioscience (R&D systems Europe, Abingdon, UK), and the optimal dose obtained from previous IC50 values14 and a dose response (data not shown).
Johnson F.L., Patel N.S., Purvis G.S., Chiazza F., Chen J., Sordi R., Hache G., Merezhko V.V., Collino M., Yaqoob M.M, & Thiemermann C. (2017). Inhibition of IκB Kinase at 24 Hours After Acute Kidney Injury Improves Recovery of Renal Function and Attenuates Fibrosis. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 6(7), e005092.
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8

Investigating Antioxidant Modulation of NF-κB Signaling

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Cell culture media and reagents were purchased from Gibco (Carlsbad, CA). Manganese (II) chloride (MnCl2), N-Acetyl-L-cysteine (NAC), α-tocopherol (AT), protease inhibitor cocktail, and dimethyl sulfoxide (DMSO) were purchased from MilliporeSigma (St. Louis, MO). The TNF-α inhibitor (E)-4-2-4-chloro-3-nitrophenyl (C-87) and the IKK-β inhibitor IKK16 were purchased from Tocris (Littleton, CO). The chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) was purchased from Life Technologies, Inc. (Carlsbad, CA). Antibodies for EAAT2 (ab41621), IKK-β (ab32135) and phospho-IKK-β (ab59195) were obtained from Abcam (Cambridge, MA); antibodies for NF-κB p65 (sc-8008), YY1 (sc-7341), β-actin (sc-47778), and histone H4 (sc-25260) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Bright-Glo luciferase reporter assay kits were purchased from Promega (Madison, WI), and an RNA isolation kit was obtained from Qiagen (Valencia, CA). The TNF-α standard tetramethylbenzidine (TMB) enzyme-linked immunosorbent assay (ELISA) development kit for human samples (900-T25) was purchased from PeproTech (Rocky Hill, NJ). All chemicals were prepared in phosphate-buffered saline (PBS), double-distilled H2O or DMSO, according to the manufacturer’s instructions, and diluted to working concentration in Opti-MEM prior to use.
Rizor A., Pajarillo E., Nyarko-Danquah I., Digman A., Mooneyham L., Son D.S., Aschner M, & Lee E. (2021). Manganese-induced reactive oxygen species activate IκB kinase to upregulate YY1 and impair glutamate transporter EAAT2 function in human astrocytes in vitro. Neurotoxicology, 86, 94-103.
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9

Pharmacological Inhibitors of Cellular Signaling

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PD98059 (2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one), YM98483 (N-[4-[3,5-Bis (trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide), SB203580 (4-[5-(4- Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine), ML-9 (1-(5-Chloronaphthalene- 1-sulfonyl)-1H-hexahydro-1,4-diazepine), SP600125 (Anthra[1-9-cd]pyrazol-6(2H)-one), IKK 16 (N-(4-Pyrrolidin-1-yl-piperidin-1-yl)-[4-(4-benzo[b]thiophen-2-yl-pyrimidin-2-ylamino)phenyl]carboxamide dihydrochloride) and TAK-242 ((R)-Ethyl 6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1- enecarboxylate) were purchased from Tocris (Minneapolis, MN). Thapsigargin ((3S,3aR,4S,6S,6AR, 7S,8S,9bS)-6-(Acetyloxy)-2,3,3a,4,5,6,6a,7,8,9b-decahydro-3,3a-dihydroxy-3,6,9-trimethyl-8-[[(2Z)-2-methyl-1-oxo-2-butenyl]oxy]-2-oxo-4-(1-oxobutoxy)azuleno[4,5-b]furan-7-yloctanoate), lipopolysaccharide (LPS), lipopolysaccharide (LPS) and 2-APB (2-Aminoethoxydiphenyl borane) were purchased from Sigma (St Louis, MO, USA). They were dissolved in Milli-Q water or dimethyl sulfoxide (DMSO) as stock solutions and further diluted to working concentrations.
Birla H., Xia J., Gao X., Zhao H., Wang F., Patel S., Amponsah A., Bekker A., Tao Y.X, & Hu H. (2022). 1Toll-like receptor 4 activation enhances Orai1-mediated calcium signal promoting cytokine production in spinal astrocytes. Cell calcium, 105, 102619.
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10

Evaluating NF-κB Modulators in RAW-Blue Cells

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RAW-Blue cells (a murine RAW 264.7 macrophage reporter cell line) were purchased from InvivoGen. This reporter cell line stably expresses a secreted embryonic alkaline phosphatase gene inducible by NF-κB activation that can be detected. The assay was performed according to the manufacturer’s instructions. Briefly, 2 × 104 RAW-Blue cells were incubated with 20 µg of GBex alone or in combination with (1) NF-κB pathway inhibitor (IKK16, 200 nmol, 2539, Tocris), (2) anti-TGF-β Ab (IDII, 100 nM, obtained from Dr Andrew Hinck, University of Pittsburgh), (3) COX inhibitor (Celecoxib, 1 µM, PZ0008-, Sigma), (4) anti-IL-2R Ab (10 µg, MAB224, Millipore), (5) CD73 inhibitor (Adenosine 5′-(α,β-methylene)diphosphate, 1 µM, M3763, Sigma), (6) phosphodiesterase inhibitor (Rolipram, 1 µM, R6520, Sigma), (7) anti-IL-6 Ab (10 µg, mabg-hil6-3, Invivogen), (8) anti-FAS Ab (10 µg, 05-338, Millipore), and (9) anti-TNF-α Ab (10 µg, htnfa-mab1, Invivogen) for 24 h under standard conditions. After incubation, 20 µL of media was collected and incubated with 200 µL QUANTI-blue reagent (Invivogen) and optical density was measured at 655 nm using TECAN spectrophotometer (TECAN).
Azambuja J.H., Ludwig N., Yerneni S., Rao A., Braganhol E, & Whiteside T.L. (2020). Molecular profiles and immunomodulatory activities of glioblastoma-derived exosomes. Neuro-oncology Advances, 2(1), vdaa056.
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