Tpca 1

Manufactured by Selleck Chemicals

Sourced in United States, Germany

TPCA-1 is a laboratory chemical compound used as a tool compound in biochemical research. It functions as a selective inhibitor of the enzyme PARP-1, which is involved in DNA repair processes. The core purpose of TPCA-1 is to serve as a research tool to study the role of PARP-1 in cellular mechanisms.

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Lab products found in correlation

21 protocols using tpca 1

Modulation of Astrocyte Inflammatory Response

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Cells were transfected with mimic pre-miRNA for miR146a or miR147b (mirVana miRNA mimics, Applied Biosystems, Carlsbad, CA, USA) for 24 hours as described previously (van Scheppingen et al. 2016b) .
Astrocyte cultures were stimulated with human recombinant (r)IL-1 β (10 ng/ml; Peprotech, Rocky Hill, NJ, USA) or lipopolysaccharide (LPS; 100 ng/ml; Sigma, St. Louis, MO, USA) for 24 hours. Viability of human cell cultures was not influenced by the treatments (as shown previously; (van Scheppingen et al. 2016a )). To examine the effect of the IκB kinase-2 (IKK-2) inhibitor TPCA-1 in astrocyte cultures, treatment with 1 or 5 µM TPCA-1 (Selleck Chemicals, Munich, Germany) in DMSO (0.05% final DMSO concentration) was started 1 hour before stimulation with IL-1β was initialized, and treatment was continued during stimulation.

van Scheppingen J., Mills J.D., Zimmer T.S., Broekaart D.W.M., Iori V., Bongaarts A., Anink J.J., Iyer A.M., Korotkov A., Jansen F.E., van Hecke W., Spliet W.G., van Rijen P.C., Baayen J.C., Vezzani A., van Vliet E.A, & Aronica E. (2018). miR147b: A novel key regulator of interleukin 1 beta-mediated inflammation in human astrocytes. Glia, 66(5).

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Screening for IFN Response Inhibitors

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Inhibitors of the IFN response, BX795,^{14 (link)} TPCA-1,^{15 (link)} and ruxolitinib (Rux)^{16 (link)} (Selleck Chemicals, Munich, Germany), were prepared as 10-mM stocks in DMSO. Actinomycin D (AMD) and cyclohexamide (CHX) (Sigma, Dorset, UK) were prepared as 10-mM stocks in DMSO and ethanol, respectively. The Small Diversity Set Compound Library (Dundee Drug Discovery Unit [DDU], University of Dundee, UK) was used for HTS and consists of 15,667 compounds at 10 mM in DMSO. Hit compounds StA-IFN-1 (4-(1-acetyl-1H-indol-3-yl)-5-methyl-2,4-dihydro-3H-pyrazol-3-one) (Chembridge, San Diego, CA), StA-IFN-2 (4-{[4-(thieno[3,2-d]pyrimidin-4-yl)-1,4-diazepan-1-yl]methyl}benzonitrile) (Enamine, Monmouth Jt., NJ), StA-IFN-4 (2-[(4,5-dichloro-6-oxo-1(6H)-pyridazinyl)methyl]-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one) (Enamine, Monmouth, NJ), and StA-IFN-5 (6-methyl-4-phenyl-N-(pyridin-4-yl)quinazolin-2-amine) (Mcule, Budapest, Hungary) were stored as 10-mM stocks in DMSO. All compounds were used at the indicated concentrations.

Gage Z.O., Vasou A., Gray D.W., Randall R.E, & Adamson C.S. (2016). Identification of Novel Inhibitors of the Type I Interferon Induction Pathway Using Cell-Based High-Throughput Screening. Journal of Biomolecular Screening, 21(9), 978-988.

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EGFR Inhibitors and TPCA-1 Effects

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EGFR inhibitors (erlotinib, gefitinib and afatinib) and TPCA-1 were purchased from Selleck Chemicals (Houston, TX, USA). Drugs were prepared in dimethyl sulfoxide (DMSO) at a concentration of 10–100mmol/L stock solutions and stored at –20 °C. Further dilutions were made in culture medium to final concentration before use. Phospho-EGFR (Tyr1068), phospho-STAT3 (Tyr705), phospho-ERK1/2 (Thr202/Tyr204) and β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA). The secondary antibodies Amersham ECL-anti-rabbit IgG horseradish peroxidase-linked species-specific whole antibody and Amersham ECL-anti-mouse IgG peroxidase-linked whole antibody were purchased from GE Healthcare UK limited (Buckinghamshire, UK).

Codony-Servat C., Codony-Servat J., Karachaliou N., Molina M.A., Chaib I., Ramirez J.L., Gil M.D., Solca F., Bivona T.G, & Rosell R. (2017). Activation of signal transducer and activator of transcription 3 (STAT3) signaling in EGFR mutant non-small-cell lung cancer (NSCLC). Oncotarget, 8(29), 47305-47316.

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MAPK Signaling Pathway Analysis

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The anti-His-tagged, anti-IκBα, anti-IL-1β, anti-PCNA, MAPK Family Antibody Sampler Kit and Phospho-MAPK Family Antibody Sampler Kit were supplied by Cell Signaling Technology (Beverly, MA, USA). Antibodies against p65 and actinin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). BMS-345541, TPCA-1, SC-514, and SP600125 were purchased from Selleckchem. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Isopropyl β-d-1-thiogalactopyranoside (IPTG) and imidazole were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Human Inflammatory Response and Autoimmunity RT² Profiler™ PCR Array (PAHS-077ZA-6) was purchased from Qiagen (Valencia, CA, USA). All human and mouse cytokines ELISA kits were obtained from eBioscience (San Diego, CA, USA). Zeba™ spin desalting columns, HisPur Ni-NTA superflow agarose, high-capacity endotoxin removal spin columns and LAL chromogenic endotoxin quantitation kit were purchased from Thermo Scientific (Rockford, IL, USA). Rosetta™ 2 (DE3) pLysS competent cells were obtained from EMD Millipore Corporation (Billerica, MA, USA).

Chen Y.J., Zhu J.Q., Fu X.Q., Su T., Li T., Guo H., Zhu P.L., Lee S.K., Yu H., Tse A.K, & Yu Z.L. (2019). Ribosome-Inactivating Protein α-Momorcharin Derived from Edible Plant Momordica charantia Induces Inflammatory Responses by Activating the NF-kappaB and JNK Pathways. Toxins, 11(12), 694.

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Investigating Cell Line Response to NF-κB and TLR4 Inhibitors

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Human colon cancer cell lines such as DLD-1, HT-29, and LS174T (ATCC, Manassas, VA, USA) were cultured and maintained in RPMI1640 or DMEM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen). Bay11-7085 (NF-κB inhibitor), TPCA-1 (NF-κB inhibitor), and TAK-242 (TLR4 inhibitor) were purchased from Selleckchem (Houston, TX, USA).

Huang H.C., Cai B.H., Suen C.S., Lee H.Y., Hwang M.J., Liu F.T, & Kannagi R. (2020). BGN/TLR4/NF-κB Mediates Epigenetic Silencing of Immunosuppressive Siglec Ligands in Colon Cancer Cells. Cells, 9(2), 397.

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TPCA-1 Inhibition of IKK2 in Mice

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Mice at 8 weeks of age were treated with intraperitoneal injection of the TPCA-1 (2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide) (Selleckchem), a selective inhibitor of IKK2 (IKKβ), at 20 mg/kg body weight, twice at 12 hr apart. TPCA-1 was given in a vehicle consisting of 0.9% DMSO, 7% dimethylacetoacetamide (DMA) and 10% Cremophor EL (both from Sigma-Aldrich) in PBS. Radiation was delivered 2 hr after the 2^nd TPCA-1 injection.

Mahdi A.H., Huo Y., Tan Y., Simhadri S., Vincelli G., Gao J., Ganesan S, & Xia B. (2018). Evidence of inter-tissue differences in the DNA damage response and the pro-oncogenic role of NFκB in mice with disengaged BRCA1-PALB2 interaction. Cancer research, 78(14), 3969-3981.

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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).

Gao Y., Yang Y., Yuan F., Huang J., Xu W., Mao B., Yuan Z, & Bi W. (2017). TNFα-YAP/p65-HK2 axis mediates breast cancer cell migration. Oncogenesis, 6(9), e383-.

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CpG Oligonucleotide Stimulation of Gastric Cells

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AGS, NUGC4, MKN28, and gp130^F/F primary gastric epithelial cells (2 × 10³) were seeded in triplicate wells in 96-well plates. After 24 hours of serum starvation, cells were treated with either ODN 2006 CpG oligonucleotide (Class B) or ODN 2006 control oligonucleotide (Invivogen, San Diego, CA) for 24 hours (proliferation) or 48 hours (viability). Where indicated, cells were also pretreated for 30 minutes with specific inhibitors against the ERK MAPK (U0126; 0.5 μmol/L; Cell Signaling Technology) and NF-κB (TPCA-1; 10 μmol/L; Selleck Chemicals, Houston, TX) pathways before stimulation with CpG or control oligonucleotides (both at 2 μmol/L). Cell proliferation was assessed with the ClickIT EdU Microplate Assay (Molecular Probes, Eugene, OR) and cell viability by using the MTT assay.⁸ (link)^,¹¹ (link)

Tang K., McLeod L., Livis T., West A.C., Dawson R., Yu L., Balic J.J., Chonwerawong M., Wray-McCann G., Oshima H., Oshima M., Deswaerte V., Ferrero R.L, & Jenkins B.J. (2022). Toll-like Receptor 9 Promotes Initiation of Gastric Tumorigenesis by Augmenting Inflammation and Cellular Proliferation. Cellular and Molecular Gastroenterology and Hepatology, 14(3), 567-586.

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Evaluating NF-κB Modulators in Cell Assays

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NF-κB antagonist and agonists: TNFα (315–01A, Peprotech, Rocky Hill, NJ, USA ), Prostratin (5739, Tocris, Bristol, UK), CGS 21680 HCl (1063, Tocris), Betulinic acid (53603, Selleck Chemicals, Houston, TX, USA), PSI (4045, Tocris), Cardamonin (2509, Tocris), Bay 11–7082 (S2913, Selleck Chemicals), Bay 11–7085 (S7352, Selleck Chemicals), RO 106–9920 (1778, Tocris), TPCA-1 (S2824, Selleck Chemicals), Ikk-16 (S2882, Selleck Chemicals), PF 184 (4238, Tocris), IMD 0354 (2483, Tocris), Andrographolide (S2261, Selleck Chemicals), Costunolide (2483, Tocris), CID 2858522 (4246, Tocris), Pictilisib (S1065, Selleck Chemicals), Luteolin (S2320, Selleck Chemicals), Celastrol (1571, Tocris), Artemisinin (2668, Tocris). Cells were plated (at a seeding density of 1x10⁴ for 96 well plates and 1x10³ for 384 well plates) 12 h before treatment. Cells were treated with drugs at a range of concentrations either with fresh media (for agonists and vehicle only control) or fresh media with 5ng/ml TNFα (for antagonists and vehicle control) and incubated for 24 h. Each drug dose was performed in triplicate or quadruplicate and experiments were repeated at least three times.

Harrold A.P., Cleary M.M., Bharathy N., Lathara M., Berlow N.E., Foreman N.K., Donson A.M., Amani V., Zuercher W.J, & Keller C. (2020). In vitro benchmarking of NF-κB inhibitors. European journal of pharmacology, 873, 172981.

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Maintaining Cell Lines in Culture

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All cells were maintained in culture at 37°C and 5% CO₂ in Dulbecco’s modified Eagle medium (Thermo Fisher) supplemented with 10% (v/v) fetal bovine serum (Thermo Fisher), 0.2% (v/v) Primocin (Thermo Fisher), 1% penicillin (100 IU/mL; Thermo Fisher), and streptomycin (100 μg/mL; Thermo Fisher). SB cells were generated previously by us.²⁴ (link) FAC cells also were generated by us (unpublished data). KPPC cells were kindly gifted by Nabeel Bardeesy, PhD (Harvard Medical School, Massachusetts General Hospital, Broad Institute).²⁵ (link)^,⁵² (link) The following siRNAs at 10 nmol/L final concentration were used: ON-TARGETplus Non-targeting Control Pool (#D-001810-10-20; Horizon) and ON-TARGETplus Mouse Cflar siRNA SMARTPool (#L-041091-00-0005; Horizon). Lipofectamine RNAiMAX Transfection Reagent (#13778150; Thermo Fisher) was used for siRNA transfection. Recombinant mouse TRAILs were purchased from PeproTech (#315-19), R&D Systems (#1121-TL-010), and Enzo Life Sciences (#ALX-201-073-C020). TPCA-1 (#S2824; Selleckchem) at 10 μmol/L final concentration was used. The CellTiter-Glo 2.0 Cell Viability Assay (#G9242; Promega) was used per the manufacturer’s protocol.

Loeuillard E.J., Li B., Stumpf H.E., Yang J., Willhite J.R., Tomlinson J.L., Rohakhtar F.R., Simon V.A., Graham R.P., Smoot R.L., Dong H, & Ilyas S.I. (2024). Noncanonical TRAIL Signaling Promotes Myeloid-Derived Suppressor Cell Abundance and Tumor Growth in Cholangiocarcinoma. Cellular and Molecular Gastroenterology and Hepatology, 17(5), 853-876.

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