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Pevonedistat

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Pevonedistat is a laboratory chemical used as a research tool. It is a small molecule inhibitor that targets the NEDD8-activating enzyme (NAE). This enzyme is involved in protein modification pathways and has been studied for its potential role in cellular processes. Pevonedistat is commonly used in experimental settings to investigate the effects of NAE inhibition on various biological systems.

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Lab products found in correlation

Anti β actin, by Cell Signaling Technology (2 mentions) Prolong gold antifade with dapi, by Thermo Fisher Scientific (2 mentions) Sybr gold, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 goat anti rat, by Thermo Fisher Scientific (1 mentions) White 384 well plate, by Corning (1 mentions) Spectramax i3x, by Molecular Devices (1 mentions) Tacs mtt cell proliferation assay kit, by Bio-Techne (1 mentions) Celltiter glo, by Promega (1 mentions) Anti β tubulin, by Thermo Fisher Scientific (1 mentions) Cisplatin, by Selleck Chemicals (1 mentions) Anti cul3, by Cell Signaling Technology (1 mentions) Anti nrf2 ab62352, by Abcam (1 mentions) Anti cd3 bv510, by BD (1 mentions) Cycloheximide (chx), by Merck Group (1 mentions) Sb431542, by Merck Group (1 mentions) Mln4924, by Selleck Chemicals (1 mentions) Anti cd107a apc, by BD (1 mentions) Anti cd16 pe cy7, by BD (1 mentions) Anti nkp46 pe 9e2, by BioLegend (1 mentions)

5 protocols using pevonedistat

1

Investigating Cellular Responses to Pevonedistat and Cisplatin

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Reagents were obtained from the following sources: Pevonedistat (#S7109) was purchased from SelleckChem (Houston, TX), and cisplatin was obtained from the hospital pharmacy. The antibodies anti-NEDD8 (ab81264), anti-p21 (ab109520), anti-p27 (ab32034), anti-γH2AX (Ser-139, ab81299), anti-CUL4A (ab92554), anti-CUL4B (ab67035), anti-phospho-Rb (Ser-780, ab173289), anti-Rb (ab181616), anti-DDB2 (ab181136), anti-E2F1 (ab179445), and anti-cisplatin-DNA adduct (ab103261) were purchased from Abcam (Cambridge, MA). Anti-NAE (CST14321), anti-WEE1 (CST4936), anti-active caspase-3 (CST9661), and anti-β-actin (CST3700) were obtained from Cell Signaling Technology (Danvers, MA). Anti-β-Tubulin (MA5-16308), goat anti-rat Alexa-Fluor 488 (#11006), goat anti-rabbit Alexa-Fluor 554 (#11010), goat anti-mouse Alexa-Fluor 488 (#11001), Prolong Gold antifade with DAPI (#P36931) and SYBR-Gold (#511494) were purchased from Thermo Fisher (Waltham, MA). Goat Anti-Rabbit HRP tagged secondary antibody (#111-035-144) was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
Jones T.M., Espitia C.M., Ooi A., Bauman J.E., Carew J.S, & Nawrocki S.T. (2022). Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism. Cell Death & Disease, 13(4), 350.
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2

Pharmacological Inhibition of Cellular Pathways

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The following drugs were used throughout the study: CPT (S1288, Selleckchem), TAK-243 E1 ubiquitin–activating enzyme inhibitor (S8341, Selleckchem), MG132 26S proteasome inhibitor (S2619, Selleckchem), and MLN4924 Nedd8-activating enzyme inhibitor pevonedistat (S7109, Selleckchem).
Meroni A., Grosser J., Agashe S., Ramakrishnan N., Jackson J., Verma P., Baranello L, & Vindigni A. (2022). NEDDylated Cullin 3 mediates the adaptive response to topoisomerase 1 inhibitors. Science Advances, 8(49), eabq0648.
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3

Multiparameter Cell Viability Assay

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Three hundred cells were plated on 384-well white plates (Corning); after 24 hours of incubation, the indicated drugs were added, and cells were incubated for 72 hours. Cell viability was evaluated using CellTiter-Glo (Promega) with SpectraMax i3x (Molecular Devices) according to the manufacturer's instructions. For the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, 1 × 104/well of H295R cells were seeded in 96-well plates and the indicated drugs were added after 24 hours and cultured for an additional 72 hours. Cell viability was measured using the TACS MTT Cell Proliferation Assay Kit (Trevigen). Drugs were obtained from the following sources: TAK-243, SJG-136, topotecan, doxorubicin, cisplatin, etoposide, and temozolomide were from Developmental Therapeutics Program (DTP, NCI); BAY-1895344, aldoxorubicin, entinostat, BMS-754807, brigatinib, ceritinib, Infigratinib, ulixertinib, berzosertib, navitoclax, capivasertib, MK-2206, XL-228, ribociclib, palbociclib, IPI-549, H-89, LY-2090314, cabozantinib, and everolimus from NCATS; bortezomib (5.04314) and mitotane (SML1885) from Sigma-Aldrich; OTS-167 (S7159) and pevonedistat (S7109) from Selleckchem.
Arakawa Y., Jo U., Kumar S., Sun N.Y., Elloumi F., Thomas A., Roper N., Varghese D.G., Takebe N., Zhang X., Ceribelli M., Holland D.O., Beck E., Itkin Z., McKnight C., Wilson K.M., Travers J., Klumpp-Thomas C., Thomas C.J., Hoang C.D., Hernandez J.M., Del Rivero J, & Pommier Y. (2024). Activity of the Ubiquitin-activating Enzyme Inhibitor TAK-243 in Adrenocortical Carcinoma Cell Lines, Patient-derived Organoids, and Murine Xenografts. Cancer Research Communications, 4(3), 834-848.
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4

Cellular Response to Pevonedistat and Cisplatin

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Reagents were obtained from the following sources: Pevonedistat and cisplatin were purchased from SelleckChem. The antibodies anti-NEDD8 (ab81264), anti-KIM-1 (ab47635), anti-TXNIP (ab188865) anti-OCT2/SLC22A2 (ab170871), and anti-NRF2 (ab62352) were purchased from Abcam. The antibodies anti-CUL3 (#2759) and anti-β-Actin (#3700) were purchased from Cell Signaling Technology. Anti-CTR1/SLC31A1 (27499-1-AP) was obtained from Proteintech. Anti-β-Tubulin (#MA5-16308) and Prolong Gold antifade with DAPI were purchased from Thermo Fisher Scientific. Goat Anti-Rabbit horseradish peroxidase (HRP)-tagged secondary antibody (#111-035-144) was purchased from Jackson ImmunoResearch Laboratories.
Jones T.M., Espitia C.M., Chipollini J., Lee B.R., Wertheim J.A., Carew J.S, & Nawrocki S.T. (2023). Targeting NEDDylation is a Novel Strategy to Attenuate Cisplatin-induced Nephrotoxicity. Cancer Research Communications, 3(2), 245-257.
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5

Immune Cell Activation and Inhibition Assay

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The NAE inhibitor MLN4924/Pevonedistat was purchased from Selleckchem.Com (Houston, Texas, USA). The TGFβ type I receptor inhibitor SB431542 and Cycloheximide (CHX) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). The final concentration of DMSO in all experiments was <0.1%. Daratumumab and Elotuzumab mAbs were provided by the “Policlinico Umberto I” hematology center pharmacy.
The following monoclonal antibodies (mAbs) were used for immunostaining: anti-CD14-APC-H7 (MφP9), anti-CD19-APC-H7 (HIB19), anti-CD45-PE-Cy™7 (HI30), anti-CD3-BV510 (HIT3a and UCHT1), anti-CD56-BV421/PE (NCAM16.2), anti-CD16-PE-Cy™7 (3G8), anti-CD107a-APC (H4A3), anti-CD138-FITC/PE (MI15), anti-CD38-APC (HIT2), anti-IFN-γ-PE (4S.B3), anti-Perforin-Alexa Fluor® 488 (δG9), anti-Granzyme B-PE (GB11), anti-NKG2D-APC (1D11), anti-DNAM1-FITC (DX11), anti-NKp30-Alexa Fluor® 647/BB700 (p30-15), anti-TIM-3-BB515 (7D3), anti-PD-1-BV421 (EH12.1) were purchased from BD Biosciences (Franklin Lakes, New Jersey, U.S.A.). Anti-NKp46-PE (9E2) and anti-SLAMF7-APC (162.1) were purchased from BioLegend (San Diego, California, U.S.A.) . Anti-TNFα-FITC (cA2), REA Control Antibody-APC (REA293), anti-TGFβ RII-APC (REA903) were purchased from Miltenyi Biotec (Bergisch Gladbach, North Rhine-Westphalia, Germany). Anti-TGFβ1/2/3 (1D11) monoclonal antibody was used as blocking antibody [R&D System (Minneapolis, MN, USA)].
Petillo S., Sproviero E., Loconte L., Cuollo L., Zingoni A., Molfetta R., Fionda C., Soriani A., Cerboni C., Petrucci M.T., Fazio F., Paolini R., Santoni A, & Cippitelli M. (2023). NEDD8-activating enzyme inhibition potentiates the anti-myeloma activity of natural killer cells. Cell Death & Disease, 14(7), 438.
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