Flavopiridol

Manufactured by Selleck Chemicals

Sourced in United States, United Kingdom

Flavopiridol is a synthetic compound that functions as a cyclin-dependent kinase (CDK) inhibitor. It is primarily used as a research tool in laboratory settings to study cell cycle regulation and related cellular processes.

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Flavopiridol HCl (2 citations)

35 protocols using flavopiridol

Flavopiridol and HDAC Inhibitor Treatments

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Cells were grown for 24 h after they were seeded onto the cell culture plates. For the flavopiridol time course experiment, 0.9 μL of 1 mM flavopiridol (Selleck Chemicals) was added to each 6 well to attain a final concentration of 300 nM. For the HDAC inhibitor time course experiment, 1 μL of 1 mM of either abexinostat (Selleck Chemicals) and pracinostat (Selleck Chemicals) was added to each 96 well to attain a final concentration of 10 μM. Similarly, for the HDAC inhibitor and dexamethasone co-treatment experiment, we added 1 μL of 100 μM or 1 μL of 1 mM of either abexinostat or pracinostat to each 96 well to attain a final concentration of 1 and 10 μM, respectively. dexamethasone (Selleck Chemicals) was added at a concentration of 100 nM (1 μL of 10 μM) two hours before the end of the HDAC inhibitor treatment. DMSO (10%) was used as a vehicle for flavopiridol and HDAC inhibitor treatments and ethanol (10%) was used as a vehicle for dexamethasone treatment. The HDACi timecourse and HDACi/DEX co-treatment experiment was performed in duplicates, and the flavopiridol timecourse experiment was only performed once.

Kim H.J., Booth G., Saunders L., Srivatsan S., McFaline-Figueroa J.L, & Trapnell C. (2022). Nuclear oligo hashing improves differential analysis of single-cell RNA-seq. Nature Communications, 13, 2666.

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Transcriptional Inhibition Assay in Halocynthia Embryos

Cited 3 times

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Actinomycin D (50-76-0; A1410; Sigma) was diluted into DMSO at 10 mg/mL stock. The stock solution was diluted into ASW to a final concentration of 40 μg/mL. This concentration was reported to block transcription in Halocynthia embryos (Miyaoku et al, 2018 (link)). Flavopiridol (146426-40-6; S1230; Selleck chemicals) was diluted into water to 10 mM stock. The stock solution was diluted into ASW to final concentration 1 and 10 μM. The transcriptional inhibitor-treated embryos were fixed by MEM-PFA (4% PFA, 0.1 M MOPS, 0.5 M NaCl, 1 mM EGTA, 2 mM MgSO₄) after 1 h inhibitor treatment, and used for in situ hybridization.
1-Azakenpaullone (S7193; Selleckchem; Feinberg et al, 2019 (link)), Ruxolitinib (INCB018424; S1378; Selleckchem), Vismodegib (GDC-0449; S1082; Selleckchem), DAPT (208255-80-5; D5942; Millipore Sigma), SB431542 (S1067; Selleckchem; Ohta and Satou, 2013 (link)), U0126 (9903; Cell Signaling Technology; (Hudson et al, 2003 (link))) and Dorsomorphin (1219168-18-9; S7306; Selleckchem; (Ohta and Satou, 2013 (link); Feinberg et al, 2019 (link))) were used to perturb define signaling pathways as described in corresponding references. These treatments were done in a final concentration of 10 μM for 2 or 4 h. The inhibitor-treated embryos were fixed by MEM-PFA after 2 h inhibitor treatment, and used for in situ hybridization.

Ohta N, & Christiaen L. (2024). Cellular remodeling and JAK inhibition promote zygotic gene expression in the Ciona germline. EMBO Reports, 25(5), 2188-2201.

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Anticancer Drug Combination Protocol

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Dinaciclib, doxorubicin, etoposide, cytarabine, flavopiridol, SNS-032, and PHA-767491 were purchased from Selleck Chemicals (Houston, TX). ABT-199 was kindly provided by AbbVie Inc. (North Chicago, IL).

Li L., Pongtornpipat P., Tiutan T., Kendrick S.L., Park S., Persky D.O., Rimsza L.M., Puvvada S.D, & Schatz J.H. (2015). Synergistic Induction of Apoptosis in High-Risk DLBCL by BCL2 Inhibition with ABT-199 Combined With Pharmacologic Loss of MCL1. Leukemia, 29(8), 1702-1712.

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Combination Therapy Evaluation Protocol

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The Bcl-2/Bcl-xL/Bcl-w antagonist ABT-737 were purchased from Chemie-Tek, Indianapolis, IN. The pan-CDK inhibitors SCH727965 (Dinaciclib), and flavopiridol (Alvocidib) were purchased from Selleck (Houston, TX, USA). The selective CDK9 inhibitor II (4-(3,5-Diamino-1H-pyrazol-4-ylazo)-phenol) and the Bcl-2 antagonist HA14-1 were purchased from Calbiochem/EMD Chemicals/Millipore and BioMol/Alexis/Enzo respectively. Bortezomib and carfilzomib were purchased from Chemie-Tek.
Drugs were dissolved in sterile DMSO, aliquoted and stored at -80°C. In all experiments, final DMSO concentrations did not exceed 0.1%.

Zhang Y., Zhou L., Leng Y., Dai Y., Orlowski R.Z, & Grant S. (2017). Positive transcription elongation factor b (P-TEFb) is a therapeutic target in human multiple myeloma. Oncotarget, 8(35), 59476-59491.

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Blastocyst Development Modulation

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Embryos from CD1 (Charles River laboratories, France) intercrosses were recovered at E2.75 and at E3.25 (expanding blastocyst) by flushing oviducts or uteri in M2 (Millipore), washed twice and cultured inside their intact zona pellucida in 400 µL KSOM (Millipore) in Nunc 4-well plates until the stages of interest at 37 °C, 8% CO₂. For treatment, we used FGF receptor inhibitor PD173074 (100 nM, Sigma Aldrich), MEK inhibitor PD0325901 (1μM, Sigma Aldrich), flavopiridol (1μM, Selleckchem), MG132 (10 μM, Sigma Aldrich) and FGF4 (1 μg/ml, R&D systems) supplemented with heparin (1 μg/ml, Sigma Aldrich). Experiments shown in Figs 2(A–C) and 3 were performed in parallel using same batches of embryos. All experiments were conducted according to the French and European regulations on care and protection of laboratory animals (EC Directive 86/609, French Law 2001–486 issued on June 6, 2001) and were approved by the Institut Pasteur ethics committee (n° 2012–0011).

Bessonnard S., Coqueran S., Vandormael-Pournin S., Dufour A., Artus J, & Cohen-Tannoudji M. (2017). ICM conversion to epiblast by FGF/ERK inhibition is limited in time and requires transcription and protein degradation. Scientific Reports, 7, 12285.

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Screening Inhibitors of Cyclin-Dependent Kinases

Cited 1 time

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CYC202 (seliciclib, R-Roscovitine), CCT68127, and CYC065 were provided by Cyclacel Ltd. Cycloheximide (C4859) and ActD (A9415) were purchased from MilliporeSigma and MG132 (1748) from Tocris Bioscience. Temozolomide, flavopiridol, palbociclib, dinaciclib, and SNS-032 were purchased from SelleckChem. BAY 1145372 was purchased from Active Biochem. Compound 3 was provided by Keith Jones (ICR). THZ1 (A8882) was purchased from Stratech. NVP-2 was obtained from Calla Olson, Baylor College of Medicine. THAL-SNS-032 was synthesized in house (27 (link)).

Poon E., Liang T., Jamin Y., Walz S., Kwok C., Hakkert A., Barker K., Urban Z., Thway K., Zeid R., Hallsworth A., Box G., Ebus M.E., Licciardello M.P., Sbirkov Y., Lazaro G., Calton E., Costa B.M., Valenti M., De Haven Brandon A., Webber H., Tardif N., Almeida G.S., Christova R., Boysen G., Richards M.W., Barone G., Ford A., Bayliss R., Clarke P.A., De Bono J., Gray N.S., Blagg J., Robinson S.P., Eccles S.A., Zheleva D., Bradner J.E., Molenaar J., Vivanco I., Eilers M., Workman P., Lin C.Y, & Chesler L. (2020). Orally bioavailable CDK9/2 inhibitor shows mechanism-based therapeutic potential in MYCN-driven neuroblastoma. The Journal of Clinical Investigation, 130(11), 5875-5892.

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Pharmacological Modulation of Cellular Signaling

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Tetrodotoxin (TTX) and flupenthixol were purchased from Tocris Bioscience (Bristol, UK), flavopiridol was from Selleckchem (Houston, TX, USA), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MI, USA). Peptides were synthesized by Biomatik (Wilmington, DE, USA). DA was made fresh every 30 min to minimize oxidation. Antagonists were administered 10 min before DA application. Concentrations of flavopiridol (100 nM) and 5,6-dichloro-1-β-D-ribobenzimidazole (DRB, 100 μM) were chosen based on previously demonstrated effective dosages (Chao and Price, 2001 (link); Bensaude, 2011 (link); Yuan and Burrell, 2013 (link); Krenz et al., 2014 (link)).

Krenz W.D., Parker A.R., Rodgers E, & Baro D.J. (2015). Monoaminergic tone supports conductance correlations and stabilizes activity features in pattern generating neurons of the lobster, Panulirus interruptus. Frontiers in Neural Circuits, 9, 63.

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Cyclin-CDK Complexes Phosphorylation Assay

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HEK 293T cells were transiently (co)transfected for 48 h with vectors expressing FFSS-tagged cyclin D1, FFSS-tagged cyclin D2, FFSS-tagged cyclin D3, HA-tagged CDK2, HA-tagged CDK4, or FH-tagged empty vector, in the combinations indicated. Complexes were purified by double immunoprecipitation in the following order: anti-Flag immunoprecipitation, elution with 3×Flag peptide, then anti-HA immunoprecipitation. Complexes bound to the beads were pre-washed in NEBuffer for Protein Kinases (PK, New England Biolabs B6022) and incubated with 0.5 μg human recombinant Rb (773-end) with an N-terminal GST tag, expressed in an Escherichia coli expression system (Sigma-Aldrich SRP0256). Reactions were carried out in NEBuffer for PK containing 0.1 mM NaV, 0.2 nM okadaic acid, 10 μM ATP in the presence or absence of either palbociclib isethionate (Sigma-Aldrich) or flavopiridol (Selleck Chemicals) at the concentrations indicated for 30 min at 30 °C, before stopping the reaction with Laemmli buffer. Samples were resolved by SDS–PAGE and immunoblotted as indicated.

Simoneschi D., Rona G., Zhou N., Jeong Y.T., Jiang S., Milletti G., Arbini A.A., O’Sullivan A., Wang A.A., Nithikasem S., Keegan S., Siu Y., Cianfanelli V., Maiani E., Nazio F., Cecconi F., Boccalatte F., Fenyö D., Jones D.R., Busino L, & Pagano M. (2021). CRL4AMBRA1 is a master regulator of D-type cyclins. Nature, 592(7856), 789-793.

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Pharmacological Inhibition of Cell Signaling

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To analyse the effects of pharmacological inhibitors, cells were seeded in 6-well plates at 2.5x10⁵ cells per well, incubated overnight to allow for adhesion and serum starved for subsequent 24 h. Serum free medium containing appropriate inhibitors was used to pre-treat cells for 30 min, unless indicated otherwise, prior to stimulation with 20 ng/mL EGF (Sigma-Aldrich, St Louis, MO, USA, Cat. no. E5036), 1 mM sodium orthovanadate – NaOVa (Na₃VO₄, Calbiochem, Cat. no. 567540), phenylarsine oxide – PAO (Sigma, Cat. no. P3075) or pervanadate – pV (obtained as described previously [22 (link)]; using Catalase, Sigma, Cat. no. C1345 and H₂O₂ Sigma, Cat. no. 31642) for 1 hour and lysed. Following concentrations of inhibitors were used: DMSO (Sigma-Aldrich, Cat. no. D2438, solvent control), 10 μM erlotinib (Tarceva, Molecula; Cat. no. 89983631), 0.5 μM afatinib (Selleckchem, Cat. no. S1011); 5 μM gefitinib (CellSignaling; 4765), 5 μM lapatinib (Selleckchem, Cat. no. S1028), 300 nM flavopiridol (Selleckchem, Cat. no. S1230), 5 μM arry380 (Gentaur Europe, Cat. no. A8366), 10 μM Nu7441 (Selleckchem, Cat. no. S2638) and 10 μM ruxolitinib (Selleckchem, Cat. no. S1378).

Stec W., Rosiak K., Treda C., Smolarz M., Peciak J., Pacholczyk M., Lenart A., Grzela D., Stoczynska-Fidelus E, & Rieske P. (2018). Cyclic trans-phosphorylation in a homodimer as the predominant mechanism of EGFRvIII action and regulation. Oncotarget, 9(9), 8560-8572.

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Comprehensive Antibody Characterization Protocol

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Antibody for total RB was purchased from BD Biosciences (San Jose, CA, clone C245, catalog #554136). Antibodies purchased from Cell Signaling Technology (Beverly, MA) included: total RB (clone 4H1, Catalog #9309), phospho-RB (Ser608) (#2181), phospho-AKT (Ser473) (#4060), cyclin D1 (#9309), PARP-1 (#9542), caspase 3 (#9665), caspase 9 (#9502), and phospho-p70 S6K (Thr389, #9205). β-actin antibody was purchased from Sigma Aldrich (St. Louis, MO, catalog #A1978).
Laboratory-grade palbociclib was generously provided by Pfizer, Inc. (New York, NY) and purchased from Selleck Chemicals (Boston, MA). Everolimus and pemetrexed were purchased from LC chemical (Woburn, MA). Decitabine was purchased from Sigma (St. Louis, MO). Selumetinib, flavopiridol, sunitinib, AZD5438, ponatinib, AZD4547, erlotinib, pazopanib, imatinib, PF-04691502, dacomitinib and rapamycin were purchased from Selleck Chemicals (Boston, MA). Depsipeptide was obtained from the NCI Repository in the Developmental Therapeutics Program (NSC #309132, Bethesda, MD). Stock solutions for all the agents were prepared in 100% DMSO (Sigma, St. Louis, MO) at 10mM concentrations and stored at -20°C. Stock solutions were diluted in fresh RPMI 1640 media prior to each experiment.

Gopalan P.K., Villegas A.G., Cao C., Pinder-Schenck M., Chiappori A., Hou W., Zajac-Kaye M., Ivey A.M, & Kaye F.J. (2018). CDK4/6 inhibition stabilizes disease in patients with p16-null non-small cell lung cancer and is synergistic with mTOR inhibition. Oncotarget, 9(100), 37352-37366.

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