Roscovitine

Manufactured by Cell Signaling Technology

Sourced in United Kingdom

Roscovitine is a highly selective cyclin-dependent kinase (CDK) inhibitor. It functions by binding to and inhibiting the activity of CDK2, CDK5, CDK7, and CDK9 enzymes.

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Lab products found in correlation

Uo126, by Selleck Chemicals (2 mentions) Silvestrol, by MedChemExpress (2 mentions) Homoharringtonine, by Bio-Techne (2 mentions) Insulin in solution, by Merck Group (2 mentions) Full length app 695 in pcax vector, by Addgene (2 mentions) Cycloheximide (chx), by Merck Group (2 mentions) Ro 3306, by Merck Group (2 mentions) Torin1, by Bio-Techne (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Azd7762, by Selleck Chemicals (1 mentions) Mk 8776, by Selleck Chemicals (1 mentions) Ly2603618, by Selleck Chemicals (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions) Mk1775 azd1775, by Merck Group (1 mentions) Hela cell, by American Type Culture Collection (1 mentions) Hek293t, by American Type Culture Collection (1 mentions) Atr inhibitor ve821, by Selleck Chemicals (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Hibernate a, by Transnetyx (1 mentions)

9 protocols using roscovitine

Investigating Gamma-Secretase Inhibitors

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γ‐secretase inhibitor MRK‐003 was a gift of Merck & Co. (Whitehouse Station, NJ). 5 μM MRK‐003 was used in most experiments; exceptions are reported in figures and/or legends. Torin 1 (1‐[4‐[4‐(1‐Oxopropyl)‐1‐piperazinyl]‐3‐(trifluoromethyl) phenyl]‐9‐(3‐quinolinyl)‐benzo[h]‐1,6‐naphthyridin‐2(1H)‐one) (Tocris Bioscience, Bristol, UK) was dissolved in DMSO and used at a final concentration of 0.250 μM. Roscovitine (Cell Signaling) was dissolved in DMSO and used at a final concentration of 20 μM. UO126 (Selleck Chemicals, Huston, TX) was dissolved in DMSO, and used at a final concentration of 10 μM. Silvestrol (Medchemexpress LLC, Princeton, NJ) was dissolved in DMSO and used at a final concentration of 0.04 μM and 0.1 μM. Homoharringtonine (Tocris) was dissolved in DMSO and used at a final concentration of 0.1 μM. Insulin in solution (Sigma–Aldrich) was diluted in sterile PBS and used at the specified concentrations. pcDNA3‐RLUC‐POLIRES‐FLUC was a gift from Nahum Sonenberg (McGill University, Montreal); pCDF1‐MCS1‐EF1‐cop GFP expressing the APP C‐terminal 59 aa was a gift from Dr. Xiao Z.C. (Institute of Molecular and Cell Biology, Singapore); full length APP 695 in pCAX vector was from Addgene (Cambridge, MA). We used the empty plasmids as transfection controls.

Sobol A., Galluzzo P., Liang S., Rambo B., Skucha S., Weber M.J., Alani S, & Bocchetta M. (2015). Amyloid Precursor Protein (APP) Affects Global Protein Synthesis in Dividing Human Cells. Journal of Cellular Physiology, 230(5), 1064-1074.

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Inhibiting LSF, FQI1, and FQI2 in Cells

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Small molecule inhibitors of LSF, FQI1 and its achiral analogue FQI2, have been described previously [12 (link)]. After initial titration, in vitro assays were performed using either 2 or 5 μM FQI1 or FQI2. As indicated, cells were pre-treated for 2 h with Cycloheximide (10 μg/ml; Sigma; C7698) or Roscovitine (30 μM; Cell Signaling; #9885) before treatment with LSF inhibitors. Control and Cyclin B1 siRNAs were obtained from Santa Cruz Biotech (sc-37007 and sc-29284, respectively).

Rajasekaran D., Siddiq A., Willoughby J.L., Biagi J.M., Christadore L.M., Yunes S.A., Gredler R., Jariwala N., Robertson C.L., Akiel M.A., Shen X.N., Subler M.A., Windle J.J., Schaus S.E., Fisher P.B., Hansen U, & Sarkar D. (2015). Small molecule inhibitors of Late SV40 Factor (LSF) abrogate hepatocellular carcinoma (HCC): Evaluation using an endogenous HCC model. Oncotarget, 6(28), 26266-26277.

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Cell Culture and Inhibitor Treatments

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Human NCI-H460 and A549 lung cancer (ATCC) and U2OS osteosarcoma cells were cultured in DMEM (Dulbecco's modified Eagle’s) medium, and SW900 lung cancer and Reh pre-B cell leukemia cells in RPMI (Roswell Park Memorial Institute) medium (both media from Life Technologies), at 37°C in a humidified atmosphere with 5% CO₂. The media were supplemented with 10% fetal bovine serum (origin South America, Life Technologies) and 1% Penicillin/Streptomycin (Life Technologies). All cell lines (except Reh) were verified by STR (short tandem repeat) technology as described previously [43 (link)]. The Wee1 inhibitor MK1775 (AZD1775) was from Merck Calbiochem. The Chk1 inhibitors AZD7762, LY2603618 and MK8776 were from Selleck Chemicals, UCN01 was a gift from R.J. Schultz, National Cancer Institute, and the Chk2 inhibitor PV1019 was from Millipore. The dual CDK1/CDK2 inhibitor Roscovitine and the CDK2 inhibitor CVT-313 were from Cell Signaling, and the CDK1 inhibitor RO-3306 from Merck Calbiochem.

Hauge S., Naucke C., Hasvold G., Joel M., Rødland G.E., Juzenas P., Stokke T, & Syljuåsen R.G. (2016). Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading. Oncotarget, 8(7), 10966-10979.

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Comprehensive Inhibitor Screening Protocol

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P38 inhibitor SB203580, JNK inhibitor SP600125, Jak2 inhibitor II, and MG132 were purchased from EMD Bioscience. Cdk inhibitor Roscovitine was purchased from Cell Signaling technology. Cycloheximide was purchased from Sigma-Aldrich. Human Stem cell factor (SCF), thrombopoietin (TPO), and FLT3-ligand (FLT3L), human GM-CSF, murine IL-3 and SCF were purchased from Peprotech. EPO was manufactured by Amgen. Fetal bovine serum (FBS) was purchased from Hyclone. TF1 human erythroid cells (American Type Culture Collection (ATCC)) were cultured in RPMI 1640 medium supplemented with 10% FBS and GM-CSF (2 ng ml⁻¹) and 1% penicillin/streptomycin. HEK293T (ATCC) and Hela cells (ATCC) were kept in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. All cell lines were authenticated by supplier. All cells were tested for mycoplasma contamination.

Hu P., Nebreda A.R., Hanenberg H., Kinnebrew G.H., Ivan M., Yoder M.C., Filippi M.D., Broxmeyer H.E, & Kapur R. (2018). P38α/JNK signaling restrains erythropoiesis by suppressing Ezh2-mediated epigenetic silencing of Bim. Nature Communications, 9, 3518.

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Reverse Transfection and Chemical Inhibition

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To knock down genes of interest, reverse transfection was performed in 6-well plates with 10nM siRNA and Lipofectamine 2000 (Life Technologies). Cells were either harvested or treated with chemicals after 48 h. siRNAs to Wee1 (s21, silencer select), Claspin #1 (s34330, silencer select), Claspin #2 (s34331, silencer select), CtIP #1 (s11849, silencer select), CtIP #2 (s11851, silencer select), p53 (s605, silencer select), Wee1 (404, silencer), Cdk1 #1 (s464, silencer select), Cdk1 #2 (s465, silencer select), Plk1 (s449, silencer select) and Negative Control No.1 siRNA (silencer select, silencer) were obtained from Ambion, Life Technologies. The following chemical inhibitors were used: Wee1 inhibitor MK-1775 (Selleckchem), ATR inhibitor VE-821 (Selleckchem), Chk1 inhibitor SB 218078 (Calbiochem, Merck), Cdk1, 2 and 5 inhibitor Roscovitine (Cell Signaling), Cdk1 inhibitor RO-3306 (Sigma Aldrich).

Saini P., Li Y, & Dobbelstein M. (2015). Wee1 is required to sustain ATR/Chk1 signaling upon replicative stress. Oncotarget, 6(15), 13072-13087.

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Visualizing Neuronal Dynamics in DRG Cells

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DRG neurons were isolated according to Perlson et al. (2009) (link) and maintained in F-12 media (Invitrogen) with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. For live-cell analysis, DRG neurons were plated on glass-bottom dishes (World Precision Instruments, Inc.) and cultured for 2 d at 37°C in a 5% CO₂ incubator. Before plating, neurons were transfected with 0.5–0.7 µg plasmid DNA or 20–60 pM siRNA as noted, using a Nucleofector (Lonza) according to the manufacturer’s specifications. 20 mM roscovitine (Cell Signaling) or DMSO was added as noted 24 hours prior to imaging. Imaging was performed in low fluorescence nutrient medium (Hibernate A; BrainBits) with 2% B27 and 2 mM GlutaMAX on an inverted epifluorescence microscope (DMI6000B; Leica) using an Apochromat 63×, 1.4 NA oil immersion objective (Leica) in an environmental chamber at 37°C. Digital images were acquired with an ORCA-R2 (Hamamatsu Photonics) using LAS-AF software (Leica). Images were taken every 3 s for a total of 3 min for GFP-LC3 and DsRed2-mito, every 368 ms for a total of 1.06 min for GFP-Rab7 and LAMP1-RFP, and every 500 ms for a total of 2 min for mRFP-TrkB.

Klinman E, & Holzbaur E.L. (2015). Stress-induced CDK5 activation disrupts axonal transport via Lis1/Ndel1/dynein. Cell reports, 12(3), 462-473.

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Combination Therapy for Melanoma

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GDC-0941 (pictilisib; PI3K inhibitor) and GDC-0973 (cobimetinib; MEK inhibitor) were obtained from the Department of Chemistry at Genentech, Inc. (South San Francisco, CA). GDC-0973 and GDC-0941 drug concentrations for melanoma cell lines were selected based on conditions described previously (3 (link)), and dosing solutions were prepared in DMSO (Sigma-Aldrich). Nocodazole (M1404) and thymidine (T1895) were purchased from Sigma-Aldrich. Cell Extraction Buffer (FNN0011, Thermo Fisher Scientific), MG132 InSolution (474791, Calbiochem), CDK2 inhibitor III (Calbiochem), roscovitine (Cell Signaling Technology), and calf intestinal alkaline phosphatase (New England Biolabs) were obtained from the sources indicated. The Click-iT® EdU Assay kit was from Thermo Fisher Scientific.

Dogan T., Gnad F., Chan J., Phu L., Young A., Chen M.J., Doll S., Stokes M.P., Belvin M., Friedman L.S., Kirkpatrick D.S., Hoeflich K.P, & Hatzivassiliou G. (2017). Role of the E3 ubiquitin ligase RNF157 as a novel downstream effector linking PI3K and MAPK signaling pathways to the cell cycle. The Journal of Biological Chemistry, 292(35), 14311-14324.

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IFN Signaling Pathway Inhibitors Assay

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Doxycycline was purchased from Sigma-Aldrich. Recombinant active human IFNγ and IFNβ were purchased from InvivoGen. Small interference RNAs targeting human c-JUN were purchased from Dharmacon. Human IFN receptor neutralization antibody anti-IFNAR2 was purchased from PDI Assay Science. The JAK kinase inhibitor Ruxolitinib was purchased from STEMCELL Technologies. The MEK and JNK inhibitors trametinib and SP600125, respectively, were purchased from Cell Signaling Technology. AZD6738 (AstraZeneca) and roscovitine (Cell Signaling) were each dissolved in DMSO and stored in aliquots at −20°C. Human APOBEC3A and scramble siRNAs were purchased from Ambion and transfected into cells by using Oligofectamine according to the manufacturer’s protocol. APOBEC3A knockdown was maximal at 3 days following transfection, at which point cells were used for additional experiments.

Zhao K., Zhang Q., Flanagan S., Lang X., Jiang L., Parsels L.A., Parsels J.D., Zou W., Lawrence T.S., Buisson R., Green M.D, & Morgan M.A. (2021). Cytidine Deaminase APOBEC3A Regulates PD-L1 Expression in Cancer Cells in a JNK/c-JUN-dependent Manner. Molecular cancer research : MCR, 19(9), 1571-1582.

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Investigating Gamma-Secretase Inhibition

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γ-secretase inhibitor MRK-003 was a gift of Merck & Co. (Whitehouse Station, NJ). 5 μM MRK-003 was used in most experiments; exceptions are reported in figures and/or legends. Torin 1 (1-[4-[4-(1-Oxopropyl)-1-piperazinyl]-3-(trifluoromethyl)phenyl]-9-(3-quinolinyl)-benzo[h]-1,6-naphthyridin-2(1H)-one) (Tocris Bioscience, Bristol, UK) was dissolved in DMSO and used at a final concentration of 0.250 μM. Roscovitine (Cell Signaling) was dissolved in DMSO and used at a final concentration of 20 μM. UO126 (Selleck Chemicals, Huston, TX) was dissolved in DMSO, and used at a final concentration of 10 μM. Silvestrol (Medchemexpress LLC, Princeton, NJ) was dissolved in DMSO and used at a final concentration of 0.04 μM and 0.1 μM. Homoharringtonine (Tocris) was dissolved in DMSO and used at a final concentration of 0.1 μM. Insulin in solution (Sigma-Aldrich) was diluted in sterile PBS and used at the specified concentrations. pcDNA3-RLUC-POLIRES-FLUC was a gift from Nahum Sonenberg (McGill University, Montreal); pCDF1-MCS1-EF1-cop GFP expressing the APP C-terminal 59 aa was a gift from Dr. Xiao Z.C. (Institute of Molecular and Cell Biology, Singapore); full length APP 695 in pCAX vector was from Addgene (Cambridge, MA). We used the empty plasmids as transfection controls.

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