Pha 767491

Manufactured by Merck Group

Sourced in Spain

PHA-767491 is a laboratory research tool used for screening and identification of small molecules. It functions as an inhibitor for the cell division cycle 7-related protein kinase (CDC7). This compound is utilized in experimental settings to study cell cycle regulation and related biological processes.

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7 protocols using pha 767491

Knockdown and Chemical Inhibition Assays

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For knockdown experiments, cells were transfected 24–96 h before sample collection with the indicated siRNA using RNAiMax transfection reagent (Life Technologies) according to the manufacturer's instructions: siLUC (100–200 nM; 5′-GGUACGCGGAAUACUUCGAdTdT-3′), siFZR1, siRad51, siAPE1, siTDG (100–200 nM siGENOME SMARTpool Dharmacon). The following reagents were used to treat ES cells for the indicated time at the indicated final concentrations before collection: ATM inhibitor (KU55933, Kudos; 8 h, 10 μM); ATR inhibitor (ETP-46464, provided by O. Fernandez-Capetillo, CNIO, Madrid; 8 h, 5 μM; VE-821, Selleckchem; 8 h, 10 μM); PARP inhibitor (Olaparib, Selleckchem; 24 h, 10 μM); Reducing/scavenging agent: (N-acetylcysteine, Sigma; 10 h, 10 mM); Transcription inhibitors (Cordycepin, Sigma; 100 min, 50 μM; Alpha-amanitin, kindly provided by P. Janscak, 3–6 h, 20 μM); Ape1 inhibitor (Methoxyamine hydrochloride, Sigma; 10 h, 1 μM); CDK4/6 inhibitor (LY2835219, Selleckchem; 4 h, 1 μM); Cdc7 inhibitors (PHA-767491, Sigma; 8 h, 10 μM; XL-413, kindly provided by C. Santocanale, 4 h, 10 μM); CDK1 inhibitor (RO-3306, Sigma; 10 h, 10 μM); PLK inhibitor (BI-6727, Selleckchem; 4 h, 500 nM); Nucleosides (EmbryoMax, Millipore; 24 h, 5 ×). Roscovitine (Seliciclib, Selleckchem; 8 h, 20 μM).

Ahuja A.K., Jodkowska K., Teloni F., Bizard A.H., Zellweger R., Herrador R., Ortega S., Hickson I.D., Altmeyer M., Mendez J, & Lopes M. (2016). A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells. Nature Communications, 7, 10660.

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Inducible KRAS Transgene Expression

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Cell lines were cultured in their respective medium with antibiotic selection as indicated in Supplementary Table 2 with 10% fetal calf serum (PAN Biotech, P30-1506, Lot No. P130902) and 1% penicillin/streptomycin (Biochrom) at 37 °C and 5% CO₂. Transgene expression was induced with 2 µg/ml of doxycycline (Applichem) where indicated. For drug treatment experiments, KRAS transgene expression was induced immediately after seeding and drugs were added one night after seeding the cells (PHA 767491, hydroxyurea, oxaliplatin—all Sigma-Aldrich).

Gastl B., Klotz-Noack K., Klinger B., Ispasanie S., Salib K.H., Zuber J., Mamlouk S., Bublitz N., Blüthgen N., Horst D., Morkel M., Schäfer R, & Sers C. (2020). Reduced replication origin licensing selectively kills KRAS-mutant colorectal cancer cells via mitotic catastrophe. Cell Death & Disease, 11(7), 499.

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Cellular Assays with Diverse Chemical Modulators

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The drugs were used at the following concentrations: Auxin (Sigma I5148), 500 µM; doxycycline (Sigma D3447), 2 µg ml⁻¹; asunaprevir (Selleckchem S4935), 3 µM; monastrol (Selleckchem S8439), 50 µM; MPI-0479605 (Selleckchem S7488), 1 µM; genistein (Sigma G6649), 10 µM; SP600125 (Selleckchem S1460), 10 µM; abemaciclib (Selleckchem S5716), 50 nM or 0.5 µM; K03861 (Selleckchem S8100), 400 nM or 1 µM; palbociclib (Selleckchem S1579), 120 nM or 1 µM; aphidicolin (Sigma A0781), 0,4 µM or 1 µM; hydroxyurea (Selleckchem S1896), 2 mM; PHA767491 (Sigma PZ0178), 1 µM; RO3306 (Calbiochem 217699), 10 µM; dihydrocytochalasin D (Sigma D1641), 0,75 µM; latrunculin B (Sigma L5288), 5 µM; 5′-chloro-2′-deoxyuridine (CIdU) (Sigma C6891), 100 µM; 5′-iodo-2′-deoxyuridine (IdU) (Sigma I7125), 100 µM.

Gemble S., Wardenaar R., Keuper K., Srivastava N., Nano M., Macé A.S., Tijhuis A.E., Bernhard S.V., Spierings D.C., Simon A., Goundiam O., Hochegger H., Piel M., Foijer F., Storchová Z, & Basto R. (2022). Genetic instability from a single S phase after whole-genome duplication. Nature, 604(7904), 146-151.

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Anticancer Drug Screening in Organoids

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Organoids were seeded into 96-well plates at 1,000 cells/well (Day 0). The indicated concentrations of Rucaparib (Selleckchem, S1098), Niraparib (MCE, HY-10619), Olaparib (Selleckchem, S1060), Gemcitabine (MCE, HY-B0003), Doxorubin (sigma, D1515), Paclitaxel (Selleckchem, S1150), Carboplatin (Sigma, 1096407), Seliciclib (MCE, HY-30237), PHA767491(Sigma, PZ0178), BAY1895344 (Selleckchem, S8666), Chloroquine (Selleckchem, S4157) and YKL-5–124 (a gift from Dr. Kwok-kin Wong) were added on the day following seeding (Day 1). Media were changed, and fresh drug was added on Day 3. Cell viability was assessed on Day 5 by adding10 μl PrestoBlue and incubating for 30 min in 37℃. Fluorescence was measured in a FlexStation® 3 Multi-Mode Microplate Reader (BOSTONind). Results were normalized to DMSO controls, and IC₅₀ values were determined using Graphpad Prism 7.

Zhang S., Iyer S., Ran H., Dolgalev I., Gu S., Wei W., Foster C.J., Loomis C.A., Olvera N., Dao F., Levine D.A., Weinberg R.A, & Neel B.G. (2020). Genetically Defined, Syngeneic Organoid Platform for Developing Combination Therapies for Ovarian Cancer. Cancer discovery, 11(2), 362-383.

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ESCC Cell Line Cultivation and Drug Treatment

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Human ESCC KYSE150 cells were purchased from the Cancer Hospital of the Chinese Academy of Medical Sciences (Beijing, People’s Republic of China), and KYSE30 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, People’s Republic of China). Both Cells were maintained in RPMI 1640 medium supplemented with 10% FBS (Hyclone, Thermo Fisher Scientific, Waltham, MA, USA) and grown at 37°C with 5% CO₂. Cis, 5-FU, and PHA-767491 were purchased from Sigma and were added to the cultures at the given time points.

Cao J.X, & Lu Y. (2018). Targeting CDC7 improves sensitivity to chemotherapy of esophageal squamous cell carcinoma. OncoTargets and therapy, 12, 63-74.

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Small Molecule Inhibitor Protocol

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The ATR inhibitor VE822 and the CDK1 inhibitor (RO-3306) were purchased from Selleck (S7102) and Calbiochem (217699) respectively. Hydroxyurea (HU) was purchased from TCI Chemicals (H0310), Cordycepin and N-Acetyl-cysteine (NAC) were purchased from Wako (017-05131), 5,6-dichloro-1-β-dribofuranosylbenzimidazole (DRB) from Cayman Chemical (10010302), and triptolide from AdipoGen (AG-CN2-0448). 5-Ethynyl Uridine (EU) was purchased from Thermo Fisher Scientific (E10345). CDC7 inhibitor PHA-767491 was purchased from Sigma-Aldrich (PZ0178). Concentrations and conditions used in each assay are listed in Figure legends.

Kurashima K., Kamikawa Y, & Tsubouchi T. (2024). Embryonic stem cells maintain high origin activity and slow forks to coordinate replication with cell cycle progression. EMBO reports.

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Cell Cycle Synchronization and DNA Replication Inhibition

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Cells were treated with 1 µM palbociclib (Cdk4/6 inhibitor, Selleckchem S1579), or with 0.5 µM abemaciclib (Cdk4/6 inhibitor, Selleckchem S5716) or with 1 µM K03861(Cdk2 inhibitor, Selleckchem S8100) for 16 h to synchronize cells at G1/S transition, and were collected (indicated by ‘G1 arrest’ in the figures). Alternatively, cells were then washed five times with PBS and released in S phase for 10 h before being collected. To extend G1 duration cells were treated with 160 nM palbociclib or with 50 nM abemaciclib or with 400 nM K03861 for 16 h and were collected (indicated by ‘G1 lengthening’ in the figures). Alternatively, cells were then washed 5 times in PBS and released in S phase for 10 h before being collected.
To inhibit DNA replication, cells were released in S phase in the presence of low doses of Aphidicolin (APH, A0781 from Sigma-Aldrich), a DNA replication polymerase inhibitor, or of PHA767491 (PZ0178 from Sigma-Aldrich), a Cdc7 inhibitor (indicated by ‘release in S phase + APH’ or ‘release in S phase + PHA’, respectively, in the figures). Doses were chosen to significantly decrease EdU incorporation without affecting the levels of DNA damage.

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