Crenolanib

Manufactured by Selleck Chemicals

Sourced in United States

Crenolanib is a laboratory chemical compound used in scientific research and development. It is a tyrosine kinase inhibitor that may be utilized in various in vitro and in vivo studies. The core function of Crenolanib is to inhibit the activity of certain cellular enzymes, which can be relevant for investigating signaling pathways and cellular processes.

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Lab products found in correlation

44 protocols using crenolanib

Isolation of Primary Mouse Mammary Fibroblasts

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Primary mouse mammary fibroblasts (MMFs) were purified as described [7] (link), [27] (link). Briefly, mammary tissue was minced and digested with an enzymatic solution [0.15% Collagenase, C0130 (Sigma, Saint Loius, MO, USA), 160 U/ml hyaluronidase (H#4001, Sigma), insulin (#I5500, Sigma), 1 μg/ml hydrocortisone (#H4001, Sigma) and 1% penicillin/streptomycin (ThermoFischer Scientific, Waltham, MA, USA)] in 5% CO₂ overnight at 37 °C. Tissue was neutralized with 10%FBS-DMEM (ThermoFisher), centrifuged and resuspended in 10%FBS-DMEM. Gravity separation was then utilized to separate out heavier epithelial organoids from the fibroblast fraction. After 24 hours, culture media was replaced with fresh 10%FBS-DMEM. For crenolanib experiments, crenolanib (Selleckchem, Houston, TX, USA) was added to culture media at a concentration of 1 μM for 24 hours.

Hammer A.M., Sizemore G.M., Shukla V.C., Avendano A., Sizemore S.T., Chang J.J., Kladney R.D., Cuitiño M.C., Thies K.A., Verfurth Q., Chakravarti A., Yee L.D., Leone G., Song J.W., Ghadiali S.N, & Ostrowski M.C. (2017). Stromal PDGFR-α Activation Enhances Matrix Stiffness, Impedes Mammary Ductal Development, and Accelerates Tumor Growth. Neoplasia (New York, N.Y.), 19(6), 496-508.

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Investigating YAP Signaling Modulation

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Crenolanib (Selleck Chemicals) and Dasatinib (TSZ Chemicals) was added to cells at final concentrations from 0.1–10μM. The following primary antisera were used for immunoblot analysis: PDGF Receptor (28E1 Cell Signaling, Danvers, MA), actin (C-11 Santa Cruz, Dallas, TX), phospho YAP^Y357 (ab62751 abcam, Cambridge, MA), phospho YAP^S127 (4911 Cell Signaling), total YAP (63.7 Santa Cruz), lamin B1 (D8P3U Cell Signaling), GAPDH (MAB374 Millipore, Temecula, CA), phospho Src^Y416 (2101 Cell Signaling), Src (L4A1 Cell Signaling), and Mcl-1 (S-19 Santa Cruz). The following primary monoclonal antibodies were used for immunofluorescence: total YAP (63.7 Santa Cruz) and anti-FLAG M2 (F1804 Sigma Adrich). For ChIP immunoprecipitation assays we employed an anti-YAP monoclonal antibody (63.7 Santa Cruz). ProLong Antifade with 4′,6-diamidino-2-phenylindole (DAPI, Life Technologies) was used for nuclear staining. The following primers used are listed in Table 1.

Smoot R.L., Werneburg N.W., Sugihara T., Hernandez M.C., Yang L., Mehner C., Graham R.P., Bronk S.F., Truty M.J, & Gores G.J. (2017). Platelet-Derived Growth Factor Regulates YAP Transcriptional Activity via Src Family Kinase Dependent Tyrosine Phosphorylation. Journal of cellular biochemistry, 119(1), 824-836.

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Glioblastoma Stem Cell Culturing and Treatments

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We have used the same clinical materials reported in our previous papers
[24 (link), 25 (link)]. In brief, the CSC cells were retrieved from adult patients affected by GBM and undergoing craniotomy at the Institute of Neurosurgery, Catholic University-School of Medicine of Rome, Italy. Dissociated cells were cultured in the presence of human recombinant EGF (20 ng/ml; PeproTech, Rocky Hill, NJ), human recombinant bFGF (10 ng/ml; PeproTech), in DMEM/F12 (1:1) serum-free medium (Invitrogen, Carlsband, CA) containing L glutamine 2 mM, glucose 0.6%, putrescine 9.6 ug/ml, progesterone 0.025 mg/ml, sodium selenite 5.2 ng/ml, insulin 0.025 mg/ml, apo-transferrin sodium salt 0.1 mg/ml, sodium bicarbonate 3 mM, Hepes 5 mM, BSA 4 mg/ml, heparin 4 ug/ml (all purchased by Sigma-Aldrich). Floating neurospheres were dissociated with Accutase at 37°C (Merck-Millipore). In some cases, neurospheres were passaged up to passage P60 and the experiments were performed between P14 and P60. Cell starvation was planned for 2 days in Stem Medium w/o EGF and bFGF. Subsequently, PDGF-AA was added (40 ng/ml, Peprotech) for different time points (5′, 10′, 30′, 120′ and 24 hours). Cells treatments were performed with GSI-X (also named L-685,458, Calbiochem), AG1478 (Calbiochem) and Crenolanib (CP-8685596, Selleckchem).

Cenciarelli C., Marei H.E., Zonfrillo M., Pierimarchi P., Paldino E., Casalbore P., Felsani A., Vescovi A.L., Maira G, & Mangiola A. (2014). PDGF receptor alpha inhibition induces apoptosis in glioblastoma cancer stem cells refractory to anti-Notch and anti-EGFR treatment. Molecular Cancer, 13, 247.

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TNBC Cell Line Cultivation Protocol

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Human normal breast epithelial cell (MCF-10A) and human TNBC cell lines (MDA-MB-231, MDA-MB-468, MDA-MB-436, HCC-1937) were available from the ATCC Cell Bank (Manassas, VA). Cell samples were cultivated in DMEM (Invitrogen, Carlsbad, CA), adding FBS (10%) and penicillin–streptomycin mixture (1%) under 37 °C and 5% CO₂. Besides, cycloheximide (CHX; 5 µM) and MG132 (10 μM) were both purchased from Sigma-Aldrich (St. Louis, MO). Crenolanib (1 μm) was bought from Selleckchem (Houston, TX).

Dong F., Ruan S., Wang J., Xia Y., Le K., Xiao X., Hu T, & Wang Q. (2020). M2 macrophage-induced lncRNA PCAT6 facilitates tumorigenesis and angiogenesis of triple-negative breast cancer through modulation of VEGFR2. Cell Death & Disease, 11(9), 728.

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Cell Proliferation Kinetics Monitoring

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Cell proliferation kinetics were monitored and recorded with the IncuCyte System (Essen BioScience, Ann Arbor, MI). Imatinib, pazopanib, and crenolanib were all obtained from Selleck (Houston, TX). For all in vitro experimentations, all three compounds were dosed at 1 μM. R1507 was dosed at 100 nM.

Heske C.M., Yeung C., Mendoza A., Baumgart J.T., Edessa L.D., Wan X, & Helman L.J. (2016). The Role of PDGFR-β Activation in Acquired Resistance to IGF-1R Blockade in Preclinical Models of Rhabdomyosarcoma. Translational Oncology, 9(6), 540-547.

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Crenolanib Modulates PDGF Signaling

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Crenolanib (CP-868596) was purchased from Selleck Chemicals LLC (USA). Recombinant human PDGF-BB protein was purchased from R&D system (USA). α-SMA, desmin, vimentin, and collagen-I, signal transducer and activator of transcription 3 (STAT3), phosphor-STAT3 (p-STAT3), signal transducer and activator of transcription 5 (STAT5), phosphor-STAT5 (p-STAT5), serine-threonine protein kinase (AKT), phosphor-AKT (p-AKT), extracellular regulated kinase (ERK), phosphor-ERK (p-ERK), PDGFRα, phosphor-PDGFRα (p-PDGFRα), PDGFRβ, phosphor-PDGFRβ (p-PDGFRβ), and β-actin were purchased from Abcam technology (USA).

Yan J.F., Huang W.J., Zhao J.F., Fu H.Y., Zhang G.Y., Huang X.J, & Lv B.D. (2017). The platelet-derived growth factor receptor/STAT3 signaling pathway regulates the phenotypic transition of corpus cavernosum smooth muscle in rats. PLoS ONE, 12(2), e0172191.

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Tyrosine Kinase Inhibitor Preparation

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PLX4720, AZD8055, GDC0941, BEZ235, Canertinib, Erlotinib (Tarceva), Gefitinib (Iressa), Crenolanib, Lapatinib (Tykerb) and Dasatinib (Sprycel) (all from Selleck Chemicals, Houston, TX, USA) were diluted in DMSO at 10 mM/ml and stored at −20°C until use; when added to cell cultures, the final DMSO concentration was 0.25–0.5%.

Dugo M., Nicolini G., Tragni G., Bersani I., Tomassetti A., Colonna V., Del Vecchio M., De Braud F., Canevari S., Anichini A, & Sensi M. (2015). A melanoma subtype with intrinsic resistance to BRAF inhibition identified by receptor tyrosine kinases gene-driven classification. Oncotarget, 6(7), 5118-5133.

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Chemical Agents for Cell Studies

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The following chemicals were used in this study: crenolanib (Selleck, CP-868596), nocodazole (Wako, 31430-18-9), MG132 (Wako, 133407-82-6), BI2536 (AdooQ, A10134), and SiR-DNA (Spirochrome, CY-SC007).

Watanabe K., Takao D., Ito K.K., Takahashi M, & Kitagawa D. (2019). The Cep57-pericentrin module organizes PCM expansion and centriole engagement. Nature Communications, 10, 931.

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Small Molecule Inhibitor Evaluation

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FF-10101 and FLA-9430 were provided by FUJIFILM. Gilteritinib (#S7754), quizartinib (#S1526), crenolanib (#S2730), and midostaurin (#S8064) were purchased from Selleckchem.

Ferng T.T., Terada D., Ando M., Tarver T.C., Chaudhary F., Lin K.C., Logan A.C, & Smith C.C. (2022). The irreversible FLT3 inhibitor FF-10101 is active against a diversity of FLT3 inhibitor resistance mechanisms. Molecular cancer therapeutics, 21(5), 844-854.

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Inhibition of Pim Kinases and FLT3-ITD in AML

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AZD1208, an orally bioavailable highly selective inhibitor with single nanomolar potency against all three Pim kinases, Pim-1, Pim-2 and Pim-3 (14 (link)), provided by AstraZeneca, was used at 1 μM based on inhibition of BAD phosphorylation at serine 112 as a pharmacodynamic endpoint (16 (link)) and on phase I clinical trial data (11 ). The FLT3 inhibitors quizartinib and crenolanib (Selleck Chemicals, Houston, TX), sorafenib (LC Laboratories, Woburn, MA) and gilteritinib (Active Biochem, Maplewood, NJ), all clinically active in FLT3-ITD AML, were used at pharmacologically relevant concentrations (17 (link)–20 (link)). The proteasome inhibitor MG132 and the USP9X inhibitor WP1130 were purchased from EMD Millipore, Billerica, MA.

Kapoor S., Natarajan K., Baldwin P.R., Doshi K.A., Lapidus R.G., Mathias T.J., Scarpa M., Trotta R., Davila E., Kraus M., Huszar D., Tron A.E., Perrotti D, & Baer M.R. (2017). Concurrent inhibition of Pim and FLT3 kinases enhances apoptosis of FLT3-ITD acute myeloid leukemia cells through increased Mcl-1 proteasomal degradation. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(1), 234-247.

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