Dasatinib (DASA), imatinib (IMA), nilotinib (NILO), and ponatinib (PONA) were purchased from Cayman Chemical Company (Ann Arbor, USA). After dissolving in DMSO, TKI were kept at – 20 °C until use. Stock concentrations were 20 mM for DASA, IMA and PONA, and 3 mM for NILO, respectively. Peak and IC50 concentrations were chosen according to the literature: DASA 150 nM (peak) and 10 nM (IC50); IMA 3000 nM (peak) and 600 nM (IC50); NILO 3000 nM (peak) and 30 nM (IC50); PONA 145 nM (peak) and 70 nM (IC50) (Peng et al. 2004 (link); Kantarjian et al. 2006 (link), 2010 (link); Rix et al. 2007 (link); Weisberg et al. 2007 (link); Cortes et al. 2012 (link); Menna et al. 2020 (link)). Direct effects of DMSO (maximum level was 0.1% v/v in NILO peak samples) were ruled out by including DMSO only as control throughout all experiments.
Dasatinib
Manufactured by Cayman Chemical
Sourced in United States
Dasatinib is a multi-targeted tyrosine kinase inhibitor. It acts by inhibiting several protein kinases, including BCR-ABL, SRC, c-KIT, PDGFR, and EPH receptor kinases.
Automatically generated - may contain errors
Lab products found in correlation
Nilotinib, by Cayman Chemical (4 mentions)
Gefitinib, by Cayman Chemical (3 mentions)
Imatinib, by Cayman Chemical (2 mentions)
Pf 573228, by Cayman Chemical (2 mentions)
Uk14304, by Merck Group (2 mentions)
Pf562271, by Adipogen (2 mentions)
Gdc 0994, by Apexbio (2 mentions)
Zstk474, by Cell Signaling Technology (2 mentions)
Ionomycin, by Thermo Fisher Scientific (2 mentions)
Anisomycin, by Merck Group (2 mentions)
Yohimbine, by Merck Group (2 mentions)
Jasplakinolide, by Cayman Chemical (2 mentions)
Bapta am, by Selleck Chemicals (2 mentions)
Ly2584702, by Selleck Chemicals (2 mentions)
Y 27632, by Enzo Life Sciences (2 mentions)
Latrunculin b, by Enzo Life Sciences (2 mentions)
Imatinib, by Phoenix Pharmaceuticals (2 mentions)
Ly294002, by LC Laboratories (2 mentions)
Ponatinib, by Cayman Chemical (1 mentions)
Ibrutinib, by Selleck Chemicals (1 mentions)
19 protocols using dasatinib
1
Tyrosine Kinase Inhibitor Dose Assessment
2
Evaluating the Efficacy of DNA-PK and KIT Inhibitors
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Cell lines were treated with the following agents, either alone or in combination as indicated in text. DNA-PK inhibitors: NU7441 (Selleckchem), M3814 (Merck); KIT signaling inhibitors: dasatinib (Cayman Chemical), ibrutinib (Selleckchem), FTY720 (Cayman Chemical), and AAL(S) (synthesized by A/Prof Jonathan Morris, School of Chemistry, UNSW as described (43 (link))). Dimethyl sulfoxide was used as the solvent for all compounds. Final vehicle concentration was below 0.1% for all experiments.
3
Identification of Antigen-Specific CD8+ T Cells
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After in vitro stimulation, PBMCs were incubated with phycoerythrin (PE)-conjugated tetramer on ice for 30 min. Following wash in Dulbecco’s phosphate-buffered saline (DPBS, GIBCO) with 0.5% bovine serum albumin (BSA, Iwai Chemicals), the cells were stained with fluorescein isothiocyanate-conjugated anti-human CD8 antibody (clone RPA-T8, BD Biosciences), allophycocyanin-conjugated anti-human CD3 antibody (clone UCHT1, BD Biosciences), and phycoerythrin-Cy7 (PE-Cy7)-conjugated anti-human CD4 antibody (clone RPA-T4, BD Biosciences) on ice for 20 min. The cells were washed in DPBS with 0.5% BSA and then stained with DPBS containing 0.1 μg/mL 4',6-diamidino-2-phenylindole (DAPI, BD Biosciences). CD8+ T cells that bound to pMHCI tetramer were identified using SH800 (Sony Biotechnology). PBMCs were treated with 450 nM dasatinib (Cayman Chemical) for inhibiting TCR downregulation from surface of T cells before tetramer staining [23 (link)].
4
Dasatinib Inhibition of Neutrophils
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SFK inhibitor Dasatinib (100 nM) (Cayman Chemical, no. 11498) were applied to cultured neutrophils as indicted in the results for 24 h. Treated neutrophils were used for subsequent analysis as described in the results section.
5
Kinase Inhibitor Library Screening
6
Antibody-based FAK and YAP Profiling
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The following antibodies were used: monoclonal FAK (13009S, Cell Signaling Technology); FAKpY397 (8556S, Cell Signaling Technology); YAP (sc-101199, Santa Cruz Biotechnology); Ki-67 (9129S, Cell Signaling Technology). Alexa Fluor 647 anti-MHCII antibody (cz11, clone 130.8E8D9) was a gift of D. Antczak, Cornell University, Ithaca, NY, USA. The FAK inhibitor was PF-573228 (5 μM; 14924, Cayman Chemical). The Src inhibitor was dasatinib (0.1 μM; 11498, Cayman Chemical).
7
Imatinib and Dasatinib Cytotoxicity Assay
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0.5 × 104 Ba/F3 cells expressing mutant Bcr-Abl proteins were plated in each well of 96-well plates in RPMI medium containing 10% FBS and lacking IL-3 (total volume, 100 μL). Imatinib (Ark Pharm) was included in the media in increasing concentrations (final concentrations ranging from 0–20 μM). Dasatinib (Cayman Chemical Company, 11498) was included in the media in increasing concentrations (final concentrations ranging from 0–10 μM). Viable cell numbers were assessed after 48 h using the WST-1 reagent (Roche), according to manufacturer’s specifications. Assays were performed in quadruplicate. A450 readings were plotted versus the logarithm of the concentration of drug and fit to a sigmoidal equation with the hill slope set to 1.0. The concentration of drug resulting in 50% maximal inhibition is reported as cellular IC50.
8
Mammary Epithelial Cell Culture and Sorting
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MECs, including nonmalignant MCF10A (American Type Culture Collection; verified by epithelial cellular morphology in two dimensions, ability to form hollow, polarized acini in 3D rBM gels, and expression of epithelial markers, including E-cadherin and cytokeratins 8 and 14) and premalignant Ha-Ras–transformed MCF10AT (Karmanos Cancer Institute; verified by epithelial cell morphology in two dimensions) were cultured as described (Rubashkin et al., 2014a (link)). Tumorigenic MEC lines PyMT Flox3 and metastatic Met1 (kind gift of the Moses laboratory, Vanderbilt University) were cultured as described previously (Borowsky et al., 2005 (link)).
The 3D multicellular spheroids were generated in rBM (Matrigel; Corning) suspension cultures as described (Rubashkin et al., 2014a (link)). MCF10A and Met1 MECs were flow sorted on a FACSAria II cell sorter (BD Biosciences) via primary and secondary antibody staining for top ∼20% or bottom ∼20% expression of α5 integrin, β1 integrin, or αvβ3 integrin. The chemokine human TGFβ at 10 nM (PeproTech or Sigma-Aldrich) was used for 3 d to induce mesenchymal behavior. The small-molecule inhibitor dasatinib was used at 20 nM (Cayman Chemical).
The 3D multicellular spheroids were generated in rBM (Matrigel; Corning) suspension cultures as described (Rubashkin et al., 2014a (link)). MCF10A and Met1 MECs were flow sorted on a FACSAria II cell sorter (BD Biosciences) via primary and secondary antibody staining for top ∼20% or bottom ∼20% expression of α5 integrin, β1 integrin, or αvβ3 integrin. The chemokine human TGFβ at 10 nM (PeproTech or Sigma-Aldrich) was used for 3 d to induce mesenchymal behavior. The small-molecule inhibitor dasatinib was used at 20 nM (Cayman Chemical).
9
Chemical Compound Preparation Protocol
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Stocks of 200 μM phorbol-12,13-dibutyrate (PDBu, EMD Millipore, #524390), 1 mg/mL anisomycin (Sigma-Aldrich, A9789), 10 mM UK14304 (Sigma-Aldrich, U104), 10 mM yohimbine (Sigma-Aldrich, Y3125), 1 mM gefitinib (Cayman, #13166), 1 mM ionomycin (Peprotech, #5608212), 2 mM jasplakinolide (Cayman, #11705), 25 mM latrunculin B (Enzo Life Sciences, BML-T110–0001), 10 mM PF562271 (AdipoGen, SYN-1064), 10 mM ZSTK474 (Cell Signaling, #13213), 10 mM dasatinib (Cayman, #11498), 10 mM GDC-0994 (APExBIO Technology, B5817), 1 mM LY2584702 (Selleck, S7698), 10 mM BAPTA-AM (Selleck, S7534) were prepared by dissolving the chemicals in DMSO; 10 mM Y27632 (Enzo Life Sciences, #ALX-270–333) in water. Stocks were diluted to the indicated final concentrations in the culture medium. The EGF stock solution was prepared by dissolving EGF (Sigma-Aldrich, E9644) in 10 mM acetic acid to a final concentration of 1 mg/ml. All drug stocks were stored at −20°C. 2-Deoxyglucose (2-DG, MilliporeSigma, #D8375) was dissolved in culture medium to 100 mM and used immediately.
10
Senolytic Drugs: Evaluation and Characterization
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Senolytic drugs used are s follows: JQ1, ARV825, and ABT263 were obtained from Medchem Express (cat#: HY-13030, HY-16954, HY-10087, respectively). OTX015 and 17-DMAG were purchased from Selleck (cat#: S7360 and S1142, respectively)). Quercetin and Dasatinib were purchased from Cayman (cat#: 10005169) and LC Laboratories (cat#: D-3307), respectively.
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