The PDGFRA mutant and isogenic control clones were seeded into 96-well plates (5000 cells/well) and allowed to adhere overnight. Cell Counting Kit-8 (WST-8, Dojindo, Kumamoto, Japan) was used to assess cell viability/proliferation. To assess drug sensitivity, culture media containing drugs at concentrations ranging from 0.5 to 8 μM was added to cells the day after seeding. Cell viability was evaluated using the WST-8 kit 4 d after treatment. The drugs used were as follows: temozolomide (Tokyo Chemical Industry, Tokyo, Japan), lenvatinib (Cayman Chemical, Ann Arbor, MI, USA), crenolanib (Abcam, Cambridge, UK), abemaciclib, and palbociclib (LKT Laboratories, St. Paul, MN, USA).
Temozolomide
Manufactured by Tokyo Chemical Industry
Sourced in Japan
Temozolomide is a white to off-white, crystalline powder. It is a chemical compound used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Astaxanthin, by Tokyo Chemical Industry (3 mentions)
Adonixanthin, by Tokyo Chemical Industry (3 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Lenvatinib, by Cayman Chemical (1 mentions)
Cck 8 assay, by Dojindo Laboratories (1 mentions)
Ab6326, by Abcam (1 mentions)
Milli q, by Merck Group (1 mentions)
Lionheart fx automated microscope, by Agilent Technologies (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Wuhan Servicebio Technology (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cisplatin, by MedChemExpress (1 mentions)
8 protocols using temozolomide
1
PDGFRA Mutant Drug Sensitivity Assay
2
Cell Proliferation Assay with Astaxanthin and Temozolomide
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U251MG, GL261, or U87MG cells were seeded onto 96-well plates at a density of 2 × 103 cells/well with DMEM supplemented with 10% FBS and then incubated for 24 h, after which the culture medium was changed to DMEM containing 10% FBS. Then, astaxanthin, adonixanthin or temozolomide (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were added to the culture. Cell proliferation was determined using the CCK-8 assay according to the manufacturer’s instructions (Dojindo, Kumamoto, Japan). After each incubation, 10 μL of CCK-8 solution were added to each well. Plates were incubated for 3 h for 37 °C, and the absorbance was read at 450 nm with a reference wavelength of 630 nm using a Varioscan Flash 2.4 microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).
3
Mutagen Activity Comparison of ACNU, BCNU, and TMZ
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The ACNU (nimustine hydrochloride; 1-[(4-amino-2-methyl-5-pyrimidinyl)-.methyl]-3-(2-chloroethyl)-3-nitrosourea hydrochloride; CAS 55661–38-6) was purchased from Wako Pure Chemicals Co. (Osaka, Japan), and the BCNU (carmustine; 1,3-bis(2-chloroethyl)-1-nitrosourea; CAS 154–93-8) and temozolomide (TMZ; 3,4-dihydro-3-methyl-4-oxoimidazo-[5,1-d][1,2,3,5]tetrazine-8-carboxamide; CAS 85622–93-1) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The structures of these compounds are shown in Fig. 1 .![]()
The structures of the mutagens used in this study
4
Astaxanthin and Temozolomide Cytotoxicity
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GL261 cells were seeded onto 96-well plates at a density of 2 × 103 cells/well with DMEM supplemented with 10% FBS and then incubated for 24 h, after which the culture medium was changed to DMEM containing 10% FBS. Then, astaxanthin, adonixanthin, or temozolomide (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were added to the culture. After 72 h of culture, the culture medium was changed to DMEM containing 10% FBS and treated with BrdU at 10 µM for 3 h. After that, immunocytochemistry was performed according to the protocol of anti-BrdU antibody (abcam, ab6326).
5
Mesoporous Silica Nanoparticle Formulation
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Medical grade chitosan (MW 33 kDa, degree of deacetylation 91.5%, viscosity 75 mPa s 20 °C) was purchased from Chitocean (Newfoundland, Canada). Temozolomide was purchased from Tokyo Chemical Industry (Tokyo, Japan) and used as received. Mesoporous silica nanoparticles colloidal aqueous dispersion (15 wt.%, pore size 4 nm, 0.5 µm particle size) was purchased from Sigma-Aldrich (St. Louis, MO). All the other chemicals are purchased from Sigma-Aldrich (St. Louis, MO) and used as received. All aqueous solutions were prepared with deionized (DI) water obtained using an ultrafiltration system (Milli-Q, Millipore) with a measured resistivity above 18 MΩ/cm.
6
Astaxanthin, Adonixanthin, and Temozolomide Cytotoxicity in GL261 Cells
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GL261 cells were seeded onto 96-well plates at a density of 2 × 103 cells/well with DMEM supplemented with 10% FBS and then incubated for 24 h, after which the culture medium was changed to DMEM containing 10% FBS. Then, astaxanthin, adonixanthin, or temozolomide (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were added to the culture. Cell death was measured by Hoechst 33,342 (Invitrogen, Carlsbad, CA, USA) and propidium iodide (Invitrogen). At 96 h after treatment, the Hoechst 33,342 and propidium iodide were added to the medium to final concentrations of 8.1 and 1.5 µM, respectively, for 15 min. Images of stained cells were captured with a Lionheart™ FX Automated Microscope (BioTek, Tokyo, Japan). The percentage of propidium iodide-positive cells was determined by distinguishing Hoechst 33,342 and propidium iodide fluorescence.
7
Cytotoxicity Assessment of Topotecan and Temozolomide
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Cytotoxicity of compounds was examined in HEK293FT WT and HEK293FT/P1-KO cells by the MTT test (Dia-m, Novosibirsk, Russia). Cells were seeded in 96-well plates (10,000 cells per well) in the DMEM/F12 medium (Servicebio) supplemented with 10% of FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) (Thermo Fisher Scientific) at 37 °C and 5% CO2 in a humidified atmosphere. The tested compounds were added to the medium at nearly 30% confluence. To determine the cytotoxicity of topotecan (Tpc, Actavis, Sindan Pharma, Romania) and temozolomide (Tmz, TCI Chemicals, Zwijndrecht, Belgium), the cells were incubated with one of the compounds at various concentrations for 72 h. All measurements were repeated at least twice.
8
Solvent-Diluted Drug Preparation
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Drugs were diluted from DMSO stocks (10–200 mm ), purchased from MedChem Express, except for temozolomide (TCI Chemicals), DOX (Selleck), and cisplatin (MedChemExpress, 3 m stock in dH20).
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