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Cytotoxicity.tab
Tabular Data - Original format: MS Excel (XLSX) - 2.7 KB - Dec 28, 2020 - 11 Downloads
License: CC BY - Creative Commons Attribution 4.0
19 Variables, 19 Observations - UNF:6:ZSQFdHjirpmZXMIran5RHg== The cytotoxicity of drugs has been assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test and the percentage of viable cells in the experimental groups in relation to untreated cells (control), which viability was taken as 100% had been determined. Exponentially growing cells were seeded into 96-well clear flat-bottom microplates and 24 h later incubated with increasing concentrations of PARPi, ATRi or CHK1i (0.5-30 µM) for 120h. After then, the medium was removed, cell monolayers were washed twice with PBS, 50 µM of MTT (0.5 mg/ml final concentration) was added, and the cells were incubated under the same conditions for 4 h. Formazan crystals resulting from the MTT reduction were dissolved in DMSO. Absorbance was measured at 570 nm.
To select the most effective PARPi/ATRi and PARPi/CHK1i ratios, several concentration ratios of the compounds (1:1; 2:1; 10:1; 1:2; 1:10) were tested at the lowest effective concentration based on
the survival curve (0.5 uM). | |
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Caspase 3_7 activity.xlsx
MS Excel (XLSX) - 1.5 MB - Dec 28, 2020 - 9 DownloadsMD5: f7c8656c51570af6d4772c838402b044
License: CC BY - Creative Commons Attribution 4.0
The activities of caspases 3 and 7 were estimated with CellEvent Caspse-3/7 Green Detection Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The cells were seeded on 96-well plates (15000/well) and after 24 h they were incubated with the appropriate drugs for 24 h or 48 h. The cells were fixed by adding a final concentration of 4% paraformaldehyde solution (10 min, room temperature). Cells were labeled with CellEvent Caspase-3/7 Green Detection Reagent (5 µM) diluted in PBS with 5% FBS to avoid fluorescence background. After activation of caspase 3/7 in apoptotic cells, the four amino acid (Asp-Glu-Val-Asp, DEVD) peptide was cleaved, enabling the dye to bind to DNA, which produced a bright fluorogenic response with absorption/emission maxima of 502/530 nm. | |
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Clonogenic assay.xlsx
MS Excel (XLSX) - 11.0 KB - Dec 28, 2020 - 3 DownloadsMD5: 9cf7a6237d1bdc284aa98ac088a10d7d
License: CC BY - Creative Commons Attribution 4.0
The effect of PARPi, CHK1i, and ATRi on cell growth was assessed using a clonogenic assay. For this analysis, 200 cells were plated onto six-well plates in a growth medium, and after overnight attachment, cells were exposed to the test compounds for 5 days. The cells were washed with a fresh medium and allowed to grow for 10–14 days under drug-free conditions. Then, the cell colonies were fixed with methanol mixed with acetic acid (7:1) for 10 min and stained with 0.5% crystal violet for 20 min. The plates were rinsed with water, air-dried, photographed, and evaluated for colony estimation. Colonies containing more than 50 cells were counted. | |
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Comet assay.xlsx
MS Excel (XLSX) - 170.9 KB - Dec 28, 2020 - 7 DownloadsMD5: b4aeb275dc38445261ef6cd8d4cd1a98
License: CC BY - Creative Commons Attribution 4.0
The alkaline version of single cell gel electrophoresis (comet assay) was used to detect alkali-labile sites, single-strand breaks (SSBs), and DSBs caused by exposure to the tested compounds. Each experiment was repeated three times. In the neutral versions, the tail DNA percentage is positively correlated with DSBs. The cells were plated on 12-well plates (100000 cells/well) and treated with PARPi, ATRi, CHKi, and their combination at the 0.5 µM dose for 2, 24, and 48 h at 37 C. Next, the cells were collected into Eppendorf tubes and rinsed with PBS. The cells were then suspended in 0.75% low melting point agarose dissolved in PBS (pH 7.4) and placed on microscope slides precoated with 0.5% normal melting point agarose. Subsequently, the slides were treated with a cooled lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100, pH 9.0) for 1–24 h at 4 C. Then, slides were placed in the developing buffer (300 mM NaOH, 1 mM EDTA) for 20 min. Electrophoresis was performed in a buffer composed of 30 mM NaOH and 1 mM EDTA at 0.73 V/cm and 290 mA for 20 min. In the neutral version of the comet assay, electrophoresis was run in a buffer consisting of 100 mM Tris and 300 mM sodium acetate, with the pH adjusted to 9.0 using glacial acetic acid. Electrophoresis was performed for 60 min after a 20 min equilibrium period at an electric field strength of 0.41 V/cm and 50 mA at 4 C. The samples were stained with 4,6-diamidino 2-phenylindole (DAPI) (1 g/mL). The slides were stored in a wet chamber at 4 C and analyzed under a fluorescence microscope. The percentage of DNA in the comet tail was chosen as an indicator of DNA damage. Thirty randomly selected cells from each slide were measured using an image analysis system (Nikon, Japan) attached to a COHU 4910 video camera, which was equipped with a UV-1 filter block consisting of an excitation filter (359 nm) and a barrier filter (461 nm) connected to the image analysis system Lucia-Comet v. 4.51 | |
