Propidium iodide rnase staining solution

Cells were fixed in ice-cold 70% ethanol and stained with Propidium Iodide/RNase Staining Solution (F10797, Thermo Fisher Scientific) with RNase A solution (Novagen) added at 100μg/ml after fixation. DNA contents were measured using Gallios flow cytometer (Beckman Coulter) using FlowJo software (FlowJo, LLC) after 24 hours of drug exposure. G1 phase synchronization was achieved in logarithmically-growing A549 lung cancer cells treated with nocodazole 20ng/ml (Sigma Aldrich) for 4 hours with cells isolated by mitotic shake-off before seeding onto tissue culture plates for 2 hours before CYC065 or vehicle treatment. Independent triplicate replicate experiments were performed.

Cells were incubated in the presence of inhibitors for the corresponding amount of time. One hour prior to fixation 10 μM ethylene-deoxyuridine (EdU, Thermofisher) was added to the media. Subsequently, the cells were trypsinized and fixed in ice-cold absolute ethanol. The cells were re-hydrated via PBS wash and permeabilized with 0.5% Triton X-100 in PBS for 15 min at room temperature with rotation. After a PBS wash, a click chemistry reaction cocktail was added to the cells (100 mM Tris HCl pH 7.6, 4 mM CuSO₄, 2.5 μM azide-fluor488 (Sigma), 100 mM Sodium ascorbate (Sigma)) and incubated for 30 min at room temperature, protected from light. After a PBS wash, propidium iodide/RNase staining solution (Thermo) was added to the cells for 30 min. The cell cycle profiles were acquired on a BD LSRII flow cytometer and analysed with the BD FACSdiva software.