Iscove s modified dulbecco s medium

HeLa cells were purchased from ATCC and grown in DMEM medium (Corning) supplemented with 10% FBS (Sigma-Aldrich) and 1% (v/v) penicillin/streptomycin (Life Technologies) in a humidified incubator at 37°C with 5% CO₂. HAP1 cells were purchased from Horizon Discovery and cultured in Iscove’s modified Dulbecco’s medium (Corning) supplemented with 10% FBS (Sigma-Aldrich) and 1% (v/v) penicillin/streptomycin (Life Technologies) in a humidified incubator at 37°C with 5% CO₂. Cells were seeded onto glass coverslips and grown for 24 h, washed twice with PBS and fixed for 20 min with 4% paraformaldehyde at room temperature. After rinsing twice with PBS, cells were permeabilized for 30 min with 0.1% saponin in PBS, treated with RNase A (Roche) and subsequently blocked in 2% BSA blocking buffer for 1 h. Next, cells were incubated with the anti-G-quadruplex DNA 1H6 (Millipore) antibody (1:200) overnight at 4°C. Coverslips were rinsed four times with PBS and subsequently incubated with anti-Mouse IgG Atto 488 (Sigma-Aldrich) (1:200) for 1 h at room temperature followed by further four washes in PBS. Cells were then stained with 100 nM Acti-stain™ 670 phalloidin (Cytoskeleton, Inc) for 30 min followed by four washes in PBS. Coverslips were mounted on glass slides using DAPI-containing mounting media (Invitrogen) and analyzed using an LSM710 confocal microscope (Carl Zeiss AG).