Isovolt titan

Cervical carcinoma HeLa cells (ATCC No. CCL-2) cells were used throughout the study. A single exposure to 10 Gy X-ray was applied (250 kV, 16 mA, 2.5 Gy min⁻¹ – GE Isovolt Titan) to induce DNA damage and trigger DDR. On exposure to IR, cells were incubated in a humidified environment, with 5% CO₂ at 37 °C as indicated. Sham-irradiated control cells were included. C2C12 (ATCC No CRL-1772) cells were used for CRISPR-Cas9 experiments. HeLa and C2C12 cells were cultured in DMEM (4.5 g l⁻¹ glucose, Biochrom AG) supplemented with 10% and 20% fetal calf serum (Biochrom AG), respectively. All media were supplemented with 2 mM L-glutamine (Sigma), 100 U per ml penicillin and 100 μg ml⁻¹ streptomycin (Sigma). All cell lines were tested for mycoplasma and found free of contamination (MycoAlert, Lonza).

Cells were irradiated at 120 kV by an X-ray machine (ISOVOLT Titan; GE, Ahrensburg, Germany) with IR doses of 2 Gy. Unirradiated samples were processed along with irradiated samples.

Three tubes were left untreated, three were ex vivo irradiated with 2 Gy, and two were irradiated with 8 Gy ionizing radiation. Cells were irradiated at a dose rate of 2 Gy per minute with a 120 kV X-ray machine (ISOVOLT Titan; GE, Ahrensburg, Germany). Furthermore, cells in two U-bottom push cap tubes were incubated for 0.5 h with 10 μg/mL Oxaliplatin (pharmacy of the University Hospial, Erlangen, Germany) and afterwards irradiated with 2 Gy ionizing radiation. Cells were incubated for further 48 h with Oxaliplatin in the 1.4 mL U-bottom tubes at 37°C before flow cytometry analysis.