Titan x ray generator

Cells were seeded into T25 flasks. The number of cells was calculated to reach a confluence of about 70%. After reaching adequate confluence, the medium was changed to a serum-reduced medium containing only 2% of FBS. Subsequently, the cells were treated with the kinase inhibitors. For the DNA-PK inhibitor CC 115 and the ATM inhibitor AZD0156, concentrations of 0, 0.5, 1 and 2 µM were used; for the ATR inhibitor VE 822 cells were treated with 0, 0.1 and 0.5 µM. After 3 h half of the flasks were irradiated with 2 Gy ionizing radiation (IR) by an ISOVOLT Titan X-ray generator (GE, Ahrensburg, Germany). After 48 h of treatment, the cells, including the supernatant, were harvested and stained with 10 µL of a mixture of equal parts APC Annexin V (BD Biosciences, Franklin Lakes, NJ, USA) and 7 AAD (BD Biosciences, Franklin Lakes, NJ, USA) and incubated on ice for 30 min. Subsequently the staining agent was removed, and the cells were centrifuged and resuspended in cold Ringer’s solution (Fresenius, Bad Homburg, Germany) in preparation for flow cytometry analysis. This was performed with the Cytoflex flow cytometer (Cytoflex, Beckman Coulter, Brea, CA, USA). For data evaluation, the Kaluza analysis software (Beckman Coulter, Brea, CA, USA) was used.

FEN1 inhibitor FEN1-IN-4 (Selleck Chemicals LLC, Huston, TX, USA) was dissolved in dimethyl sulfoxide (DMSO; Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and stored in aliquots at −80 °C. During experiments, the required amount was thawed immediately before use. A concentration of 25 μM was selected for the experimental setup.
The irradiation of cells with 2 Gy of IR was performed by an ISOVOLT Titan X-ray generator (GE, Ahrensburg, Germany). In all experiments, there were four experimental conditions. Cells were treated either with 2 Gy of IR or 25 μM FEN1-IN-4 or a combination therapy of both. For the combination therapy the cells were irradiated 3 h after treatment with the inhibitor. The control group was treated analogously only with DMSO, unless otherwise noted.