Figure 4. Knock down of CCDC6 increases vulnerability to genotoxic stress by UV induced DNA damage.

HCT116 cells were transduced with CCDC6 shRNA expressing lentivirus or empty control followed by UV irradiation (0.002 J/cm²), 48 hours after transduction. Cells were harvested at 2, 6 and 24 hours after irradiation and analyzed for the cell cycle using flow cytometry. The percentages of the cell populations in each phase of the cell cycle for every time point are depicted as bars in the diagram (A). In control cells, UV irradiation results in G₁ and S phase increase while in CCDC6 knock down cells UV irradiation causes a reduction of cell population in S phase and an increase in G₂ phase. A representative experiment is shown. (B) The apoptotic levels were measured by flow cytometric assessment of the subG₀/G₁ population. Knock down of CCDC6 increase UV-mediated cell death. (C) Cell survival analysis by Po-PRO and 7-ADD staining (excluding the double positive cells) revealed a significantly decreased cell survival of cells lacking CCDC6. Error Bars represent 3 independent experiments. (D) Cell lysates of HCT116 cells treated with etoposide (20 µM) or radiated with UV (0.002 J/cm²) for 2, 6 or 24 hours and the untreated controls were resolved on a SDS-PAGE and probed for pH2Ax Ser139. Upon UV irradiation, pH2Ax Ser139 levels arise earlier and to a higher extent in CCDC6 knock down cells compared to the control. UV irradiation is causing high levels of pH2Ax Ser139 even in 2 hours after irradiation in CCDC6 knock down cells. The effect is similar upon etoposide treatment, although less dramatic. (E) Increased basal levels and nuclear foci of pH2Ax Ser139 are present in CCDC6 knock down cells, even in the absence of any additional treatment.