Fig. 4

Cell cycle and Reactive Oxygen Species. a) Representative flow cytometry results of cell cycle analysis of CaSki and C-4I cells 24 h post-irradiation. b) Stacked percentages of cells in the G1, S and G2/M cell cycle phases (n ≥ 3). *P < 0.05 and **P < 0.01 refer to IR-treated vs NT samples. §P < 0.05 refers to the comparison between CaSki and C-4I cells. Stacked percentages of cell cycle phases with/without 2 Gy IR for the different silenced conditions in CaSki (c) or C-4I (d) cells (n ≥ 3). Statistical significances have been evaluated only for the G2/M phases through a paired t-test. *P < 0.05 and **P < 0.01 refer to IR-treated vs NT samples. §P < 0.05 refers to the silenced gene condition vs the control (siC).  e) Representative pictures of morphological features of mitotic catastrophe in C-4I cell line: micronucleation and multipolar mitotic spindles (indicated by arrows) were clearly evident 24 h after 2 Gy IR. Staining of α- tubulin (green) and DAPI (blue). f) ROS-associated DCFH-DA fluorescence in CaSki and C-4I cells at 2 h and 4 h after 2 Gy IR, compared to the corresponding non-treated cells (n = 3). To establish statistically significant differences, unpaired t-test was carried out. **P < 0.01 and ***P < 0.001 refer to IR-treated (at different time points) vs NT samples. §P < 0.05 and §§§P < 0.001 refer to the comparison between CaSki and C-4I cells, for each time point. g) Comparison of DCFH-DA fluorescence in silenced CaSki and C-4I cells 2 h post-IR (n ≥ 3). To establish statistically significant differences, unpaired t-test was carried out. *P < 0.05 refers to IR-treated vs NT samples. §§P < 0.01 refer to the silenced gene condition vs the control (siC), for each treatment