Figure 1.

GCV-induced nuclear DNA damage triggers cellular senescence or death. (a) HCA2 cells expressing 3MR were given the indicated concentrations of GCV 48 h after plating; media+or −GCV was replenished every 2 days. Cell number was determined 5 and 12 days later and is plotted as the log of the number of cell divisions. (b) HCA2-3MR cells were treated as in (a) and collected 7 days after GCV addition for cell-cycle analysis by flow cytometry. Cell-cycle distribution is plotted as the percentage of cells in each phase of the cell cycle for each concentration of GCV. (c) HCA2-3MR cells were treated as in (a) and collected 4 days after GCV treatment for live-dead apoptosis analysis using flow cytometry. Representative samples are shown and the data are plotted in the right graph as the percentage of cells in the live (Q4), early apoptotic (Q3) or late apoptotic/necrotic (Q1 and Q2) quadrants for each concentration of GCV. (d) HCA2 or HCA2-3MR cells were plated on glass slides and fixed for immunofluorescence following 48 h treatment with the indicated doses of GCV. Left panels: 53BP1 (green) shows both diffuse nuclear staining and punctate focal staining (DNA damage foci). Nuclear DNA was stained with DAPI (4,6-diamidino-2-phenylindole; blue). The red arrowheads show cells with ≥3 53BP1 foci. Right panel: The percentage of nuclei with ≥3 53BP1 foci was determined in at least 200 cells per GCV concentration. Light gray: HCA2-control cells; dark gray: HCA2-3MR cells. (e) HCA2 or HCA2-3MR cells were plated and counted 24 h later (day 0) and 7 days following GCV addition at the indicated concentrations (media+or −GCV were replenished every 2 days). (f) HCA2 cells were treated as in (a) and collected 4 days after GCV treatment for live-dead apoptosis analysis by flow cytometry. Scatter plots are shown in Supplementary Figure S2A. The data is plotted as percentage of cells in the live (Q4), early apoptotic (Q3) or late apoptotic/necrotic (Q1 and Q2) quadrants for each concentration of GCV