FIGURE 6.

The apoptotic nuclear morphology observed in STP-treated IMR-5 cells overexpressing mCAD correlates with a higher percentage of cells carrying 3′-OH SSBs and DNA damage. IMR-5 cells stably transfected with empty pcDNA3 (Neo) or pcDNA3-mCAD wild-type (mCAD WT) were left untreated (Control) or treated with 1 μm STP for 4 h. A, flow cytometry analysis of ssDNA damage using the monoclonal antibody F7-26. Data are shown as density plots representing size (y axis, in a logarithm scale) versus intensity of red fluorescence (x axis, in a logarithm scale). FSC, forward scatter of light. Cell density (events) is shown on a pseudocolor scale from minimum density (blue) to maximum density (red). The circled populations correspond to cells presenting ssDNA damage. The percentage of gated cells in each condition is indicated. The experiment was repeated three times with a low variability (< 10%). B, TUNEL reactivity and Hoechst nuclear staining of IMR-5 Neo or IMR-5 mCAD WT cells left untreated (Control) or treated with STP. The right panels are magnifications of the insets in the center panels. The arrowheads indicate representative apoptotic stage II and TUNEL-positive nuclei. Scale bar = 40 μm. C, quantification of the data presented in B representing TUNEL-positive nuclei (left panel) and apoptotic nuclei (right panel) in IMR-5 Neo (white bars) or IMR-5 mCAD WT (black bars) cells upon STP treatment. The means ± S.E. of three independent experiments are represented. Asterisks indicate p ≤ 0.01.