Overexpression of H2AX leads to aberrant chromatin aggregation and apoptosis. (A) Fluorescence microscopic analysis of U-2 OS cells transiently overexpressing empty GFP vector (control) or GFP-H2AX for 24 h. Note the chromatin aggregation in cells transfected with H2AX. Nuclei stained with DAPI. Scale bar indicates 10 μm. (B,C) Quantification of U-2 OS cells with chromosome aggregation 24 h after transfection with empty GFP vector (control in B), GFP-H2AX, GFP-H2AX-S139A, empty myc-tag vector (control in C) or myc-H2A. Each bar indicates mean + standard error of at least three independent experiments with at least 100 cells counted per experiment. (D) TUNEL staining of U-2 OS cells transiently transfected with GFP-H2AX and either showing an apoptotic morphology (top) or chromatin aggregation (bottom). Note that the cell with chromatin aggregation is TUNEL-negative. Nuclei stained with DAPI. Scale bar indicates 10 μm.