Endogenous PARP1 colocalizes with the DSB-flanking proteins γ-H2AX and ATM following laser microirradiation. a, human SK-N-SK cells were labeled for immunofluorescence immediately after laser microirradiation using anti-PARP1, anti-PAR, phospho-specific anti-H2AX S139, and phospho-specific anti-ATM S1981 antibody. Line profiles of background-corrected intensities were plotted for PARP1, PAR, γ-H2AX, ATM pS1981, and DAPI in each nucleus (right). The arrows indicate the direction of individual line scans from overlapping tracks in DAPI-stained nuclei. Bar, 10 μm. b, microirradiated cells were immediately fixed and labeled for immunofluorescence using anti-PARP1 and anti-Rad51 antibody. c, because laser microirradiation also generated SSBs, cells were labeled to visualize the SSB protein XRCC1 immediately after laser microirradiation. Synthesis of PAR was also labeled using anti-PAR antibody. Colocalization is shown in the right panel. DNA was stained with Hoechst 33258. d, schematic representation of temporal dynamics of SSB and DSB proteins recruitment to DNA damage induced by laser microirradiation.
