FIGURE 4:

Analysis of HS-induced DNA damage by TdT and PolI nucleotide analogue incorporation assays. Control (untreated) and heat-stressed (45.5°C, 30 min) mcf-7 cells were fixed, permeabilized, and subjected to a fluorescein-labeled nucleotide analogue (green channel) incorporation assay using either E. coli DNA polymerase I (A) or terminal deoxynucleotidyl transferase (B). As a positive control, mcf-7 cells fixed and then treated with DNase I (0.1 U/ml, 30 min) were used. After incorporation of a nucleotide analogue, the cells were immunostained with an antibody against γH2AX (red). The DNA was stained with DAPI (blue). A superimposition of the green and red channels is shown as “Merge.” Scale bars: 20 and 5 μm (for last row).