Fig. 5. Correlation between DNA replication and the UV-induced H2AX phosphorylation in A549 cells.

Exponentially growing cells were pulse-labeled with EdU for 60 min then were exposed to UV light and subsequently incubated for 30 min before culture termination. The incorporated EdU was detected by the “click-iT™” methodology using Alexa Fluor 488 azide (green fluorescence), γH2AX was detected using Alexa Fluor 633 secondary Ab (far red fluorescence), DNA was counterstained with DAPI. Using paint-a-gate analysis, the EdU incorporating cells (above the skewed dashed line, panel A) were colored red. As shown in panel B the red colored cells demonstrate expression of γH2AX. Panel C shows the bivariate analysis of γH2AX expression vs EdU incorporation. A strong correlation (R²=0.98) between the extent of H2AX phosphorylation (intensity of γH2AX IF) and the level of EdU incorporation is seen among the EdU incorporating cells outlined with the oval dashed line (the regression plot is shown by the solid line). The subpopulation of cells with increased expression of γH2AX but not incorporating EdU (Sel) was secondarily gated and electronically painted green. These cells were characterized by high intensity of maximal pixel of DAPI fluorescence revealing their highly condensed chromatin. Imaging of these cells by iCys (“Compu-sort”) revealed that they were pre-mitotic or mitotic (M) cells (panels D,E).