dSTORM microscopy evidences in HeLa cells clustered and scattered γH2AX nanofoci sensitive to ATM, DNA-PK, and ATR kinase inhibitors.

In response to DNA double-strand breaks (DSB), histone H2AX is phosphorylated around the lesion by a feed forward signal amplification loop, originating γH2AX foci detectable by immunofluorescence and confocal microscopy as elliptical areas of uniform intensity. We exploited the significant increase in resolution (~ × 10) provided by single-molecule localization microscopy (SMLM) to investigate at nanometer scale the distribution of γH2AX signals either endogenous (controls) or induced by the radiomimetic bleomycin (BLEO) in HeLa cells. In both conditions, clustered substructures (nanofoci) confined to γH2AX foci and scattered nanofoci throughout the remnant nuclear area were detected. SR-Tesseler software (Voronoï tessellation-based segmentation) was combined with a custom Python script to first separate clustered nanofoci inside γH2AX foci from scattered nanofoci, and then to perform a cluster analysis upon each nanofoci type. Compared to controls, γH2AX foci in BLEO-treated nuclei presented on average larger areas (0.41 versus 0.19 µm