Restoration of telomerase activity in the late PD HMEC samples does not rescue their delayed DSB disappearance phenotype.
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Results obtained from donor 1 samples. A. Western blot showing that transduction of late PD HMECs with hTERT gene restituted telomerase protein subunit expression. B. Metaphase plates of a late PD non-transduced (top) and hTERT-transduced (bottom) HMECs. Telomeric (red) and centromeric (green) sequences detected with fluorescent in situ hybridization procedures. Telomerase restoration resulted in a reduction of telomere-signal-free ends (white arrow heads) and end-to-end fusions (Dic) as a consequence of telomere elongation. C. Bar diagram showing average frequencies of telomere-signal-free ends in non-transduced and hTERT-transduced HMECs. D. Histogram showing the kinetics of γH2AX appearance and disappearance measured by flow cytometric analysis. hTERT-transduced late PD HMECs (brown) reach its maximum percentage of γH2AX positive cells 90 min post-irradiation, which is delayed as compared to the early PD HMECs (green), but is the same time point as non-transduced late PD HMECs (red). A minimum of 10,000 cells and two replicas were analyzed by bivariate flow cytometry for each time-point and cell subpopulation.