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Fig. 4. Photorepair performed by the phr-EGFP recombinant enzyme prevents UV-induced apoptosis in XP12RO-SV and diploid human fibroblasts. (A) Apoptosis analysis by flow cytometry in UV-irradiated XP12RO-SV (XPA mutated) cells. Adphr-EGFP and mock-infected cells were UV-irradiated at the indicated doses and exposed immediately to PRL or dark conditions. The percentage of apoptosis was accessed 48 hours after UV irradiation by flow cytometry analysis. In the graphics presented, `counts' corresponds to the number of cells and `FL2-H' to the amount of DNA measured by P.I. fluorescence. M1 represents the percentage of cells in sub-G1 region of the cell cycle, that is apoptotic cells. (B) Photorepair performed by phr-EGFP prevents UV-induced morphological apoptotic markers in XP12RO cells. Mock and Adphr-EGFP-infected samples were UV-irradiated and treated as described in (A). The morphology of cells was analyzed 48 hours after UV-irradiation by fluorescence microscopy using acridine orange/ethidium bromide staining. Shaded bars represent control samples (UV=0 J/m2), black bars irradiated samples kept in the dark, and white bars, irradiated samples exposed to PRL. (C) Apoptosis analysis by flow cytometry in UV-irradiated human primary cells. 198VI (Normal), XP456VI (XPA mutated) and XP148VI (XPC mutated) were treated as described in (A), the samples being analyzed 72 hours after UV irradiation by flow cytometry. The frequency of subdiploid nuclei was calculated and plotted for the three cell lines employed. Shaded bars represent control samples (UV=0 J/m2), closed bars irradiated samples kept in dark, and open bars irradiated samples exposed to PRL. The data for each cell line represent the average of two independent experiments.
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