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Fig. 2. Detection of DNA damage by phosphorylation of H2AX at ser 139, TUNEL, and comet assay. (A) Western blot analyses of H2AX-P. HCT116 cells were treated with increasing concentration of BEL for 8 or 28 hours. H2AX-P and p53-P, p21, total p53 and PCNA levels were analyzed by western blotting. Cells treated with doxorubicin (Dox) (1 µg/ml) for 28 hours were used as a positive control for dsDNA breaks. (B) Western blot analysis of H2AX-P in HCT116-p21–/– cells. HCT116-p21–/– cells were treated with increasing concentrations of BEL for 8 hours and H2AX-P levels were analyzed by western blotting. HCT116-p21–/– cells were next incubated with and without caspase inhibitor (Z-VAD-FMK, 20 µM) for 30 minutes as indicated before being continuously cultured in the presence or absence of 12.5 µM BEL for 6 hours. H2AX-P levels in these cells were analyzed by western blotting. (C) Immunofluorescent staining of H2AX-P in multiple HCT116 cells. Cells were treated with vehicle (control), Dox (0.2 µg/ml) for 8 hours, BEL (12.5 µM) for 8 hours. Samples were stained for DAPI (blue) and H2AX-P (red) and analyzed by a confocal microscope at x20 magnification. Merged cells are shown in pink. (D) Immunofluorescent staining of H2AX-P in a single nucleus. BEL (12.5 µM, 8 hours) and Dox (0.2 µg/ml, 8 hours) treated cells were stained with anti-H2AX-P antibody (red) and DAPI (blue), and individual cells were analyzed using a confocal microscope at x100 magnification. Merging of H2AX-P and DAPI staining in the nucleus appears pink. (E) TUNEL analysis in HCT116 cells. HCT116 cells were treated with 50 J/m2 UV light and BEL (10 and 15 µM) for 8 hours, and stained using TUNEL (green) and DAPI (blue) followed by the confocal fluorescence microscopy analysis at x63 magnification. Merging of TUNEL and DAPI staining in the nucleus appear light blue. (F) Comet assay to detect DNA damage in individual cells. HCT116 cells were treated with vehicle (control), 50 J/m2 of UV light, and 10 µM BEL for 8 hours, respectively. The cells were subjected to an analysis by comet assay. The upper panel showed the results visualized by a fluorescent microscope. The cells with UV light treatment showed the typical `comet pattern' of damaged DNA. The lower panel showed the plot of tail moments (TM) analyzed by the CometScore. More than 50 cells were scored in each condition. There was no significant difference between the BEL treatment and the control. *, significant difference of UV-light treatment from the control or BEL treatment.
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