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Fig. 2. ssDNA focus formation in response to UV-irradiation depends on PML-NB integrity. (A) Western blot of nuclear matrix extracts prepared from GM-847 cells at 48 hours following transfection with PML- or GL2 (control)-specific siRNAs. The band indicated by an asterisk either represents a non-specific background band or a PML-isoform that is resistant to siRNA-mediated depletion. The ß-actin protein is used as a loading control. (B,C) Cells were transfected with the indicated siRNAs on day 1, and on day 2 the growth medium was supplemented with 10 µM BrdU. On day 3 (24 hours following addition of BrdU) cells were UV irradiated (or non-irradiated) and subsequently grown in the presence or absence of 4 mM caffeine for 1 or 2 hours before fixation. (B) Representative immunofluorescence images of siRNA-transfected GM-847 cells at 2 hours following irradiation. (C) Quantitative determination of cells containing ssDNA foci. For each sample, more than 500 cells were scored. The average of two independent experiments ± s.d. is shown. (D) GM-847 cells were transfected with the plasmid pYFP-PML + carrier plasmid (see Materials and Methods) and immediately after subjected to BrdU-labeling for 24 hours. Cells were then treated with UV light (or left non-irradiated) followed by incubation in the presence of caffeine for 90 minutes prior to fixation. The YFP signal and the immunofluorescently labeled BrdU are shown. (E) Quantitative determination of YFP-positive cells that contain ssDNA foci following UV-irradiation and caffeine treatment (same experiment as D). For each sample, 100 YFP-positive cells were scored. The average of two independent experiments ± s.d. is shown. (NI) Non-irradiated.
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