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Figure 2


Fig. 2. Early recruitment of p21 to DNA repair sites. (A) LF1 human fibroblasts were irradiated with UV-C (10 J/m2) and 30 minutes later samples were extracted in situ and fixed for indirect immunofluorescence determination, or biotin-streptavidin amplification of chromatin-bound PCNA (green fluorescence) and p21 (red fluorescence), respectively. DNA (blue fluorescence) was stained with Hoechst 33258. (B) HeLa cells were cotransfected with p21-GFP and RFP-PCNA expression vectors, and 24 hours later exposed to UV-C radiation (10 J/m2). After 30 minutes, control (C) and irradiated (UV) cells were extracted in situ and fixed for detection and counting of cells showing only chromatin-bound RFP-PCNA (empty bars), p21-GFP (solid bars), or both (dashed bars). The percentages of cells in a representative experiment are shown. (C) Confocal sections of merged green and red fluorescence signals, of untreated control and UV-C-irradiated cells. (D) HeLa cells expressing p21-GFP and RFP-PCNA were exposed to local UV-C radiation (10 J/m2) through 3 µm pores. Confocal sections of green (p21-GFP) and red (RFP-PCNA) fluorescence signals are displayed, together with the merged image showing also the blue fluorescence (Hoechst) of DNA counterstaining. Bars, 10 µm (A,C); 5 µm (D).





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