(Downloading may take up to 30 seconds.
If the slide opens in your browser, select File -> Save As to save it.)
Click on image to view larger version.


Figure 2


Fig. 2. (A) Survival and growth of HeLa cells treated with MMC. Data from untreated cells and cells exposed to MMC (0.5 and 0.2 µg ml-1) for 16 hours are shown. Each point represents six parallel wells. Error bars are undetectable as they are smaller than the symbols. (B) Co-localization of WRN with RAD51, RAD54B and RAD54 after treatment with MMC. Co-localization of ECFP-RAD51 and EYFP-WRN (upper row), ECFP-RAD54B and EYFP-WRN (middle row), ECFP-RAD54 and EYFP-WRN (lower row) in HeLa cells treated with MMC (0.5 µg ml-1) over night. (C) Re-localization of WRN, RAD54 and RAD54B to PCNA foci after MMC treatment. HeLa (pictures 1-16) and U2OS cells (pictures 17-20) were treated with MMC (0.5 µg ml-1) over night to arrest the cells in S-phase. HeLa cells co-transfected with ECFP-RAD54B, EYFP-WRN, HcRed-PCNA: untreated (pictures 1-4) and treated with MMC (pictures 5-8). HeLa cells co-transfected with ECFP-RAD54, EYFP-WRN, HcRed-PCNA: untreated (pictures 9-12) and treated with MMC (pictures 13-16). Stable EYFP-WRN expressing U2OS cells co-transfected with ECFP-RAD54B and HcRed-PCNA and treated with MMC (pictures 17-20). (D) FRETN values between WRN and the RAD52 epistasis proteins after MMC treatment over night, and between RAD51-RAD54 and RAD51-RAD54B. aRepresents previously published data (Baynton et al., 2003) that was taken using the same conditions and settings as the current experiments. It is presented here for comparative purposes. Uuntreated cells. FRETN [FRETN=FRET/(I1 x I3)]1/2 represents FRET normalized against protein expression levels measured from intensities (I) (given as arbitrary units below 250). FRET is calculated from the mean of intensities within a region of interest containing more than 25 pixels. Within the region of interest, all pixels had intensities below 250, and the I levels were between 85-190 for the donor (ECFP) and between 55-155 (EYFP) for the acceptor, respectively. Corresponding pictures from which the data were derived are shown in Fig. 2B and Fig. 1, pictures 19-24 (RAD54-RAD51U and RAD54B-RAD51U). The background level outside foci, i.e. the co-localizing area, is 0, which is also the FRET value determined for more than 95% of foci with co-localizing UNG2-ECFP and UNG2-EYFP proteins.