FIGURE 5.

FANCI forms nuclear foci and colocalizes with PCNA and FANCD2 in the absence or presence of DNA damage. Methanol-fixed cells with or without Triton X-100 pre-extraction were stained pairwise with either FANCI- and PCNA-specific antibodies (A), or FANCI- and FANCD2-specific antibodies (B). The stained cells were subject to confocal microscopy. Because the Triton X extraction dramatically reduced the fluorescence signal of FANCI in the absence of MMC treatment, we presented a two-dimensional view of a three-dimensional reconstruction of multifocal plane images to enhance the signal. The images in the −MMC +Triton panels of both A and B reflect overall signal level of the whole cell. Other panels represent a two-dimensional view of one focal plane image. Green, FANCI. Red, PCNA or FANCD2. Yellow and arrows, colocalization. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue).