Colocalization of activated FANCD2 and BRCA1 in discrete nuclear foci during S phase.
(A) HeLa cells were synchronized at G1/S with mimosine and released into S phase. S-phase cells were double-stained with the monoclonal anti-BRCA1 antibody D-9 (green; i,iv) and the rabbit polyclonal anti-FANCD2 antibody (red; ii,v), and stained cells were analyzed by immunofluorescence microscopy. Where green and red signals overlap, a yellow pattern is seen, indicating colocalization of BRCA1 and FANCD2 (iii,vi). (B) Differential effect of DNA damage on BRCA1 foci and FANCD2 foci during S phase. HeLa cells were synchronized with mimosine and released into S phase. Enriched S-phase–synchronized cell populations were analyzed by DNA flow histograms to ensure cell synchrony (data not shown). S-phase cells were untreated or were exposed to IR (5 Gy), MMC (20 ng/mL), or UV (50 J/m2) as indicated. One hour after treatment, cells were fixed and immunostained with antibodies to FANCD2 and BRCA1. Original magnification, × 600.
