The FA proteins form foci at G₁-S and during S phase. HeLa cells transfected with either EGFP-FANCA/ECFP-FANCG or ECFP-FANCG/EYFP-FANCC were grown on slides and synchronized by double thymidine block to the G₁-S border. Whole cells and chromatin were visualized at the indicated times after release: (A) G₁-S, 0 hours; (B) S phase, 4 hours; (C) early G₂ phase, 6 hours; (D) late G₂ phase, 8 hours; (E) mitosis, after 16 hours in 1 μM nocodazole; and (F) early G₁, after collection of floating mitotic cells, followed by 4 hours in regular media to allow for anchorage and cytokinesis. (G) Representative FACScan analysis of transfected, synchronized cells. (H) Asynchronous HeLa cells cotransfected with GFP-FANCA and CFP-FANCG were sorted by FACScan into G₁, S, and G₂-M groups. Microscopy revealed that FA proteins were cytoplasmic during G₁ and G₂-M and nuclear during S phase.