Fig. 6. Progression of the replication sites of the HSR subchromosomal domain. Clone-22 cells were pulse-labeled with IdU for 10 minutes, chased for 40 minutes in fresh medium, and then pulse-labeled with CldU for 10 minutes. The cells were immediately fixed with PFA, and the site of IdU or CldU labeling was detected with specific antibodies, which are shown in red and green pseudo-color, respectively. The HSR was detected by FISH and is shown in blue pseudo-color. (A-C) Whole nuclear images show that cells were in late S phase. In the images, the approximate nuclear rim was outlined based on the faint background FISH-signal (cyan circles). The enlarged images for the HSR domain show that the replication site changed from the periphery of the HSR domain to the internal region during the IdU and CldU labeling. For clarity, two-color (IdU plus CldU) and three-color (IdU, CldU plus HSR) merged images are shown (A'-C' and A''-C'', respectively). Serial confocal images of 0.64-µm intervals in the z-axis that intercept the center of the HSR domain are shown. Arrowheads point to the replication of the telomere of the same arm as the HSR. Bars, 5 µm.