Sister telomere cohesion is lost prematurely (or not established) in S phase in SA1- and TIN2-depleted cells. (A–C) FISH analysis of BrdU-positive cells. (A) siRNA-treated HeLaI.2.11 cells were incubated with BrdU for 60 min before harvest, stained with anti-BrdU antibody (red), and hybridized with a telomere-specific fluorescently labeled probe 16pter (green). DNA was stained with DAPI (blue). (B) Table showing the number of FISH signals scored in BrdU-positive cells as singlet or doublets from three independent experiments with SDs. (C) Graphical representation of the frequency of doublets in BrdU-positive cells. Bar graphs represent the average values with SDs. (D–K) FISH analysis of late S phase synchronized cells. (D) Schematic representation of the experimental protocol to synchronize siRNA-treated cells. (E) FACS analysis and (F–H) FISH analysis of cells 4 h after release from the second thymidine arrest. Cells were hybridized with a telomere 16pter (F, green), arm 20p12 (G, white), or centromere 6cen (H, red) probe. Asterisks indicate a centromere that has lost cohesion. DNA was stained with DAPI (blue). Bars, 5 μm. (I) Tables showing the FISH signals scored as singlets or doublets from exp. 1 for the telomere (16pter), arm (20p12), or centromere (6cen) probe. (J) Tables showing the FISH signals scored as singlets or doublets from exp. 2 for the telomere (20qter), arm (10p14), or centromere (10cen) probe. (K) Graphical representation of the frequency of doublets from two independent experiments.