Figure 2.

Stn1 deficiency impairs efficient replication of telomeres. (A) FTs induced by Stn1 KD. Metaphase chromosomes from HeLa 1.2.11 cells depleted of Stn1 were hybridized to PNA TTAGGG telomere probe. Representative telomere FISH images of shStn1 and control cells (shLuc) are shown. Three individual chromosomes are shown in amplified images to illustrate FTs (white arrows). Quantitation of FTs was performed by analyzing a total of > 75 metaphase spreads from each sample. At least 3 000 chromosomes were scored in each sample. Two-tailed t-test was used to calculate statistical significance. Results were from three independent experiments. (B) In-gel hybridization of separated leading and lagging telomeres from asynchronous HeLa cells with C-rich probe. Cells were labeled with BrdU continuously for 19 h and leading and lagging daughter telomeres were separated by CsCl gradient ultracentrifugation. (C) Quantitation of the abundance of ss G-rich DNA. Signals were normalized to the total telomere signals. Results were from three independent experiments. Error bars: S.E.M. (D) FACS analysis of DNA content in synchronized HeLa cells expressing shLuc and shStn1. Note that ∼10% cells failed to enter the cell cycle (Supplementary information, Figure S3B). (E) Separation of leading/lagging/unreplicated telomeres from cells collected at 6, 8, and 10 h after releasing from double thymidine block. Note the density shift of lagging telomere at 6 h (dashed lines) but not leading telomere in shStn1 cells. Results were from 6 independent experiments. P = 0.005. Two-tailed t-test was used to calculate statistical significance. (F) Percentage of unreplicated telomeres among the total telomeres at 6, 8, and 10 h after release. Two-tailed t-test was used to calculate statistical significance. P = 0.015 at 6 h, P = 0.019 at 8 h. Results were from at least 4 independent experiments. Error bars: S.E.M.