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Fig. 4 | Genome Biology

Fig. 4

From: A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression

Fig. 4

TERT localizes to telomeres in S but not G1 phase of the cell cycle. a IF analysis of fixed HeLa cells expressing FLAG-SNAP-TERT, synchronized in G1 and S phase of the cell cycle (scale bar = 5 μm). Cells expressing FLAG-SNAP-TERT displayed telomere-localized TERT foci in S but not G1 phase of the cell cycle, while parental cells never showed telomere-localized TERT foci. b FACS analysis of the DNA content of cells synchronized in S phase showed a peak between the 2 N and 4 N peaks of asynchronous cells, confirming that they were in S phase. The G1 cell population contained 2 N and 4 N peaks but was depleted for cells with intermediate DNA content. 4 N cells, which failed to release from their mitotic arrest, were easily distinguished from G1 cells by their morphology. c Quantification of the number of TERT foci which co-localized with TRF2 signals in edited HeLa cells synchronized at different stages of the cell cycle. Data were generated from two independent experiments, each analyzing 50 cells per condition (mean ± standard deviation). d FACS analysis of the DNA content of edited HeLa cells released from a double thymidine block as they transition through S phase. Prior to release, the cell population contained mostly cells with 2 N DNA content, which progressively increased as the cells underwent DNA replication. Nine to ten hours after release, DNA replication was complete, as indicated by the majority of cells having 4 N DNA content. e Quantification of the number of TERT foci co-localized with TRF2 signals at different time points during S phase (50 cells per time point, mean ± standard error of the mean; for corresponding images see Fig. S4 in Additional file 1). A.U. arbitrary units, Propidium Iodide (PI)

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