Fig. 3.

Ape1 is required for stabilization of telomeric DNA. (A) Ape1 depletion causes TRF2 loss at telomeres. Control (Ctr.) and APE1 siRNA transfected U2OS cells were fixed at 72 h posttransfection and immunostained for Ape1 (green) and TRF2 (red). DNA was stained with DAPI. (Scale bar, 10 µm.) (B) Telomere-ChIP assay was performed in U2OS cells at 72 h of siRNA transfection. The recovered DNA from chromatin immunoprecipitation with Ape1 or TRF2 antibodies was submitted to quantitative real-time PCR analysis. Telomeric DNA was amplified using a telomere-specific primer pair, and the results are presented as the percentage of input DNA. Error bars correspond to SDs from three independent experiments. (C) Ape1 depletion induces DNA damage response at telomeres. BJ-hTERT cells transfected with control (Ctr.) or APE1 siRNAs were fixed at 72 h posttransfection and immunostained for TRF1 (green) or γH2AX (red). DNA was stained with DAPI. (Scale bar, 5 µm.) Cells showing more than three TRF2/γH2AX colocalizing foci from three independent, blinded experiments were quantified by counting at least 100 cells per experiment. *P ≤ 0.01 (Student t test).