Telomere DNA FISH were performed as described (20 (link)). Briefly, Hela cells grown on eight-well chambered slide (Nunc Lab-Tek II, 155409) were transfected with ATM-SPARK plasmid. Thirty minutes after challenged with NCS, cells were fixed in 4% (w/v) paraformaldehyde for 10 min at room temperature. After fixation, cells were permeabilized in 1× phosphate-buffered saline (PBS) with 0.5% Triton X-100 for 10 min at room temperature. Cells were incubated in 0.1 N of HCl for 5 min and washed twice in 2× saline-sodium citrate (SSC) with 0.1% Tween 20 (SSCT) for 2 min. After incubated in 2× SSCT with 50% formamide for 1 hour at 60°C, the well was loaded with 150 μl of ISH solution [2× SSCT, 50% (v/v) formamide, 10% dextran sulfate, ribonuclease A (400 ng/μl), and 100 nM probe]. After denaturation at 80°C for 3 min, cells were incubated overnight at 44°C.
After hybridization, cells were washed four times in 2× SSCT at 60°C and rinsed in 1× PBS for 1 min. Cells then incubated with 1 μM Atto 550–labeled imager in PBS and washed twice in 1× PBS. The cells were blocked with 2% bovine serum albumin and 10% goat serum in PBS. Then stained with anti-GFP antibody (1:200 dilution; Abcam, Ab290) 3 hours at room temperature. After washing three times with phosphate-buffered saline with 0.1% Tween 20 (PBST), the cells incubated with Alexa Fluor 488–conjugated secondary antibodies (1:200 dilution; Cell Signaling Technology, #4412) at room temperature for 1 hour. The telomere probe sequence is ACATCATCATGGGCCTTTTGGCCCATGATGATGTATGATGATG/3InvdT/, and the Imager sequence is ATGATGATGTATGATGATGT.