Ab13579

The human embryonic kidney cell line HEK293T, the human hepatoma cell line HepG2 and 7721 cells, and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, in 5% CO₂ at 37 °C. The antibodies used in this study were as follows: anti-TRF2 (ab13579, Abcam, Massachusetts, USA), anti-NOLC1 (sc-374033, Santa Cruz Biotechnology, California, USA and ab184550, Abcam, Massachusetts, USA), anti-Flag (F1804, Sigma, California, USA), UBF (sc-9131, Santa Cruz Biotechnology, California, USA), and anti-actin (SC-130300, Santa Cruz Biotechnology, California, USA). The GFP–TRF2 expression plasmid was purchased from Addgene (ID no. 19798). The Flag-NOLC1 expression vector was constructed by inserting the full-length NOLC1 complementary DNA (cDNA) into the pCMV-Flag vector. The Flag-TRF2 and mutant TRF2 expression vectors were constructed by inserting the corresponding TRF2 cDNA into the pcDNA3.1 vectors. The primers were listed in Table S1.

Cells grown on glass coverslips were fixed in 4% paraformaldehyde/PBS for 15 min, then permeabilized with 0.1% triton-X100/PBS at 37°C for 30 min, and finally blocked with 5% goat serum/PBS at 37°C for 3 h. Immunofluorescence was performed using standard methods, and the slides were incubated alternately with BG4 (80 ng/μL), anti-FLAG antibody (#2368, Cell Signaling Technology), γH2AX antibody (#9718, Cell Signaling Technology), TRF2 antibody (ab13579, Abcam), or POT1 antibody (ab21382, Abcam) at 37°C for 3 h. The glass coverslips were washed six times with blocking buffer and were then incubated with anti-rabbit Alexa 488-conjugated antibody (A21206, Life Technology), anti-mouse Alexa 555-conjugated antibody (A21427, Life Technology), and 2 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen) at 37°C for 3 h. The glass coverslips were again washed six times with blocking buffer, and then, digital images were recorded using an LSM710 microscope (Zeiss, GER) and analyzed with ZEN software. Fifty nuclei were counted in each group, and the s.e.m. was calculated from three replicates. Frequency distribution graphs were plotted using GraphPad Prism (GraphPadSoftware Inc.).

Primary antibodies used for immunostaining were anti-TRF1 (Abcam, ab192629, 1:100), anti-BubR1 (Abcam, ab28193, 1:100), anti-Zw10 (Abcam, ab21582, 1:100), anti-acetylated-α-tubulin (Sigma Aldrich, T7451, 1:500; Abcam, ab179484, 1:500), anti-centromere (Antibodies Incorporated, 15-234, 1:100), anti-CENP-A (Cell Signaling, #2048, 1:100), anti-Survivin (Cell Signaling, #2808, 1:100), anti-Hec1 (Santa Cruz Biotechnology, sc-515550, 1:100), anti-H3T3ph (Upstate, 07-424, 1:100), anti-SMC3 (Abcam, ab128919, 1:100), anti-SMC4 (Novus Biologicals, NBP1-86635, 1:100), and anti-TRF2 (Abcam, ab13579, 1:100). Secondary antibodies were Alexa Fluor 488-conjugated anti-mouse (Jackson ImmunoResearch, 115-545-144 1:500), Alexa Fluor 594-conjugated anti-mouse (Jackson ImmunoResearch, 111-585-146, 1:500), Alexa Fluor 488-conjugated anti-rabbit (Jackson ImmunoResearch, 115-545-144 1:500), Alexa Fluor 594-conjugated anti-rabbit (Jackson ImmunoResearch, 111-585-144, 1:500), and Alexa Fluor 488-conjugated anti-sheep antibodies (Abcam, ab150177, 1:500).

Post-transfected (24 hours after transfection) cells were fixed with 4% ice cold paraformaldehyde for 15 min, then permeated with 0.25% Triton X-100 for 30 min at room temperature. Blocking was performed with PBS solution containing 5% goat serum and 0.3% Triton X-100 for 1 h. Mouse monoclonal anti-TRF2 antibody (ab13579, Abcam) diluted 1:500 was incubated overnight with the cells at 4°C. After washing with PBS, goat anti-mouse secondary antibody conjugated with Alexa647 diluted 1:1000 was applied for 1 h at room temperature. Cells were completely rinsed with fresh PBS prior to imaging. Confocal imaging was performed with the PicoQuant system where a 465 nm laser was used to excite TRF1-GFP and a 633 nm laser was used to excite Alexa647.

HeLa1.3 cells were maintained at 37 °C (5% CO₂) in DMEM (D5796, Sigma) containing 10% FBS. For polyamide staining, cells were grown on coverslips coated with poly-lysine. The cell coverslips were washed twice in phosphate-buffered saline (PBS) and fixed with 1.85% formaldehyde in PBS for 15 min at room temperature. For blocking, cells were treated with 10% normal goat serum (NGS) (S26-100, Millipore) in TE buffer (10 mM Tris–HCl pH 7.5, 1 mM EDTA) for 30 min at room temperature. After a brief rinse with TE buffer, the cells were incubated with 10% NGS, 100 nM TH59-DB in DMF, mouse anti-TRF2 (ab13579, Abcam), and 0.5 μg/mL DAPI (4′,6-diamidino-2-phenylindole) (10236276001, Roche) in TE buffer at 37 °C for 1 h. After washing with TEN200 buffer (10 mM Tris–HCl, pH7.5, 1 mM EDTA, and 200 mM NaCl), cells were incubated with 10% NGS, streptavidin Alexa488 (s11223, Invitrogen) and anti-mouse Alexa594 (AA11032, Invitrogen) in TE for 1 h at room temperature. The cells on the coverslips were washed with TEN200 buffer (five times for 3 min), then mounted. Cell images were recorded with a DeltaVision microscope and deconvolved to eliminate out-of-focus blur to obtain clearer pictures. The deconvolved images were projected (‘Quick Projection’ tool) to obtain the maximum intensity of telomere signals.