Img 124a

U-2 OS^ATRX cells grown on coverslips for 4 days with or without 0.4 μg ml⁻¹ doxycycline were prepared for IF by standard procedures. Cells were prepermeabilized with ice cold 0.5% Triton X-100 for 5 min before fixation with 4% paraformaldehyde. The following antibodies were used for immunostaining: anti-alpha tubulin (1:50,000, Abcam ab7291); anti-ATRX (1:200 for WB; 1:500 for IF, Santa Cruz sc-15408); anti-DAXX (1:500, Sigma D7810); anti-MRE11 (1:200, Abcam ab214); anti-MRE11 (1:100, Calbiochem PC388); anti-RAD50 (1:200, Abcam ab89); anti-PML (1:200, Santa Cruz sc5621); anti-TRF2 (1:200, Imgenex IMG-124A) and anti-TRF1 (1:50, Santa Cruz sc-1977). For cell cycle analysis anti-BrdU (Abcam ab6326) was used at 1 μg ml⁻¹. Secondary antibodies for IF and for cell cycle analysis (Invitrogen Alexa-fluor conjugated) were used at 1:3,000. Secondary antibodies for western blots (Sigma anti-mouse IgG A4416 or Sigma anti-rabbit IgG A6667) were used at 1:10,000. Cells were treated for 48 h with 2 μM PDS (kind gift from Shankar Balasubramanian) and 0.4 μg ml⁻¹ doxycycline for the specified number of days. All images were taken on an Olympus BX51 confocal microscope at 100 × magnification. Uncropped images are shown in the Supplementary Information.

The SNAP-tag was labeled using 1–2 μM SNAP-Cell® 647-SiR (New England Biolabs, S9102S) for 2–3 h in DMEM containing 10 % FBS, 2 mM GlutaMAX^TM-I, 100 units/ml penicillin and 100 μg/ml streptomycin at 37 °C with 5 % CO₂. Following SNAP-labeling, cells were washed with PBS and pre-extracted with Triton X Buffer (20 mM HEPES pH 7.9, 50 mM NaCl, 3 mM MgCl₂, 300 mM sucrose, 0.5 % Triton X-100), for 1 min on ice, rinsed with PBS and fixed with formaldehyde (4 % formaldehyde, 2 % sucrose in PBS) for 10 min at room temperature. Cells were then re-permeabilized using Triton X Buffer for 10 min at room temperature, and incubated in blocking buffer (3 % BSA in PBS) for 30 min. After blocking, cells were incubated with primary antibodies for TRF2 (Imgenex, IMG-124A, 1:500) and coilin (Santa Cruz, sc-32860, 1:100) in blocking buffer for 1 h. Next, cells were washed with PBS and incubated with secondary antibodies (Life Technologies, A-31556, and Abcam, ab150117, 1:500) in blocking buffer for 1 h. After a final wash, cells were mounted using ProLong® Diamond Antifade Mountant (Life Technologies, P36970).