Pc388

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.