Metacyte module

Cells were fixed with 4% paraformaldehyde (Sigma Aldrich), permeabilized with 0.2% Triton-X and blocked in PBS/BSA 1%. Samples were then co-immunostained over night at 4 °C, using a rabbit telomeric protein TRF1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA) in combination with mouse yH2AX (Millipore) or a mouse 53BP1 antibody (Millipore). After washes in PBS/BSA1% samples were incubated with the secondary antibodies (anti-mouse Alexa 546 and anti-rabbit Alexa 488, respectively, Invitrogen, Carlsbad, CA, USA). Finally, slides were counterstained with DAPI and analyzed with fluorescence microscopy using an Axio-Imager Z1 microscope (Zeiss) equipped with the Metacyte module of the Metafer automated capture software and a CCD camera (MetaSystems). The frequency of foci and colocalization dots per cell were scored in 100 nuclei in at least two independent experiments.

After treatment, cells were seeded on a glass in petri dish and left to grow up the fixation. Slides were treated as described for the immunofluorescence staining, with the same antibodies. After the incubation with the secondary antibody, slides were washes in PBS/Triton X-100 0.05% and then fixed in 4% formaldehyde for 10 min and dehydrated through graded alcohols (70, 80, 100%). Slides and probes (Cy3 linked telomeric peptide nucleic acid (PNA) probe, Panagene, Daejeon, South Korea) were co-denatured at 80 °C for 3 min and hybridized for 2 h at RT in a humidified chamber. After hybridization, slides were washed twice for 15 min in 50% formamide, 10 mM Tris, pH 7.2, and 0.1% BSA followed by three 5-min washes in 0.1 M Tris, pH 7.5, 0.15M NaCl and 0.08% Tween 20. Slides were then dehydrated with an ethanol series and air dried. Finally, slides were counterstained with DAPI (Sigma Aldrich) in Vectashield (Vector Laboratories, Burlingame, CA, USA). Cells, in particular colocalization between γH2AX or 53BP1 foci and telomere, were analyzed using an Axio Imager Z1 microscope (Carl Zeiss, Oberkochen, Germany) equipped with the Metacyte module of the Metafer automated capture software and a CCD camera (MetaSystems, Milano, Italy). The frequency of colocalization dots per cell were scored in 100 nuclei in at least two independent experiments.

At different times after irradiation, cells were fixed for 20 min with 4% paraformaldehyde (Sigma Aldrich) in PBS at 4 °C, permeabilized with 0.2% Triton X-100 in PBS and blocked with PBS/BSA 1%. Cells were incubated with a rabbit polyclonal antibody against PML (H-238:sc5621, Santa Cruz Biotechnology) or a mouse mono-clonal anti-RPA2 antibody (Abcam, Cambridge, UK) overnight at 4 °C. After washing cells were incubated with Alexa 488 anti-rabbit antibody for PML or Alexa 488 anti-mouse antibody for RPA2 (Invitrogen). After immunostaining, telomeric FISH was performed as described above. Images were captured with fluorescence microscopy using an Axio-Imager Z1 microscope (Zeiss) equipped with the Metacyte module of the Metafer automated capture software and a CCD camera (MetaSystems). For PML analysis, a cell was considered positive when it showed at least three PML/telomere co-localization events. At least 50 nuclei in two independent experiments for PML and RPA2 were analyzed to identify events of possible co-localization.