Pna telc fitc probe

WISH experiments were performed as previously described25 (link). In situ hybridization of the telomeres was performed using a metaphase spread and a PNA-TelC-FITC probe (Panagene) as previously reported14 (link). Visualization was performed on the Zeiss SteREO Discovery V.20. The WISH probes were produced using the DIG RNA Labelling kit (T7/SP6) (Roche). The probe templates were produced by PCR with a T7 promoter sequence added to the 5′ end. The antisense probes for SmedOB1 and Smedwi-1 were generated with the following primers: SmedOB1 FP: AAGGAATGTTTTGAACG; SmedOB1 RP: TAATACGACTCACTATAGGGTACTGCTGACTTTCCTT. Smedwi-1 FP: TACAGCCTGATACAGTTAC; Smedwi-1 RP: taatacgactcactatagggTTGTAGTAGAATACCCTCC.

Western blotting and IF-FISH were carried out as previously described39 (link)52 (link). For IF-FISH, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, P6148), and then permeabilized with permeabilization buffer (0.5% triton X-100, 20 mM HEPES, 50 mM NaCl, 3 mM MgCl₂, 300 mM sucrose)(Sigma-Aldrich,USA), blocked with 5% goat serum (Gibco, 16210–072) and incubated with primary and secondary antibodies. After incubation, the cells were fixed with 4% paraformaldehyde, then washed with. PBS and dehydrated with ethanol, followed by hybridization with a PNA-TelC-FITC probe (F1002,1:500,Panagene) and FITC-labeled (TTAGGG) peptide nucleic acid (PNA) probe (Panagene, Korea). Figs 1B and 2A used TTAGGG probe and other FISH analysis was performed by telomeric probe CCCTAA. More than 300 cells were counted in each cell line for APB scoring. The following antibodies and their working concentrations were used for Western blotting: anti-Flag (F7425, 1:8000, Sigma-Aldrich); anti-DAXX (sc-7152, 1:400, Santa Cruz Biotechnology); anti-ATRX (sc-15408, 1:400, Santa Cruz Biotechnology); anti-GAPDH (M20006M, 1:6000, Abmart). The following antibodies and their working concentrations were used for IF-FISH: anti-γH2AX (05–636, 1:500, EMD Millipore) and anti-PML (sc-966, 1:400, Santa Cruz Biotechnology).