This work was supported by National Natural Science Foundation of China Grants [31271472; 31322033; 31301110], National Basic Research Program of China [2014CB964703], the Science and Technology Planning Project of Guangdong Province (2015B020228002), and Pearl River Scholar Funded Scheme?(2013). Notes EMBO Reports (2017) 18: 1412C1428 [PMC free article] [PubMed] [Google Scholar]. Triton X\100 and blocked with 5% goat serum. Fixed cells were incubated with primary antibody at 4C overnight and incubated with secondary antibody at room temperature for 2?h. Coverslips were counterstained with DAPI for observation under Rabbit Polyclonal to PPP1R2 a Zeiss Axioplan II microscope. For IF\FISH assays, following secondary antibody incubation, coverslips were fixed again with 4% paraformaldehyde and sequentially dehydrated with 75, 95, and 100% ethanol. The coverslips were then incubated with hybridization mix containing Cy3\(TTAGGG)3 (Panagene, F1006\5). DNA was heat\denatured at 85C for 5?min, and the slides were incubated at 37C for 2?h protected from light. The coverslips were washed, dehydrated as described above, and counterstained with DAPI. Antibodies used in this assay are as follows: anti\TRF2 (EMD Millipore, 05\521), anti\53BP1 (Novus Biologicals, NB100\304), anti\PCNA (Gene Tex, GTX100539), anti\Topo II (Abcam, ab52934), anti\pDNA\PKcs (S2056; Abcam, ab18192), anti\Rad51 (Santa Cruz, SC\8349), DyLight 488\conjugated goat anti\mouse IgG H&L (Multisciences, 4120353), DyLight EPI-001 488\conjugated goat anti\rabbit IgG H&L (Multisciences, 4220431), and DyLight 549\conjugated goat anti\mouse IgG H&L (Multisciences, 4120816). Native FISH Native FISH was performed as described 73 with a minor modification. Briefly, coverslips were fixed in 2% paraformaldehyde at room temperature for 10?min, permeabilized for 10?min in KCM buffer [0.1% Triton X\100, 10?mM TrisCHCl (pH 7.5), 120?mM KCl, and 20?mM NaCl] and treated with RNase A and RecJf (20?U) at 37C for at least 2?h. Cells not treated with RecJf were used as a control. Hybridization was performed in hybridization buffer containing 10?mM TrisCHCl (pH 7.5), 85.6?mM KCl, 0.5% blocking reagent (Roche, 11096176001), 70% formamide, and 40?nM Cy3\(TTAGGG)3 (Panagene, F1006\5). Slides were incubated at room temperature for 2?h, washed, and counterstained with DAPI. Images were acquired using 100 objective on a Leica TCS SP5 microscope. Two\dimensional gel electrophoresis 2D agarose gel electrophoresis was performed as described 74, 75. Briefly, 10?g genomic DNA was digested with RsaI and HinfI (Fermentas, Thermo Scientific) and loaded onto a 0.4% agarose gel. Electrophoresis was carried out in 1??TBE at 1?V/cm for 12?h at room temperature. The lane containing DNA was excised from the gel and the gel buffer was exchanged with 1??TBE with 0.3?g/ml ethidium bromide (EB; Sigma). The gel slice was transferred and cast with 1% agarose gel in 1??TBE containing 0.3?g/ml EB. The gel was run at 4C for 6?h at 3?V/cm. Detection of subtelomeric DNA HinfI\ and RsaI\digested HeLa DNA was EPI-001 subjected to 2D agarose gel as described above. The gel with em t /em \circle\tail signal was excised and DNA in gel was purified with QIAquick Gel Extraction Kit (QIAGEN). Purified DNA was used EPI-001 as a template for PCR with primers targeting subtelomeric sequence of specific chromosomes. The primer pairs were referred to that previously described 43. Telomere restricted fragment (TRF) assay The telomere length assay was performed as previously described 35. Plug assay/Constant\field gel electrophoresis of embedded cells The plug assay was performed as previously described 76. Briefly, 1??106 cells were rinsed twice with 1??PBS, embedded in 50?l 0.7% prewarmed agarose (45C), and solidified in a 1?ml decapitated injector. Agarose plugs were incubated with digestion buffer (10?mM TrisCHCl pH8.0, 10?mg/ml Protease K, 0.5% SDS, 40?mg/ml RNase A, 100?mM EDTA) at 37C for 20?h. Plugs were placed into the wells of a 0.7% agarose gel and sealed with 0.7% agarose. Electrophoresis was EPI-001 carried out at 1?V/cm at room temperature for 8?h. Native/denatured in\gel hybridization In\gel hybridization analysis of telomeric DNA was performed as described 77 with a minor modification: Agarose gels were pumped dry at room temperature. For native in\gel hybridization, gels were hybridized in Denhart’s hybridization buffer with 32P\labeled C\/G\probe after prehybridization. The C\/G\probes were generated as previously described. Gels were washed three times with 2??SSC?+?0.5% SDS twice, and 2??SSC?+?0.1% SDS once. For denatured in\gel hybridization, gels were denatured with 0.5?M NaOH and neutralized with 1?M TrisCHCl (pH EPI-001 8.0) prior to following the procedure for native hybridization. The gels were exposed to a PhosphorImager screen (GE Healthcare) and scanned on a Typhoon imager (GE Healthcare). Calculations of the relative abundance of em t /em \circle\tail were made using Image Quant software. Western blot analysis Western blotting was.
This work was supported by National Natural Science Foundation of China Grants [31271472; 31322033; 31301110], National Basic Research Program of China [2014CB964703], the Science and Technology Planning Project of Guangdong Province (2015B020228002), and Pearl River Scholar Funded Scheme?(2013)