Histone h3 tri methyl k9

Glioma cells were plated in 24-well plates on coverslips and one day later were exposed to X-rays (8 Gy) and TMZ (100 μM). At different times following treatments, cells were fixed with 4% PFA. Non-specific bindings were blocked with a solution of 3% bovine serum albumin (BSA) (Sigma-Aldrich, France)-PBS-0.1% Tween (Sigma-Aldrich, France) for 1 hour at room temperature. Then, cells were incubated overnight at 4°C with a primary antibody. The following primary antibodies were used: histone H3 (trimethylK9) (1/500; Abcam, ab8898); phospho-histone H2AX (ser139) (1/200; Cell Signalling Technology, D175, 2577S) and cleaved-caspase-3 (1/1600; Cell Signalling Technology, 9661S) in 1% BSA-PBS-0.1% Tween. The revelation was achieved by an Alexa-555-conjugated anti-rabbit secondary antibody (1/200; Molecular Probes, A21429). Cells were counterstained with Hoechst 33342 (10 μg/ml; Sigma-Aldrich, France) for nuclear staining. A micronucleus assay was performed from Hoechst 33342 staining and a cell with at least a micronucleus was considered positive. All immunocytochemical markers were observed on a Leica DM6000 microscope with a 40X objective. For each condition, at least 3 coverslips were analysed. Images from 3 representative high-power fields per slide were acquired.

ChIP assays were performed as described previously [20 (link)]. DNA transfection of HeLa cells was performed as described above. At 120 h after the first transfection, cells were crosslinked with 1% formaldehyde for 10 min at room temperature. Fixation was completed following the addition of glycine with a final concentration of 200 mM. The crosslinked genomic DNA was sheared by sonication using a Digital Sonifier Model 250 (Branson). The DNA-protein complexes were immunoprecipitated with Dynabeads Protein G (Invitrogen). An antibody directed against Histone H3 (tri methyl K9) (Abcam) was used. The precipitated DNA was analyzed by real-time PCR using the primers 5'-GTTCGCCAAAGGAAAAGCAGG-3' and 5'-GTGTCTGTCTCTCCCGGATGTC-3', which target a region located upstream of the transcription start site of the SERPINE1 promoter, or primers 5'-GCCCCTGTTTACGGAGCATTTC-3' and 5'-TGCGTGATACTGGGCTAGGAAC-3' which target the genomic region (ch10:79154928–79155075).