α brdu

ES and OS cell lines were treated with cladribine or clofarabine at different concentrations for 48 h. For evaluation of the cell cycle, cell cultures were incubated with 10 μmol/L bromodeoxyuridine (BrdU) (Sigma-Aldrich) for 1 h in a CO₂ atmosphere at 37°C. Harvested cells were fixed in ice-cold 70% ethanol for 30 min. After DNA denaturation with 2 N HCl for 30 min at room temperature, cells were washed with 0.1 mol/L Na₂B₄O₇, pH 8.5 and processed for indirect immunofluorescence staining, using α-BrdU (BD Biosciences) diluted 1:4 as a primary MAb and α-mouse FITC (1:100 – Thermo Scientific) as a secondary antibody. After treatment with 0.5 mg/mL RNase and staining with 20 μg/mL propidium iodide, cells were and analyzed by flow cytometry (FACSCalibur; Becton Dickinson) for cell cycle evaluation and for assessing cell death by DNA content analysis.

The following antibodies were used for ChIP: α-macroH2A1.2 (Millipore MABE61), α-phospho-S139-H2AX (Millipore 05–636), α-H2B (Abcam ab52484), α-H2A (Abcam ab18255), TRF2 (Novus Biologicals IMG-124A) and normal mouse IgG (Millipore 12–371). Primary antibodies for western blotting were: α-macroH2A1.2 (Millipore MABE61), α-phospho-S139-H2AX (S139) (Millipore 05–636), α-BRCA1 (Santa Cruz sc-6954), α-ATRX (Cell signaling 14820s), α-GAPDH (Santa Cruz sc-32233), α-H2AX (Abcam ab20669). Secondary antibodies were goat anti-mouse IgG (H+L)-HRP (Invitrogen 31430) and goat anti-rabbit IgG (H+L)-HRP (Invitrogen 31460). Primary antibodies for IF were α-BRCA1 (Santa Cruz sc-6954), α-γ-H2AX (Millipore 05–636, Abcam ab11174), α-BrdU (BD Biosciences 555627), and TRF2 (Novus Biologicals IMG-124A), secondary antibodies were goat-anti-mouse or goat-anti-rabbit IgG (H+L) coupled to Alexa Fluor 488 or 647 (Life Technologies).