Phospho histone h2a x ser139

Cells were fixed in ice-cold methanol for 5 min, permeabilized with 0.3% Triton X-100 in PBS, and blocked in 5% bovine serum albumin (BSA). Samples were incubated overnight at 4°C in primary antibody in 1% BSA and 0.3% Triton X-100 in PBS, followed by washing in PBS and incubation in secondary antibody in 1% BSA and 0.3% Triton X-100 in PBS. Antibodies against the following proteins were used: H3K9me3 (Cell Signaling 13969; 1:5000), H3K27me3 (Cell Signaling 9733; 1:5000), phospho-histone H2A.X (Ser139) (Cell Signaling 9718; 1:5000), phospho-histone H2A.X (Ser139) (Merck Millipore 05-636; 1:1000), lamin A/C (Cell Signaling 4777; 1:5000), Sox9 (Cell Signaling 82630; 1:5000), RNAPII-S2p, (Abcam 5095; 1:5000), collagen 2A1 (Thermo Fisher Scientific MA5-12789; 1:5000), and cleaved caspase-3 (Cell Signaling 9664; 1:1000). The EdU incorporation assay was performed using a Click-iT™ EdU Cell Proliferation Kit (Thermo Fisher Scientific); 1 μM EdU was added to the culture medium filled in the plastic bag prior to HP and detected after fixation.

Cells were treated with 10 nM trabectedin (MedChemExpress) for 4 h. During the last 15 min, 20 µM 5-ethynyl-2′-deoxyuridine (5-EdU; Jena Bioscience) was added. Cells were then washed with 300 mM sucrose (Merck) in PBS on ice, pre-extracted with 300 mM sucrose and 0.25% Triton X-100 in PBS for 2 min on ice and fixed with 3.7% formaldehyde in PBS for 15 min at room temperature. Cells were then permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature and blocked in 3% BSA (Thermo Fisher Scientific) in PBS. Dividing cells were visualized by click-it chemistry, labeling the cells for 30 min with a mix of 60 µM Atto azide-Alexa 594 or Atto azide-Alexa 647 (Atto Tec), 4 mM copper sulfate (Sigma-Aldrich) and 10 mM ascorbic acid (Sigma-Aldrich) in a 50 mM Tris buffer. After washing with PBS, cells were blocked with 100 mM glycine (Sigma-Aldrich) in PBS for 10 min at room temperature and subsequently with 0.5% BSA and 0.05% Tween 20 in PBS for 10 min at room temperature. To visualize γH2AX, cells were incubated with a primary antibody for phospho-Histone H2A.X Ser139 (JBW301, Merck) for 2 h at room temperature and then with a secondary antibody, anti-mouse Alexa 555 (A-21424, Thermo Fisher Scientific) or anti-mouse Alexa 647 (A-21235, Thermo Fisher Scientific), and DAPI for 1 h at room temperature and mounted in Polymount (Brunschwig).