Anti γh2ax

Cells were lysed in 1× SDS sample buffer supplemented with the protease inhibitor mixture (Sigma‐Aldrich, St Louis, MO, USA). Equal amounts of protein (30 μg) were separated on SDS/polyacrylamide gels and then transferred onto membrane filters (Merck Millipore, Amsterdam, the Netherlands). After blocking with 5% non‐fat dry milk, the membranes were probed with anti‐p53 (Santa Cruz Biotechnology, Dallas, TX, USA), anti‐phospho‐p53 at Ser‐15 (Cell Signaling Technology, Danvers, CA, USA), anti‐acetyl‐p53 at Lys‐373/382 (Upstate, Lake Placid, NY, USA), anti‐p21^WAF1 (Santa Cruz Biotechnology), anti‐Bcl‐2‐associated X protein (BAX; Cell Signaling Technology), anti‐NOXA (Cell Signaling Technology), anti‐HDAC2 (Cell Signaling Technology), anti‐poly (ADP‐ribose) polymerase (PARP; Cell Signaling Technologies), anti‐γH2AX (BioLegend, San Diego, CA, USA), anti‐ATM (Santa Cruz Biotechnology), anti‐phospho‐ATM at Ser‐1981 (Merck Millipore) or with anti‐actin antibody (Santa Cruz Biotechnology) followed by an incubation with horseradish peroxidase‐conjugated secondary antibodies (Invitrogen). Immunodetection was performed with enhanced chemiluminescence (ECL; GE Healthcare Life Science, Piscataway, NJ, USA).

Cells attached to a glass coverslip or tissue section in O.C.T. compound (Sakura Finetek) were used for immunostaining. Tissue sections were prepared at 10 µm thickness using a cryostat (NX70, Leica) and dried at 37 °C for 10 min. The samples were washed twice in 2 mL PBS (–) and fixed in 2% paraformaldehyde (Nacalai Tesque) for 5 min. The samples were then dehydrated with acetone (Nacalai Tesque) for 5 min. Permeabilization was performed in 50 µL 0.5% Triton X-100 (Nacalai Tesque) for 5 min. Samples were then blocked with 10% goat serum (143-06561, Wako) in BlockAce solution (KAC Co., Ltd.) at room temperature for 1 h, followed by washing in 0.1% BSA/PBS (Sigma) twice for 5 min each. Primary antibody (for DNA DSBs analysis, anti-γH2AX, 1:50, Biolegend, 613402; for immunohistochemistry of thymus, anti-K8, 1:50, Biolegend, 904804, and anti-K5, 1:50, Biolegend, 905504) was incubated at 4 °C overnight under humid conditions. The secondary antibody (Alexa Fluor 555 conjugated anti-mouse IgG, Thermo Fisher, A21425; Alexa Fluor 647 conjugated anti-rabbit IgG, Thermo Fisher, A21246) was incubated at room temperature for 1 h. Nuclei were stained with Hoechst33342 (1:1000; KV072, Wako) for 3 min before imaging with a fluorescent microscope (EVOS^® FL, Invitrogen). Intensity of γH2AX signals were accessed using ImageJ software^{46 (link)}.

Cells were fixed with 0.34% Schaudinn’s fixative (2:1 ration of saturated HgCl₂ [Sigma-Aldrich]: ethanol) and membrane-permeabilized with cold methanol on ice for 10 min. Cells were then spread onto slides coated with poly-l-lysine (Sigma-Aldrich) and air dried. After rehydration in PBS (4.3 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, pH 7.4), cells were incubated for 2 hr at room temperature with primary antibody: anti-γH2AX (1:500; BioLegend, San Diego, CA, RRID:AB_315794), anti-GFP (1:500; mouse monoclonal; Sigma-Aldrich, RRID:AB_390913), or anti-H3K56 (1:500; rabbit polyclonal; Active Motif, Carlsbad, CA, RRID:AB_2661786) antibody. After washing with PBS, cells were incubated with FITC-labeled goat anti-mouse (1:500; Merck Millipore, Temecula, CA, RRID:AB_92634) or Rhodamine-labeled goat anti-rabbit (1:2000; Merck Millipore, RRID:AB_90296) secondary antibody for 1 hr at room temperature in the dark. After washing with PBS, cells were stained with 1 μg/μl 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) and observed under fluorescence microscopy.