Primary antibodies against γ h2ax

For γ-H2AX staining, HT29 cells were seeded on glass coverslips in 12-well dishes and incubated for 24 h. The cells were treated with 10 μM of K1586 for 1 h before irradiating with 2 Gy IR. After incubating for 1 h, the cells were fixed with a 3.7% formaldehyde solution for 10 min at room temperature (RT), permeabilized in ice-cold PBS with 0.1% Triton X-100 (PBST) for 10 min, and blocked with 5% bovine serum in PBST for 1 h at RT. Cells were incubated overnight at 4 °C with primary antibodies against γ-H2AX (Cell Signaling Technology, Danvers, MA, USA). Cells were washed and incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (Life Technologies Corporation, Carlsbad, CA, USA) for 1 h at RT. Glass coverslips were attached to the glass slides with mounting solution (Vectashield; Vector Laboratories, Burlingame, CA, USA) containing 6-diamidino-2-phenylindole (DAPI).

The knee joint sections from DMM and sham mice were incubated overnight at 4° C with primary antibodies against γH2AX (Cell Signaling Technology, Danvers, MA, 1:100) and Col2a1(Abcam, Cambridge, UK). Then, the sections were incubated with species-matched Alexa-488 or Alexa-594-labeled secondary antibodies (Life Technologies, Grand Island, NY, USA). Subsequently, the sections were mounted with PBS containing DAPI (Thermo Fisher Scientific), and photographed using a FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan). The proportions of positively stained cells relative to the total number of cells were determined for each sample.