Sc 517348

To evaluate γH2AX and RAD51 foci formation, PaCa44 and Panc1 cells were grown on slides, treated as explained above, washed with PBS and air dried. The cells were then incubated with 2% paraformaldehyde (Electron Microscopy Science, Hatfield, PA, USA) for 30 min and permeabilized with 0.1% Triton X-100 (Sigma Aldrich, Burlington, MA, USA) for 5 min. After 3 washes, the cells were incubated with 1% glycine, 3% BSA for a further 30 min. Then, the cells were incubated with the primary monoclonal antibody against p-H2AX (Ser 139) (1:100) (Santa Cruz Biotechnology Inc., Dallas, TX, USA, sc-517348) or RAD51 (1:100) (Santa Cruz Biotechnology Inc., Dallas, TX, USA, sc-398587) for 1 h at room temperature. Slides were then washed 3 times with PBS and the cells were further incubated with a polyclonal conjugated-Cy3 sheep anti-mouse antibody (1:2000) (Jackson ImmunoResearch, UK) for 30 min at room temperature. After 3 washes in PBS, the cells were stained with DAPI (1:5000) (SIGMA) for 1 min at room temperature. Slides were further washed in PBS, mounted with glycerol:PBS (1:1) and analyzed with an Apotome Axio Observer Z1 inverted microscope (Zeiss, Germany) equipped with an AxioCam MRM Rev.3 (Germany) at 40 magnification. Foci amount was counted by Image J software (USA).

Fractured bone was harvested from mice after euthanasia, fixed in 10% formalin for 12 h, and decalcified in 10% EDTA for 21 days. For histochemistry staining, the fractured bones were embedded in paraffin. Five-millimeter-thick longitudinally oriented bone sections were stained with Safranin O-Fast green (SOFG) to quantify the cartilage area and woven bone area. For immunofluorescence staining, we prepared cryo-embedding medium composed of 2% polyvinylpyrrolidone (PVP), 20% sucrose and 8% gelatin in 1× PBS. The fractured bones were embedded with cryo-embedding medium, and 7 µm cryosections were obtained. Bone sections were processed for antigen retrieval by digestion with 0.05% trypsin at 37 °C for 15 min and then incubated with primary antibodies against Ctsk (ab19027, 1:200, Abcam), p21 (sc-6246, 1:100, Santa Cruz), γ-H2AX (sc-517348, 1:100, Santa Cruz), GFP (ab6673, 1:200, Abcam), Arg2 (sc-393496, 1:100, Santa Cruz), GCA (PA5–77127, 1:200, Invitrogen), F4/80 (ab6640, 1:400, Abcam) and Ocn (M137, 1:500, Takara) overnight at 4 °C, followed by incubation with FITC- or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch, 1:200). Nuclei were counterstained with DAPI (Sigma).

Cells were homogenized with a Tris-SDS-EDTA Lysis buffer containing protease and phosphatase inhibitors and beta-mercaptoethanol. Samples were boiled and run on SDS-PAGE for western blotting according to standard procedures. Nitrocellulose membranes were probed for p21 (1:1000, CST 2947, Cell Signaling Technology), p16 (1:1000, CST 80772, Cell Signaling Technology), β-Actin (1:200, sc-81178, Santa Cruz Biotechnology), Vinculin (1:200, sc-73614, Santa Cruz Biotechnology), γH2A.X (1:200, sc-517348, Santa Cruz Biotechnology), H2A.X (1:200, sc-517336, Santa Cruz Biotechnology), pAkt (1:1000, CST 9018, Cell Signaling Technology), PP2A-A (1:1000, CST-9780, Cell Signaling Technology), Akt (1:1000, CST 2938, Cell Signaling Technology), and Phospho-Akt1 (Ser473) (D7F10) (1:1000, CST-9018, Cell Signaling Technology). Blots were detected with the Azure Biosystems ECL detection kit (AC2103). All gels derived from the same experiment and were processed in parallel (Supplementary Fig. 13). Quantification of band intensities by densitometry was carried out using ImageJ.

Immunofluorescence staining of γH2AX was performed according to the IF method described above. The anti‐γH2AX (1:100 dilution, sc‐517348, Santa Cruz, USA) antibody was used for γH2AX foci staining. Cells were then counted by staining with DAPI solution and images were captured by using Leica SP8 LIGHTNING confocal microscopy.

Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature (RT) for 10 min, washed three times with PBS, and then permeabilized for 15 min with 0.1% Triton-X 100 (Sigma-Aldrich, St. Louis, MO) in PBS at RT. Cells were blocked with PBS supplemented with 4% bovine serum albumin for 30 min and incubated with primary antibodies against NRF2 (1:200, D121053, Sangon Biotech, China), and γH2AX (1:200, sc-517348, Santa Cruz, USA) at 4 °C for 16 h. After washing with PBS three times, cells were incubated with secondary antibodies for 1 h at 37 °C in the dark. Following another three washing steps in PBS, nuclear counterstaining was performed with 1 μg/mL Hoechst 33342 (Sigma Aldrich). Fluorescence images were obtained by Evos f1 fluorescence microscope (AMG, USA).

Cell extracts were prepared, resolved on gels, transferred to nitrocellulose and incubated with antibodies against the N terminus of p53, which can recognize mutant and wild type of p53 (1:1,000; monoclonal antibody; ab1101; abcam), β-actin (1:500; monoclonal antibody; M1210-2; Huaan), BCL-2 (1:1,000; monoclonal antibody; sc-7382; Santa Cruz), and PUMA (1:500; monoclonal antibody; sc-374223; Santa Cruz). γH2AX-139 (1:1,000; monoclonal antibody; sc-517348; Santa Cruz).