Phospho γh2ax

Mouse intestinal tissue samples were collected and fixed in 10% formalin and were processed for paraffin embedding. Sectioned samples were immunostained according to standard protocols: samples were deparaffinized, blocked and incubated with primary, and fluorescence- or horseradish peroxidase (HRP)-conjugated secondary antibodies. For HRP-conjugated secondary antibody, 3,3'-Diaminobenzidine substrate was used, followed by haematoxylin nuclear counterstaining. Antibodies against the following were used for immunohistochemistry: LIG4 (Sigma (HPA001334); 1:250 dilution); phospho-γH2AX (Cell Signaling (20E3); 1:250 dilution); RFP (Thermo (RF5R); 1:250 dilution); cleaved caspase 3 (Cell Signaling (5A1E); 1:500 dilution); β-catenin (Cell Signaling (D10A8); 1:500 dilution); CD44 (BD Pharmingen (G44-26); 1:250 dilution); and CD133 (eBioscience (13A4); 1:200 dilution). Immunostained tissues were photographed using a dissection microscope (Zeiss; AxioVision). For comparison, images were captured with the same exposure time. CRC tissue microarray slides were purchased from Biomax (Co2086).

Placental tissues (n = 6; n = number of placental samples per condition) and cell lysates (n = 10; n = number of experiments performed; 10–20 μg) were separated on a 4–12% Bis-Tris gel and transferred to a nitrocellulose membrane. Membranes were incubated overnight with primary antibodies (phospho-γ-H2AX-Cell Signaling Technology, Danvers, MA, USA, rabbit #9718, RAGE-R&D, Minneapolis, MN, USA, goat #AF1145, p-ATM-Cell Signaling Technology, Danvers, MA, USA, mouse #4526, MRE11-Cell Signaling Technology, Danvers, MA, USA, rabbit #4895, β-Actin-Cell Signaling Technology, Danvers, MA, USA, rabbit #4967 or mouse #3700 and Lamin B1-Cell Signaling Technology, Danvers, MA, USA, rabbit#15068). The membranes were then incubated with fluorescent secondary antibodies for an hour and washed ×3 with TBST the next day prior to imaging.
Membranes were developed on a Li-COR Odyssey CLx. Fluorescence densities were determined, and comparisons were made between treated and control groups.

Tumor mass was snap-frozen, pulverized, collected and resuspended in lysis buffer (50 mM HEPES, 250 mM KCl, 0.1 mM EDTA, 0.1 mM, 0.1% NP-40, 10% glycerol) as previously described [18 ]. Extracts were centrifuged at 12,000 rpm for 30 min at 4°C and supernatants were collected for protein quantitation. A total of 15 μg of total protein was analyzed by SDS-PAGE and blotted onto PVDF membranes to detect Bcl-2 (2870, Cell signaling), Mcl1 (5453, Cell signaling), Bcl-XL (2764, Cell Signaling, Danvers, MA), Bax (sc-493, Santa Cruz Biotechnology), FAK (sc-557, Santa-Cruz Biotechnology), cleaved caspase-3 (9661, Cell Signaling), cleaved PARP (5625, Cell Signaling), E-cadherin (610181, BD Biosciences, San Jose, CA), phospho-histone 3 (9713, Cell Signaling), and phospho γ-H2AX (9718, Cell Signaling). β-actin (4970, Cell Signaling) was used as a loading control. HRP-conjugated anti-mouse (sc-2005, Santa Cruz Biotechnology) or anti-rabbit (sc-2004, Santa Cruz biotechnology) were used as secondary antibodies and signals were detected using a Lumigen TMA-6 reagent (GE healthcare, Pittsburgh, PA).

For total protein extraction, cells were lysed by a lysis buffer containing protease inhibitor cocktail (Roche; USA) and PMSF (Roche; USA). To separate nuclear fraction, we used NE-PER nuclear and cytoplasmic extraction kit (Piece, Rockford, USA) according to the manufacturer’s instruction^{39 (link)}. Western blotting was then performed according to the routinely protocol including protein separation, membrane-transferring, block and primary and secondary antibody incubation. The primary antibodies were against β-actin (abcam; 1:5000; UK), GAPDH (abcam; 1:5000; UK), c-myc (proteintech; 1:1000; USA), RAD51 (abcam; 1:10000; UK), KU80 (abcam: 1:10000; UK), β-catenin (proteintech; 1:1000; USA) and phospho-γH2AX (Cell Signaling, 1:1000; USA), HMGB1 (abcam, 1:10000; UK) and p-chk1, p-chk2, p-cdc25C, ATRIP (Cell Signaling; 1:1000; USA), acetyl-H3 (Lys9) (absin, 1:5000; China), GFP (proteintech, 1:2000, USA). Finally, proteins were visualized with ECL and Kodak film without light.

Before or after IR exposure, cells or sections from xenografts were fixed with 4% paraformaldehyde for 20 min and permeabilized by 0.5% triton for 10 min. The cells were then soaked in normal goat serum for 30 min and incubated with primary antibodies: c-myc (proteintech; 1:50; USA), RAD51 (abcam; 1:1000; UK), KU80 (abcam; 1:1000; UK), β-catenin (proteintech; 1:50; USA), MDC1 (proteintech; 1:50; USA), and phospho-γH2AX (Cell Signaling (20E3); 1:250; USA) overnight at 4 °C. After washed by PBS for three times, the cells were then incubated with Alexa Flura® 488 conjugated, goat anti-rabbit IgG at room temperature for 2 h. Then the cells’ nuclei were stained with DAPI. Finally, images were obtained with a fluorescent microscope (Zeiss) and images were captured under the same exposure time.