P atm

The following antibodies were used: Actin (Sigma), ATM (D2E2, Cell Signaling Technologies), P-ATM (Ser1981, Abcam), CHK2 (checkpoint kinase 2; Cell Signaling Technologies), P-CHK2 (Thr68, Cell Signaling Technologies), MELK (Sigma), p21 (cyclin-dependent kinase inhibitor 1; Upstate), p53 (PAB 1801, Santa Cruz), p53 DO-1 (Santa Cruz), P-p53 (Ser15, Cell Signaling Technologies) and Vimentin (Santa Cruz).
BrdU and γH2A.X (phospho-histone H2AX) immunostainings were performed following a treatment for 2 h with the indicated compounds. Cells were fixed in 10% formalin solution (Sigma–Aldrich) for 10 min at room temperature and 20 min in ice-cold methanol, after which they were washed 3× in PBS. Subsequently, the cells were blocked for 1 h in PBSAT (PBS+1% BSA+0.1% Tween) and incubated overnight with the primary antibody in PBSAT. The cells were washed 3× with PBSAT and incubated with the secondary antibody for 1 h at room temperature, again washed 3× with PBSAT and incubated with the Hoechst staining solution (1/2000 in PBSAT).

After the cells were treated with different concentrations of HS-173 and irradiated for various times, they were collected and washed with cold phosphate-buffered saline (PBS). The cells were then lysed with RIPA buffer containing protease and phosphatase inhibitor cocktails (GenDEPOT, Barker, TX, USA). Proteins in whole-cell lysates were resolved by sodium dodecyl sulfate protease and phosphatase inhibitor (SDS-PAGE), and transferred onto nitrocellulose membranes. The blots were first incubated with the appropriate primary antibodies, and then with horseradish peroxidase (HRP)-conjugated secondary antibodies. Antibody binding was detected using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA, USA). Primary monoclonal antibodies against the following proteins were used: cleaved PARP, cleaved caspase-3, survivin, p-AKT, AKT (Cell Signaling Technology, MA, USA) p-Cdc2, p-ATM, ATM, p-DNA-PKcs, DNA-PKcs, p-KAP1, p-53BP1 (Abcam, Cambridge, UK) KAP1, 53BP1(Santa Cruz, Dallas, TX, USA) and β-actin (Sigma-Aldrich, OH, USA) Secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Whole cell lysates were prepared by RIPA kits (Atto, Tokyo, Japan) while the protein concentration was accomplished by a SMART^TM micro BCA kit (Intron biotechnology). Proteins were separated on Bis-Tris pre-cast gels and transferred to PVDF membrane (Bio Rad, CA, USA). Primary antibodies against PARP (BD), caspase-3, CD133, VEGF, Chk1, p-Chk1, Chk2, p-Chk2, PCNA, MCM2, p-ATM (Abcam, Cambridge, UK), mTOR, p-mTOR, AMPKα, p-AMPKα (Cell signaling, MA, USA), HIF-1α (Sigma-Aldrich), β-actin, and β-tubulin (Santa Cruz, CA, USA) were used at 1:1000. Peroxidase conjugated secondary antibody were against rabbit or mouse (Jackson Immunoreasearch, MD, USA) diluted 1:5000. Chemiluminescent solution was obtained from Intron biotechnology and detected using Fuji RX film.

Cells were grown on cover slides and treated with arginine starvation as described. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature and incubated in a blocking buffer (PBS; 5% goat serum; 0.3% Triton™ X-100) for 60 min. Antibodies used were TOM20 (1:200, 42406), γH2AX (1:400, 2577), p-53BP1 (S1778) (1:100, 2675), p65 (1:400, 8242) and V5 (1:500, 80076) from Cell Signaling; H3K9me3 (1:1000, 39765) and H3K27me3 (1:500, 39155) from Active Motif; p-DNA PKcs (S2056) (1:200, ab124918) and p-ATM (S1981) (1:100, ab81292) from abcam. Alexa 488 and Cy3 conjugated secondary antibodies (Jackson Laboratories) were used in 1:1000 dilution at room temperature for 1 h. Nuclei were stained with 0.5 μg/mL DAPI for 10 min. Immunofluorescence images were taken using a Leica TCS SP5 confocal microscope.

The proteins from tissues and cells were separated by standard 10% SDS‐PAGE followed by transferring the proteins to a PVDF membrane. The proteins were detected by the following primary antibodies: AIMP3 (Proteintech, 10805‐1‐AP), p53 (Proteintech, 10442‐1‐AP), N‐cadherin(Cell Signaling, 13116), E‐cadherin (Cell Signaling, 3195), Vimentin (Cell Signaling, 5741), Snail (Cell Signaling, 3879), Slug (Cell Signaling, 9585), ATM (Proteintech, 27156‐1‐AP), p‐ATM (Abcam, ab81292), β‐actin (Cell Signaling, 3700) and GAPDH (Proteintech, 60004‐1‐Ig) and followed by incubation with a secondary antibody. Staining was performed with ECL western blot detection reagent. Antibody to GAPDH or β‐actin was served as the endogenous control. All experiments were performed in triplicate.
For immunoprecipitation, cells were collected with lysis buffer and subjected to IP with HA antibodies and protein‐G beads at 4°C overnight with gentle rolling. Bound proteins were analysed by immunoblotting.

Hematoxylin and eosin (H&E) staining was performed on the paraffin-embedded sections from formalin-fixed colonic tissues. Histologic analysis was carried out independently by two pathologists, according to previously validated criteria (Ameho et al., 1997 (link)). For IHC analysis, colon sections obtained from patients with UC or CAC, and DSS-induced models were stained with the indicated antibodies, including γH2Ax (CST, Danvers, MA, United States, #9718), p-ATM (Abcam, ab36810) and APC7 (Santa Curze, sc-365649).