P histone h2ax

DNA double strand breaks were detected by both flow cytometry [48 (link)] and Western blotting [35 (link)] as described previously. For flow cytometry, TFB-treated cells were fixed, washed, and incubated at 4 °C for 1 h in 2 μg/mL of p-Histone H2AX (Ser 139) (γH2AX) monoclonal antibody (sc-101696; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing, cells were suspended for 1 h in a secondary antibody (Jackson Laboratory, Bar Harbor, ME, USA) for 30 min at room temperature. Finally, the cells were resuspended in 20 μg/mL of PI for flow cytometry analysis (BD Accuri™ C6; Becton-Dickinson).
For Western blotting of γH2AX expression, 30 μg protein lysates were resolved in 10% SDS-PAGE. Except when p-Histone H2AX (Santa Cruz Biotechnology) was chosen for the primary antibody, procedures were the same as those employing Western blotting for apoptosis proteins, mentioned above.

40 μg of cell lysates obtained from ground-frozen tissue or cells were used for assessing protein expression. Primary antibodies used for detection were FOXM1 (1:1000, Abcam, ab207298), MAD2 (1:1000, BD biosciences, 610678), HA (1:1000, Covance, MMS-101R), Cyclin B (1:200, Santa Cruz, SC-245), Cleaved-Caspase 3 (1:1000, Cell signaling, 9661), p-Histone H2AX (1:250, Santa Cruz, SC-517348), CDC20 (1:250, Santa Cruz, SC-13162), Actin (1:3000, Sigma, A2066). Protein band quantification was carried out using ImageJ.

Antibodies for western blotting were purchased from Cell Signaling (Danvers, MA): p-EGFR (Tyr1068) (2234, 1:1000), EGFR (4267, 1:1000), p-MEK1/2 (Ser217/221) (9154, 1:1000), MEK1/2 (8727, 1:1000), p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (9101, 1:1000), p44/42 MAPK (Erk1/2) (9102, 1:1000), p-p38 MAPK (Thr180/Tyr182) (9211, 1:1000), p38 MAPK (9212, 1:1000), α-tubulin (2144, 1:2000), vimentin (3390, 1:1000), TACE (3976, 1:1000), Snail (3879, 1:1000), E-cadherin (3195, 1:1000), p-FAK (Tyr397) (3283, 1:1000), FAK (3285, 1:1000), p-STAT3 (Tyr705) (9131, 1:1000), STAT3 (9139, 1:1000), GAPDH (2118, 1:2000), β-actin (3700, 1:2000), p-DNA-PK (68716, 1:1000), DNA-PK (38168, 1:1000), PARP (9532, 1:1000), p-Histone H2A.X (Ser139) (9718, 1:1000), Histone H2A.X (2595, 1:1000), p-Chk2 (Thr68) (2197, 1:1000), Chk2 (2662, 1:1000), p53 (2524, 1:1000), caspase-7 (9492, 1:1000), caspase-3 (14220, 1:1000), AURK-A (14475, 1:1000); Santa Cruz Biotechnology (Dallas, TX): integrin β1/ITGB1 (sc-374429, 1:1000). Selumetinib (AZD6244, MEK1/2 inhibitor), osimertinib (AZD9291, EGFR^T790M), M3814 (DNA-PK inhibitor, DNA-PK-I) and ZM-447439 (AURK-A inhibitor, AURK-A-I), MK-4827 (PARP inhibitor, PARP-I), M4076 (ATM inhibitor, ATM-I), M6620 (ATM/ATR inhibitor, benzosertib) and M4344 (ATR inhibitor, ATR-I) were purchased from Selleck Chemicals (Selleckchem).