P chk2

Protein samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, UK). Blots were blocked for 1 h in 5% milk/0.1% Tween 20 in phosphate buffered saline (PBS-T) and then incubated with primary antibodies (1: 1000) at 4°C overnight. Blots were then washed three times for 15 min in PBS-T, followed by incubation with secondary antibody (according to different primary antibodies, HRP-conjugated goat anti-mouse, anti-rabbit, and rabbit anti-goat IgG were used (1: 5000, Santa Cruz, Dallas, TX)) in 5% milk/PBS-T for 1 h, and then washed three times for 15 min in PBS-T. The membranes were briefly incubated with ECL detection reagent (Amersham Biosciences, Castle Hill, Australia) to visualize the proteins and were then exposed on X-ray film. Primary antibodies used were as follows: ATM (Cat# 600-401-398), and p-ATM (Cat# 600-401-400) were purchased from Rockland Immunochemical (Gilbertsville, PA, USA); cleavage-caspase-3 (Cat# 9661), cleavage-PARP (Cat# 5625P), γ-H2Ax (Cat# 9718), Bax (Cat# 2772s), and Bcl-2 (Cat# 2870), ATR (Cat# 2790), p-ATR (Cat# 2853S) were from Cell Signaling Technology (Beverly, MA); PARP (Cat# 7150), pro-caspase-3 (Cat# 7272), β-Actin (Cat# 1615), Chk1 (Cat# 377231), p-Chk1 (Cat# 2341), Chk2 (Cat# 8813), p-Chk2 (Cat# 2661) were from Santa Cruz (Dallas, Texas, US).

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).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma (Sigma/Aldrich Chemical Company, St. Louis MO, USA). 7-KC was obtained from Cayman (Cayman Chemical Company, Ann Arbor, MI, USA). Cell culture biologicals (Dulbecco's modified Eagle's medium [DMEM], fetal bovine serum [FBS], trypsin/EDTA etc) were obtained from Gibco (Life Technologies, Taipei, Taiwan). Eahy926 (EAHY) endothelial cells were kindly given by Professor Cora-Jean S. Edgell (North Carolina University, NC, USA) [32 ] and studied in my laboratory before [33 , 34 (link)]. EAHY cells are hybrid cells from human umbilical vein endothelial cells and have the differentiation function of endothelium and express factor VIII-related antigens [32 ]. They were cultured in DMEM containing 10% FBS. Enzyme-linked immunosorbant assay (ELISA) kits for IL-8 were obtained from PeproTech (PeproTech Company, Rocky Hill, NJ, USA). Antibodies against Cdk1, cyclin B1, p-ATM, p-ATR, p-Chk1, p-Chk2, p-p53, p-Akt and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Santa Cruz (Santa Cruz Biotechnology Inc., Dallas, Texas, USA).