Atm inhibitor ku55933

pWZL Neo Myr Flag Syk was purchased from addgene and subcloned into plvx3 vector. GFP-CtIP were generously provided by Dr. Junjie Chen (MD Anderson Cancer Center, TX) and subcloned into plvx3. Flag–CtIP–T847E was purchased from Addgene. Anti-FLAG agarose, 3×FLAG peptide and ATM inhibitor KU55933 were purchased from Sigma Aldrich. Olaparib was purchased from LC-lab. Syk inhibitor R406 was purchased from selleckchem.
Syk(D3Z1E, 13198) antibody, anti-SQ/TQ motif (6966) and anti-pS345 Chk1 (2348) was purchased from CST. anti-CtIP (Thr847) (p1012–847, 1:2000), and anti-CtIP (Thr327) (p1012–327, 1:2000) were purchased from PhosphoSolutions. anti-RPA32 (sc-56770) and anti-CtIP (sc-271339 for WB) were purchased from Santa Cruz; anti-FLAG (F1804) was purchased from Sigma; anti-CtIP (61141, 1:1000 for IF) was purchased from Active Motif; anti- γ-H2AX antibody(05–636) was purchase from Millipore. Anti-GAPDH (60004–1-lg) was purchased from Proteintech; anti-Rad51 (GTX100469 for IF) was purchased from Genetex. Anti-H3(17168–1, AP) was purchased from Proteintech. All the secondary antibody were purchased from Jackson ImmunoResearch.

Epirubicin, Aclarubicin, Doxorubicin, Daunorubicin, Doxorubicinone, and Dimethyl-doxorubicin were dissolved in phosphate-buffered saline (PBS) at 1 mg/ml, stored at −80°C and used at the indicated final concentrations. Anthracyclines were from various commercial sources except for Dimethyl-doxorubicin, a gift from Jacques Nefjees. Etoposide (Sigma-Aldrich) was dissolved in DMSO and used at indicated final concentrations. MG-132 (Sigma-Aldrich) was dissolved in DMSO and used at a final concentration of 10 µM. ATM inhibitor KU-55933 (Sigma-Aldrich) was dissolved in DMSO and used at a final concentration of 5 µM. Human recombinant TNF (PeproTech) was dissolved in RPMI and used at a final concentration of 10 ng/ml for the indicated times. Actinomycin D (ActD, Sigma-Aldrich) was dissolved in DMSO and used at a final concentration of 5 μg/ml for the indicated times. LPS (from E. coli 055:B5, Santa Cruz) was dissolved in RPMI and used at a final concentration of 100 ng/ml. PFA (Paraformaldehyde)-fixed E. coli were prepared as before (Moura-Alves et al., 2011 (link)) in house and added to the medium at a ratio of 20 bacteria per cell. AlamarBlue cell viability assay (Invitrogen) was used to determine cell viability according to the manufacturer’s instructions.