Olaparib

GFP-CtIP, full-length and truncated FLAG–CtIP were generously provided by Dr. Junjie Chen (MD Anderson Cancer Center, TX). FLAG–ASF1A was generously provided by Dr. Zhiguo Zhang (Columbia University, NY). Full-length and truncated FLAG–USP52 and Myc-USP52 were generously provided by Dr. Lei Shi (Tianjin Medical University, Tianjin, China). FLAG–USP52 S1003A and S469A, FLAG–CtIP K62R, K760R, K782R, and 2KR mutants were generated by site-directed mutagenesis (Stratagene). Flag–CtIP–T847E was purchased from Addgene. CHX, IgG agarose, anti-FLAG agarose, 3×FLAG peptide and ATM inhibitor KU55933 were purchased from Sigma Aldrich. Olaparib was purchased from Toronto Research Chemicals. Anti-USP52 (ab241505, 1:1000) was purchased from abcam; anti-CtIP (Thr847) (p1012-847, 1:2000), and anti-CtIP (Thr327) (p1012-327, 1:2000) were purchased from PhosphoSolutions; anti-RPA32 (sc-56770, 1:2000), anti-Ub (sc-8017, 1:2000) and anti-CtIP (sc-271339, 1:1000 for WB) were purchased from Santa Cruz; anti-FLAG (F1804, 1:2000) was purchased from Sigma; anti-pS345 Chk1 (2348, 1:1000), anti-Myc Tag (2276, 1:2000) and anti-SQ/TQ motif (9607, 1:1000) were purchased from CST; anti-CtIP (61141, 1:1000 for IF) was purchased from Active Motif; anti-GAPDH (60004-1-lg, 1:2000) was purchased from Proteintech; anti-Rad51 (GTX100469, 1:1000) was purchased from Genetex.

Culture media and supplements (Dulbecco’s Modified Eagle’s Medium low glucose (DMEM); fetal bovine serum (FBS); penicillin/streptomycin, trypsin/EDTA; antimycotic) and Trypan Blue were all purchased from Gibco (Life Technologies do Brasil Ltda., São Paulo, SP, Brazil). 5-fluorouracil (Cas number 51–21-8), MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Cas number 298-93-1), propidium iodide (PI; Cat number 25535–16-4) and Dimethyl Sulfoxide (DMSO; Cas number 67685) were all purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Olaparib was purchased from Toronto Research Chemicals (Cat number 763113–22-0, Toronto Research Chemicals, North York, ON, Canada). PE Annexin V Apoptosis Detection Kit I was purchased from BD Bioscience (Cat number 559763, BD Bioscience, Franklin Lakes, NJ, USA). All other chemicals and solvents used were of analytical or pharmaceutical grade.