Ml216

The RWPE-1, LNCaP, 22RV1, PC3, and DU145 cell lines were obtained from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China). HEK-293T cells were purchased from Kunming Cell Bank (Kunming, China). The cells were cultured and aliquots were stored in liquid nitrogen for future use. RWPE-1 cells were cultured in customized medium (ZQXZ Bio, Shanghai, China). LNCaP, 22RV1, and DU145 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (P/S) at 37 °C in a 5% CO₂ environment. PC3 cells were cultured in ATCC-formulated F-12K medium supplemented with 10% FBS and 1% P/S at 37 °C in a 5% CO₂ environment. HEK-293T cells were cultured in DMEM/HIGH GLUCOSE (HyClone, Logan, Utah, USA) supplemented with 10% FBS and 1% P/S at 37 °C in a 5% CO₂ environment. Olaparib (AZD2281) and ML216 (CID-49852229) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

3D-outgrowth quantification and in vivo bioluminescence imaging were carried out as described (Gooding et al, 2017 (link)). Cells were cultured in appropriate media supplemented with 5% Cultrex, as well as 5-FdU or 5-fluorouracil (5-FU; Sigma-Aldrich), the ATR inhibitors AZ20 (4-{4-[(3R)-3-Methylmorpholin-4-yl]-6-[1-(methylsulfonyl)cyclopropyl]pyrimidin-2-yl}-1H-indole; MedChem Express) or VE-821 (3-amino-6-[4-(methlsulfonyl)phenyl]-N-phenyl-2-pyrazinecarboxamide; Sigma-Aldrich), the BLM inhibitor ML216 (1-(4-fluoro-3-(trifluoromethyl)phenyl)-3-(5-(pyridine-4-yl)-1,3,4-thiadiazol-2-yl)urea; MedChem Express), or the telomerase inhibitor BIBR1532 (Selleckchem) as indicated. For U2OS cells, Cultrex cushions were supplemented with type I collagen (3 mg/ml; BD Biosciences). In mice, 5-FdU and 5-FU were administered by slow intravenous injection (0.1 ml at a rate of 0.4 ml/min). Mice were randomly assigned to receive cell lines or treatments. Endpoints for 3D-outgrowth and pulmonary tumor assays were determined prospectively. Growth was normalized to an initial reading taken 24 h post-plating (in vitro) or immediately after inoculation (in vivo).

Roswell Park Memorial Institute (RPMI) 1,640 culture medium was purchased from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). Olaparib (AZD2281, Ku-0059436) was purchased from Selleck (Houston, TX, USA). ML216 (CID-49852229) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Antibodies against pBRCA1 (Ser1524), p53BP1 (Ser1778), pATM (Ser1981), pATR (Ser428), Rad50, Mre11, pDNA-PKcs (Ser2056), Ku80, pChk2 (Thr68), pChk1 (Ser317), pRb (Ser807/811), pRb (Ser795), p21, and γH2AX were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against RPA70, Rad51, 53BP1, CyclinA, and Ki-67 were purchased from Abcam (Cambridge, UK). Antibodies against β-tubulin, β-actin, and glyceraldehyde 3-phosphate dehydrogenase were purchased from Proteintech (Rosemont, IL, USA).