Hydroxyurea

Hoxb8 immortalized [23 (link)] R26-CreER^T2Recql4^fl/+ (control) and R26-CreER^T2Recql4^fl/K525A cells were maintained in IMDM, 10% FBS (non-heat inactivated) and 1% GM-CSF containing media (BHK-HM5 cell conditioned media). The cells were treated for 4 days with 400nM 4-hydroxy tamoxifen (Merck Millipore) then genotyped to confirm complete recombination. Cells were then plated at 10,000 cells/well in 96 well plates (Corning, CLS3610) and incubated for 48 hours with the indicated concentration of drugs in triplicates per dose (dose range Doxorubicin: 0–0.5μM, Hydroxyurea: 0–0.5mM, 4-Nitroquinoline: 0–2μM and Topotecan: 0–0.5mM). Doxorubicin was obtained from St. Vincent’s Hospital Pharmacy. Hydroxyurea was purchased from Selleck. 4-Nitroquinoline and Topotecan were purchased from Sigma-Aldrich. Cell viability was measured using ATP-Lite (Perkin Elmer) as directed by the manufacturer and measured on an EnSpire plate reader (Perkin Elmer). Data were plotted and the IC₅₀ value calculated using Prism 7 software. The dose-response curve was plotted as mean±SEM.

Cytotoxic chemical or inhibitors were added to cell culture for indicated period of time for respective assays and at following concentrations: Cisplatin (4 μg/ml; Supertrack Bio-pharmaceutical, 131,102), MMS (0.5 mg/ml); Camptothecin (2 uM; Selleck, S2423), Mitomycin C (0.2μg/ml; Selleck, S8146), Paclitaxel (35 uM; Selleck, S1150), Nocodazole (100 ng/ml; Sigma, M1404) and hydroxyurea (5 mM; Selleck, S1896); Caffeine (20 μg/ml; Sigma, 58-08-2). Ionizing radiation was performed at 1 Gray/min using custom-made X-ray machine (Wandong Ltd., Beijing) and dosages were described in respective experiments.

In vitro cultivation and drug treatment of E. multilocularis metacestode vesicles were performed as previously described (Cheng et al., 2017 (link)). Briefly, for the growth assay, vesicles were cultured in the host cell conditioned medium supplemented with drug and the growth of vesicles was analyzed at indicated time. For the vesicle formation assay, protoscoleces were collected from parasite material and in vitro cultured in conditioned medium supplemented with drugs. The initial process of vesicle formation, in which protoscoleces dilate and vacuolate, were examined after 21 days of culture. The CDK4/6 inhibitor Palbociclib, EGFR inhibitor CI-1033, MEK inhibitor U0126 and hydroxyurea were supplied by Selleck Chemicals. Recombinant human EGF was supplied by PeproTech. Drugs were added into the culture medium at a final concentration as required. The vesicles were then used for RNA isolation, whole protein extraction, or EdU labeling at the indicated time.

The following compounds were provided by Selleck Chemicals (Houston, TX): 5-fluorouracil (#S1209), adavosertib (#S1525), B02 (#S8434), berzosertib (#S7102), campthotecin (#S1288), cisplatin (#S1166), etoposide (#S1225), gemcitabine (#S1714), GSK'872 (#S8465), hydroxyurea (#S1896), irinotecan (#S2217), KU-55933 (#S1092), KU-60019 (#S1570), mirin (#S8096), nocodazole (#S2775), NU7026 (#S2893), olaparib (#S1060), oxaliplatin (#S1224), prexasertib (#S7178), Q-VD-Oph (#S7311), rabusertib (#S2626), rucaparib (#S1098), talazoparib (#S7048), triapine (#S7470), UPF 1069 (#S8038) VE-821 (#S8007), and veliparib (#S1004). Verapamil (#V4629) were provided by Sigma-Aldrich, CCT241533 (#HY-14715B) by MedChem Express (Monmouth Junction, NJ), NP-004255 (#PC-61922) by ProbeChem (Shanghai, China), and PV1019 (#220488) by Calbiochem-Merck-Millipore (Billerica, MA). The appropriate amount of DMSO (#5879) was employed for negative control conditions.

All NSC compounds were obtained from the Developmental Therapeutics Program (DTP) repository at NCI/NIH. JA1-5 (JS-2088), JA5-8 (BAS 05169959), and JA5-9 (AO-476/12797006) were purchased from Ryan Scientific. JA2-9 (Z57032584), JA5-10 (EN300-63858), and JA5-11 (Z385453050) were purchased from Enamine. JA2-8 (JFD03560SC) was purchased from Maybridge, and JA5-7 (F3350-0573) was purchased from Life chemicals. All inhibitors were dissolved at 40 mM concentrations in DMSO. Hydroxyurea, Nedaplatin, Doxorubicin, Veliparib, and Olaparib were purchased from SelleckChem. PDD00017273 was from Tocris.

Cisplatin (S1166), Adriamycin (S1208), Hydroxyurea (S1896), Rucaparib (S1098), 5-Fluorouracil (S1209), DMXAA (S1537), BFA (S7046), Rapamycin (S1039), and 3-MA (S2767) were from Selleck. Doxycycline hyclate (D9891), Phenylarsine oxide (P3075), GTP-Agarose suspension (G9768), Protein A agarose (P3476), mouse monoclonal Anti-Flag M2 Affinity Gel (A2220), and mouse monoclonal Anti-HA-Agarose antibody (A2095) were from Sigma-Aldrich. DiABZI STING agonist-1 (Tautomerism) (HY-112921) and Digitonin (HY-N4000) were from MCE. HaloTag® Ligands (GA1110) for super-resolution microscopy were from Promega. 2'3'-cGAMP (tlrl-nacga23-1) was from Invivogen. Pierce™ Anti-DYKDDDDK Magnetic Agarose (A36797) was from Thermo Fisher Scientific.

The concentrations used for the MAPK pathway inhibitors (MEKi-1/2 and ERKi) were determined experimentally to be the doses at which phosphorylation of ERK1/2 and RSK1/2/3 returned to baseline despite the presence of oncogenic BRAF expression. The reagents used in these studies are as follows: MEKi-1: U0126 (Selleck Chemicals), 10 µM; MEKi-2: Trametinib (GSK1120212), 20 nM (Selleck Chemicals); ERKi: SCH772984, 20 nM (Selleck Chemicals); Hydroxyurea, 1 mM (Selleck Chemicals); doxycycline, 1 µg/mL (Sigma-Aldrich D9891); thymidine, 2.5 mM (Sigma-Aldrich T1895); RO-3306, 7 µM (Sigma-Aldrich SML0569)