Azd1152 hqpa

Cell viability was assessed over 96 hr using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) absorbance (Promega, Madison, WI, USA) at 490 nm with at least 8 repeats per treatment condition per time point in each experiment. For cell count experiments, 1 × 10⁶ cells were cultured on a 3-cm Petri dish for 24 hr prior to exposure to drug inhibitor and grown for a total period of 96 hr with varying concentrations of AZD1152-HQPA (Selleck Chemicals LLC, Houston, TX, USA) dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA). The final DMSO concentration was 0.01% v/v in media. The live cell number was determined by trypan-blue exclusion using a Vi-Cell XR counter (Beckman-Coulter, Mississauga, Ontario). Experiments were independently repeated three times.

Parasite was maintained by in vivo propagation of the parasite material in mice (supplied by Xiamen University Laboratory Animals Center, XMULAC). Mature and developing protoscoleces were collected from parasite material, manually picked under the microscope, and then immediately used for RNA isolation or EdU labeling. In vitro cultivation of metacestode vesicles was performed using host cell conditioned medium as previously described [26 (link)]. The growth of metacestode vesicles and the process of vesicle formation from protoscoleces were examined after 21 days and 14 days of culture, respectively as described by Cheng et al. [27 (link)].
Aurora inhibitors (MLN8237, MLN8054, MK-5108 and AZD1152-HQPA), nocodazole and hydroxyurea were supplied by Selleck Chemicals. Drugs were added into the culture medium at a final concentration as indicated. All of the drug experiments were carried out under axenic culture conditions as described before [26 (link)]. For longer periods of treatment, experiments were performed with exchange of the medium containing the same ingredients every three days.

For knockdown and replacement experiments, Borealin 3′ UTR siRNA (AGGUAGAGCUGUCUGUUCAdTdT) was transfected using RNAiMAX (Invitrogen) according to the manufacturer’s protocol. Mitotic phenotypes were analyzed in the first mitosis after complementation. Borealin stable cell lines were plated in the presence of 2 mM thymidine for 24 h. Next, the fresh DMEM/10% FBS media were added, and cells were transfected with siRNA and incubated for 12 h. Next, media were replaced, and cells were incubated with DMEM/10% FBS with 2 mM thymidine, transfected with siRNA, and incubated for 12 h. Next, cells were released from thymidine in fresh media DMEM/10% FBS for 9 h and fixed. For Aurora B inhibition experiments, 1 μM AZD1152-HQPA (Barasertib; SelleckChem) was added for 30 min before fixation in glutaraldehyde after cells were treated with siRNA following the protocol described previously.