Alisertib mln8237

Alisertib (MLN8237), Aurora A Inhibitor Ⅰ and ponatinib were purchased from Selleck Chemicals (Houston, TX, USA) and MedKoo Biosciences (Chapel Hill, NC, USA); imatinib was provided by Novartis Pharma AG (Basel, Switzerland). Stock solutions of alisertib and ponatinib were prepared in dimethyl sulfoxide, and imatinib was dissolved in distilled water, aliquoted, and stored at −20° C. Other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Dulbecco's modified Eagle's medium (DMEM) was from Mediatech (Manassas, VA); Opti-MEM, Lipofectamine and Plus reagent were from Invitrogen (Carlsbad, CA), [³²P]γATP was from Perkin-Elmer (Waltham, MA); the synthetic peptide substrate for AURA (T288) (APSSRRTTLCGT) was from Bio-synthesis (Lewisville, TX), ECL reagent was from GE Healthcare (Piscataway, NJ); phosphatidic acid was from Avanti Polar Lipids (Alabaster, AL). The plasmids used in this study were as follows: pCMV6-mycDDK-AURA, pcDNA3.1-mycPLD2-WT, pEGFP-Spo20PABD (phosphatidic acid sensor) and pRK5-mycS6K-WT. The AURA inhibitor Alisertib, MLN 8237, was from Selleck Chemical (Houston, TX).

For proliferation assays cells were plated in 384-well plates at a density of 800 cells per well in 40 µl total volume. One day later, a serial dilution of each inhibitor was performed using a D300e dispenser (Tecan). 96 h post-treatment, cell viability was determined using CellTiterGlo reagent (Promega) and luminescence quantified on an Envision MultiLabel Plate Reader (PerkinElmer). To calculate the fraction cell viability drug-treated cells were normalized to average cell viability of DMSO-only treated cells. Curve fitting was performed using GraphPad Prism v9.1.1 four-parameter inhibitor response with variable slope. AUC values were calculated by GraphPad Prism v9.1.1. Inhibitors were obtained from SelleckChem: Erlotinib-OSI-744 (S1023), Osimertinib-AZD9291 (S7297), Torin 1 (S2827), Alisertib-MLN8237 (S1133), Barasertib-AZD1152 (S1147). DMSO (Sigma-Aldrich).

Human breast cancer cell lines (MDA-MB-468, MDA-MB-231 and MCF-7), lung cancer cell lines (A549 and H460), and colon cancer cells (HCT-116 and HT-29) were obtained from ATCC (the American Type Culture Collection, Manassas, VA, USA). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) or RPMI 1640, containing 10% heat-inactivated fetal bovine serum. Alisertib (MLN8237) and INCB-18424 (Ruxolitinib) were purchased from Selleck chemicals (Houston, TX). Nocodazole and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO). VX-680 (Tozasertib) was purchased from BioVision incorporated (Milpitas, CA). AJI-214 and AJI-100 were synthesized as described previously [30 (link)]. All drugs for cell culture were dissolved in DMSO (Sigma).

Cells were harvested at a confluent rate of 80–90% and seeded into 96-well plate with 5000 (L929), 10.000 (BT-12, CHLA-02, CHLA-04) and 15.000 (CHLA-266, Re1-P6) cells per well. Cell growth inhibition assay was assessed after 72 h of exposure to a serial dilution of BTZ (2.8–100 nM) (Selleckchem, Houston, TX, USA), alisertib (MLN8237) (0.2–200 µM) (Selleckchem), suberoylanilindehydroxamic acid (SAHA) (2.6–100 µM) (Selleckchem), lenvatinib (0.1–100 µM) (Selleckchem, Houston, TX, USA). Proliferation assay was performed by incubating the cells with MTT 5mg/mL (Bionovas, Taipei, Taiwan) for adherent cells or with MTS (ab197010, abcam) for suspension cells at 37 °C for 4 h. Then dark blue formazan crystals were solubilized in dimethyl sulfoxide (DMSO) (Bionovas, Taipei, Taiwan) for MTT assay. The formazan dye’s absorbance was measure at the optical density of 570 nm for MTT and of 490 nm for MTS with a spectrophotometer (SpectraMax 190, Molecular devices, San Jose, CA, USA). All experiments were triplicated in two independent biological replicates.