Azd4573

A total number of 12,000 cells per well were seeded in a 96-well plate and then treated with different concentration of drugs (SNS-032 and AZD4573, obtained from Selleck, Houston, TX, United States) for 72 h. Cell viability was evaluated using CTG (Promega CellTiter-Glo^TM Luminescent Cell Viability Assay Kit) according to the manufacturer’s protocol. The absorbance optical density of 405 nm was recorded using a microplate reader (Synerge2; BioTek Instruments, Winooski, VT, United States), and the half maximal inhibitory concentration (IC50) was calculated using GraphPad Prism.

Cell proliferation was measured by a Counting Kit-8 (CCK-8) detection kit (Catalog No. CK04, Dojindo Molecular Technologies, Japan). The cells were seeded in a 96-well plate with 3000 cells per well and treated with siRNA transfection. At the indicated time points, 10 μL of CCK-8 solution was added, followed by incubation at 37°C for two hours, and absorbance at 450 nm was determined.
For in vitro inhibitor assays, AZD4573 (S8719, Selleck) was dissolved to 10 mM in DMSO and then dosed to yield a final DMSO concentration of ≤ 0.3%. Chordoma cells was treated with different concentration of AZD4573 from 0.4 nM to 4000 nM (ten-folds dilution) for nine days and the cell proliferation was tested by CCK-8 as well.

The antibodies used for ChIPs and immunoblot analyses are summarized in S2 Table. PTC209 and AZD4573 were purchased from Selleckchem.com (Houston, TX, United States).