Untreated SHEP-21N cells (750 cells/well) and SHEP-21N cells under doxycycline pressure for ≥3 days (1,000 cells/well) were seeded in 384-well microplates (Greiner Bio-One, #781080) and incubated overnight. Next, cells were exposed to 14.6 nmol/L to 15 μmol/L adavosertib in 2-fold serial dilutions using the Tecan D300e Digital Dispenser. DMSO and thonzonium bromide (20 μmol/L) were included as negative and positive control, respectively. Readout was conducted on days 1 and 3 after treatment initiation using the CellTiter-Glo 2.0 Luminescent Cell Viability Assay (Promega) and the EnSpire Alpha multimode plate reader (PerkinElmer) for luminescence detection. Cell viabilities were calculated as described for in vitro HTS and dose–response curves were generated using GraphPad Prism (version 9.3.1). All experiments were conducted in triplicate in three independent experiments. Each data point represents the mean ± standard deviation (SD). The DepMap GDSC dataset (https://depmap.org/portal/ ; ref. 30 (link)) was utilized to explore the efficacy of adavosertib (i.e., MK-1775) in an independent cohort. A one-sided Wilcoxon test was performed to statistically compare the AUC (31 (link)) difference between MYCN amplified (N = 6) versus MYCN wild-type (N = 14) classical neuroblastoma cell lines.
Protocol detail
Adavosertib Dose-Response in Neuroblastoma
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Adavosertib
After treatment
Cell lines
Cell viabilities
Cells
Dilutions
Dmso
Doxycycline
Luminescence
Luminescent assay
Mk 1775
Mycn
Neuroblastoma
Pressure
Prism
Promega
Thonzonium bromide
Corresponding Organization : Princess Máxima Center
Other organizations : Children's Cancer Institute Australia, Sydney Children's Hospital, Prince of Wales Hospital, Royal Children's Hospital, Murdoch Children's Research Institute, Peter MacCallum Cancer Centre, University of Melbourne
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Variable analysis
independent variables
- Adavosertib concentration (14.6 nmol/L to 15 μmol/L in 2-fold serial dilutions)
- Cell viability
- Cell type (Untreated SHEP-21N cells, SHEP-21N cells under doxycycline pressure for ≥3 days)
- Cell seeding density (750 cells/well for untreated SHEP-21N cells, 1,000 cells/well for SHEP-21N cells under doxycycline pressure)
- Incubation time (overnight)
- Thonzonium bromide (20 μmol/L)
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