Cyquant direct red cell proliferation assay kit

Cells were treated with PTC-209 (Tocris) and PD325901 (Tocris) both dissolved in DMSO (Sigma) at the indicated concentrations. At specific time points and after appropriate treatment, cells were harvested, and the number of viable cells was counted with a hemocytometer and Trypan Blue staining or with CyQUANT Direct Red Cell Proliferation Assay Kit (ThermoFisher Scientific). Synergy/antagonist effect of the combined treatment was analyzed as previously described.^{16 (link)} See Supplementary Methods.

For growth curve and cell viability assays, MB cells or hNSCs were seeded in 24-well plates at the same density. Cells were treated for 72 h with mTOR (rapamycin, Sigma; Torin1, Calbiochem), FLT3 (TCS359, SelleckChem) or MET (PHA665752, Tocris) inhibitors or with inositol hexakisphosphate (IP6, Sigma) at the indicated concentrations. At specific time points or after appropriate treatment, cells were harvested, and the number of viable cells was counted with a haemocytometer and Trypan Blue staining or with CyQUANT Direct Red Cell Proliferation Assay Kit (ThermoFisher Scientific). Syenrgy/antagonist effect of the combined treatment was identified by Loewe Model with Combenefit software^{88 (link)}. Apoptosis was assessed with Caspase-3 Colorimetric Assay kit (ab39401, Abcam) following the manufacturer’s protocol. Briefly, cells treated with increasing concentrations of IP6 were lysed, and 50 µg of protein lysate was incubated with DEVD-AFC substrate and reaction buffer for 2 h at 37 °C.