Luminescence assay

Stably transduced Ba/F3 cells were cultured in RPMI medium supplemented with IL-3 (1 ng ml⁻¹). For the cell proliferation assay, Ba/F3 cells were transferred into IL-3 depleted RPMI medium, and cell growth was quantified in quadruplicates every 2–4 days by a luminescence assay (Promega). For cell viability assays and ALK inhibitor–dose-response curves, 2,000 Ba/F3 cells were plated in triplicates in 96-well plates with increasing concentrations of the ALK inhibitors crizotinib (LC laboratories), TAE-684 (ChemieTek), or ceritinib (ChemieTek) as indicated. All drugs were re-suspended in DMSO. The cell viability was assessed after 72 h by a luminescence assay (Promega). Results were normalized to cell growth in medium containing an equivalent concentration of DMSO. The inhibition curve was determined with GraphPad Prism 6.0 software using the ‘log(inhibitor) vs response – variable slope’ nonlinear regression model. For immunoblots, 10 million Ba/F3 cells were harvested after 2 h treatment with crizotinib, washed in ice-cold PBS, and lysed in RIPA buffer. All assays were independently performed at least twice and a representative experiment is shown.

WT cortical neurons were treated with vehicle (0.001% DMSO) or different concentrations of CP2 for 24 h before NAD⁺/NADH levels were determined using Luminescence assay (Promega) according to the manufacturer’s instructions.

Neuroblastoma cells were transduced with GPC2 targeting shRNA as above and after 48-72 hours of selection in puromycin, GPC2 depleted or control shNTC transduced cells were plated in at least triplicate in a Real-time Excelligence system (RT-CES; F Hoffman La-Roche, Basal, Switzerland) and growth was monitored every 30 minutes. For CellTiter-Glo^® Luminescent Cell Viability Assays (Promega) or Caspase Glo^® 3/7 Assays (Promega), shRNA transduced neuroblastoma cells were seeded in 96-well plates in parallel to the above after 48-72 hours of selection in puromycin and assayed after 72 hours of additional growth. Luminescence was measured with the respective luminescence assay according to manufacturer's instructions (Promega) and quantified relative to shNTC transduced cells. For the Caspase Glo^® 3/7 Assays, luminescence values were quantified relative to shNTC transduced cells and further normalized to the cell number using the CellTiter-Glo^® Assay luminescence values performed in parallel. Data presented are representative of at least two independent experiments.