Quantifying Cell Viability Responses

Cells in single-cell suspension were plated with culture medium in 96-well clear-bottom black microplates (Cat #3917; Corning Costar, Corning, NY, USA) at a density of 2500 cells per well for adult GBM cell lines (GBM6, GBM 10, GBM14, GBM 39, GBM43, and GBM 108) or 5000 cells per well for DMG cell lines (SU-DIPG XIII-P [47 (link)], SU-DIPG XVII [48 (link)], SF8628, SF8628-B23 (H3F3A K27M knockout of SF8628), and PED17) and cultured overnight at 37 °C with 5% CO₂. The next day, cells were treated in triplicate with either vehicle (ddH₂O or PBS) or serial dilutions of IL-13 (to final concentrations of 100, 50, 20, 10, 5, 1, and 0.5 ng/mL) or GB-13 (to final concentrations of 320, 100, 32, 10, 3.2, 1, 0.32, 0.1, 0.032, 0.01, 0.0032, and 0.001 ng/mL). Cells were incubated for 72 h and then assayed with CellTiter-Glo Luminescent Cell Viability Assay (Cat #G7570; Promega, Madison, WI, USA) according to the manufacturer’s recommendations. Luminescence was measured using an Infinite M200 PRO multimode microplate reader (Tecan Group, Männedorf, Switzerland), normalized to control wells (ddH₂O or PBS only), and relative luminescence treatment was plotted as a function of drug concentration. The potency (50% inhibitory concentration, IC₅₀) of each treatment was calculated by non-linear least-squares curve fitting using Prism 9 (GraphPad, San Diego, CA, USA).