Incucyte system

Percentage confluence over time was measured in an Incucyte system (Sartorius, Göttingen, Germany). Cell proliferation was also measured using a luciferase-based assay (Cat#G9711, RealTime Glo, Promega).

The procedure was adapted from previous work [28 (link)]. Cells (i.e., U87MG, U118MG, U138MG, and GL261) were plated in 96-well CellCarrier Spheroid ULA Microplates™ (Perkin Elmer^®) at 2000 cells/well in culture medium, including 3–4 replicates per experimental condition. Spontaneously formed tumor spheroids appeared after 48 to 74 h of growth. Then, spheroids were treated with TMZ or Fingolimod in culture medium containing fluorescent DNA intercalating agent, which detects dead cells that are stained when their plasma membrane is compromised (Sytox™ green Dead Cell Stain, reference S7020, ThermoFisher Scientific™). Confluence and the positive fluorescence area were monitored by image-based analysis using the Incucyte™ system (Sartorius^®, Goettingen, Germany). One image every 4 h was acquired. Spheroid growth was determined by analyzing the total spheroid surface (in pixel²). All data were normalized to the control condition per experiment. Cytotoxicity was determined as the fluorescent positive area in pixel² among the spheroid area. Cytotoxicity was defined as a percentage (%). Two to three independent experiments were performed per test substance. All experiments were performed with 3 to 4 technical replicates.

Bright-field images of organoids were acquired every 8 h over a period of 120 h by the Incucyte SX5 Live-Cell Analysis Instrument (Essen Bioscience). Processing and merging of z-stacks was performed by the organoid module of Incucyte system according to the manufacturer’s instructions (Sartorius AG). The TissueFAXSiPlus system (TissueGnostics) was used for endpoint analysis. Z-stacks from four focal planes were merged to generate bright-field images of organoids. Bright-field image analysis and subsequent quantification of organoid size and number was performed by StrataQuest (version 7.1.1.129; TissueGnostics; Stüve et al., 2023 ).

CVB3 mutants were generated by site-directed mutagenesis using the mCherry-CVB3 fluorescent infectious clone mentioned above^{49 (link)}. For each mutant, non-overlapping primers containing the desired mutation in one of the primers were used to introduce the mutation with Q5 polymerase, followed by DpnI (Thermo Scientific) treatment, phosphorylation, ligation, and transformation of chemically competent bacteria (NZY5α Competent Cells, NZY Tech). Successful mutagenesis was verified by Sanger sequencing. Subsequently, plasmids were linearized with SalI-HF (ThermoFisher), and 600 ng of plasmid were transfected into 5 × 10⁴ HEK293T cells, together with 600 ng of a plasmid encoding the T7 polymerase (Addgene 65974) using Lipofectamine 2000 (ThermoFisher) according to manufacturer’s instructions of use. Cells were then incubated until cytopathic effect (CPE) was observed and passage 0 virus was collected. Infectious virus titer was determined using an Incucyte system (Sartorius) to quantify the red fluorescence from the mCherry reporter signal as detailed above. If needed, mutants with low titers were amplified for an additional passage. The capsid region of the mutant virus populations was reverse transcribed, PCR-amplified, and sequenced to ensure no compensatory mutations or reversions arose during replication.