Fig. 1

oHSV-1 replicates and induces cell death in GL261 glioblastoma cells in vitro. (A) GL261 cells were seeded in a 24-well plate (7.5 × 10⁴ cells/well), infected with oHSV-1 (MOI = 1) for 1 h in serum-free DMEM medium, then maintained in DMEM medium supplemented with 2% fetal bovine serum (FBS). The medium was refreshed daily, and EGFP expression, serving as an indicator of viral infection, was monitored at 1, 2, 6, and 10 days post-infection. Fluorescence was monitored using a Leica EpiFluorescence microscope revealing a progressive spread of the infection to the entire monolayer. Concurrently, brightfield microscopy demonstrated an increasing cytopathic effect, characterized by cellular rounding. All images were captured at 10x magnification and represent multiple microscopic fields from a triplicate experiment. (B) GL261 cells were seeded in a 24-well plate (7.5 × 10⁴ cells/well), infected with oHSV-1 (MOI = 1) for 1 h in serum-free DMEM medium, then maintained in DMEM medium supplemented with 2% FBS. The supernatant was collected and replaced with fresh 2% FBS medium every 24 h. Infectious viral particles were quantified as plaque forming units (PFU)/mL by plaque titration assay on green monkey Vero cells. The experiment was performed in triplicate. Y axis is in logarithmic scale. (C) 10 days after infection, surviving infected GL261 cells were trypsinized and counted using a hemocytometer and Trypan blue exclusion staining. Uninfected GL261 cells seeded and cultured in the same conditions for 10 days were counted in a similar way. The experiment was performed in triplicate and the difference in the number of live cells between uninfected and oHSV-1-infected GL261 cells was evaluated by Student’s t-test. ****P < 0.0001. In all panels, error bars represent standard deviation