Cytotoxic effect of CNF1 on GL261 cells. (A) Percentage of living GL261 cells after continuous exposure to CNF1 (18 nM). Data represent means ± SEM (n = 3). Inset: Morphology of GL261 cells treated with CNF1 for 9 days. Scale bar = 5 μm. (B) Percentage of cells stained only by Annexin V (Ann V + PI-, left columns), stained only by Propidium Iodide (Ann V- PI+), positive for both markers (Ann V + PI+) and unlabeled (right columns). The analysis was performed in untreated GL261 cells (Control, open bars), in GL261 cells serum deprived for 2 days (grey bars), and in GL261 cells treated with CNF1 for 10 days (green bars). CNF1 induces a very significant upregulation in the frequency of Annexin V-Propidium Iodide double labelled cells (one way ANOVA followed by Holm-Sidak test, p < 0.05).