Fig. 1

Effect of siramesine in cell lines. The standard human glioma cell lines U87, A172, T98G and U251 were grown as adherent cell cultures and exposed to siramesine. a Images of the glioma cell line T98G showed cells which rounded up and shrinked upon siramesine exposure (arrow). b Upon exposure to siramesine (0–50 μM) cell proliferation (WST-1 assay) cell death (LDH assay) was measured. U87 and A172 were more sensitive towards siramesine compared to U251 and T98G after both 24 and 48 h as seen in both the WST-1 assay (upper panel) and the LDH assay (lower panel). c Inhibitors of cathepsins (z-FA-FMK and Ca-074-Me) and caspases (z-DEVD-FMK and z-DEVD-CMK) were used to identify mediators of siramesine-induced cell death. Cell proliferation (WST-1 assay) and cell death (LDH assay) was measured for all four cell lines, suggesting differential mechanisms in the different cell lines. d-e Lysosomal involvement was investigated with acridine orange which accumulates in acidic cellular compartments, primarily in lysosomes resulting in red fluorescence. Acridine orange staining in the glioma cell lines appeared as red dot-like staining corresponding to the presence of intact lysosomes (0 μM siramesine). Confocal imaging identified loss of red fluorescence in the lysosomes upon siramesine exposure in all of the glioma cell lines after only 1 h of exposure to siramesine (5–30 μM). This suggested that siramesine exposure lead to compromised/ruptured lysosomal membranes. Scalebar 100 μm (a), Scalebar 50 μm (e). Data are displayed as mean values ± SEM, and *P < 0.05, **P < 0.01, ***P < 0.001 were assessed by one-way ANOVA. AU, arbitrary units