Fig 2: Caspase-3 or -7 are responsible for the second A-SMase activation peak in CD95-receptosomesa. A-SMase activity in magnetic and non-magnetic fractions isolated from control or DEVD (inhibitor of caspase-7 / -3) treated SKW6.4 cells. DEVD decreases the second activation peak of A-SMase in the magnetic fraction demonstrating the potential role of caspase-7 and -3 in the activation of the A-SMase. Represented are the mean values of four independently performed assays with control or DEVD treated cells (2way Anova with Bonferroni post-test). b. Western blot analysis of CD95 magnetic and non-magnetic fractions isolated from SKW6.4 cells with anti-pro-caspase-7, anti-cleaved-caspase-7, anti-pro-caspase-3 and anti-cleaved-caspase-3 antibodies. c and d. Merged confocal microscopic images of SKW6.4 cells shows a co-localization (yellow) between SFF-CD95L/CD95 (green) and pro-caspase-7, cleaved-caspase-7, pro-caspase-3 or cleaved-caspase-3 (red) at indicated times of CD95 internalization.

Fig 3: ASM overexpression does not sensitize glioma cells to alkylating agents or irradiation in vitro.Human glioma wt (open bars), SIEW (dark grey bars) or ASM (light grey bars) cells of (A, C, E) LNT-229 or (B, D, F) T98G were treated with increasing concentrations of (A, B) TMZ or (C, D) CCNU for 24 h or increasing doses of (E, F) irradiation. Statistically significant changes compared to untreated cells are marked with asterisks (*, p<0.05; **, p<0.01; t-test).

Fig 4: Biphasic activation of A-SMase in CD95-receptosomesa. Merged confocal microscopic images of SKW6.4 cells stimulated with 100 ng/ml SFF-CD95L. The CD95L was visualized by an anti-FLAG antibody and anti-mouse Alexa-Fluor 488. A-SMase stained by a polyclonal anti-A-SMase antibody and anti-rabbit Alexa-Fluor 555 was detected in CD95-receptosomes between 1 min and 60 min internalization. Co-localization of CD95 and A-SMase is indicated in yellow. b. Magnetic and non-magnetic fractions were used to measure A-SMase activation in SKW6.4 cells after indicated time points of CD95 internalization. Three independent preparations were pooled and measured in triplicate. (1way-Anova with Bonferroni's multiple comparison test) c. Western blot analysis of CD95 magnetic and non-magnetic fractions with anti-CD95 and anti-A-SMase antibodies. Corresponding to the activity assay, Western blot analysis demonstrated a biphasic appearance of A-SMase in the magnetic fractions.

Fig 5: Apoptosis inducing SFF-CD95L activates the A-SMase in SKW6.4 cell lysates in a biphasic mannera. Induction of apoptosis in SKW6.4 cells after treatment with 100 ng/ml SFF-CD95L analyzed by AnnexinV/propidium iodide staining. b. Western blot analysis of SFF-CD95L induced activation of caspases. Initiator caspase-8 as well as effector caspases-3 or -7 showed a type I cell line dependent activation kinetic. c. Time course of A-SMase activity determined in SKW6.4 cell lysates after SFF-CD95L treatment. The activity maximums appeared after 15 and 90 minutes SFF- CD95L stimulation (n=3, 1way-Anova with Bonferroni's multiple comparison test).