Fig. 8
Effects of eculizumab on U-87MG-AQP4-ECFP cells after complement and E5415A treatment. Cells were treated with active or heat-inactivated human complement (AC/IC) in combination with E5415A (10 µg/mL). In addition to E5415A + AC, complement inhibitors against C5 (eculizumab, 30 µg/mL in (A–C), 1–100 µg/mL in D + E) were added to the complement 15 min before the application on cells. (A) LDH assay results with cell lysis normalized to lysis buffer-treated cells and additional AC/IC-only background absorbance subtraction. (B) IL6 ELISA results of IL6 levels in cell supernatants of treated samples. IL6 production of untreated cells was subtracted. (C) qPCR results of genes differently expressed in U-87MG-AQP4-ECFP cells after complement and E5415A treatment. Log2-fold changes of E5415A + AC/IC with or without eculizumab-treated cells were compared to untreated controls. GAPDH served as a housekeeping gene, and all ΔCt values of all genes were calculated before the normalization to untreated cells. After treatments, cells were stained for (D) terminal complement complex deposition on their surface or (E) complement component C3/C3b. The mean fluorescence of complement component-bound fluorescent antibodies was measured. Fluorescence of untreated samples was subtracted. Additionally, the fluorescence levels of all samples were normalized to E5415A + AC-only treated cells. All experiments (A-E) were performed in biological triplicates and technical duplicates. (A + B) 2-way ANOVA with Šídák’s multiple comparisons test between treatments with and without eculizumab. (D + E) An ordinary one-way ANOVA with Dunnett’s multiple comparisons test was performed, comparing all complement inhibitor treatments to E5415A + AC-only treated samples * p < 0.05 **p < 0.01, ***p < 0.001.
