Fig. 9

Effects of treatment with AQP4-IgG positive and negative NMOSD serum samples on U-87MG-AQP4-ECFP. U-87MG-AQP4-ECFP cells were treated with human NMOSD patient sera #1–6 in combination and active or heat-inactivated human complement. After the treatment, cells were harvested for RNA isolation (A), and the cell supernatant was used for cytotoxicity (B) and IL6 (C) assessment. (A) Log2-fold changes of 2^−ΔΔCt values of human sera plus complement (AC/IC). ΔCt values were calculated with the Ct values of the respective genes minus the Ct values of the housekeeping gene GAPDH. To assess serum-specific effects (ΔΔCt), ΔCt values of complement-only samples were subtracted from those with serum plus complement. (B) Cytotoxicity was assessed with the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega). Afterwards, each mean value of active or inactive complement only was subtracted from the respective values with serum. These values were normalized to those of lysis buffer-treated cells (set to 100%), to obtain serum-specific impact on cell lysis. (C) IL6 levels of the cell supernatants after treatment were measured by human IL6 ELISA (R&D Systems). Afterwards, each median value of active or inactive complement-only was subtracted from the respective values with serum. Negative values were set to zero. (A) The heat map was created with GraphPad Prism 10.4. (B,C) Bar charts (means, standard deviation, and individual values) were created with GraphPad Prism 10.4. Groups (n = 3) were compared by 2-way ANOVA with Šídák’s multiple comparisons test * p < 0.05, **p < 0.01, ***p < 0.001. AC,  active complement; AQP4, aquaporin-4; IC,inactive complement.