Fig. 2

rAQP4 IgG promotes extracellular ATP release. HEK293 cells (A, B) or HEK293 cells expressing AQP4 (C, D) were stimulated with control IgG (A, C) or rAQP4 IgG (B, D) in the presence of 5% normal human serum (NHS). Only HEK-AQP4 incubated with rAQP4 IgG underwent a balloon-like morphological change (arrowheads) (D). Scale bar = 100 μm. (E) Extracellular ATP level in the culture medium. HEK293 cells (HEK) or HEK293 cells expressing AQP4 (HEK-AQP4) were incubated with either control IgG or rAQP4 IgG in the presence of various concentrations of complement. Data are expressed as means ± SEM and were analyzed by factorial ANOVA with three between-subjects factors [NHS, 0%, 5%, or 10%; group: rAQP4 IgG or control IgG; HEK-AQP4, HEK or HEK-AQP4]. The main effects of NHS (P < 0.001), group (P < 0.001), and HEK-AQP4 (P < 0.001) and the interactions of NHS times group (P < 0.001), NHS times HEK (P < 0.001), and group times HEK (P < 0.001) were statistically significant. For the visualization, the corresponding two-group comparisons were indicated by the asterisks, *P < 0.05 and **P < 0.01. F HEK and HEK-AQP4 were immunostained with anti-AQP4 antibody. HEK-AQP4 were confirmed to express AQP4 antigen. Scale bar = 25 μm. Rat astrocytes were stimulated with control IgG (G, I) or rAQP4 IgG (H, J) in the presence of 10% NHS (G, H) or 2% rabbit complement (I, J). Only rat astrocytes incubated with rAQP4 IgG exhibited a balloon-like morphological change (arrows). G, H Scale bar = 50 μm. I, J Scale bar = 100μm. K Rat astrocytes were stimulated with human IgG or rAQP4 IgG in the presence of 10% NHS. Data are expressed as means ± SEM and were analyzed by Student’s t test and **P < 0.01