Figure 2. MAC induces nuclear translocation and coordinate expression of IL-15/IL-15Rα on the cell surfaces of IFN-γ-primed human ECs.

(A) Representative images of confocal microcopy analysis of ECs that were pretreated with IFN-γ for 48 hours before being treated with PRA for 30 minutes or 4 hours, fixed and permeabilized, and stained for intracellular IL-15 and IL-15Rα. Scale bars: 5 μm. (B) IFN-γ–pretreated ECs were treated with either gelatin veronal buffer (GVB) control or PRA sera treatment, fixed and permeabilized prior performing PLA between IL-15 and IL-15Rα. Representative images of confocal microscopy analysis. Scale bars: 30 μm. (C) IFN-γ–pretreated ECs were treated with either GVB or PRA for 30 minutes and 4 hours. Extracts from 4 specific cellular compartments (cytoplasmic [C], membrane [M], soluble nuclear [SN], and chromatin-bound nuclear [CN]) were isolated by stepwise lysis and analyzed for IL-15 and IL-15Rα protein by immunoblotting. (D) ELISA measurements of IL-15 in culture supernatants of IFN-γ–primed ECs treated with PRA sera. (E) ECs were pretreated with IFN-γ for 48 hours before PRA treatment and analysis of surface staining IL-15 and IL-15Rα by flow cytometry (n = 4). (F) Representative images of confocal immunofluorescence analysis of IL-15 and IL-15Rα surface staining on unpermeabilized IFN-γ–pretreated ECs treated with either PRA sera or control GVB. Scale bars for top row: 30 μm; scale bars for “Zoom” bottom row: 5 μm. (G) IFN-γ–pretreated ECs were treated with either GVB control or PRA sera treatment. Proximity ligation assay (PLA) was performed between surface IL-15 and IL-15Rα and analyzed by confocal microscopy. Scale bars: 30 μm. Data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; 1-way ANOVA and Tukey’s multiple comparisons test in D and unpaired 2-tailed Student’s t test in E. Representative of 3 independent experiments using 3 HUVEC donors.