Figure 6.

Signaling of newly synthesized IL-6 in transit to the plasma membrane. (A) Confocal images of moDCs co-expressing IL-6 fused to GFP (green in merge; IL-6-GFP) and IL-6RA fused to mCherry (magenta; IL-6RA-mCh) stimulated without or with LPS for 4 h. DAPI is in blue in merge. Graphs: fluorescence cross-sections as indicated. Yellow regions: overlap of IL-6-GFP and IL-6RA-mCherry. Bar graph: quantification of overlap by Pearson correlation coefficient (four donors; >7 cells/donor). Scale bar, 20 μm. (B) Stills from time-lapse TIRF microscopy of moDC overexpressing IL-6-GFP (green in merge) and IL-6RA-mCherry (magenta). Details of two exocytosis events indicated by the yellow arrowheads are shown. Graphs: quantification of the signals over time. Scale bar, 10 μm. Yellow regions: occurrence of exocytosis events (see also Supplementary Movie S3). (C) Scheme of the experimental setup for STAT3 activation under constant medium flow with or without LPS for 4 h. The graph shows the IL-6 concentrations in the supernatant from moDCs cultured in the presence or absence of LPS and with or without flow by ELISA. (D) Quantification of pY-STAT3 activation from moDCs in C for individual donors (mean ± SEM). Data were normalized to the highest pY-STAT3/STAT3 ratio per donor. (E) Quantification of VAMP3 after siRNA gene silencing (siV3) or non-targeting siRNA control (siCntrl) with or without 4 h LPS stimulation (mean ± SEM from 3 donors). GAPDH: loading control. (F) Quantification by western blot of pY-STAT3 activation after siV3 incubated with or without LPS for 4 h (mean ± SEM from 3 donors). (G) Quantification of pY-STAT3 activation upon incubation with 1 μg/ml LPS for 4 h or 15 ng/ml IL-6 for 20 min, in the absence or presence of an IL-6-neutralizing antibody (anti-IL-6) or IgG control (IgG-cntrl) (mean ± SEM from three donors). Representative western blots are shown.