Fig. 4: Subcellular localization of IL-1β and LAMP2A in LPS and LRZ-stimulated monocytes. | Cell Death & Disease

Fig. 4: Subcellular localization of IL-1β and LAMP2A in LPS and LRZ-stimulated monocytes.

From: Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways

Fig. 4

a Confocal microscopy of representative monocyte stimulated 6 h with LPS (upper panel) or LRZ (lower panel), stained for IL-1β and LAMP2A as indicated (N = 6). Scale bar, 5 μm. One single stack of Z-stack series is shown (Z = 4 for LPS and Z = 5 for LRZ). b Left-hand panels: TIRF microscopy of representative monocyte stimulated with LPS (upper panel) or LRZ (lower panel) and stained as in a (N = 6). Penetration depth = 110 nm. Scale bar, 5 μm. Right-hand panel: quantification of LAMP2A+/IL-1β+ vesicles found by TIRF analysis, in 50 LPS or LRZ stimulated monocytes, using Laplacian plugins for the extraction of image features (ImageJ) (N = 6), mean ± SEM. c Direct stochastic optical reconstruction microscopy (dSTORM) of IL-1β and LAMP2A as indicated (N = 4). A representative LPS (upper panel) and LRZ-stimulated cell (lower panel) is shown. Scale bar, 2μm. Insets show magnifications to better highlight structures where IL-1β and LAMP2A molecules are close to each other. d Cross-correlation (C) between the two channels (green and red, corresponding to IL-1β and LAMP2A fluorescence) as a function of distance. Six cells from 4 independent experiments were analyzed. For each cell, 10 representative ROIs (2 × 2 μm2) were chosen in the peripheral region of the cells for the analysis. The closeness of LAMP2A and IL-1β molecules is not random (C > 1) and increased co-clustering at the plasma membrane is higher in LPS-stimulated monocytes (y axis, amplitude of the cross correlation). Error bars indicate standard error of C on each ROI in the individual cell. e The image shows a representative Western blot (out of 3 performed) comparing the intracellular pools of LAMP2A and GAPDH in monocytes stimulated for 3 or 6 h with LPS or LRZ. f Surface expression of LAMP2A increases upon a 6 h stimulation with LPS relative to LRZ (monocytes from a representative subject out of 3 tested are shown). Data information: In b (right-hand panel) data are expressed as Mean ± SEM. Unpaired t-test was used for significance (***P < 0.001)

Back to article page