Figure 4.
Neutrophils deliver CatG into EC in vivo and in vitro. (A) Representative 3D super-resolution confocal images (Zeiss Airyscan) of HUVEC incubated with cell-free neutrophil supernatant (cf-NS) of non-stimulated (ice control) or stimulated (50 ng/ml TNFα + 1 µM fMLP) neutrophils pre-treated with DMSO or NEI20; scale bar = 10 µm; magnification = 4 µm. (B) 3D-rendered isosurface of the endothelial monolayer from the 0.35 µm z-stack images using Imaris software showing that the CatG (magenta) appears along the strictly intracellular cortactin signal (green); scale bar = 5 µm. (C) Quantitative analysis of CatG inside HUVEC per field of view (FOV). (D) Quantification of the cortactin MFI of the images in A using Imaris software (n = 3 independent experiments). (E and F) Representative 3D confocal super-resolution images (Leica SP8) of PBS (E) or TNFα-treated (F) cremasteric PCVs (scale bars = 20 μm; magnification = 3 µm). The middle panels have the green channel (MRP-14) removed for clarity. The bottom panels show CatG signals extracted from venules using the CD31 signal. Orange zoomed-in panels show rendered 3D isosurface of endothelial CD31 (cyan) with internalized CatG signal (magenta). (G) Quantification of endothelial CatG MFI in PCV; n = 3 mice/group. Data are represented as means ± SEM; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗∗P < 0.0001. Refer to the image caption for details. Panel A: Three sets of 3D super-resolution confocal images of endothelial cells (HUVEC) incubated with cell-free neutrophil supernatant (cf-NS) under different conditions. The images show staining for Hoechst (blue), cortactin (green), and cathepsin G (CatG, magenta). The conditions include ice control, cf-NS with DMSO, and cf-NS with NEI20. Each set includes a zoomed-in view. Panel B: A 3D-rendered isosurface image showing CatG (magenta) along the cortactin signal (green) within the endothelial monolayer. Panel C: A bar graph quantifying CatG inside HUVEC per field of view (FOV) under the different conditions. Panel D: A bar graph quantifying the mean fluorescence intensity (MFI) of cortactin under the different conditions. Panel E: 3D confocal super-resolution images of PBS-treated cremasteric postcapillary venules (PCVs) showing staining for CD31 (cyan), MRP-14 (green), and CatG (magenta). The middle panels remove the green channel for clarity, and the bottom panels show CatG signals extracted from venules using the CD31 signal. Panel F: Similar images as Panel E but for TNF-treated PCVs. Panel G: A scatter plot quantifying the endothelial CatG MFI in PCVs for PBS and TNF-treated groups.

Neutrophils deliver CatG into EC in vivo and in vitro. (A) Representative 3D super-resolution confocal images (Zeiss Airyscan) of HUVEC incubated with cell-free neutrophil supernatant (cf-NS) of non-stimulated (ice control) or stimulated (50 ng/ml TNFα + 1 µM fMLP) neutrophils pre-treated with DMSO or NEI20; scale bar = 10 µm; magnification = 4 µm. (B) 3D-rendered isosurface of the endothelial monolayer from the 0.35 µm z-stack images using Imaris software showing that the CatG (magenta) appears along the strictly intracellular cortactin signal (green); scale bar = 5 µm. (C) Quantitative analysis of CatG inside HUVEC per field of view (FOV). (D) Quantification of the cortactin MFI of the images in A using Imaris software (n = 3 independent experiments). (E and F) Representative 3D confocal super-resolution images (Leica SP8) of PBS (E) or TNFα-treated (F) cremasteric PCVs (scale bars = 20 μm; magnification = 3 µm). The middle panels have the green channel (MRP-14) removed for clarity. The bottom panels show CatG signals extracted from venules using the CD31 signal. Orange zoomed-in panels show rendered 3D isosurface of endothelial CD31 (cyan) with internalized CatG signal (magenta). (G) Quantification of endothelial CatG MFI in PCV; n = 3 mice/group. Data are represented as means ± SEM; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗∗P < 0.0001.

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