Figure 1.
Figure 1. Human DCs express cell-surface CLEC-1. (A) Western blot analysis of CLEC-1 expression in human moDCs, HAECs, HUVECs, and HEKs. Cell extracts were immunoprecipitated with anti-human CLEC-1 mAb (D6 clone) and then analyzed by western blot with a second in-house anti-human CLEC-1 mAb (IgG1). Arrows indicate human CLEC-1 and IgG HC and LC at the expected size of 32, 50, and 25 kDa, respectively. M line represents molecular-weight size markers. (B) Representative dot plots and histograms of IgG1 isotype or CLEC-1 (IgG1) staining in non-permeabilized (non-perm) and permeabilized (perm) conditions, evaluated by flow cytometry for human blood: (i) CD16+ and CD16− subpopulation of CD45+CD14−CD11c+HLA-DRhigh DCs; (ii) CD45+CD14+CD16+ monocytes; (iii) SSChighCD16+ neutrophils; and (iv) HAECs. Histograms represent the overlay image of CLEC-1 staining (gray filled histogram) matching the isotype control IgG1 staining (open histogram). (C) Representative dot plots or histograms of IgG1 isotype or CLEC-1 (IgG1) staining vs DC-SIGN or CD16 staining for human moDCs in non-perm or perm conditions and evaluated by flow cytometry (i-ii). Histograms represent the overlay image of CLEC-1 staining (gray filled histogram) matching the isotype control IgG1 staining (open histogram). (iii) Cell-surface expression of CLEC-1 vs HLA-DR on unstimulated (US), TLR 4-L (LPS), TLR 3-L (Poly I:C), TLR 7-L (R848), and TGF-β–stimulated moDCs. (iv) Histogram represents MFI ± standard error of the mean (SEM) of CLEC-1 staining of 6 independent experiments. Statistical analysis of CLEC-1 MFI staining was performed between US and each stimuli. Panel Di shows representative confocal microscopy images, and (ii) quantitation of CLEC-1 protein in non-perm and perm conditions for human HUVECs, moDCs, CD16+ monocytes and neutrophils. Panels exhibiting DAPI (blue) and CLEC-1 (green) staining revealed by anti-human CLEC-1 mAb (D6 clone) followed by secondary anti-mouse Alexa-488 antibody. Original magnification ×600. Images are representative of 4 independent experiments. CLEC-1 protein quantitation was performed by velocity software and expressed as histogram of mean ± SEM of numbers of fluorescent spots per cell (n ≥7). *P < .05; **P < .01; ***P < .001. IgG HC, IgG heavy chain; IgG LC, IgG light chain; MFI, mean fluorescence intensity; mono, monocytes; neutro, neutrophils.

Human DCs express cell-surface CLEC-1. (A) Western blot analysis of CLEC-1 expression in human moDCs, HAECs, HUVECs, and HEKs. Cell extracts were immunoprecipitated with anti-human CLEC-1 mAb (D6 clone) and then analyzed by western blot with a second in-house anti-human CLEC-1 mAb (IgG1). Arrows indicate human CLEC-1 and IgG HC and LC at the expected size of 32, 50, and 25 kDa, respectively. M line represents molecular-weight size markers. (B) Representative dot plots and histograms of IgG1 isotype or CLEC-1 (IgG1) staining in non-permeabilized (non-perm) and permeabilized (perm) conditions, evaluated by flow cytometry for human blood: (i) CD16+ and CD16− subpopulation of CD45+CD14−CD11c+HLA-DRhigh DCs; (ii) CD45+CD14+CD16+ monocytes; (iii) SSChighCD16+ neutrophils; and (iv) HAECs. Histograms represent the overlay image of CLEC-1 staining (gray filled histogram) matching the isotype control IgG1 staining (open histogram). (C) Representative dot plots or histograms of IgG1 isotype or CLEC-1 (IgG1) staining vs DC-SIGN or CD16 staining for human moDCs in non-perm or perm conditions and evaluated by flow cytometry (i-ii). Histograms represent the overlay image of CLEC-1 staining (gray filled histogram) matching the isotype control IgG1 staining (open histogram). (iii) Cell-surface expression of CLEC-1 vs HLA-DR on unstimulated (US), TLR 4-L (LPS), TLR 3-L (Poly I:C), TLR 7-L (R848), and TGF-β–stimulated moDCs. (iv) Histogram represents MFI ± standard error of the mean (SEM) of CLEC-1 staining of 6 independent experiments. Statistical analysis of CLEC-1 MFI staining was performed between US and each stimuli. Panel Di shows representative confocal microscopy images, and (ii) quantitation of CLEC-1 protein in non-perm and perm conditions for human HUVECs, moDCs, CD16+ monocytes and neutrophils. Panels exhibiting DAPI (blue) and CLEC-1 (green) staining revealed by anti-human CLEC-1 mAb (D6 clone) followed by secondary anti-mouse Alexa-488 antibody. Original magnification ×600. Images are representative of 4 independent experiments. CLEC-1 protein quantitation was performed by velocity software and expressed as histogram of mean ± SEM of numbers of fluorescent spots per cell (n ≥7). *P < .05; **P < .01; ***P < .001. IgG HC, IgG heavy chain; IgG LC, IgG light chain; MFI, mean fluorescence intensity; mono, monocytes; neutro, neutrophils.

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