Fig 1: Assessing candidate L-EV cargo S100A9 in the context of lymphatic trafficking.(a and b, magnification) Immunohistochemical analysis identified cytoplasmic and nuclear S100A9 staining of predominantly keratinocytes in Patients 1 and 2 with additional weak cytoplasmic staining of melanoma cells in Patient 1. (c) By electron microscopy, immunogold labeling with S100A9 (arrows) associated with isolated patient-matched L-EVs (scale bar 250 nm) compared to secondary only stained L-EVs (Supplemental Figure 2). (d and e, magnification) Patient matched SLNs showed expression of S100A9 in a gradient pattern with strongest staining present in the subcapsular sinus as well as S100A9 positive staining in macrophage-enriched areas. Co-localization of S100A9 staining with CD68 macrophage marker was observed on serial tissue section (f).
Fig 2: Representative images of S100A9 staining patterns in SLN tissue. IHC staining of S100A9 was observed in all SLN cases assessed and scored by clinical pathologist. Two distinct staining patterns emerged, (a) a cytoplasmic stain correlating with histiocyte regions within the SLN (SLN2) and (b) a nuclear staining of cells consistent with dendritic cells (SLN10).
Fig 3: MMP-9, clusterin, and S100A9 expression in EAT and S100A9 and NF-?B p65 expression in SAT from CAD and non-CAD groups as measured by western blot. Western blot analysis compared MMP-9, clusterin, and S100A9 in EAT and S100A9 and NF-?B p65 in SAT from CAD and non-CAD groups. (a) and (b), gel images and relative level of protein expression in SAT between CAD and non-CAD groups. (c) and (d), gel images and relative level of protein expression in EAT between CAD and non-CAD groups. ß-actin was used as a loading control. The blot was repeated three times with n = 4 for each group. (b) Densitometric analysis of the blot in (a). *P<0.05 vs non-CADs group; #P<0.01 vs non-CADs group. a.u.: arbitrary unit.
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