Figure 6

Lymphatic endothelial heparan sulfate is required for receptor-dependent display of CCL21 on the lymphatic surface. A. Binding of human CCL21 (hCCL21) to heparin was examined using heparin affinity chromatography followed by silver staining of salt-eluted fractions on SDS-PAGE (upper panel). "Pre," recombinant human CCL21 sample directly loaded onto the silver-stained gel; "FT," flow-through from the column. Basic fibroblast growth factor (FGF-2) was run as a positive control (lower panel). B. hLEC were treated as indicated and the binding of CCL21 to hLEC was examined by immunofluorescence (IF) using anti-CCL21 antibody. hLEC, untreated cells; H'ase, Heparin: hLEC pre-treated with heparinase or heparin, respectively; H'ase+CCL21, Heparin+CCL21: hLEC pre-treated with heparinase or heparin, respectively, followed by addition of exogenous human recombinant CCL21 (+CCL21). Representative merged images showing CCL21 signal (green) and DAPI nuclear stain (blue) were taken under 100× magnification. (Scale bar, 100 μm.) C. hLEC were transfected with either control RNA (siDS) or siRNA targeting HS biosynthetic enzymes Ndst1 or XylT2. Binding CCL21 to CCR7 on hLEC was detected by proximity ligation assay (PLA). Representative merged images showing PLA signal (red) and nuclear DAPI stain (blue), imaged using fluorescence microscopy (400×; Scale bar, 50 μm). PLA signal from each field was quantified and indexed to total nuclear area within the same field, and mean values for each condition were graphed (bottom). At least 5 random fields from each group were included for analysis. Mean data was normalized to control signal (siDS). *P < 0.01, as compared to control.