Expression of components of the plasmin activation pathway and CCL21 cleavage activity of cultured cells. (A and B) Flow cytometry–based analysis of uPA, uPAR, and plasminogen expression in conditionally immortalized LECs and primary LN LECs. (A) Representative histogram plots and corresponding (B) summary of the delta mean fluorescent intensity (ΔMFI; specific-isotype staining) values measured in 4–5 different experiments. (C) CCL21 cleavage assay performed in presence or absence of LECs and plasminogen (plg), revealing the dependence of CCL21 cleavage on both factors (i.e., LECs and plg). One representative out of two similar experiments is shown. (D and E) CCL21 cleavage assay performed with (D) bone marrow–derived DCs and (E) primary keratinocytes, revealing their ability to cleave CCL21 in presence of plg. One representative out of two similar experiments is shown in D and E. (F) qRT-PCR–based analysis of mRNA from LN LECs isolated from WT, uPA^mut, and uPA^−/− mice. (G) Absolute CT values and (H) relative expression levels. Data from four LN LEC isolations are shown. One-way ANOVA. (H and I) Impact of heparitinase treatment on the CCL21 gradient in uPA^mut mice. (H) Representative images showing LYVE-1 and the immobilized perilymphatic CCL21 gradient in the steady-state ear skin of uPA^mut mice upon in vitro treatment with heparitinase (HEP) or in untreated CTRs. Scale bar: 50 μm. (I) Quantification of the CCL21 staining intensity as a function of the distance from the nearest LYVE-1⁺ LV. n = 3 mice per condition, two-way ANOVA. Source data are available for this figure: SourceData FS3.