Fig. 5
Vegfr3-deleted LECs induce proliferation of neighboring VEGFR3+ cells in a cell-contact-dependent manner. a VEGF-C concentration measured by ELISA in the ear skin at indicated stages. Bars represent mean (n = 2–8 mice, as indicated) ± s.d. b Experimental set up for co-cultures of WT and VEGFR3− (KO) primary LECs from Vegfr3flox/flox;R26-mTmG;Prox1-CreERT2 mice (left) and immunofluorescence showing VEGFR3 depletion in Cre− targeted (GFP+) LECs (right, arrows). c Left: proliferating WT cells were frequently associated with KO cells. Right: quantification of the proportion of EdU+ WT cells that were in direct contact with KO cells (mean (n = 6 biological replicates) ± s.e.m). d Assessment of cell proliferation in co-cultures of primary human LECs treated with control (siCTRL) or VEGFR3 (siVEGFR3) siRNA. Cell tracker-labeled siCTRL cells (purple) were mixed with unlabeled siVEGFR3 cells in 40:60%, or they were cultured alone. e Quantification of proliferating EdU+ cells in co-cultures of siCTRL and siVEGFR3 LECs mixed in different ratios. Data are normalized to the proliferation rate (0.8 ± 0.2%) in siCTRL alone and represent mean (n = 5 biological replicates) ± s.e.m. f Predicted and observed proportions of siCTRL LECs (all vs. EdU+) that are in direct contact with siVEGFR3 LECs in 80:20% siCTRL:siVEGFR3 co-cultures (mean (n = 5 biological replicates) ± s.e.m). Predicted proportion was calculated based on random distribution and n = 5 (5.2 ± 0.2, quantified from n = 172 LECs stained for VE-cadherin) neighbors. g Quantification of proliferating EdU+ cells in siCTRL LECs cultured alone (−), or co-cultured without direct cell–cell contacts with siVEGFR3 cells seeded on Transwell inserts (+). Data are normalized to the proliferation rate in siCTRL alone and represent mean (n = 6 biological replicates) ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Two-tailed unpaired Student’s t test (a, e, g) and Fisher’s exact test (f). Scale bars: 50 µm (b, c), 200 µm (d). ns: not significant
