Effect of FGF2 on endogenous LYVE-1 expression in endothelial cells. (A) Endogenous LYVE-1 expression in FGF2-stimulated endothelial cells. LECs and HUVECs were stimulated with 10 ng/mL FGF2 in a time-dependent manner 24, 48, and 72 hours. Endogenous LYVE-1 was measured by qPCR. Results are expressed in fold-change of LYVE-1 mRNA in FGF2-stimulated cells versus nonstimulated cells on each time-point using the GAPDH reference gene. Reversion of the TNFβ-dependent down-regulation of LYVE-1 by FGF2 in LECs (B) and HUVECs (C). LECs and HUVECs were stimulated with 10 ng/mL FGF2 for 24, 48, 72 hours, and 10 ng/mL TNFβ was added to the culture medium. Endogenous LYVE-1 was measured by qPCR as described. Values obtained by qPCR are the means ± SD (n = 3). (D) Immunofluorescence of LECs incubated with TNFβ in the absence or presence of FGF2. Cells were incubated for 48 hours with TNFβ (20 ng/mL) in the absence or presence of FGF2 (20 ng/mL). Immunofluorescence was performed using anti-podoplanin (positive control for lymphatic cells, i-iv) or anti–LYVE-1 (v-viii) antibodies. (ix-xii) Represent the merge of both labeling. (E) Effect of FGF2 on the down-regulation of LYVE-1 in an ex vivo explant culture assay. Explants from male BALB/c mice were cultured in a humidified atmosphere at 37°C in 5% CO₂ in the presence of recombinant human 200 ng/mL TNFβ and/or FGF2 at 200 ng/mL for 48 hours. Immunostaining using anti-podoplanin or anti–LYVE-1 antibodies were carried out as indicated in “Methods.” (i-iv) Podoplanin staining; (v-viii) LYVE-1 staining; and (ix-xii) merge.