Up-regulation of VEGFR-3 and Prox1 precedes new lymphatic vessel formation during inflammation. (A) Double immunostaining of VEGFR-3/LYVE-1 and Prox1/LYVE-1 in serial diaphragm sections derived from mice treated with saline or TG (n = 3-4 mice per group) and harvested 1, 2, 3, 4, and 7 days after onset of treatment. Scale bars represent 50 μm. Lymphatic vessels shown are representative of whole diaphragm sections from 3 to 4 mice per group. (B-D) Quantification of Prox1-positive (B), VEGFR-3—positive (C), and LYVE-1—positive (D) vessels normalized per area of the entire diaphragm section measured in square millimeters. Quantitative analysis was performed on diaphragms harvested from 3 to 4 mice per group at indicated days after the first TG or saline injection. Data are presented as the mean number of vessels per diaphragm section ± SEM; ns denotes nonsignificant changes; *P < .05 and ***P < .01 versus control, as determined by Student unpaired t test. (E) Protein expression of Prox1, VEGFR-3, LYVE-1, NF-κB p50 phosphorylated on Ser337, nonphosphorylated NF-κB p50, NF-κB p65 phosphorylated on Ser276, nonphosphorylated NF-κB p65, and β-actin was determined by Western blot of combined lysates (100 μg of total protein per lane) derived from 3 to 4 mice per group. (F) Protein expression in Western blots was determined by band densitometry. Values were normalized to β-actin and are shown as fold increase relative to expression of corresponding proteins in untreated control mice at day 0.