Raw Data

Raw dat

IL-4 and IL-13 blockade does not increase corneal angiogenesis.

<p><b>A.</b> Representative immunofluorescent cornea whole mount images stained for CD31 and LYVE-1. Scale bar = 100μm. <b>B.</b> Quantification of blood vessel area per 0.25mm². Control vs. IL-4mAb (n = 8 in each; p = NS); control vs. IL-13mAb (n = 8 in each; p = NS); IL-4mAb vs. IL-13mAb (n = 8 in each; p = NS). <b>C.</b> Quantification of blood vessel volume per 0.25mm². Control vs. IL-4mAb (n = 8 in each; p = NS); control vs. IL-13mAb (n = 8 in each; p = NS); IL-4mAb vs. IL-13mAb (n = 8 in each; p = NS).</p

IL-4 or IL-13 blockade increases inflammatory lymphangiogenesis.

<p><b>A.</b> Representative gross images of corneas 14 days after suture placement. <b>B.</b> Representative immunofluorescent cornea whole mount images for LYVE-1. Scale bar = 100μm. <b>C.</b> Quantification of lymphatic vessel area per 0.25mm². Control vs. IL-4mAb (n = 8 in each; *p<0.001); control vs. IL-13mAb (n = 8 in each; *p<0.001); IL-4mAb vs. IL-13mAb (n = 8 in each; p = NS). <b>D.</b> Quantification of lymphatic vessel volume per 0.25mm². Control vs. IL-4mAb (n = 8 in each; *p<0.001); control vs. IL-13mAb (n = 8 in each; *p<0.001); IL-4mAb vs. IL-13mAb (n = 8 in each; *p<0.05).</p

rhIL-4 and rhIL-13 decrease LEC proliferation and downregulate expression of Prox-1.

<p><b>A.</b> Flow cytometry analysis for incorporated EdU by cultured hLECs after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 24 hours. Representative histograms. Quantification of EdU positive hLECs. Control vs. rhIL-4 (n = 5 in each; *p<0.01); control vs. rhIL-13 (n = 5 in each; *p<0.01); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS). <b>B.</b> Quantification of hLEC proliferation using a WST-1 cell assay kit. Control vs. rhIL-4 (n = 10 in each; *p<0.01 for both concentrations); 50 ng/ml rhIL-4 vs. 100 ng/ml rhIL-4 (n = 10 in each; *p<0.05); control vs. rhIL-13 (n = 10 in each; *p<0.01 for both concentrations); 50 ng/ml rhIL-13 vs. 100 ng/ml rhIL-13 (n = 10 in each; p = NS). <b>C.</b> Representative immunofluorescent staining for Ki67 and VEGF-R3 in cultured hLECs after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 24 hours. Scale bar = 100μm. Quantification of Ki67⁺ cells per high-power field. Control vs. rhIL-4 (n = 5 in each; *p<0.001); control vs. rhIL-13 (n = 5 in each; *p<0.001); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS). <b>D.</b> Representative immunofluorescent staining for Prox-1 in cultured hLECs after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 24 hours. Scale bar = 100μm. Quantification of Prox-1 signal intensity using MetaMorph analysis per high-power field. Control vs. rhIL-4 (n = 5 in each; *p<0.001); control vs. rhIL-13 (n = 5 in each; *p<0.001); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS).</p

hLECs express IL-4Rα and respond to rhIL-4/IL-13 with increased apoptosis.

<p><b>A.</b> Representative immunofluorescent images of IL-4Rα and Prox-1 staining from cultured hLECs. Scale bar = 20μm. <b>B.</b> Flow cytometry analysis for Annexin V and propidium iodide of cultured hLECs after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 24 hours. Representative contour plots. Quantification of apoptotic hLECs. Control vs. rhIL-4 (n = 5 in each; *p<0.001); control vs. rhIL-13 (n = 5 in each; *p<0.001); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS).</p

rhIL-4 and rhIL-13 inhibit hLEC tubule formation.

<p><b>A.</b> Representative images of cultured hLECs tubule formation on matrigel after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 12 hours. <b>B.</b> Quantification of tubule formation per high-power field. Control vs. rhIL-4 (n = 5–8 in each; *p<0.01); control vs. rhIL-13 (n = 5–8 in each; *p<0.01); rhIL-4 vs. rhIL-13 (n = 5–8 in each; p = NS).</p

rhIL-4 and rhIL-13 inhibit hLEC migration.

<p><b>A.</b> Representative images of cultured hLECs after scratch wound and treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) at 0, 12 and 24 hours. White dotted lines represent LEC migration borders. Black solid lines represent initial gap created by scratch. <b>B.</b> Quantification of hLEC migration at 12 and 24 hours. Control vs. rhIL-4 (n = 5 in each; *p<0.01 for both time points); control vs. rhIL-13 (n = 5 in each; *p<0.01 for both time points); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS for both time points).</p

IL-4 and IL-13 blockade increases LEC proliferation <i>in vivo</i>.

<p><b>A.</b> Representative immunofluorescent cornea whole mount images stained for Ki67 and LYVE-1. Scale bar = 100μm. <b>B.</b> Quantification of Ki67⁺/LYVE-1⁺ cells (n/0.25mm²). Control vs. IL-4mAb (n = 5 in each; *p<0.01); control vs. IL-13mAb (n = 5 in each; *p<0.01); IL-4mAb vs. IL-13mAb (n = 5 in each; *p<0.01).</p