Journal: Genes & Development

Article Title: Angiopoietin 2 regulates the transformation and integrity of lymphatic endothelial cell junctions

doi: 10.1101/gad.237677.114

Figure Lengend Snippet: ANG2 regulates lymphangiogenesis during embryonic development. ( A–H ) Pregnant females were injected intraperitoneally (i.p.) with 30 mg/kg ANG2-blocking antibody or a control antibody at E12.5, E14.5, and E16.5, and the ventral skin of embryos was analyzed at E18.5 by immunofluorescence for LYVE1, VEGFR3, Ki67, and PROX1, as indicated. Dashed lines indicate the umbilicus. Vessels inside the red squares are quantified in I . (Yellow arrows) Sprouts; (white arrows) Ki67 + LECs; (arrowheads) Ki67 − LECs. Bars: A , E , 200 µm; B , C , F , G , 20 µm; D , H , 10 µm. ( I ) Quantification of the migration, branch points, and sprouts; the number of LECs per 1-mm vessel; and the percentage of Ki67 + LECs out of all LECs. N = 4–5. ( J–L ) The mothers of VE-cadherin-tTA; TetO-Ang2 embryos were provided with tetracycline water until E10.5, and the ventral skin of the embryos was analyzed at E18.5. The anti-VEGFR3 antibody (30 mg/kg) was injected i.p. at E14.5 and E16.5. (DTG) Double transgenic for VE-cadherin -tTA and TetO -Ang2 ; (Ctrl) control embryos (wild type [WT] or single transgenic). N = 4–7. Boxed regions are magnified in the bottom panel. Bars: top panel, 200 µm; bottom panel, 20 µm. All values are mean ± SEM. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.

Article Snippet: The following primary antibodies were used: FOXC2 (1:2500) , GFP (1:1500; Torrey Pines Biolabs, #TP401), integrin-α9 (1:500; R&D Systems, #AF3827), Ki67 (1:200; Abcam, #ab16667), LYVE1 (1:2000) , 2–3 µg/mL PROX1 (1:500; R&D Systems, #AF2727) , SMA (1:500; Sigma-Aldrich, #6198), VE-cadherin (1:100 [BD Biosciences, #555289] and 1:100 [eBioscience, #14-1441]), VE-cadherin P-Y685 , and VEGFR3 (1:50; R&D Systems, #AF743).

Techniques: Injection, Blocking Assay, Immunofluorescence, Migration, Transgenic Assay