Figure 5: Constitutive Rab11a activation leads to ectopic angiogenic sprouting.
A,B. Quantification of Rab7 (A) and Rab11a+bb (B) positive vesicles in siDYNC1LI1 HUVECs versus siControl-treated cells. C,D. Representative images of HUVECs immunostained for Rab7 (C) and Rab11a+bb (D) following siControl or siDYNC1LI1 transfection and treatment with 40 ng/mL VEGF-A for 10 min. E. Western blot analysis shows increased expression of Rab11 in siDYNC1LI1 HUVECs compared to siControl cells. F. qPCR reveals no change in the transcript levels of either Rab7 or Rab11 in siDYNC1LI1 cells, suggesting that the increased protein levels of Rab11 occur post-transcriptionally. G,H. Confocal images of Tg(fli:Gal4); Tg(UAS:Kaede) zebrafish embryos injected with a Tol2-integratable UAS:CA-rab11a-mCherry DNA plasmid to express constitutively active Rab11a protein in the endothelium in a mosaic fashion (H) versus carrier injected controls (G). Blood vessels are shown in green; mosaic expression of UAS:CA-rab11a-mCherry is shown in magenta. Endothelial cells expressing UAS:CA-rab11a-mCherry and Tg(fli:Gal4); Tg(UAS:Kaede) appear white when the images are merged. Sites of UAS:CA-rab11a-mCherry expression (white arrows) phenocopy the increased angiogenesis noted in dync1li1y151/y151 mutants. I. Percentage of ISVs displaying ectopic sprouts in carrier injected controls versus UAS:CA-rab11a-mCherry-injected embryos at 96 hpf. n=5 embryos. For panels A,B, each dot represents an individual cell. Panels A,B are representative of three independent experiments and statistics were calculated using an unpaired t-tests. For panel F, n=3. Statistics for panel F were calculated using one-sample t-tests to compare siDYNC1LI1 fold change values to hypothetical value 1. Data are presented as the mean ± S.D. Statistics for panel I were calculated using an unpaired t-test with Welch’s correction. Data are presented as the mean ± S.D. Scale bars: 10um (C,D) and 50uM (G,H).