Deletion of arl4aa and ARL4A Caused Fragmentation of Golgi Complex in VDA Endothelial Cells in Zebrafish and Human HeLa Cells (A) Electron microscopy of endothelial cells of VDA of un-injected (control [CON]) and F0 embryos injected with TALEN E2-2 at 30 hpf. (i–ii) Endothelial cells of VDA are shown in the rectangles. (iii–vi) High power magnification of control (iii) and F0 embryos (iv–vi). Golgi complex shows typical configuration in control embryos (9/10 cells from 4 control embryos) but aberrant morphology in F0 embryos being unusually elongated with disruption (iv, 6/10 cells from 6 embryos), under-developed (v, 1/10 cells from 6 embryos), and “onion-skin” appearance (vi, 2/10 cells from 6 embryos). NC, notochord; VDA, ventral wall of dorsal aorta; PCV, posterior cardinal vein; N, nucleus. (B) Electron microscopy of neural tube in CON (i) and F0 embryos (ii) injected with TALEN E2-2, showing normal Golgi complex configuration (iii and iv). Scale bars, 2 μm (Ai,ii and Bi,ii) and 200 nm (Aiii–vi and Biii, iv). Orange arrowheads: abnormal morphology of the Golgi complex. (C) Immunostaining of Golgi membrane Giantin in control and arl4aa morphant (MO) embryos at 30 hpf. The insert on top shows the morphology of 30-hpf embryos with the rectangle highlighting the region of the DA under detailed examination. The dotted red line in the bright field defines the DA. Immunostaining: Giantin was stained with anti-Giantin followed by Alexa Fluor 488-conjugated antibody and the nuclear outline is shown by DAPI staining. The yellow arrowhead denotes the fragmentation of the Golgi complex in endothelial cells from the VDA. The white arrowhead denotes normal expression of Giantin at the Golgi complex. Scale bar, 100 μm. (D) Immunostaining of Giantin followed by Alexa Fluor 594-conjugated antibody in scramble sequence control, ARL4A shRNA-1, and ARL4A shRNA-2 transfected HeLa cells. GFP+ labeled the successfully transfected cells. White arrowheads indicate normal Golgi complex and yellow arrowheads denote the fragmented and dispersed Giantin staining. Scale bar, 20 μm. (E) Both ARL4A shRNA-1 and shRNA-2 significantly increased the proportion of cells with fragmented Golgi complex as shown by Giantin expression, data are shown as means ± SEM (n = 3 experiments, p < 0.001). See also Figures S4–S6.
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