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Fig. 2 Myosin Vb Exon D knockdown results in increased trans-Golgi branching and accumulation of post-TGN vesicles in the peridermal cells. (A) Brightfield images of control and Exon D morphants (Exon D MO) at 48 hpf show that knockdown of Exon D isoform (Rab10 binding) does not result in rounded up cells in the head region. (B) Confocal images of peridermal cells of the control (Control MO) and Exon D morphants (Exon D MO) at 36 hpf immunostained for GalT-GFP and trans-Golgi using WGA. (C,D) 3D rendering of TGN by Huygen's software (C) followed by quantification (D) revealed an increase in both surface area and volume of the TGN. (E,F) Confocal stacks of control and Exon D morphants stained for trans-Golgi (WGA) and Rab10 (E) and trans-Golgi (WGA) and Rab11 (F). (G-I) Confocal micrographs of BODIPY-FL-C5-Ceramide-labeled peridermal cells from control and Exon D morphants at 36 hpf, imaged using λex=488 nm and λem at ∼515 nm (green) and ∼620 nm (red) (G) followed by quantification of number of vesicles (H), and intensity ratio of red:green fluorescence of large Ceramide-rich compartments [‘red’ appearing vesicles in overlays (G)] as well as the TGN (I). White arrows in B, E-G point to trans-Golgi vesicles in overlay panels. White arrowheads in C point to trans-Golgi branches. Data are median±interquartile range. ***P<0.001 (Mann–Whitney U test; Table S1). Scale bars: 20 µm (B,E-G); 10 µm (C).