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. Author manuscript; available in PMC: 2020 Aug 17.
Published in final edited form as: Nat Chem Biol. 2020 Feb 17;16(3):327–336. doi: 10.1038/s41589-020-0474-4

Figure 3. Retro-2 treatment slows the anterograde transport of Syn5.

Figure 3

(a) Confocal acquisitions of HeLa cells expressing Syn5-RUSH construct (green) or (c) ManII-RUSH construct (green) and labeled with anti-giantin (red). Cells were pre-treated for 60 min at 37 °C with 25 μM Retro-2, or 0.05% DMSO as control (pre-release). Trafficking was triggered by incubation with biotin for 20 min at 37 °C. Dashed lines indicate nuclei. Scale bars = 10 μm. Representative images from (a) 3 (pre-release) or 4 (Release and Retro-2 + release) independent experiments and (c) 2 independent experiments. (b) Quantification of Syn5-GFP intensity in the Golgi area, normalized to Release conditions. Graph shows mean +/- SD of 28 (Pre-release), 24 (Release), 56 (Release + Retro-2) cells per experiment from 3 (pre-release) or 4 (Release and Retro-2 + release) independent experiments, each point represents one experimental replicate. Unpaired two-tailed t-test performed between Release and Retro-2 + Release conditions, *** P = 0.001. (d) Quantification of ManII-GFP intensity in the Golgi area. Graph shows mean +/- SD for 48 (pre-release), 71 (release), 81 (release + Retro-2) cells per experiment from 2 independent experiments. Each point represents a single cell. Unpaired, two-tailed t-test, performed between Release and Retro-2 + Release conditions, NS = not significant, P = 0.936. (e) HeLa cells transfected with siSar1A/B, incubated for 45 min at 37 °C with STxB-Cy3 (green) and labeled with giantin (red) and EEA1 (blue in right panels). Nuclei were labeled with Hoechst (blue in left panels). Dotted lines indicate areas shown in zoom panels. Scale bar = 10 μm. Representative images from 3 independent experiments. (f) Quantification of STxB colocalization with Golgi area defined by giantin. Graph represents mean +/- SD for 40 (scrambled) and 39 (siSar1A/B) cells from 3 independent experiments. Each point represents one cell. Unpaired two-tailed t-test, ** P = 0.0055. (g) Quantification of number of EEA1 spots positive for STxB. Graph represents mean +/- SD for 39 (scrambled) and 37 (siSar1A/B) cells from 3 independent experiments. Each point represents one cell. Unpaired two-tailed t-test, *** P < 0.0001.