Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure S1: Chemical structures of inhibitors, route to synthesis and solubility of LG186. A) Structure of brefeldin A, (B) structure of Exo2, (C) structure of LG186. D) Route to synthesis of LG186. E) Solubility of LG186 was measured (by absorbance at 348 nm) before (circles) and after (crosses) centrifugation.

Figure S2: Effective concentration and speed of action of LG186. COPI (green in merge) and GM130 (red in merge) localization in HeLa cells treated with for 1 h BFA, GCA or LG186 at the concentrations indicated. (B) RPE1 cells incubated for 30 min with LG186 at the concentrations indicated were fixed and labeled to show giantin and GalT distribution. Each panel is 30-µm wide. Indistinguishable results were obtained using HeLa cells (not shown). (C, D) Live-cell imaging was performed on HeLa cells stably expressing ɛCOP-GFP (C) or NA-GFP (D) immediately after addition of the indicated chemicals (BFA 10 µm, LG186 and GCA 50 µm or DMSO as vehicle only). Bar = 10 µm.

Figure S3: LG186 inhibits secretory transport but not transferrin recycling. A) HeLa cells were infected with adenovirus to express tsO45-G-YFP at 39.5°C for 16 h to accumulate the protein in the ER. Cells were subsequently incubated at 39.5°C for 15 min in the presence of DMSO (upper panel) or 50 µm LG186 (lower panel). Cells were then shifted to 32°C to allow export of tsO45-G-YFP from the ER for the times indicated (45 or 120 min), fixed and processed for immunofluorescence with an anti-giantin antibody. The nuclei were counterstained with DAPI (blue in merge). Representative single z-slice is shown to enhance the ER pattern and bar = 10 µm. B, C) TIRF imaging of cells expressing NPY-Venus (neuropeptide Y fused to the Venus fluorescent protein) preincubated for 1 h in DMSO (B) in 50 µm LG186 (C) in the presence of LG186. Dissipation of fluorescence in (B) indicates fusion of transport vesicles with the plasma membrane. In 5 min of imaging 45 ± 15 fusion events are seen per cell. In (C) epifluorescence image shows the NPY-Venus distributed throughout the ER. This is also visible under the TIRF illumination but no fusion events are seen. D) HeLa cells were grown on glass-bottom live-cell dishes, incubated in serum-free medium containing Alexa-568-transferrin at 25 µg/mL and supplemented by BFA (10 µm), Exo2 (50 µm), LG186 (50 µm) or DMSO only, for 1 h at room temperature to allow accumulation of labeled transferrin in the recycling endosomes. After a quick wash in PBS, cells were incubated in complete medium in the continuous presence of the chemical at 37°C to allow recycling of the labeled transferrin, and PFA-fixed at the indicated times. Cells were then imaged using a 60× oil immersion lens (top panel, bar = 10 µm). Images were processed using ImageJ. E) After applying the same threshold to all images, total fluorescence intensity was measured for two to three fields for each condition, normalized against DMSO t = 0 and plotted against time. Results are presented as mean ± SEM from two independent experiments (i.e. four to six different fields for each condition) (bottom panel). When Exo2 seems to increase the uptake of Alexa-568-transferrin (t = 0, p = 0.00182), its recycling in all conditions appears comparable to the DMSO control.

Figure S4: LG186 does not induce dissociation of AP1 from TGN membranes. RPE-1 cells incubated with BFA (10 µm), Exo2 (50 µm), LG186 (50 µm) or DMSO for the indicated times were processed for immunofluorescence to label giantin (green in merge), TGN46 (red in merge) and AP1 (blue in merge). BFA induces collapse of the Golgi and dissociation of AP1 from TGN membranes. GCA, Exo2 and LG186 disperse the Golgi but at short incubation times, AP1 remains associated with TGN membranes.

Figure S5: LG186 disrupts the TGN in MDCK cells. The TGN in MDCK cells (TGN46-GFP, green in merge) is dispersed by LG186 (50 µm) but not BFA (10 µm) or Exo2 (50 µm); this correlates with Golgi collapse (giantin, red in merge). Tubulation of TGN46-GFP-positive structures in the presence of BFA is presumably caused by targeting of BIG2, thereby inducing tubulation of endosomal compartments through which TGN46-GFP traffics. Exo2 and LG186 do not target BIG2 and consequently do not induce this tubulation. Bar = 10 µm.

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