Figure 2. Depletion of Sec16A phenocopies Retro-2 effects.

(a) HeLa cells transfected with the indicated siRNAs, or treated for 30 min with 25 μM Retro-2, followed by incubation for 45 min at 37 °C with STxB-Cy3 (red). Cells were labeled for giantin (green, left panels) and EEA1 (blue, right panels). Nuclei were labeled with Hoechst (blue, left panels). Arrows indicate spots of STxB colocalized with EEA1, dotted lines indicate areas of zoomed panels. Scale bar = 10 μm. Representative images from 6 independent experiments (Giantin colocalisation) and 3 independent experiments (EEA1 colocalisation). (b) Quantification of STxB colocalization with Golgi area defined by giantin. Graph represents mean +/- SD for 46 (scrambled), 35 (Retro-2), 39 (siSec16A), 42 (Retro-2+siSec16A) cells from three independent experiments. Each point represents an individual cell. One-way ANOVA was performed overall (P < 0.0001) with Dunnett’s multiple comparison test to compare Scrambled to treated conditions (*** P = 0.0002, ** P = 0.0021). (c) Quantification of number of EEA1 spots positive for STxB. Graph represents mean +/- SD for a minimum of 40 cells from 3 independent experiments. One-way ANOVA was performed overall (P < 0.0001) with Dunnett’s multiple comparison test to compare Scrambled to treated conditions (*** P < 0.0001). (d) Intoxication of HeLa cells with SLT1 in the indicated conditions. HeLa cells were transfected with either scrambled siRNA, or siRNAs against Sec16A. Where indicated, cells were incubated 30 min with 25 μM Retro-2 or vehicle (0.05% DMSO, “control”), before the addition of SLT1 for 1 h at the indicated concentrations. Medium was removed and replaced for 1 h with Ca²⁺ and Mg²⁺-supplemented PBS containing [³⁵S]-methionine. Each data point represents the mean of a representative experiment, from 2 independent experiments. (e) HeLa cells were transfected with siSec16A and where indicated treated for 30 min with 25 μM Retro-2 or DMSO control. Cells were labeled for TGN46 (green) and Syn5 (red), and nuclei labeled with Hoechst (blue). Representative images from 3 independent experiments. (f) Quantification of Syn5 at Golgi area as defined by TGN46. Graph represents mean +/- SD for 48 (Scrambled), 39 (Retro-2), 41 (siSec16A), 43 (Retro-2 + siSec16A) cells from 3 independent experiments. Each point represents an individual cell. One-way ANOVA was performed overall (P < 0.0001) with Dunnett’s multiple comparisons test to compare Scrambled to treated conditions (*** P < 0.0001).