UNC50 is required for the early endosome-to-Golgi trafficking of STx2. (A) WT or ΔUNC50 cells were treated with indicated amounts of STx2 holotoxin for 16 h. Cell viability was then assessed using the MTT assay described in the Viability assays section of Materials and methods. Viability without toxin exposure was normalized to 100 for each cell line (n = 3). (B) Depiction of the mean LD₅₀ of STx2 from A. Error bars represent 95% confidence intervals (CIs). *, P < 0.05 by t test. (C) Trafficking of His-tagged STx2B was assayed in WT or ΔUNC50 cells. Cells were fixed at 0 or 60 min after start of transport. STx2B was detected using a polyclonal antibody against the His tag. Golgi was demarcated using a monoclonal antibody against giantin. (D) Quantification of the Pearson’s coefficient for colocalization between STx2B and giantin from C (n = 25 cells per group). *, P < 0.05 for the comparison between WT cells at 60 min and ΔUNC50 cells at 60 min by one-way ANOVA and Tukey-Kramer post hoc test. (E) Quantification of STx2B fluorescence intensities from C. Intensity of WT cells at 0 min was set to 100. Intensities of other groups were expressed relative to WT at 0 min (n ≥ 25 cells per group). *, P < 0.05 for the difference between WT cells at 0 min and other groups using one-way ANOVA and Dunnett’s post hoc test. (F) ΔUNC50 cells were transfected with GFP-tagged Rab5. 1 d after transfection, transport of His-tagged STx2B was assayed. Cells were fixed 10 min after start of transport and imaged to detect STx2B using a polyclonal antibody against the His tag and GFP. Arrowheads show overlap between STx2B and GFP. (G) ΔUNC50 cells were treated with leupeptin (100 µg/ml) and pepstatin (50 µg/ml) for 24 h or left untreated. After this, transport of STx2B was assayed. Cells were fixed at 0 min or 4 h after start of transport (0 min time-point is not depicted). STx2B was detected using a polyclonal antibody against the His tag. Golgi was demarcated using a monoclonal antibody against giantin. (H) Quantification of STx2B fluorescence intensities from G. For each treatment condition, intensity at 0 min was normalized to 100, and intensity at 4 h was expressed relative to 0 min (n = 25 cells per group). *, P < 0.05 by t test. (I) Transport of STx2B was assayed in ΔUNC50 cells treated with leupeptin (100 µg/ml) and pepstatin (50 µg/ml) as described in G. Cells were fixed at the 4-h time point and imaged to detect STx2B, using the polyclonal anti-His antibody, and Lamp2, using a monoclonal antibody. Arrowheads show overlap between STx2B and Lamp2. (J) ΔUNC50 cells were transfected with CRISPR-sensitive myc-tagged UNC50. 1 d after transfection, transport of STx2B was assayed. Cells were fixed at 0 or 60 min after start of transport and imaged to detect His and myc tags. Asterisks denote transfected cells. Bars: (C, G, and J) 25 µm; (F and I) 5 µm. (K) Quantification of STx2B fluorescence intensities from J. For each transfection condition, intensity at 0 min was set to 100, and intensity at 60 min was expressed relative to 0 min (n = 20 cells per group). *, P < 0.05 by t test. (L) Quantification of the Pearson’s coefficient for colocalization between STx2B and myc-UNC50 at 0 or 60 min in transfected cells from J. The signal from myc-UNC50 demarcated the Golgi in transfected cells because in ΔUNC50 cells, CRISPR-sensitive myc-UNC50 was enriched in the Golgi (Fig. 2, H–J; n = 25 cells per group). *, P < 0.05 by t test; error bars show means ± SEM.