Fig. 3. Quantification of the defect in endosome-to-TGN transport in SNX1-suppressed cells. Toxin-uptake assays using control and SNX1-suppressed HeLa cells were performed as described. After fixation and immunolabeling with anti-SNX1 (Alexa594, red channel), samples were inspected by epifluorescence microscopy and scored blindly for either a TGN-like enrichment (x) or a vesicular appearance (^*). Cells marked `o' were not scored because of low toxin levels. (A,B) Scoring phenotypes; FITC-STxB in green channel and TGN46 (Alexa594) in red channel for control and SNX1-suppressed cells after 30 minutes of uptake. (C) Representative data for a set of control and SNX1-suppressed cells (n>600) after 30 minutes of toxin uptake. (D) TGN-like accumulation of FITC-STxB over time (n>600 for each sample; scored for 30, 60 and 120 minutes) for SNX1-suppressed and control cells. (E) Colocalization of FITC-STxB and TGN46, digitally quantified from confocal images using MetaMorph software (see Materials and Methods for details). In control cells, 37±0.4% of FITC-STxB colocalized with TGN46 after 30 minutes of internalization, whereas SNX1-suppressed cells showed 12±1% colocalization. Values given are ± the standard deviation and were determined from the mean of individual visual fields for a total of n>50 cells. Bar, 20 µm.