Fig. 3. Internalization of CTX in CaCo-2 and HeLa cells. CTX-FITC was internalized for 30 minutes at 37°C in CaCo-2 (A) and HeLa (F,J) cells and after fixation the Golgi apparatus was labeled for GM130 (B,G,K) and endosomes for TfR (C,H,L). Minimal internalization of CTX to either the Golgi apparatus or endosomes was detected in CaCo-2 cells (A-D). Internalized CTX was localized primarily to the Golgi in HeLa cells expressing high levels of GM1 (F-I). Increasing the sensitivity of confocal detection of cell-associated GM1 permitted the visualization of CTX in cells expressing low GM1 levels and overlap with the Golgi was not observed (J-M). The merged confocal images (D,I,M) present CTX-FITC in green, GM130 in red and TfR in blue with colocalization between CTX-FITC and GM130 in yellow and between CTX-FITC and TfR in cyan. (E) A DIC image of a CaCo-2 cell island. Arrows indicate colocalization of internalized CTX with TfR-positive endosomes. Scale bars: 8 µm (A-E) and 16 µm (F-M).