Fig. 3. MßCD blocks CTX delivery to the Golgi but not to endosomes. NIH-3T3 cells were either left untreated (A-C) or pretreated with 5 mM of mßCD (D-L) for 30 minutes then pulse labeled with FITC-CTX (A,D,G) and Rh-Tf (B,E,H) for 30 minutes at 37°C. Images presenting FITC-CTX internalization in the absence or presence of mßCD (A,D) were obtained using the same acquisition parameters. A smaller pinhole (0.6 Airy units) and increased zoom (G-L) were used to assess the overlap between internalized FITC-CTX (G), Rh-Tf-labeled endosomes (H) and the GM130-labeled Golgi apparatus (I). Merged confocal images present FITC-CTX in green and either Rh-Tf in red (C,F,K) or GM130 in red (L) and colocalization in yellow. A triple merge shows FITC-CTX in green, Rh-Tf in red and GM130 in blue (J). In mßCD-treated cells, cell-surface FITC-CTX labeling is significantly increased, and CTX is still internalized with transferrin to endosomes but not to the Golgi. Bars: A-F in F, 20 µm; G-L in L, 8 µm.

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