Supplemental Material

This article contains the following supporting material:

• Figure 1S - Cholesterol depletion or enrichment has no significant impact on the integrity of MDCK cell monolayer and endosomal morphology.
  PTR-MDCK cells cultured on 12 mm filters were treated with mβCD or mβCD/chol for 60 min at °37 C. Confocal visualization of the tight junction marker ZO1 (panel A), and determination of trans-epithelial electrical resistance (TER; panel B) were used as measures of the integrity and health of the cells. Immunostaining of hTfnR (using H68.4 monoclonal antibodies) was performed to estimate changes in endosomal structures. Panel C presents an optical section imaging the hTfnR (green) at the level of ZO1 (red). Additional sections recorded from basolateral regions showed an intense hTfnR signal concentrated at cell's perimeter in mβCD-treated cells (not shown). This phenomenon can be attributed to cholesterol-depletion mediated inhibition of hTfnR endocytosis, resulting in receptor accumulation on the cell surface. However, the morphology of intracellular hTfnR-labeled organelles in the same optical sections was not much different in mβCD than in untreated cells. Bar, 5μm.