Lipid Raft Aggregation in Epithelial Cells and Mtb Figure 10. Filipin-treated A549 cells have significantly fewer viable intracellular bacteria after uptake. Filipin- and non-Filipin treated epithelial cells were infected with CDC1551, HN878 and H37Rv at MOI 100 as described. Intracellular bacterial viability over time (amikacin protection assays) was quantified by lysing host cells and plating on Middlebrook 7H10 medium at T0, after 6 hr of bacterial uptake, and 24 hpi (T24)(A). Numbers of viable bacteria were significantly decreased in Filipin-treated compared to non-treated host cells infected with HN878 and H37Rv at T0 (**p-value ,0.001; ,0.01, respectively) and T24 (***p-value ,0.001) (A). No significant difference was found between drug-treated and non-drug treated host cells infected with CDC1551 at T0 (p-value .0.05). A significant difference was found between treatments for CDC1551 infections at T24 (***p-value ,0.01). The calculated fold change from T0 to T24 indicates a significant decrease in the number of viable intracellular bacteria over time (B) (*p-value ,0.001). Infections were performed in triplicate and repeated three times. doi:10.1371/journal.pone.0045028.g010 similar to macrophages, Mtb bacilli require cholesterol aggregation for internalization and survival during infection of the epithelial cell. Cholesterol-dependent inhibition of lysosomal fusion may explain previous observations demonstrating the absence of PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e45028