Lipid Raft Aggregation in Epithelial Cells and Mtb and 24 hpi demonstrated a time-dependent increase in LR aggregation for CDC1551 (B), H37Rv (C) and HN878 (D ) (*p-value ,0.001 and **,0.05). Dose-dependent increases in LR aggregation were also significant for H37Rv and HN878 at 6 and 24 hpi (*p-value ,0.001 and **p-value ,0.05). At MOI 100, strains HN878 and H37Rv consistently produced higher levels of LR aggregation compared to CDC1551. Infections were performed in triplicate and experiments repeated three times. A total of 15 fields were imaged per coverslip, incorporating approximately 30–50 host cells per field. Images were captured at 63x magnification and LR aggregation assessed using ImageJ software. Results represent the average of three experiments. doi:10.1371/journal.pone.0045028.g002 Overall, CFP treatments produced significantly fewer aggregates compared to infections with live bacteria for all three strains at 6 and 24 hpi (p-value ,0.001). Collectively, these data suggest that the mycobacterial compo- nents, TL and CFP from broth-grown cultures, do not demon- strate the degree of LR aggregation observed during infection with live Mtb strains. Figure 3. Treatment of A549 cells with total lipids or culture filtrate proteins from Mtb strains produces limited LR aggregation. A549 cells treated with total lipids (TL) or culture filtrate proteins (CFP) from all three strains were processed for confocal microscopy at 6 and 24 hr post-treatment as described. At 6 hr post-treatment, a significant increase in LR aggregation was observed in TL treated A549 cells compared to uninfected controls (*p-value ,0.05) (A). At 24 hr post-treatment HN878 TL produced significantly higher aggregate numbers compared to uninfected controls (**p-value ,0.001) (A). It should be noted that LR aggregation for HN878, CDC1551 and H37Rv at 24 hr post-treatment was not significantly different from uninfected and DMSO controls (p-value 0.065, 0.977 and 0.737, respectively) (A). Compared to controls, treatment with CFP from CDC1551 did not produce statistically different LR aggregation values at 6 and 24 hr post-treatment (p-value = 0.598 and 0.379, respectively) (B). LR aggregation for HN878 and H37Rv was significantly different from controls at 6 hr post-treatment (*p-value = 0.033 and 0.012, respectively) (B). Experiments were performed in triplicate and repeated three times. A total of 15 fields were imaged per coverslip, incorporating approximately 30–50 host cells per field. Data presented are the average of three experiments. Images to obtain LR data were captured at 63x magnification. doi:10.1371/journal.pone.0045028.g003 PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e45028