detergent-resistant aggregates was performed using Cholera toxin B (CT-B) 488 (Invitrogen) at a dilution of 1:200. Filipin-treatment experiments were conducted and labeled with CT-B and caveolin- 1 as described. Images were obtained with either the Zeiss Axiovert 200 M and Apotome or a Nikon A1R confocal laser microscope system. For Toll-like receptor experiments, A549 cells were seeded onto coverslips as described. Positive controls for TLR-2 stimulation were conducted using 1 mg/ml of Pam3CSK4 (InvivoGen) for 6 hr at 37uC. TLR-4 stimulation was performed using E. coli 0111:B4 strain LPS (InvivoGen) at 10 mg/ml for 6 hr at 37uC. Experimental wells of A549 cells were infected with Mtb strains CDC1551, HN878 and H37Rv at an MOI of 100. At 6 and 24 hpi specimens were fixed and prepared for labeling as described. Cells were incubated with either mouse monoclonal anti-TLR-4 (Santa Cruz) or mouse monoclonal anti-TLR-2 (Abcam) antibodies at 1:200 dilution for 1 hr at RT. Both Primary antibodies were detected using goat-anti-mouse 633 antibodies (Invitrogen) at 1:500 for 1 hr at RT. Separate experiments were conducted using mouse monoclonal transferrin receptor antibody (Zymed) at 1:200 as a negative control for colocalization with LR. The antibody was detected using donkey-anti-mouse 633 (Invitro- gen) at a dilution of 1:500. CT-B 488 staining was also performed as described for each well during the secondary antibody incubation period. Images were obtained with a Nikon Eclipse TiE confocal microscope. All cells in each experiment were also stained with Dapi at 1:500 for 1 hr at RT. All infections were performed in duplicate and experiments repeated three times. Supernatant Treatment A549 cells were seeded onto 6-well plates as described and infected with Mtb strains CDC1551, HN878 and H37Rv. Mycobacterium tuberculosis strains were preincubated at 37uC for 1 hr in EMEM 5% FBS and 50 mg/ml amikacin to inhibit protein synthesis. Pretreated bacterial cells were applied to epithelial cell monolayers in a manner previously described in parallel with non- drug treated bacteria. Amikacin (50 mg/ml) was maintained on infected and control cells for the duration of the time course. Supernatants were pipetted into a 3 ml syringe and passed through Millex-GV 0.22 mm PVDF filters (Millipore) to remove bacteria. The filtered supernatants were applied to a new mono- layer of A549 cells seeded onto coverslips and incubated at 37uC for 24 hr. Aliquots of filtered supernatants were applied to 7H11 agar plates supplemented with 0.5% glycerol, 0.05% T80, 10% ADC (CDC1551) or OADC (HN878; H37Rv) and 50 mg/ml hygromycin to verify removal of bacteria. Specimens were processed for confocal microscopy as described and imaged with a Zeiss Axiovert 200 M and Apotome. Infections were performed in duplicate and experiments repeated twice. Intracellular Bacterial Viability Epithelial cell monolayers were seeded onto 24-well plates at 2.56104 cells per well and allowed to adhere for 12 hr prior to infection. Filipin- or non-Filipin-treated A549 cells were infected with Mtb strains at an MOI of 100 as described. After 6 hr at 37uC, medium was removed from each well, monolayers washed 3 times with 1x PBS and incubated for 2 hr in EMEM with amikacin (200 mg/ml), +/2 Filipin and 5% FBS as described previously [6]. The medium again was removed, and monolayers washed with PBS and EMEM with amikacin, +/2 Filipin and 5% FBS was applied; this was defined at time point 0 (T0). At T0 and T24, the cells were washed and lysed with 0.1% Triton X-100. Infections were performed in triplicate and experiments repeated three times. Lipid Raft Aggregation in Epithelial Cells and Mtb Assessment of Colocalization Confocal images were obtained as described and imported into ImageJ 1.451/Java 1.60_20 software and analyzed using the JACoP plugin. LR aggregates were first quantified using ImageJ as described. Colocalization of TLR labeling with LR aggregates was evaluated in JACoP using Pearson and Manders coefficient. Bacteria colocalization with LR aggregates and/or caveolin-1 antibodies alone were analyzed in a similar manner. Quantification of LR Aggregates Images of specimens were obtained as described and a total of 15 fields were imaged per coverslip for each experiment. Three coverslips were obtained for all three Mtb isolates, at each timepoint for all three experimental replicates (total = 54 coverslips per experiment). Once colocalization of CT-B and caveolin-1 was verified using Image J, JACoP plugin, aggregates were quantified and analyzed for size and area using ImageJ 1.451/Java 1.60_20 [76]. Numbers of aggregates were normalized to the number of host cells imaged per field to acquire an average number of aggregates per host cell/per field for each treatment condition. Statistical Analysis Statistical significance of aggregate numbers and viable bacterial counts was examined by ANOVA and Tukey’s HSD post-hoc comparison (a = 0.05) using SPSS 17.0H statistical software. Supporting Information Figure S1 Infection with Mtb strains (MOI = 1) induce LR aggregation. A549 cells were infected with H37Rv, HN878 and CDC1551 (MOI = 1) and incubated at 37uC, 5% CO2 for 6 hr (A) or 24 hr (B) as described. An increase in CT-B/caveolin-1 puncta is observed at 24 hpi (Quantification in Fig 2). Images were obtained at 63x magnification with a Nikon A1R confocal system equipped with a Nikon Eclipse TiE confocal microscope. Infections were performed in triplicate and repeated three times. Fifteen fields were imaged per coverslip for each experiment. (TIF) Figure S2 Infection with Mtb strains (MOI = 100) induce LR aggregation. A549 cells were infected with H37Rv, HN878 and CDC1551 (MOI = 100) and incubated at 37uC, 5% CO2 for 6 hr (A) or 24 hr (B) as described. An increase in CT-B/caveolin-1 puncta is observed at 24 hpi (Quantification in Fig 2). Images were obtained at 63x magnification with a Nikon A1R confocal system with a Nikon Eclipse TiE confocal microscopy. Infections were performed in triplicate and repeated three times. Fifteen fields were imaged per coverslip for each experiment. (TIF) Figure S3 A magnified confocal image of A549 cells infected with CDC1551 demonstrates colocalization of LR markers. A549 Type II alveolar epithelial cells were infected with CDC1551 (MOI = 10) and prepared for confocal microscopy as described. Images were obtained at 63x magnification with a Nikon A1R confocal system with a Nikon Eclipse TiE confocal microscope (A). The circle indicates the area from which the magnified image was obtained. The encircled area has been cropped and magnified to illustrate the colocalization of CT-B, Caveolin-1 and bacteria for the purposes of quantification as described in the methods (B). (TIF) Figure S4 Treatment of A549 cells with Filipin III does not impact endocytosis of dextran. Filipin- (A) or non-Filipin (B) treated A549 cells were incubated with 10,000 MW dextran- PLOS ONE | www.plosone.org 13 September 2012 | Volume 7 | Issue 9 | e45028