2 volumes of 0.9% NaCl. After vigorous vortexing, the mixture was centrifuged (1,150 × g, 5

min) and the organic phase (containing GPLs) was collected and evaporated to dryness. The dried lipid extracts were dissolved in 20 μl of CHCl3/CH3OH (2:1) and subjected to TLC using aluminum-backed, 250-μm silica gel F254 plates developed with CHCl3/CH3OH (100:7). After chromatography, TLC plates were sprayed with orcinol/sulfuric acid (0.1% orcinol in 40% sulfuric acid) and glycolipids were detected by charring at 140°C. Preparation and gas chromatography–mass spectrometry (GC-MS) analysis of alditol acetate derivatives Alditol acetate derivatives of glycosyl units from SN-38 cell line GPLs were prepared and analyzed as reported [47, 61]. Briefly, lipid samples prepared by extraction as noted above were Sapitinib supplier acid-hydrolyzed in 250 μl of 2 M trifluoroacetic acid learn more for 2 hr at 120°C. After cooling down to room temperature, samples were hexane-washed (250 μl) and dried on air bath after adding 1 μg of 3,6-O-dimethyl-glucose as an internal standard. The hydrolyzed sugars were reduced overnight at room temperature by adding 250 μl of NaBD4 (prepared at 10 mg/ml in 1 M NH4OH in C2H5OH). After reduction, glacial acetic acid (20 μl) was added to remove excess NaBD4 and the samples were dried. CH3OH (100 μl) was added to each sample, and after resuspension the solvent was evaporated

to dryness (this step was repeated twice). The samples were per-O-acetylated with 100 μl of acetic anhydride at 120°C for 2 hr. After cooling, the samples were dried on air bath and suspended in 3 ml of CHCl3/H2O (2:1) by vortexing. The organic layer was extracted after centrifugation (2,500 × g, 5 min, 4°C) and dried on air bath. GC-MS analysis was performed using a Varian CP-3800

gas chromatograph (Varian Inc., Palo Alto, CA) equipped with a MS-320 mass spectrometer and using helium gas. The alditol acetate derivatives were dissolved in 50 μl of CHCl3 before injection on a DB 5 column (30 m × 0.20 mm inner diameter) with an initial oven temperature of 50°C for 1 min, followed by an increase of 30°C/min to 150°C and finally to 275°C at 5°C/min. Congo red agar plate assay The assay was carried out using reported methodologies [23]. Briefly, mycobacterial cultures (5 ml, OD600 = 1.5) PDK4 were shortly vortexed with glass beads to increase homogeneity and then centrifuged (4,700 × g, 15 min) for cell collection. The collected cells were washed with PBS (5 ml) and subsequently resuspended in PBS to an OD600 of 1. The cell suspensions were spotted (2 μl) on congo red agar plates [23] (7H9 basal medium, 1.5% agar, 100 μg/ml congo red (sodium salt of 3,3′-([1,1'-biphenyl]-4,4′-diyl)bis(4-aminonaphthalene-1-sulfonic acid), Sigma Aldrich Co.), 0.02% glucose, 30 μg/ml kanamycin). Colony morphology was examined using an Olympus SZX7 stereo microscope after plate incubation (37°C, 3 days). Sliding motility test The test was performed by standard methods [19].