Fig. 8. Inhibitors of MVBs disrupted trafficking of sphingomyelin to the chlamydial inclusion. HEp-2 cells infected with C. trachomatis were cultured in the absence or presence of 5 mM 3-methyladenine or 10 µM U18666A. Post infection, at the times indicated, cells were labeled with NBD-ceramide and analyzed 1 hour after back-exchange. Infected cells were fixed, immunonlabeled with anti-golgin-97 or anti-CD63 antibody (anti-mouse Alexa Fluor 594), and 0.5-µm-thick optical sections were acquired by confocal microscopy. (Left panels) Control cells, 18 and 48 hours post infection. The NBD probe was incorporated in the Golgi complex and TOPRO-3-positive chlamydial inclusions. (Right panels) Treatment of infected cells with 3-methyladenine or U18666A resulted in the incorporation of fluorescent probe into swollen CD63-positive compartments but was excluded from TOPRO-3-positive chlamydial inclusions. Arrowheads indicate representative swollen NBD- and CD63-positive compartments. Arrows indicate chlamydial inclusions. Insert in lower left panel shows NBD-labeled Chlamydia (untreated, 18 hours post infection) in inclusions of equivalent size to 3-methyladenine- and U18666A-treated inclusions (48 hours post infection) that lacked NBD incorporation. Bar, 20 µm.