FIG 6.

Production of sphingomyelin, not glycosphingolipids, is critical for efficient Mtb uptake. (A) Wild-type and two independent SGMS1^−/− knockout clones of U937 cells were infected with mCherry-Mtb (red) for 2 h, stained with phalloidin Alexa Fluor 488 (green), and then imaged by confocal fluorescence microscopy. (B) Quantification of Mtb uptake by cells treated as for panel A. (C) Wild-type and two independent UGCG^−/− knockout clones of THP-1 cells were infected with mCherry-expressing Mtb (red) for 2 h, stained with phalloidin Alexa Fluor 488 (green), and then imaged by confocal fluorescence microscopy. Bar, 5 μm. (D) Quantification of Mtb uptake by cells treated as for panel C. U937 and THP-1 cells were activated with 50 nM PMA for 24 h before Mtb infection. Three independent experiments were performed, each in triplicate with at least 1,000 cells quantified per replicate. Data are means ± SD. ***, P < 0.001; ns, not significant (two-tailed unpaired t test).