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. 2021 Jan 26;12(1):e03141-20. doi: 10.1128/mBio.03141-20

FIG 4.

FIG 4

Segregation of CD45 from dectin-1 at the phagocytic cup is affected in Sptlc2−/− cells. (A) Time-lapse confocal images of wild-type RAW264.7 cells transfected with GFP-tagged dectin-1 (green) and infected with mCherry-expressing M. marinum (red). Arrows indicate sites where mycobacteria trigger clustering of dectin-1. Bar, 5 μm. (B) Wild-type and Sptlc2−/− RAW264.7 cells transfected with GFP-tagged dectin-1 (green) were incubated with zymosan A particles (dashed circles) on ice, warmed to 37°C for 2 min to initiate phagocytosis, fixed, and then stained with antibodies against CD45 (purple). Representative slices of z-stacks are shown. Insets show fluorescence intensity profiles of CD45 and dectin-1-GFP signals in a single slice. Bar, 3 μm. (C) Violin plots showing the level of CD45 segregation from dectin-1-enriched phagocytic cups in wild-type and Sptlc2−/− RAW264.7 cells, defined as the percentage of dectin-1 volume containing no CD45 voxels. More than 100 individual cups were analyzed per cell line. ****, P = 0.00006 by the two-tailed unpaired t test.