Figure 2.
Lipid accumulation in CGs of Npc1−/−.CL4 CTLs. (A) Confocal immunofluorescence microscopy shows GzmB-containing cytotoxic granules (green; top). Three-dimensional reconstruction of CGs (Imaris vesicles) based on GzmB staining, which allowed assessment of their diameter. Diameter of CTL CGs (pooled data from n = 3 Npc1+/+ and n = 4 Npc1−/− mice), diameter of CGs per cell (pooled data), and mean biological replicates (right). DAPI is shown in blue. (B) Confocal microscopy shows vesicles containing unesterified cholesterol stained with filipin III in Npc1−/− and Npc1+/+ CTLs. (C) Confocal immunofluorescence microscopy shows colocalization of GzmB (green) and fluorescent cholera toxin B (CTxB; magenta) in activated Npc1−/− and Npc1+/+ CTLs. Pearson correlation coefficient was used to quantify colocalization of GzmB and CTxB. (D) Total area of CTxB-labeled compartment (as shown in panel C) was assessed by accumulation of monosialotetrahexosylganglioside (GM1); mean biological replicates (inset; n = 6 Npc1+/+ and n = 7 Npc1−/−). (E) Colocalization of GzmB and CTxB (as shown in panel C) using overlap coefficient; biological replicates (inset; n = 3). Detailed description of microscopy and analysis provided in the data supplement. *P < .05, ***P < .001, ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole.

Lipid accumulation in CGs of Npc1−/−.CL4 CTLs. (A) Confocal immunofluorescence microscopy shows GzmB-containing cytotoxic granules (green; top). Three-dimensional reconstruction of CGs (Imaris vesicles) based on GzmB staining, which allowed assessment of their diameter. Diameter of CTL CGs (pooled data from n = 3 Npc1+/+ and n = 4 Npc1−/− mice), diameter of CGs per cell (pooled data), and mean biological replicates (right). DAPI is shown in blue. (B) Confocal microscopy shows vesicles containing unesterified cholesterol stained with filipin III in Npc1−/− and Npc1+/+ CTLs. (C) Confocal immunofluorescence microscopy shows colocalization of GzmB (green) and fluorescent cholera toxin B (CTxB; magenta) in activated Npc1−/− and Npc1+/+ CTLs. Pearson correlation coefficient was used to quantify colocalization of GzmB and CTxB. (D) Total area of CTxB-labeled compartment (as shown in panel C) was assessed by accumulation of monosialotetrahexosylganglioside (GM1); mean biological replicates (inset; n = 6 Npc1+/+ and n = 7 Npc1−/−). (E) Colocalization of GzmB and CTxB (as shown in panel C) using overlap coefficient; biological replicates (inset; n = 3). Detailed description of microscopy and analysis provided in the data supplement. *P < .05, ***P < .001, ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole.

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