Figure 5.
Antigen Import Enhancement Is a Consequence of Lysosomal Trapping
(A) Physicochemical properties (according to DrugBank data) of all the compounds present in the FDA library and of those active in the antigen import assay (yEC50 <40 μM, see Figure 1F). All but one of the hits has physicochemical properties similar to those of lysosomotropic compounds (see also Figure S5A).
(B) MutuDCs cells were pulsed with 10 μg/mL WGA-Alexa Fluor 633 for 45 min and imaged immediately after addition of 5 μM prazosin-BODIPY or 10 μg/mL BODIPY. Representative data from one of two independent experiments. Scale bar, 10 μM.
(C and D) MutuDCs were pre-treated with 10 mM NH4Cl and imaged immediately following addition of prazosin-BODIPY.
(C) Representative images, scale bar 10 μM.
(D) Number of prazosin-BODIPY spots per cell (80 cells per condition per experiment, three independent experiments). p value was calculated using Mann-Whitney U test. Box and whiskers plot visualizes median, first and third quartiles (hinges) and the smallest/largest observation no further than 1.5 * IQR from the respective hinge (whiskers).
(E) Antigen import assay (Figure 1A) was performed in the presence of prazosin, importazole, tamoxifen, and DbeQ with or without NH4Cl. Representative plots are shown (prazosin, n = 3; tamoxifen, importazole, and DbeQ, n = 2).
(F and G) MutuDCs were pulsed with 3K dextran-TRITC for 45 min and incubated with 20 μM prazosin, 10 mM NH4Cl, or both for 45 min.
(F) Representative images; scale bar, 20 μM.
(G) Quantification of cytosolic fluorescence. Each dot represents one cell; fluorescence was quantified for 100 cells per condition per experiment with four independent experiments (including data in Figure 4E). p values were calculated using Mann-Whitney U test with Bonferroni correction (comparing with the control group). Box and whiskers plot visualizes median, first, and third quartiles (hinges) and smallest/largest observation no further than 1.5 * IQR from the respective hinge (whiskers).
See also Figure S5.