Fig. 2
Phagosomal acidification and proteolysis are compromised in 3d/3d DCs. a BM-DCs were incubated with OVAb for 15 min (pulse), and cargo fate (OVA Alexa-488 staining) was monitored by flow cytometry on phagosomes for the indicated chase points. The data displayed on the left panel are representative of one experiment and on the right panel is the quantification of percentage of OVA degradation (mean ± S.E.M., n = 4; *P < 0.05; **P < 0.01 using unpaired t-test). b For endosomal pH measurement, WT and 3d/3d DCs were pulsed for 10 min with FITC and Alexa-647-coupled dextrans (40 kDa) and chased for 50 min. Graph shows mean ± S.E.M. (n = 3) *P < 0.05 by unpaired t-test. c Phagosomal pH (90 min) was measured using ratiometric imaging by exposing cells to FITC-coupled OVA-coated zymosan (mean ± S.E.M., n = 3, *P < 0.05 via unpaired t-test). d Phagosomes from WT or 3d/3d DC were purified after 20 min or 2 h of particle internalization. Protein expression of gp91phox, V-ATPase and Cystatin C was detected either in total lysate (TCL, 50 μg) or in phagosomes (5 μg). Data are representative of three experiments. e Proteases’ activity in early (20 min) or late (120 min) phagosomes from WT (blue histogram) and 3d/3d (red histogram) DCs were measured with specific fluorescent substrates. Graphs show mean ± S.E.M., n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using unpaired t-test. f Phago-lysosome fusion (P-L) was measured by exposing cells loaded with lysosomal FRET acceptor Alexa-594-HA to donor Alexa-488 OVAb (n = 4). Cells were treated or not with ConcA as a negative control(n = 2) (*P < 0.05, unpaired t-test mean ± S.D.). g Percentages of β-lactamase transport to the cytosol were measured by loading WT and 3d/3d DCs with CCF4 dye followed by incubation with β-lactamase for the indicated time points (left panel, each dot corresponds to one experiment, n = 5). Representative histogram (right panel) of CCF4 cleaved product at 120 min in WT (blue solid line) and 3d/3d (red solid line). DCs incubated with CCF4 alone are shown in dashed lines **P < 0.01 by two-way ANOVA comparing all time points
