Figure 4. Exophagy is upregulated with DC maturation.

(A–D) GM-CSF BM iDCs and mDCs were incubated with Alexa546-agLDL for 60 min followed by fixation and labeling of F-actin with Alexa488-phalloidin. After 60 min, an enrichment of F-actin was detected near the sites of contact with agLDL in both GM-CSF BM iDCs (A and B) and mDCs (C and D). (E) Quantification of the average local F-actin intensity per cell in both murine and human monocyte derived iDCs and mDCs. (F–H) GM-CSF BM iDCs and mDCs were incubated with biotin-fluorescein-dextran overnight to deliver it to lysosomes. Cells were then exposed to streptavidin-Alexa546-labeled agLDL for 90 min, fixed and permeabilized. Lysosome exocytosis was observed to aggregate containing compartments in both GM-CSF BM iDCs (F–H) and mDCs (I–K). (L) Quantification of the amount of biotin-fluorescein-dextran exocytosed in both murine and human monocyte derived iDC and mDC. (M) GM-CSF BM iDCs, mDCs and J774 macrophage-like cells were incubated with CypHer 5E, a pH sensitive fluorophore, and Alexa488, a pH insensitive fluorophore, dual-labeled agLDL and the pH surrounding the aggregate measured. Quantification of the lowest pH achieved in the compartment at a single timepoint in iDCs, mDCs and J774 macrophage-like cells. The central mark on each box is the median, while the edges of the box represent the 25^th and 75^th percentiles. Error bars (E and L) represent the sem. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 student’s t test. Data are pooled from 3 independent experiments.