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Fig. 6. Blockade by vacuolin1 of the ionomycin-induced discharge of macropinosomes. Panel A shows merged images of DCs pretreated with Vac1 and loaded for 10 minutes with Bds (green) and immunolabelled for EEA1 (red), showing extensive co-localization of the two signals (yellow) in fluorescent puncta distinctly larger than those of non-pretreated cells (Fig. 1E and Fig. 5A). Panel B shows DCs as in A but stimulated with IONO for 1 minute. The puncta positive for both Bds and EEA1 appear unaffected by the ionophore. Morphometric quantification of the data in the VAC1-pretreated cells before and after IONO (D) reveals an almost doubling of the percentage of dually positive puncta, with respect to DCs non-pretreated with the drug (Fig. 5C), and no significant decrease induced by the ionophore. At the ultrastructural level (panels E,F), the VAC1-pretreated cells show swollen macropinosomes. Their Bd density is similar in IONO-treated and untreated cells and distinctly lower than that of macropinosomes of non-pretreated DCs (Fig. 1H,I and Fig. 5D). Panel C shows VAC1-pretreated DCs loaded with Bds as in A, immunolabelled for Lamp1 (red). Lamp-1-positive puncta are larger than in non-pretreated cells, however their lack of co-localization with the tracer is as in untreated cells (supplementary material Fig. S1B,D). Bar, 10 µm (A), also valid for B and C. Bar, 0.5 µm (F), also valid for E.
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