Release of luminal content of exosomes and acquisition of exosome-shuttle miRNAs by DCs. (A) Content-mixing assay between 10⁶ BMDCs transduced with RAd-LUC (Luciferase BMDCs or with RAd-Empty, control BMDCs) and 10 μg exosomes loaded with luciferin (or not, Control-Exosomes) added to the DCs 3 minutes later. The assays were done in a final volume of 2 mL of 2 g/L glucose Ca/Mg HBSS. Luciferase BMDCs incubated alone were included as negative controls. (B) Content-mixing assay between 10⁶ control BMDCs and 10 μg of exosomes loaded with luciferin (or not, Control-Exosomes) added to the DCs 3 minutes later. (C) Ultrastructural analysis of the interaction of exogenously added (BALB/c) BMDC exosomes surface labeled with 5 nm of gold particles (IA^d+, arrowheads) with the plasma membrane of an acceptor BMDC (B6, IA^d−). The arrow shows the tight contact between the surface membrane of the BMDCs and the membrane of the labeled exosome (×100 000). Bar = 100nm. (D) Internalized 5 nm of gold-labeled (arrowheads) exosomes (arrows) inside a BMDC phagosome (×100 000). Bar = 100nm. The area within the dotted line is shown at higher detail on the right. (E) Luciferase reporter construct with 3 tandem copies of the target sequence to miR-451 or miR-148a located in the 3′-untranslated region and the control vector with the inverted sequences. (F-G) Normalized expression of luciferase in 10⁶ DC2.4 cells transfected with pCMV-Luc/3 × PT-miR-451 or control pCMV-Luc/3 × PT-inverted-miR-451 (F), or with pCMV-Luc/3 × PT-miR-148a or control pCMV-Luc/3 × PT-inverted-miR-148a (G), after 18 hours incubation alone, or with increasing concentrations of exosomes in complete medium. Shown results are representative of 4 (A-B), 12 (C-D), and 3 (F-G) independent experiments.