Biological activities of KRS-containing exosomes. (A) Effects of KRS WT (6 µg), ΔN12 (6 µg), and KRS exosomes (5 µg by total proteins) on the secretion of the indicated factors from RAW264.7 cells were determined using a multiplex bead array. (B) To assess the functional importance of KRS for exosomes, we reduced the KRS level in exosomes by siRNA knockdown and confirmed by immunoblotting. (C and D) TNF-α–inducing (C) and transwell migration (D) activities of RAW264.7 cells were compared for purified KRS ΔN12 mutant (6 µg) and exosomes (normal and KRS suppressed; 5 µg total proteins) obtained from HCT116 cells. HPF, high-power field. (E) Exosomes (normal and KRS suppressed; 0.1 µg/µl) from HCT116 cells and BSA were stained with DiI (Potter et al., 1996) and Alexa Fluor 647, respectively (red fluorescence), and injected into mouse ear dermis. To visualize macrophage/neutrophil (green fluorescence) recruitment to the injection sites, time-lapse intravital images of macrophage/neutrophil (green fluorescence) recruitment were taken in 30-s intervals up to 120 min after injection. (F) These experiments were repeated three times, and the results are represented as bar graph. Green fluorescence signal was measured by ImageJ. (G) B16F10 cells expressing empty vector (EV), KRS WT, and D12A mutant were stained with DiD (Sutton et al., 2008) and injected into mouse ear dermis (4 × 104 cells/µl; red fluorescence). Bars, 100 µm. (H) These experiments were repeated three times, and the results are shown as bar graph. Green fluorescence signal was measured by ImageJ. Error bars show means ± SD from the mean of three independent experiments. n = 3. *, P < 0.05; **, P < 0.01.
