Journal: Nature Communications
Article Title: Efficient cellular transformation via protein delivery through the protrusion-derived extracellular vesicles
doi: 10.1038/s41467-025-66351-1
Figure Lengend Snippet: A Schematic illustration of EV endocytosis, endosomal transport, and cargo release. B 3D-PALM/STORM live cell imaging of the CIP4-mEos4b (magenta)-expressing CIP4 KO PANC-1 cells with l-EV fraction having SF650T-Halo-MIM I-BAR (green). Images were recorded every 10 s. Scale bar, 500 nm. C Colocalization of CIP4-mEos4b with SF650T-Halo-MIM I-BAR in ( B ). The percentages of voxels co-localized in each frame were calculated (“RAW”). Co-localization of the CIP4 images with the 180°-rotated SF650T-Halo-MIM images is shown as “Randomized”. The 13 super-resolution images from independently seeded 5 cells, which resulted in 4, 1, 4, 2, and 2 images, respectively, were analyzed. D Colocalization of EVs from the l-EV fractions having both Halo-Rac1 and mCherry-MIM I-BAR with EEA1 (Top) or Lamp1 (Bottom) in WT cells after 3 h of incubation, in which 10 6 cells were treated with 1.6 × 10 9 EV/ml in 1 ml. Arrowheads in the enlarged images show their colocalization. Scale bars, 10 μm. E Quantification of the colocalization of Halo-Rac1 with EEA1 (Top) or Lamp1 (Bottom) in WT and CIP4 KO cells using Mander’s coefficients after EV addition. The EV fraction and the number of cells and EVs were the same as ( D ). The red lines indicate the means of 54 cells from 3 independent experiments. F PALM imaging of the l-EVs having SF650B-Halo-Rac1 in the late endosome, having Lamp1-mEos4b (Left). Enlargements of the square regions in the left images are on the Right. Arrowheads indicate possible release of SF650B-Halo-Rac1 from the endosome. Scale bars, 10 μm (Left) and 100 nm (Right). G Quantification of the Halo-Rac1 release from the endosome in ( F ). The percentage of EVs with crossover of Halo-Rac1 and Lamp1 in a cell is shown (17 cells from 3 independent experiments). All EVs with Lamp1-positive endosomes were counted. The full range of images with the marking of the counting of the EVs is shown in Supplementary Fig. . The percentage of Halo-Rac1-releasing EVs was calculated as the percentage of “Halo-Rac1-releasing EVs” among the Halo-Rac1 EVs in the Lamp1-positive endosomes (endosome-colocalizing EVs). The means ± SD are shown. Statistical significance was determined by a two-tailed unpaired Mann–Whitney U test ( C , E ) or a two-tailed t-test with Welch’s correction ( G ). Source data are provided as a file.
Article Snippet: To label Halo7-tagged proteins in the EVs, HaloTag ligands SaraFluor 650 T (SF650T) or SaraFluor 650B (SF650B) ligands (Goryochemical, #A308-01 or #A201-01) were used.
Techniques: Live Cell Imaging, Expressing, Incubation, Imaging, Two Tailed Test, MANN-WHITNEY
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