Figure 2

Encapsulation of exogenously added soluble cytosolic materials into ILVs and EVs in resealed HeLa cells. (a) Super-resolution images showing encapsulated 10 kDa CF568-labeled dextran (CF568-dex) in an intraluminal vesicle (ILV)-like structure. After introducing cytosol containing 100 µg/mL of CF568-dex, intact and resealed HeLa cells were post-incubated for 120 min and subjected to immunofluorescence (IF) analysis using anti-CD63 antibody (− SLO as “intact” and + SLO as “reseal”). The cells were observed by using an N-STORM super-resolution system. Green and red represent CF568 and CD63 signals, respectively. Arrows indicate CF568-dex incorporated in ILV-like structures. Inset: supposed models of ILV-like ultrastructure on background noise signals (dots). Scale bar = 200 nm. (b) Super-resolution images showing similarity of the ultrastructure of MVEs between intact and resealed HeLa cells. HeLa cells were incubated in DMEM without phenol red and observed using LSM980 with Airyscan2 at 37 °C (live-cell imaging). Inset: similar MVE-like ultrastructure observed in both intact and resealed HeLa cells. (c) Quantification of the amount of exogenously added synthesized miRNA in whole cells and EVs. The bar plot shows the relative amount of cellular- and EV-miRNA. After introducing cytosol containing a 1.0 nM synthesized cel-miR-39, intact and resealed HeLa cells were post-incubated for 120 min and further incubated in a 5% CO₂ incubator for 48 h at 37 °C. Total cellular- and EV-RNA were extracted and miR-39-3p (guide strand) amount was measured by real-time PCR. Fluorescent signal values of the target (FAM) were normalized by those of references (ROX). Data represent results from three independent experiments (n = 3), expressed as the mean ± SD. *P < 0.05; **P < 0.01. (d) Super-resolution images showing Cy3-labeled miR-39-3p in the MVE-like structure. After introducing cytosol containing a 1.5 µM fluorescently labeled double-stranded miRNA mimic (Cy3 for cel-mir-39, guide strand; Cy5 for cel-mir-39*, passenger strand), resealed HeLa cells were post-incubated for 120 min and subjected to IF analysis using anti-CD63 antibody. The cells were observed using an LSM980 with Airyscan2. Arrows indicate miRNA accumulated in MVEs. The right panels are a magnified image of the white boxes in the left panel. hm: high magnification. Scale bar (upper images) = 500 nm. Scale bar (bottom images and cropped images) = 200 nm.