Laser confocal microscopy

Cells were seeded on coverslips in 12-well plates, and the density of planking cells was about 2 × 10⁴ cells/mL. After a period of culture for 24 h, cells were treated with 2 × 10⁻⁸ M PLTX for 10 min, followed by fixation for 20 min with 4% multi-local methanol (PFA). The cells were incubated with permeable solution of 0.3% TritonX-100 for 30 min. Then, cells were blocked with 5% BSA Quick Block (Beyotime, Shanghai, China) for 10 min. The fixed cells were covered with 1 µM red fluorescent 5′-Cy5-labeled specific aptamer (Sangon Biotech, Shanghai, China) or 1 µM random aptamer for 30 min. Then 5 µM DiO (Beyotime, Shanghai, China) was added to stain the cell membrane for 20 min. Next, 5 μg/mL DAPI (Beyotime, Shanghai, China) was added to counterstain the nucleus for 5 min. Finally, the coverslips were mounted on the glass slides with anti-fluorescence quenching sealing plate reagent. The cells were imaged on a single plane via laser confocal microscopy (Zeiss, Oberkochen, Germany). A 63× magnification objective was used.

The subcellular localization of GsMYB7 protein was analyzed using the method described by Tamara et al.and and Yan et al.[59 (link), 60 (link)]. The GsMYB7 protein subcellular localization was predicted using a web server Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/). The recombinant fragment containing the CDS sequence of GsMYB7 gene (without termination codon) was amplified and purified with the restriction sites of NcoI and SpeI at both ends (Additional file 1: Table S3). The expression vector pCAMBIA1302-eGFP was linearized with NcoI and SpeI restriction enzymes, and the recombinant fragment was ligated to construct the fusion expression vector pCAMBIA1302-eGFP-GsMYB7. The plasmids of pCAMBIA1302-eGFP and pCAMBIA1302-eGFP-GsMYB7 were transformed into the cells of Agrobacterium tumefaciens GV3101, respectively. The transformed cells of GV3101 and viral protein P19 were resuspended in the prepared osmotic buffer (The main components are 10 mM MgCl₂, 10 mM MES- KOH and 100 μM acetyleugenone) for culture and then mixed in the same volume. After sitting at room temperature for 3 h, the bacterial fluid was injected into the lower epidermis cells of 4-week-old leaf in Nicotiana tabacum, and the transformed leaves after being cultured for 48 h were observed by laser confocal microscopy (Carl Zeiss, Jena, Germany).

Purified exosomes were labeled with PKH26 red fluorescent membrane linker dye (Sigma–Aldrich) according to the manufacturer's instructions. After removal of excess dye, the labeled exosome pellets were resuspended and added to the cultured macrophages for the studies of exosome uptake. After incubation for 24 h and two washes with PBS, the cells were fixed with 4% paraformaldehyde, stained with DAPI to visualize nuclei and immediately observed by laser confocal microscopy (Zeiss).