Phdr1

The culture medium of H9c2 cells transfected with the LBH-eGFP plasmid or pEGFP-C1 plasmid was collected for exosome isolation. The isolated exosome samples were subjected to nanoflow cytometry to detect potential LBH-eGFP fusion protein or supplemented into the culture medium for CFs to observe cellular uptake of the labeled exosomes. For simulation of the entire crosstalk process without exosome isolation, the coculture of LBH-eGFP-transfected H9c2 cells and rat CFs was implemented by Transwell inserts (Corning #3450, 0.4 μm pore size) and glass bottom 6-well plates (Nest #801004). Also, the exosomes isolated from mouse CMs were labeled with PKH26 membrane dye (Sigma-Aldrich MINI26, USA) before being applied to the culture medium for CFs. After 6 hours of coculture with exosomes (20 μg total protein for each 35 mm petri dish according to BCA assay) or 24 hours of coculture with H9c2 cells, the treated CFs were fixed with 4% paraformaldehyde (PFA), stained with phalloidin-rhodamine (Santa-Cruz PHDR1, USA)/phalloidin-FTIC (Sigma-Aldrich P5282, USA) and 4′,6-diamidino-2-phenylindole (DAPI, FluoroPure™ grade, Invitrogen), respectively, and imaged by a Leica SP8 confocal microscope to confirm the internalization of labeled exosomes and the intracellular distribution of LBH-eGFP. The 3D images were remodeled from z-stack series with the 3D viewer module of Las X software.