Limb-Bud and Heart (LBH) Upregulation in Cardiomyocytes under Hypoxia Promotes the Activation of Cardiac Fibroblasts via Exosome Secretion

Figure 2

The exosomal distribution of LBH protein is elevated in the exosomes secreted by LBH-upregulated CMs under hypoxia. (a) Representative confocal microscopy images of dual staining with anti-LBH (red) and anti-EEA1 (green) in mouse primary CMs and H9c2 cells. Colocalization of LBH and EEA1 was observed in the perinuclear cytoplasm of both primary CMs and H9c2 cells (indicated by magenta arrows). (b) The distribution of particle size/concentration of exosome samples analyzed by NTA. (c) Representative TEM images of exosome samples derived from mouse primary CMs and H9c2 cells (exosomal structures were indicated by red arrows). (d) Protein expression of CD9, CD63, CD81, ALIX, syntenin-1, and calnexin in cell lysates and the corresponding CM-derived exosomes from both mouse primary CMs and H9c2 cells. (e) Western blotting measuring LBH protein levels in CM^LBH+ exosomes and CM^NC exosomes ( vs. mouse CM-normoxia-exosomes/H9c2-NC-exosomes). (f) Exosome samples derived from mouse primary CMs under normoxia or hypoxia were tested by nanoflow cytometry after being stained with anti-LBH-FITC.