Fig. 3

The effects of short-term activated HSC (3dHSC)- and long-term activated HSC (14dHSC)-derived conditioned medium (CM) and sEVs on bone marrow-derived monocyte (MO) differentiation. Primary rat MOs were cocultured with 3dHSC- and 14dHSC-CM or sEVs; untreated cells served as controls. (A) Morphology of HSC-sEV-cocultured MOs, scale bar = 100 μm. (B) Principal component analysis (PCA) based on the gene expression profiles of 3dHSC- and 14dHSC-sEV-treated MOs as well as untreated MOs obtained by RNA-Seq. Each dot represents one RNA-Seq dataset of primary cells from one rat. (C) Heatmaps were generated on a set of 28 representative genes that were differentially expressed between 3dHSC- and 14dHSC-derived sEV-treated Kupffer cells (KCs), as listed in Fig. 2C (P value < 0.05 and fold change > 2.0 or fold change < 0.5); red, highly expressed in 3dHSC-sEV-cocultured KCs; green, highly expressed in 14dHSC-sEV-cocultured KCs. Each row represents an individual gene, and each column represents an individual sample. The scale represents normalized log2 gene expression levels. (D) The gene expression levels of key macrophage biomarkers in MOs cocultured with 3dHSC- or 14dHSC-sEVs were determined by RT‒qPCR using the same batch of samples in RNA-Seq. The relative gene expression levels were normalized to β-Actin and untreated MOs. (E) The protein expression of INOS and CD206 in MOs cocultured with 3dHSC- or 14dHSC-sEVs was detected by western blotting. (F) Surface marker expression in MOs cocultured with 3dHSC- or 14dHSC-sEVs was determined by flow cytometry. Statistical significance was determined by Student’s t test relative to untreated MOs, *** p < 0.001, ** p < 0.01, * p < 0.05; Student’s t test relative to 3dHSC-sEV-cocultured MOs, ### p < 0.001, ## p < 0.01, # p < 0.05, ns, not significant