Fig. 2

The effects of short-term activated HSC (3dHSC)- and long-term activated HSC (14dHSC)-derived conditioned medium (CM) and sEVs on liver macrophage (KC). (A) Morphology of HSC-sEV-cocultured KCs. Gene expression profiles of 3dHSC- and 14dHSC-sEV-treated KCs were obtained by RNA-Seq. (B) Principal component analyses (PCA) based on the gene expression profiles of 3dHSC- and 14dHSC-derived sEV-treated liver resident macrophages as well as untreated KCs obtained by RNA-Seq. Each dot represents one RNA-Seq dataset of primary cells from one rat. (C) A heatmap was generated on the set of 388 differentially expressed genes between the 3dHSC- and 14dHSC-sEV-treated KCs. Representative genes are listed on the right side (P value < 0.05 and fold change > 2.0 or fold change < 0.5). The scale represents normalized log2 gene expression levels. (D) The gene expression levels of key macrophage biomarkers in KCs cocultured with 3dHSC- or 14dHSC-sEVs were determined by RT-qPCR using the same batch of samples used in RNA-Seq. (E) The protein expression of INOS and CD206 in KCs cocultured with 3dHSC- or 14dHSC-sEVs was detected by western blotting. (F) Surface marker expression in KCs cocultured with 3dHSC- or 14dHSC-sEVs was determined by flow cytometry. (G) The major biological activities for the 388 differentially expressed genes between 3dHSC- and 14dHSC-sEV-cocultured KCs are provided as a summary graph by Ingenuity Pathways Analysis. Orange, activated in KC3dE; blue, inhibited in KC3dE. (H) Upstream regulators of the 388 differentially expressed genes between 3dHSC- and 14dHSC-sEV-cocultured KCs. Red, activated KCs cocultured with 3dHSC-sEVs; green, activated KCs cocultured with 14dHSC-sEVs (p < 0.05 and activation z score > 2 or < -2). Statistical significance was determined by Student’s t test relative to untreated KCs, *** p < 0.001, ** p < 0.01, * p < 0.05; and Student’s t test relative to 3dHSC-sEV-cocultured KCs, ### p < 0.001, ## p < 0.01, # p < 0.05