Fig. 6

miR-21 enriched in Hyp/Exo from ASCs contributed to M2 polarization. a Profiling of miRNAs in Nor/Exo and Hyp/Exo was performed (n = 2). The relative average levels of each miRNA in Nor/Exo and Hyp/Exo are shown. Each dot represents one miRNA. The miRNAs with a ratio of Hyp/Exo to Nor/Exo greater than 2 are marked red, and those less than 0.5 are marked blue. All the other miRNAs in between are marked black. b The levels of miRNAs that satisfy both marked red in panel (a) and reported to promote M2 polarization were further examined with RT-PCR analysis. c Downregulation of miR-21 in Hyp/Exo from ASCs. Hypoxic ASCs were untransduced (PBS) or transduced with Lenti/ZipmiR-Cont (ZipmiR-Cont) or with Lenti/ZipmiR-21 (ZipmiR-21) to knockdown miR-21. Hyp/Exo secreted from these cells were isolated. The level of miR-21 in the different Hyp/Exo groups was examined with RT-PCR to verify the downregulation. U6 was used as an internal control. d–g M1 macrophages were treated with PBS or Hyp/Exo in the presence of BLZ945 or miR-21-silenced Hyp/Exo (ZipmiR-21). Vehicle and ZipmiR-Cont were used as controls for BLZ945 and ZipmiR-21, respectively. The levels of iNOS, Arg-1, P-Akt (Ser473), and P-Akt (Thr308) in the treated M1 macrophages were examined by immunoblotting analysis. GAPDH and Akt (pan) were used as loading controls (d). The percentage of CD86⁺ or CD206⁺ cells among F4/80⁺ cells in the treated M1 macrophages was quantified by using flow cytometry analysis (e). The concentrations of M-CSF (f) and IL-10 (g) in the CdM of the treated M1 macrophages were examined by using ELISA. *p < 0.05, **p < 0.01, and ***p < 0.001. All data are expressed as the mean ± SD of n = 3