Figure 7.

Exosomes with downregulated hsa_circ_00074854 suppressed macrophage M2 polarization in vitro. (A and B) TEM of L02 and HepG2 cell CM secreted exosomes (Red arrow, exosome). Scale bar, 200 nm. NTA was used to detect the size and number of exosomes. (C) Western blot analysis for exosomal proteins TSG101 and CD63. (D) The level of hsa_circ_00074854 was analyzed by RT-qPCR in exosomes isolated from L02 cells and HepG2 cells. **P < 0.01 vs L02-Exo group. (E) Confocal microscopy of the internalization of PKH26-labeled HepG2 cell-derived exosomes (red) by macrophages. (F) Macrophages were treated with exosomes (0, 25, 50 μg/mL) isolated from CM of HepG2 cells. The percentage of CD86- and CD206-positive macrophages were analyzed by flow cytometry. *P < 0.05, **P < 0.01 vs control group. (G) RT-qPCR analysis of hsa_circ_00074854 levels in exosomes isolated from the CM of hsa_circ_00074854 knockdown HepG2 cells. **P < 0.01 compared with NC group. (H) Flow cytometry analysis of the expressions of CD86/CD206 in macrophages treated with exosomes with downregulated hsa_circ_00074854 levels. **P < 0.01 vs Control group; ^##P < 0.01 vs Exo^shRNA-NC group. (I) RT-qPCR analysis of the levels of IL-10 and TNF-a in macrophages treated with exosomes with downregulated hsa_circ_00074854 levels. **P < 0.01 vs Control group; ^##P < 0.01 vs Exo^shRNA-NC group.

Abbreviations: CM, conditioned medium; Exo, exosomes; Exo^{circRNA shRNA1}, exosomes derived from hsa_circ_00074854 shRNA1-transfected HepG2 cells; Exo^shRNA-NC, exosomes derived from shRNA NC-transfected HepG2 cells; NC, negative control.