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. 2023 May 23;14:1104890. doi: 10.3389/fimmu.2023.1104890

Figure 4.

Figure 4

ACO1 protein mediates miR-503 packaging into macrophage-derived sEVs (A) The specific interaction between the miR-503 sequence and motifs of RNA-binding proteins (RBPs) was analyzed using the database of RBP specificities (RBPDB, http://rbpdb.ccbr.utoronto.ca/; threshold 0.7). Among 8 candidates, ACO1 was overexpressed in macrophages after HG treatment according to GSE86298. (B) ACO1 expression in HG-treated or non-treated macrophages according to GSE86298. (C) ACO1 knockdown was achieved in M0 macrophages by transfecting short hairpin RNA targeting ACO1 vector (#1 sh-ACO1 and #2 sh-ACO1) and confirmed by qRT-PCR. (D) sEVs were extracted from M0 macrophages transfected with sh-NC, #1 sh-ACO1, or #2 sh-ACO1 under HG or normal glucose condition for 72 h and examined for the expression of miR-503 by qRT-PCR. (E) M0 macrophages were treated with HG, transfected with sh-NC, #1 sh-ACO1, or #2 sh-ACO1, and examined for the cellular expression of miR-503 by qRT-PCR. (F) ACO1 levels were determined by Immunoblotting in samples derived by miRNA pulldowns using biotinylated miR-503 or mutated miR-503 in nuclear, cytoplasmic, or sEVs lysates. the biotinylated poly(G) was used as a negative control. (G) RIP assay of anti-IgG (negative control) or anti-ACO1 antibody was performed on cell or sEVs lysates from M0 macrophages. The level of miR-503 in the immunoprecipitated samples was measured by qRT-PCR and reported as a percentage relative to the input sample. (H) RIP assay of anti-IgG or anti-ACO1 antibody was performed on cell or sEVs lysates from M0 macrophages under normal glucose or HG condition. The level of miR-503 in the immunoprecipitated samples was measured by qRT-PCR and reported as a fold change of anti-ACO1/anti-IgG. N=3, ** p<0.01 vs. sh-NC group, IgG group or control group. * p<0.05; ns, no significance.