Fig. 4

Nuclear miR-143/145 transcriptionally increases Siglec-G expression to repress macrophage efferocytosis. a Model of P. gingivalis-induced secretion of apoEVs-miR-143/145 captured by macrophages. b Analysis of Siglec-10 expression (Siglec-G in mouse) in human early (n = 13)- and advanced (n = 16)-plaque by RNA-seq from a public database. c Efferocytosis ability of macrophages that were treated with miR-143/145 mimics, IgG, or anti-Siglec-G antibodies was detected by phagocytosis assay, with quantitative data at right. Scale bars: 20 μm. d Macrophages that were transfected with fluorescently labeled miR-143/145 mimics were observed at different time points, with quantitation of nuclear miR-143/145 foci at right. e The relative enrichment of these five putative binding sites of Ago2 in macrophages transfected with negative control (NC) or miR-143/145 mimics. The red dotted line indicates that Site 1 corresponded to miR-143 binding Site a, and Site 2 corresponded to miR-145 binding Sites (d) and (e). f Wild type plasmid based on Site a region of Siglec-G promoter was subjected to dual-luciferase reporter assay in response to NC mimics, miR-143 mimics, NC inhibitor or miR-143 inhibitor. Another wild-type plasmid based on Sites (d) and (e) region of Siglec-G promoter was subjected to dual-luciferase reporter assay in response to NC mimics, miR-145 mimics, NC inhibitor, or miR-145 inhibitor. g Validating the effect of the si-Ago2 on Ago2 expression and nuclear localization of Ago2nls. Scale bars: 10 μm. h Western blot analysis shows that miR-143/145 failed to activate Siglec-G expression in the absence of Ago2, but retained Siglec-G expression once with nuclear Ago2 re-expression in Ago2 knockdown macrophages. i Western blot showing co-IP between Ago2 and POL II in macrophages with miR-143/145 mimics treatment. Results are presented as the mean ± S.D. by one-way ANOVA followed by Tukey multiple comparisons tests. *P < 0.05; **P < 0.01, ***P < 0.001; #P > 0.05