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. Author manuscript; available in PMC: 2021 Jan 7.

Published in final edited form as: Circulation. 2019 Dec 30;141(1):42–66. doi: 10.1161/CIRCULATIONAHA.119.041460

A, Illustration of the transwell co-culture system. The upper schematic shows that the transwell comprised a 24-well culture insert with a 0.4-μm pore size in the upper part and a 24-well plate in the lower part. The lower schematic shows the experimental procedure. PMA, phorbol 12-myristate 13-acetate. B, Immunofluorescence staining showing the presence of DNA from Edu-labeled H₂O₂-treated SMCs in the cytosol of macrophages (CD68) after using a transwell co-culture system. Cells are outlined with white dots. Nuclei are outlined with yellow dots. C, Western blot showing that MMP-9 expression and the phosphorylation of STING and IRF3 were increased in macrophages that were co-cultured with H₂O₂-treated SMCs. D-G, Macrophages were transfected with DNA isolated from H₂O₂-treated SMCs. SMC-derived DNA increased the expression and phosphorylation of STING and IRF3. (D), SMC-derived DNA also increased levels of MMP-9 protein (E), MMP9 mRNA (F), and MMP-9 activity (G) (n=5 biologic repeats). H-K, Macrophages were transfected with STING or IRF3 plasmid DNA or transfected with STING or IRF3 siRNA, followed by stimulation with SMC DNA. MMP-9 protein levels (n=4 biologic repeats) (H), MMP9 mRNA levels (n=3 biologic repeats) (I), and MMP-9 activity (J) were enhanced by the overexpression of STING or IRF3 or reduced by the knockdown of STING or IRF3. K, The results of a chromatin immunoprecipitation assay showing that IRF3 bound to the MMP9 promoter in unstressed macrophages. This binding was increased in cells treated with SMC DNA and was abolished by inhibiting STING expression with STING siRNA (n=4 biologic repeats). An unpaired, two-tailed t-test was used in (F) and (I). Two-way ANOVA with the Bonferroni post-hoc test for pairwise comparisons was used in (I). **P<0.01, ***P<0.001. Data are presented as the mean ± standard error of the mean.

A, Illustration of the transwell co-culture system. The upper schematic shows that the transwell comprised a 24-well culture insert with a 0.4-μm pore size in the upper part and a 24-well plate in the lower part. The lower schematic shows the experimental procedure. PMA, phorbol 12-myristate 13-acetate. B, Immunofluorescence staining showing the presence of DNA from Edu-labeled H₂O₂-treated SMCs in the cytosol of macrophages (CD68) after using a transwell co-culture system. Cells are outlined with white dots. Nuclei are outlined with yellow dots. C, Western blot showing that MMP-9 expression and the phosphorylation of STING and IRF3 were increased in macrophages that were co-cultured with H₂O₂-treated SMCs. D-G, Macrophages were transfected with DNA isolated from H₂O₂-treated SMCs. SMC-derived DNA increased the expression and phosphorylation of STING and IRF3. (D), SMC-derived DNA also increased levels of MMP-9 protein (E), MMP9 mRNA (F), and MMP-9 activity (G) (n=5 biologic repeats). H-K, Macrophages were transfected with STING or IRF3 plasmid DNA or transfected with STING or IRF3 siRNA, followed by stimulation with SMC DNA. MMP-9 protein levels (n=4 biologic repeats) (H), MMP9 mRNA levels (n=3 biologic repeats) (I), and MMP-9 activity (J) were enhanced by the overexpression of STING or IRF3 or reduced by the knockdown of STING or IRF3. K, The results of a chromatin immunoprecipitation assay showing that IRF3 bound to the MMP9 promoter in unstressed macrophages. This binding was increased in cells treated with SMC DNA and was abolished by inhibiting STING expression with STING siRNA (n=4 biologic repeats). An unpaired, two-tailed t-test was used in (F) and (I). Two-way ANOVA with the Bonferroni post-hoc test for pairwise comparisons was used in (I). **P<0.01, ***P<0.001. Data are presented as the mean ± standard error of the mean.