Fig. 4: HG via ROS induces mitochondrial DNA (mtDNA) leakage and activates STING signaling in macrophages.

A BMDM were treated with HG (30 mmol/L) for 24, 36, and 48 h or normal control (NC, 5.5 mmol/L) for 24 h, then examined for STING using western blot analysis. B The mRNA levels were examined using RT-qPCR. C BMDM from STING^−/− and WT C57BL/6 J mice (WT-BMDM and STING^−/−-BMDM) were treated with HG and NC for 24 h, then examined for STING using western blot analysis. D WT-BMDM and STING^−/−-BMDM were treated with or without DMXAA (75 mg/mL) and C176 (5 nmol/mL) for 24 h in the absence or presence of HG for the last 24 h, then examined for inflammatory signaling using western blot analysis. BMDM were treated with HG and NC for 24 h, then examined for the displacement of STING toward Golgi apparatus (GM130) and mtDNA leakage using laser confocal microscopy. Anti-GM130 antibody labeled Golgi apparatus (red), anti-STING antibody labeled STING (green) and DAPI labeled nucleus (blue) (E), F Anti-Mitofilin antibody labeled Mitochondria (red), anti-dsDNA antibody labeled mtDNA (green) and DAPI labeled nucleus (blue). WT-BMDM and STING^−/−-BMDM were treated with HG and NC for 24 h, then examined for ROS production using living cell imaging microscopy (G), cGAS protein expression using western blot analysis (H), mRNA levels using RT-qPCR (I), downstream NF-κB and IRF3 activation using living cell imaging microscopy (J). For all bar graphs, Measurement Data were represented as mean ± SD. One-way analysis of variance (ANOVA) and Tukey’s post hoc test were used for comparing data among multiple groups. The cell experiment was repeated 3 times. *P < 0.05, **P < 0.01 and ***P < 0.001, NC vs HG (in A, B, C, H, and I), NC vs NC + DMXAA, HG vs HG + C176 and WT-BMDM vs STING^−/−-BMDM in HG condition (in D).