Journal: JCI Insight

Article Title: Activated cGAS/STING signaling elicits endothelial cell senescence in early diabetic retinopathy

doi: 10.1172/jci.insight.168945

Figure Lengend Snippet: Representative immunoblots ( A ) and densitometry graphs show that the protein levels of cGAS ( B ) and STING ( C ) are increased in the retina of human DR donor cadaver tissue (Ctl, n = 8 DR, n = 13). Immunostaining of STING in human PDR donor retinal sections shows that ( D ) STING is present (arrow) in endothelial cells, ( E ) intraretinal neovascularization, ( F ) faintly in an occluded capillary and strongly in surrounding microglia/macrophages, and ( G ) in endothelial cells and surrounding microglia/macrophages of a hyalinized capillary microaneurysm. Scale bars: 25 μm. The levels of IFN-α ( H ) and IFN-β ( I ) in the aqueous humor, and IFN-α ( J ) and IFN-β ( K ) in the vitreous humor of patients without (Ctl) and with DR or PDR. (Ctl, n = 20, DR, n = 23; NPDR, n = 10; PDR, n = 13 for H and I ; Ctl, n = 13, PDR, n = 30 for J and K ). ( L ) The levels of free DNA in the vitreous humor of patients without ( n = 11) and with PDR ( n = 17). Data represent mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 by Mann-Whitney U test for B , C , H (left), I (left), and J – L , or Kruskal-Wallis test for H (right) and I (right). Ctl, control; NPDR, nonproliferative diabetic retinopathy; PDR, proliferative diabetic retinopathy.

Article Snippet: After being blocked with 5% milk (170-6404, Bio-Rad) for 1 hour, the membranes were incubated overnight with primary antibodies (diluted 1:1000) against iNOS (2982S, Cell Signaling Technology), ICAM-1 (10020-1-AP, Proteintech), p-IκBα (Ser32) (2859T, Cell Signaling Technology), IκBα (4812S, Cell Signaling Technology), STING (NBP2-24683, Novus Biologicals), cGAS (15102S, Cell Signaling Technology), p-TBK1 (5483S, Cell Signaling Technology), TBK1 (3013S, Cell Signaling Technology), p-IRF3 (29047S, Cell Signaling Technology), IRF3 (4302S, Cell Signaling Technology), p-NF-κB (3033S, Cell Signaling Technology), NF-κB (6956S, Cell Signaling Technology), GAPDH (5174S, Cell Signaling Technology), or vinculin (ab129002, Abcam).

Techniques: Western Blot, Immunostaining, MANN-WHITNEY, Control

Journal: JCI Insight

Article Title: Activated cGAS/STING signaling elicits endothelial cell senescence in early diabetic retinopathy

doi: 10.1172/jci.insight.168945

Figure Lengend Snippet: Representative immunoblots ( A ) and densitometry graphs show that the protein levels of ( B ) cGAS and ( C ) STING expression was increased in the retina of diabetic (D) mice (diabetes induction for 2 months) compared with nondiabetic (N) controls. Immunofluorescence study shows that ( D ) STING is localized in the retinal nerve fiber layer (asterisk) of both nondiabetic and diabetic mice, and more were observed in retinal endothelial cells in diabetic retina. Red, STING + cells; green, CD31 + endothelial cells. Scale bar: 50 μm. ELISA shows that ( E ) IFN-α (not significant) and ( F ) IFN-β (significant) were increased in the diabetic retina compared with nondiabetic controls, which is correlated with pericytes loss ( G and H ; arrowheads indicate pericytes). ( I ) Flat-mount micrographs from WT diabetic mice (2 months of diabetes, 4 months of age) and age-matched nondiabetic (N) controls showing blood vessel hyperpermeability; arrows indicate leakage sites. Scale bars: 50 μm. ( J ) Quantification of FITC-BSA leakage into the retina. Representative immunoblots ( K ) show increased inflammatory factors iNOS ( L ) and ICAM-1 ( M ) and superoxide production ( N ) in diabetic mice. In A – N , n = 6 for each group. Data are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 versus nondiabetic controls by unpaired, 2-tailed Student’s t test.

Article Snippet: After being blocked with 5% milk (170-6404, Bio-Rad) for 1 hour, the membranes were incubated overnight with primary antibodies (diluted 1:1000) against iNOS (2982S, Cell Signaling Technology), ICAM-1 (10020-1-AP, Proteintech), p-IκBα (Ser32) (2859T, Cell Signaling Technology), IκBα (4812S, Cell Signaling Technology), STING (NBP2-24683, Novus Biologicals), cGAS (15102S, Cell Signaling Technology), p-TBK1 (5483S, Cell Signaling Technology), TBK1 (3013S, Cell Signaling Technology), p-IRF3 (29047S, Cell Signaling Technology), IRF3 (4302S, Cell Signaling Technology), p-NF-κB (3033S, Cell Signaling Technology), NF-κB (6956S, Cell Signaling Technology), GAPDH (5174S, Cell Signaling Technology), or vinculin (ab129002, Abcam).

Techniques: Western Blot, Expressing, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: Activated cGAS/STING signaling elicits endothelial cell senescence in early diabetic retinopathy

doi: 10.1172/jci.insight.168945

Figure Lengend Snippet: ( A ) Representative immunoblots and quantification of ( B ) STING, ( C ) p-TBK1, and ( D ) IRF3. Induction of diabetes in WT mice resulted in increased STING compared with appropriate controls; STING was not expressed in STING -KO and STING GT mice. p-TBK1 and p-IRF3 were decreased in STING -KO and STING GT diabetic mice compared with WT diabetic controls. ELISA analysis ( E ) shows that IFN-β was increased in WT diabetic retina compared with nondiabetic controls, but this was inhibited in STING -KO and STING GT diabetic mice. n = 6 mice for each group; the data are expressed as mean ± SD. Statistical differences were examined by ordinary 1-way ANOVA followed by Tukey’s multiple-comparison test. ** P < 0.01, *** P < 0.001, **** P < 0.0001 versus nondiabetic WT control. N, nondiabetic; D, diabetic.

Article Snippet: After being blocked with 5% milk (170-6404, Bio-Rad) for 1 hour, the membranes were incubated overnight with primary antibodies (diluted 1:1000) against iNOS (2982S, Cell Signaling Technology), ICAM-1 (10020-1-AP, Proteintech), p-IκBα (Ser32) (2859T, Cell Signaling Technology), IκBα (4812S, Cell Signaling Technology), STING (NBP2-24683, Novus Biologicals), cGAS (15102S, Cell Signaling Technology), p-TBK1 (5483S, Cell Signaling Technology), TBK1 (3013S, Cell Signaling Technology), p-IRF3 (29047S, Cell Signaling Technology), IRF3 (4302S, Cell Signaling Technology), p-NF-κB (3033S, Cell Signaling Technology), NF-κB (6956S, Cell Signaling Technology), GAPDH (5174S, Cell Signaling Technology), or vinculin (ab129002, Abcam).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: JCI Insight

Article Title: Activated cGAS/STING signaling elicits endothelial cell senescence in early diabetic retinopathy

doi: 10.1172/jci.insight.168945

Figure Lengend Snippet: STING-mediated induction of the retinal type I IFNs secretion through TBK1/IRF3 and inflammatory molecules via the TBK1/NF-κB pathway in diabetes results in increased endothelial cell senescence and production of superoxide. These factors combine to cause retinal microvascular injuries in DR. Hyperglycemia evokes various other pathological mechanisms such as retinal neurodegeneration, accumulation of AGEs, and induction of PKC, the polyol, and the hexosamine pathways also critical in DR, independently of STING. STING may serve as a hub that stimulates multiple pathways (also promoted by these other factors) that collectively promote this multifaceted disease. All processes are interconnected to the development and progression of DR. AGEs, advanced glycation end products; PKC, protein kinase C; ROS, reactive oxygen species.

Article Snippet: After being blocked with 5% milk (170-6404, Bio-Rad) for 1 hour, the membranes were incubated overnight with primary antibodies (diluted 1:1000) against iNOS (2982S, Cell Signaling Technology), ICAM-1 (10020-1-AP, Proteintech), p-IκBα (Ser32) (2859T, Cell Signaling Technology), IκBα (4812S, Cell Signaling Technology), STING (NBP2-24683, Novus Biologicals), cGAS (15102S, Cell Signaling Technology), p-TBK1 (5483S, Cell Signaling Technology), TBK1 (3013S, Cell Signaling Technology), p-IRF3 (29047S, Cell Signaling Technology), IRF3 (4302S, Cell Signaling Technology), p-NF-κB (3033S, Cell Signaling Technology), NF-κB (6956S, Cell Signaling Technology), GAPDH (5174S, Cell Signaling Technology), or vinculin (ab129002, Abcam).

Techniques: