Journal: Nature

Article Title: Tonic prime-boost of STING signalling mediates Niemann-Pick disease type C.

doi: 10.1038/s41586-021-03762-2

Figure Lengend Snippet: Fig. 1 | NPC1 deficiency chronically activates STING-mediated type I IFN signalling independently of cGAS. a, c, Quantitative PCR with reverse transcription (qRT–PCR) analysis of the mRNA expression of resting-state ISGs (Oas3, Oas1a and Cxcl10) in Npc1WT and two independent Npc1KO MEF cell lines (a) or in Npc1WT, Npc1KO and Npc1KOSting1KO MEFs (c) (n = 3). b, d, qRT–PCR analysis of Ifnb1 mRNA expression in Npc1WT and two independent Npc1KO MEF cell lines (b) or in Npc1WT, Npc1KO and Npc1KOSting1KO MEFs (d) after stimulation with the STING agonist DMXAA (50 μg ml−1) for 2 h (n = 3). e, qRT–PCR analysis of the baseline mRNA expression of ISGs (Oas3, Oas1a, Ifi30) in Npc1WT, Npc1KO and Npc1KOCgasKO MEFs (n = 3). f, Immunoblot analysis of the indicated proteins in Npc1WT, Npc1KO and Npc1KOCgasKO MEFs. a–e, Data are mean ± s.d. a, c, e, Unpaired two-tailed Student’s t-test. b, d, Two-way ANOVA. NS, not significant. Data are representative of at least three independent experiments.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Human research participants Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Streptavidin-HRP (1:1000, Life Technologies, S-911), Anti-NPC1 (1:1000, Abcam, rabbit ab134113), Anti-NPC1 (1:100, for endogenous co-IP, ThermoFisher scientific, rabbit PA1-16817), Anti-NPC1 (1:1000, LSbio, mouse B3437), Anti-cGAS (Mouse Specific) (1:1000, D3O8O, CST31659), Anti-STING (1:1000, D2P2F, CST13647), STING Rabbit mAb (Rodent Preferred) (1:1000, D1V5L, CST50494), AntiSTING (1:200, for endogenous STING staining, Proteintech 19851-1-AP), Anti-pSTING (Ser365) (1:1000 for WB, 1:100 for IF, D8F4W, CST 72971s), Anti-TBK1 (1:1000, D1B4, CST 3504), Anti-pTBK1 (1:1000 for WB, 1:100 for IF, D52C2, CST 5483), Anti-IRF3 (1:1000, D83B9, CST 4302), Anti-pIRF3 (Ser396) (1:1000 for WB, 1:100 for IF, 4D4G, CST 4947), Anti-α-Tubulin (1:5000, Sigma T5168), AntiGAPDH (1:5000, ThermoFisher scientific, PA1-987), Anti-NPC2 (1:1000, Abcam 186829), Goat anti–rabbit IgG-HRP conjugate (1:5000, BioRad Laboratories 1706515), Goat anti–mouse IgG-HRP conjugate (1:5000, BioRad Laboratories 1706516), Monoclonal antiCalbindin-D-28K antibody produced in mouse (1:200, Sigma-Aldrich C9848), Anti-CD68 (1:200, Abcam, ab125212), ERGIC-53 Antibody (B-9) (1:100, Santa cruz, sc-271517), Anti-SREBP2 (1:1000 for WB, 1:100 for IF, Abcam, ab30682), Anti-SREBP2 (1:1000, Novus, NB100-74543), Anti-LAMP1 (1:1000, D2D11, CST 9091), Anti-GFAP (1:200, Abcam, ab53554), Donkey anti-mouse IgG(H+L) Alexa fluo-568 (1:500, ThermoFisher, A10037), Donkey anti-mouse IgG(H+L) Alexa fluo-647 (1:500, ThermoFisher, A31571), Donkey anti-rabbit IgG(H+L) Alexa fluo-488 (1:500, ThermoFisher, A21206), Donkey anti-goat IgG(H+L) Alexa fluo-568 (1:500, ThermoFisher, A11057) Validation All antibodies are from commercially available sources and have been validated from the manufacturer with supporting publications found on the manufacturer's websites.

Techniques: Real-time Polymerase Chain Reaction, Reverse Transcription, Quantitative RT-PCR, Expressing, Western Blot, Two Tailed Test

Journal: Nature

Article Title: Tonic prime-boost of STING signalling mediates Niemann-Pick disease type C.

doi: 10.1038/s41586-021-03762-2

Figure Lengend Snippet: Fig. 2 | SREBP2 trafficking primes STING trafficking and activation in Npc1-deficient cells. a, b, qRT–PCR analysis of SREBP2 and STING activation. SREBP2 activation induces cholesterol-synthesis genes (a; Srebf2 and Hmgcs1) and STING activation induces ISGs (b; Cxcl10, Usp18 and Ccl8). Npc1WT and Npc1KO MEFs were mock-treated or treated with HP-β-CD (0.3 mM) overnight (n = 3). c, Immunoblot analysis of SREBP2 and STING activation. SREBP2 cleavage indicates activation. STING activation induces pSTING and pTBK1. d, Fluorescent microscopy analysis of SREBP2 and STING activation. Cleaved SREBP2 translocates to the nucleus (red). The STING activation markers pSTING, pTBK1 and pIRF3 are shown in green. The nucleus dye DAPI is shown in blue. Scale bars, 10 μm. e, STING activation in Npc1KO cells requires SREBP2. qRT–PCR analysis of ISG expression in Npc1WT and Npc1KO MEFs transfected with control siRNA (siCtrl) or siSrebf2 for 48 h (n = 3). f, g, qRT–PCR analysis of cholesterol-synthesis genes (f) and ISGs (g) in wild-type, Sting1−/− and Cgas−/− BMDMs that were mock-treated or treated with triparanol (Trip, 14 μM) for 12 h (n = 3). a, b, e–g, Data are mean ± s.d. Unpaired two-tailed Student’s t-test. Data are representative of at least two independent experiments.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Human research participants Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Streptavidin-HRP (1:1000, Life Technologies, S-911), Anti-NPC1 (1:1000, Abcam, rabbit ab134113), Anti-NPC1 (1:100, for endogenous co-IP, ThermoFisher scientific, rabbit PA1-16817), Anti-NPC1 (1:1000, LSbio, mouse B3437), Anti-cGAS (Mouse Specific) (1:1000, D3O8O, CST31659), Anti-STING (1:1000, D2P2F, CST13647), STING Rabbit mAb (Rodent Preferred) (1:1000, D1V5L, CST50494), AntiSTING (1:200, for endogenous STING staining, Proteintech 19851-1-AP), Anti-pSTING (Ser365) (1:1000 for WB, 1:100 for IF, D8F4W, CST 72971s), Anti-TBK1 (1:1000, D1B4, CST 3504), Anti-pTBK1 (1:1000 for WB, 1:100 for IF, D52C2, CST 5483), Anti-IRF3 (1:1000, D83B9, CST 4302), Anti-pIRF3 (Ser396) (1:1000 for WB, 1:100 for IF, 4D4G, CST 4947), Anti-α-Tubulin (1:5000, Sigma T5168), AntiGAPDH (1:5000, ThermoFisher scientific, PA1-987), Anti-NPC2 (1:1000, Abcam 186829), Goat anti–rabbit IgG-HRP conjugate (1:5000, BioRad Laboratories 1706515), Goat anti–mouse IgG-HRP conjugate (1:5000, BioRad Laboratories 1706516), Monoclonal antiCalbindin-D-28K antibody produced in mouse (1:200, Sigma-Aldrich C9848), Anti-CD68 (1:200, Abcam, ab125212), ERGIC-53 Antibody (B-9) (1:100, Santa cruz, sc-271517), Anti-SREBP2 (1:1000 for WB, 1:100 for IF, Abcam, ab30682), Anti-SREBP2 (1:1000, Novus, NB100-74543), Anti-LAMP1 (1:1000, D2D11, CST 9091), Anti-GFAP (1:200, Abcam, ab53554), Donkey anti-mouse IgG(H+L) Alexa fluo-568 (1:500, ThermoFisher, A10037), Donkey anti-mouse IgG(H+L) Alexa fluo-647 (1:500, ThermoFisher, A31571), Donkey anti-rabbit IgG(H+L) Alexa fluo-488 (1:500, ThermoFisher, A21206), Donkey anti-goat IgG(H+L) Alexa fluo-568 (1:500, ThermoFisher, A11057) Validation All antibodies are from commercially available sources and have been validated from the manufacturer with supporting publications found on the manufacturer's websites.

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot, Microscopy, Expressing, Transfection, Control, Two Tailed Test

Journal: Nature

Article Title: Tonic prime-boost of STING signalling mediates Niemann-Pick disease type C.

doi: 10.1038/s41586-021-03762-2

Figure Lengend Snippet: Fig. 5 | IRF3, but not cGAS, is required for disease pathogenesis in Npc1−/− mice. a, Body weight of mice of the indicated genotypes. All mice are on the C57BL/6J background (Npc1+/+ mice, n = 5; Npc1−/− mice, n = 10; Npc1−/−Irf3−/− mice, n = 10; Npc1−/−Cgas−/− mice, n = 10). b, Heat map showing ISG expression in the cerebellum of mice of the indicated genotypes (n = 3; 8 weeks old). The mRNA expression of each ISG was measured by qRT–PCR. c, Cerebellar ataxia scores of mice of the indicated genotypes (8 weeks old). All mice are on the C57BL/6J background. Mice were scored as in Fig. 4b (Npc1+/+ mice, n = 9; Npc1−/− mice, n = 9; Npc1−/−Irf3−/− mice, n = 13; Npc1−/−Cgas−/− mice, n = 12). d, Fluorescent IHC staining of the cerebellum of mice (C57BL/6J) of the indicated genotypes. Calbindin is shown in red, CD68 is shown in green and DAPI is shown in blue. Representative images are shown (n = 3). The bottom images are enlarged views. Scale bars, 200 μm (top); 30 μm (bottom). e, Fluorescent IHC co-staining of neural markers with STING in the cerebellum of wild-type mice (C57BL/6J; 4 weeks old). Top, STING is shown in green and calbindin (Purkinje cell marker) is shown in red. Bottom, STING is shown in green and GFAP (astrocyte marker) is shown in red. DAPI is shown in blue. Representative images are shown (n = 3). Scale bars, 200 μm (main panels); 30 μm (enlarged views; far right). f, Graphical abstract of the study. Chol., cholesterol. a, c, Data are mean ± s.d. a, Two-way ANOVA. c, Unpaired two-tailed Student’s t-test.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Human research participants Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Streptavidin-HRP (1:1000, Life Technologies, S-911), Anti-NPC1 (1:1000, Abcam, rabbit ab134113), Anti-NPC1 (1:100, for endogenous co-IP, ThermoFisher scientific, rabbit PA1-16817), Anti-NPC1 (1:1000, LSbio, mouse B3437), Anti-cGAS (Mouse Specific) (1:1000, D3O8O, CST31659), Anti-STING (1:1000, D2P2F, CST13647), STING Rabbit mAb (Rodent Preferred) (1:1000, D1V5L, CST50494), AntiSTING (1:200, for endogenous STING staining, Proteintech 19851-1-AP), Anti-pSTING (Ser365) (1:1000 for WB, 1:100 for IF, D8F4W, CST 72971s), Anti-TBK1 (1:1000, D1B4, CST 3504), Anti-pTBK1 (1:1000 for WB, 1:100 for IF, D52C2, CST 5483), Anti-IRF3 (1:1000, D83B9, CST 4302), Anti-pIRF3 (Ser396) (1:1000 for WB, 1:100 for IF, 4D4G, CST 4947), Anti-α-Tubulin (1:5000, Sigma T5168), AntiGAPDH (1:5000, ThermoFisher scientific, PA1-987), Anti-NPC2 (1:1000, Abcam 186829), Goat anti–rabbit IgG-HRP conjugate (1:5000, BioRad Laboratories 1706515), Goat anti–mouse IgG-HRP conjugate (1:5000, BioRad Laboratories 1706516), Monoclonal antiCalbindin-D-28K antibody produced in mouse (1:200, Sigma-Aldrich C9848), Anti-CD68 (1:200, Abcam, ab125212), ERGIC-53 Antibody (B-9) (1:100, Santa cruz, sc-271517), Anti-SREBP2 (1:1000 for WB, 1:100 for IF, Abcam, ab30682), Anti-SREBP2 (1:1000, Novus, NB100-74543), Anti-LAMP1 (1:1000, D2D11, CST 9091), Anti-GFAP (1:200, Abcam, ab53554), Donkey anti-mouse IgG(H+L) Alexa fluo-568 (1:500, ThermoFisher, A10037), Donkey anti-mouse IgG(H+L) Alexa fluo-647 (1:500, ThermoFisher, A31571), Donkey anti-rabbit IgG(H+L) Alexa fluo-488 (1:500, ThermoFisher, A21206), Donkey anti-goat IgG(H+L) Alexa fluo-568 (1:500, ThermoFisher, A11057) Validation All antibodies are from commercially available sources and have been validated from the manufacturer with supporting publications found on the manufacturer's websites.

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining, Marker, Two Tailed Test

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: