Journal: Nature

Article Title: Clathrin-associated AP-1 controls termination of STING signalling

doi: 10.1038/s41586-022-05354-0

Figure Lengend Snippet: a , b , Confocal imaging ( a ) or quantification of pSTING area in bright-field fluorescent microscopy images ( b ) of HeLa STING cells stimulated for 0, 0.5, 1, 2, 3, 6, 9, 12 or 24 h with 1 µM diABZI. Cells were fixed and stained for TGN46 ( trans -Golgi), pSTING and LAMP1 (endolysosomal compartment) as well as with Hoechst dye (nuclei, in dark blue). Mean ± s.e.m. of n = 3 independent experiments with at least 84 fields of view each per condition. Scale bars, 4 µm. c , d , Airyscan imaging ( c ) of HeLa STING cells stimulated for 2.5 h with 1 µM diABZI. Cells were fixed and stained for TGN46 ( trans -Golgi, not shown here), pSTING and the indicated marker as well as with Hoechst dye (nuclei, in dark blue). Colocalization of pSTING with the indicated marker is quantified by Manders’ colocalization coefficients ( d ). One representative cell is shown, and quantification is the mean ± s.e.m. of n = 8 cells examined in 1 of 4 independent experiments. Scale bars, 4 µm in larger left panel, 1 µm in zoomed-in panels. e , Airyscan imaging of HeLa STING cells stimulated for 20, 150 (see Fig. ) or 360 min with 1 µM diABZI. Cells were fixed and stained for TGN46 ( trans -Golgi), pSTING and CHC (clathrins) as well as with Hoechst dye (nuclei, in dark blue). One representative cell is shown of at least n = 6 cells from 3 independent experiments. White arrows point at occurrences of pSTING. Scale bars, 4 µm in larger left panel, 1 µm in zoomed-in panels.

Article Snippet: The next day, the cells were stimulated by adding diABZI at 1 μM (MedChemExpress, HY-103665) for 2.5 h. They were then fixed and stained for pSTING as described for the AiryScan microscopy samples.

Techniques: Imaging, Microscopy, Staining, Marker

Journal: Nature

Article Title: Clathrin-associated AP-1 controls termination of STING signalling

doi: 10.1038/s41586-022-05354-0

Figure Lengend Snippet: a , Whole-cell lysate (WCL) and CCV fractions from HeLa cells expressing Flag-tagged STING (HeLa STING ) were analysed by western blot. Clathrin heavy chain (CHC) and GAPDH were used as loading and processing controls. b , Airyscan imaging of HeLa STING cells stimulated with diABZI. One representative cell of n = 7 cells. White arrows point at occurrences of pSTING. Scale bars, 4 µm (left) or 1 µm (magnified panels). c , Quantification of the colocalization of pSTING with CHC described in b , using Manders’ colocalization coefficients. Mean ± s.e.m. of n = 7 cells. d , STED images showing pSTING enclosed in CCVs from cells transfected with mCherry–clathrin and Flag–STING and stimulated with diABZI. Regions 1 and 2 are magnified from a large-field-of-view STED image (see Extended Data Fig. ). Scale bars, 200 nm. e , CLEM of HeLa GFP–STING cells stimulated with diABZI. The images depict box 3 of Extended Data Fig. in Airyscan microscopy (left) or electron microscopy at different Z -heights (three right panels). White arrows indicate CCVs. Scale bars, 0.5 µm. f , WCL and CCV fractions from HeLa cells that were treated with non-targeting control (NC) small interfering RNA (siRNA) or AP-1 siRNA and stimulated with diABZI were analysed by western blot. CHC was used as a loading control. g , Quantification of the area of pSTING in bright-field fluorescent microscopy images of HeLa STING cells that were treated with siRNAs and stimulated with diABZI. Mean ± s.e.m. of n = 3 independent experiments with 99 fields of view per condition. h , HeLa cells transfected with siRNAs and stimulated with diABZI were analysed by western blot. Ratios of STING versus loading control (GAPDH) normalized to the 0-h time point of each condition. One representative example of three ( a – c , h ) or two ( f ) independent experiments is shown.

Article Snippet: The next day, the cells were stimulated by adding diABZI at 1 μM (MedChemExpress, HY-103665) for 2.5 h. They were then fixed and stained for pSTING as described for the AiryScan microscopy samples.

Techniques: Expressing, Western Blot, Imaging, Transfection, Microscopy, Electron Microscopy, Control, Small Interfering RNA

Journal: Nature

Article Title: Clathrin-associated AP-1 controls termination of STING signalling

doi: 10.1038/s41586-022-05354-0

Figure Lengend Snippet: a , Large-field-of-view STED image showing the colocalization of pSTING and CCVs. HeLa cGAS/STING double KO cells were transfected with mCherry-clathrin and Flag-STING. One day later they were stimulated with 1 µM diABZI for 2.5 h and then fixed and stained for pSTING. Two representative events highlighted in the boxes were magnified (see Fig. ). Scale bar, 2 μm. The image depicts n = 1 cell out of 62 cells imaged over the course of 2 independent experiments, including 25 cells showing clear colocalization and 3 exhibiting clathrin ring structures. b , c , CLEM of HeLa STING KO cells stably reconstituted with GFP-hSTING stimulated for 2.5 h with 1 µM diABZI ( b ) or left untreated ( c ). For the stimulated cell ( b ), some regions with accumulation of high GFP–STING intensity (yellow boxes) were re-imaged by electron microscopy (EM) in higher resolution at relevant Z-heights. Zoom-in and higher-resolution electron microscopy slices from box 3 is shown in Fig. . LM – light microscopy (Airyscan). White arrows indicate CCVs. Scale bars in ( b ), 2 µm (un-zoomed top images) and 0.5 µm (zoom-in box images). Scale bars in ( c ), 1 µm. n = 1 out of 3 stimulated cells ( b ) and n = 1 out of 2 non-stimulated cells imaged from one sample per condition prepared for CLEM.

Article Snippet: The next day, the cells were stimulated by adding diABZI at 1 μM (MedChemExpress, HY-103665) for 2.5 h. They were then fixed and stained for pSTING as described for the AiryScan microscopy samples.

Techniques: Transfection, Staining, Stable Transfection, Electron Microscopy, Light Microscopy

Journal: Nature

Article Title: Clathrin-associated AP-1 controls termination of STING signalling

doi: 10.1038/s41586-022-05354-0

Figure Lengend Snippet: a , Western blot showing levels of AP-1 subunits after NC siRNA or AP-1 siRNA transfection in HeLa cells. GAPDH was used as a loading control. b , Bright-field fluorescent microscopy images and corresponding quantification of AP-1γ signal intensity of HeLa STING cells treated for 3 days with NC siRNA or AP-1 siRNA and then stimulated for 0, 0.5, 1, 2, 3, 6, 9, 12 or 24 h with 1 µM diABZI. Cells were then fixed and stained for TGN46 ( trans -Golgi), pSTING and AP-1γ as well as with Hoechst dye (nuclei, in dark blue). Images shown here are from the 2-h time points. Mean ± s.e.m. of n = 3 independent experiments with 99 fields of view each per condition. Scale bars, 4 µm. c , HeLa cGAS KO cells incubated with NC siRNA or siRNAs targeting AP-1σ1 and AP-1 σ3 for 3 days and reconstituted with empty vector or HA-tagged AP-1σ1 were treated with 2.5 µM diABZI for 0, 2, 4 h before analysis by western blot. Vinculin was used as a loading control. d , AP-1 μ1 KO MEFs and μ1 KO + μ1A MEFs were treated with 0.5 μg/mL DMXAA for 2 h and analysed by western blot. Vinculin was used as a loading control. One representative of three ( b , c ) or at least two ( a , c , d ) independent experiments is shown. Ratios of target proteins versus loading control normalized to the untreated sample of each condition ( c , d ).

Article Snippet: The next day, the cells were stimulated by adding diABZI at 1 μM (MedChemExpress, HY-103665) for 2.5 h. They were then fixed and stained for pSTING as described for the AiryScan microscopy samples.

Techniques: Western Blot, Transfection, Control, Microscopy, Staining, Incubation, Plasmid Preparation

Journal: Nature

Article Title: Clathrin-associated AP-1 controls termination of STING signalling

doi: 10.1038/s41586-022-05354-0

Figure Lengend Snippet: a , Airyscan imaging of HeLa STING cells that were stimulated for 2.5 h with diABZI. The colocalization of pSTING with AP-1γ is quantified by Manders’ colocalization coefficients. One representative cell is shown, and the quantification is the mean ± s.e.m. of n = 7 cells from one out of four independent experiments. White arrows point at occurrences of pSTING. Scale bars, 4 µm (left) or 1 µm (magnified panels). b , HeLa STING cells were stimulated with diABZI. After immunoprecipitation (IP) with anti-Flag antibody, cells were analysed by western blot. c , HeLa STING cells were stimulated with diABZI for 2 h, infected with HSV-1 (multiplicity of infection (MOI) = 10) for 6 h or transfected with 90mer dsDNA (1 µg) for 3 h or 1 µM 2’3’-cGAMP for 6 h. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. d , HeLa cells transfected with siRNAs were stimulated with diABZI and analysed by western blot. Vinculin was used as a loading control. e , Induction of IFNB1 , IFIT1 , IFIT2 and IFIT3 expression was assessed by quantitative PCR with reverse transcription (RT–qPCR) in HeLa cells transfected with siRNAs and treated with diABZI for 3 h. Ratios of IFNB1 , IFIT1 , IFIT2 and IFIT3 mRNA versus GAPDH mRNA normalized to the untreated groups of each condition. Data are mean ± s.d. of three technical replicates. P values were obtained by two-tailed Student’s t -test. f , WI-38 human fibroblasts transfected with siRNAs for three days were stimulated with diABZI and analysed by western blot. GAPDH was used as a loading control. One representative example of three ( a , d – f ) or two ( b , c ) independent experiments is shown. Ratios of target proteins versus loading control normalized to the 0-h time point of each condition ( d , f ).

Article Snippet: The next day, the cells were stimulated by adding diABZI at 1 μM (MedChemExpress, HY-103665) for 2.5 h. They were then fixed and stained for pSTING as described for the AiryScan microscopy samples.

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

Journal: Nature

Article Title: Clathrin-associated AP-1 controls termination of STING signalling

doi: 10.1038/s41586-022-05354-0

Figure Lengend Snippet: a , b , HaCaT cells ( a ) or THP-1 cells ( b ) transfected with NC siRNA or AP-1 siRNA for 3 days were treated with 2.5 µM diABZI for indicated time and analysed by western blot. GAPDH was used as a loading control. c , d , Human primary epithelial cells incubated with NC siRNA or AP-1 siRNA for 3 days were treated with 2.5 µM diABZI and were analysed by RT–qPCR ( c ) and western blot ( d ). Vinculin was used as a loading control. e – j , Fibroblast cells derived from patients with SAVI characterized by STING1 N154S mutation ( e , f ), H72N ( g , h ) or V147M ( i , j ) were incubated with NC siRNA or AP-1 siRNA for three days and then analysed by western blot. Vinculin was used as a loading control in f , h , j . e , g , i , Induction of IFNB1 , IFIT1 , IFIT2 , IFIT3 expression was assessed by RT–qPCR in HeLa cells transfected with NC siRNA or AP-1 siRNA for 3 days and then stimulated with 1 µM diABZI. Mean ± s.d. of three ( c , e , g , i ) technical replicates. P values based on two-tailed Student’s t- tests ( c , e , g , i ).

Article Snippet: The next day, the cells were stimulated by adding diABZI at 1 μM (MedChemExpress, HY-103665) for 2.5 h. They were then fixed and stained for pSTING as described for the AiryScan microscopy samples.

Techniques: Transfection, Western Blot, Control, Incubation, Quantitative RT-PCR, Derivative Assay, Mutagenesis, Expressing, Two Tailed Test

Journal: Nature

Article Title: Clathrin-associated AP-1 controls termination of STING signalling

doi: 10.1038/s41586-022-05354-0

Figure Lengend Snippet: a , Schematic diagram of the CTT of human STING (hSTING) and sequence logo of the CTT as indicated from 50 species. b , HeLa STING-knockout (KO) cells transfected with Flag-tagged STING ΔCΤΤ (Δ1–341), wild-type (WT) STING, STING E (E360A), STING LI (L364A/I365A) or STING ELI (E360A/L364A/I365A) were treated with diABZI for 2 h. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. c , WCL and extracted CCV fractions from HeLa STING KO cells reconstituted with Flag-tagged wild-type STING or STING LI (L364A/I365A) and treated or not with diABZI were analysed by western blot. CHC was used as a loading control. d , Glutathione sepharose pull-down assays of wild-type LBD-STING or LBD-STING ELI by glutathione S -transferase (GST)-tagged AP-1 core with or without ARF1. e , HeLa STING cells transfected with NC siRNA or siRNAs against TBK1 or IRF3 were treated with or without diABZI. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. GAPDH was used as a loading control. f , HeLa wild-type cells, HeLa TBK1 KO cells and HeLa IRF3 KO cells stimulated with diABZI for 0, 1, 2 or 4 h were analysed by western blot. Ratios of target proteins versus loading control normalized to the 0-h time point of each condition. Vinculin was used as a loading control. g , Bio-layer interferometry binding studies of LBD-STING (top) or TBK1-phosphorylated LBD-STING (pSTING) (bottom) with AP-1 ΔμCTD. The right graphs show the binding affinity of STING (top) and pSTING (bottom). One representative example of at least three ( b , d , f ) or two ( c , e , g ) independent experiments is shown.

Article Snippet: The next day, the cells were stimulated by adding diABZI at 1 μM (MedChemExpress, HY-103665) for 2.5 h. They were then fixed and stained for pSTING as described for the AiryScan microscopy samples.

Techniques: Sequencing, Knock-Out, Transfection, Immunoprecipitation, Western Blot, Control, Binding Assay

Journal: Nature

Article Title: Clathrin-associated AP-1 controls termination of STING signalling

doi: 10.1038/s41586-022-05354-0

Figure Lengend Snippet: a , HeLa cGAS/STING double KO cells transfected with FLAG-tagged STING WT , STING 1–317 or STING LI(L364A/I365A) were treated or not with 2.5 µM diABZI for 14 h and analysed by western blot. Vinculin was used as a loading control. b , HeLa cGAS/STING double KO cells transfected with FLAG-tagged STING WT or STING L364A were treated or not with 2.5 µM diABZI for 14 h and analysed by western blot. Vinculin was used as a loading control. c , HeLa cGAS/STING double KO cells transfected with FLAG-tagged STING L363A , STING L364A or STING I365A were treated or not with 2.5 µM diABZI for 14 h and analysed by western blot. GAPDH was used as a loading control. d , HEK293T cells transfected with FLAG-tagged STING WT , STING L374A or STING L374F were treated with 2.5 µM diABZI for 2 h, immunoprecipitated with anti-FLAG antibody, and analysed by western blot. e , HeLa cGAS/STING double KO cells transfected with FLAG-tagged STING WT or STING L364F were treated or not with 2.5 µM diABZI for 14 h and analysed by western blot. GAPDH was used as a loading control. f , IFN-β luciferase assay in HEK293T cells transfected with plasmids expressing indicated STING constructions and stimulated with diABZI (2.5 µM). g , Glutathione Sepharose pull-down assays of LBD-STING WT or LBD-STING ELI(E360A/L364A/I365A) by GST-tagged AP-1 ΔμCTD core. One representative of three ( a – c , e – f ) or two ( d , g ) independent experiments is shown. Ratios of target proteins versus loading control normalized to the untreated sample of each condition ( a – c , e ). Mean ± s.d. of three ( f ) technical replicates. P values based on two-tailed Student’s t- tests ( f ).

Article Snippet: The next day, the cells were stimulated by adding diABZI at 1 μM (MedChemExpress, HY-103665) for 2.5 h. They were then fixed and stained for pSTING as described for the AiryScan microscopy samples.

Techniques: Transfection, Western Blot, Control, Immunoprecipitation, Luciferase, Expressing, Two Tailed Test

Journal: Nature

Article Title: Clathrin-associated AP-1 controls termination of STING signalling

doi: 10.1038/s41586-022-05354-0

Figure Lengend Snippet: a , HeLa STING KO cells transfected with FLAG-tagged STING WT or STING LR(L374A/I375A) were treated with 2.5 µM diABZI for 0, 1 or 2 h, immunoprecipitated with anti-FLAG antibody, and analysed by western blot. b , HeLa TBK1 KO cells reconstituted with an empty plasmid or with plasmids expressing TBK1 WT or enzyme-dead TBK1 S172A were treated with 2.5 µM diABZI for 2 h and analysed by western blot. GAPDH was used as a processing control. c , HeLa cells pretreated with DMSO or 2 µM BX795 for 24 h were stimulated with 2.5 µM diABZI or not (2 h) and analysed by western blot. Vinculin was used as a loading control. One representative of at least two ( a – c ) independent experiments is shown. Ratios of target proteins versus loading control normalized to the untreated sample of each condition ( b , c ). d , Mass spectrometry detected molecular weight of SUMO LBD-STING and TBK1-phosphorylated LBD-STING (pSTING). e , Bio-layer interferometry binding studies of LBD-STING ELI(E360A/L364A/I365A) with AP-1 ΔμCTD. f , Bio-layer interferometry binding studies of LBD-STING 3S(S355D/S358D/S366D) with AP-1 ΔμCTD. One representative of at least two ( e , f ) independent experiments is shown.

Article Snippet: The next day, the cells were stimulated by adding diABZI at 1 μM (MedChemExpress, HY-103665) for 2.5 h. They were then fixed and stained for pSTING as described for the AiryScan microscopy samples.

Techniques: Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Expressing, Control, Mass Spectrometry, Molecular Weight, Binding Assay

Journal: Nature

Article Title: Clathrin-associated AP-1 controls termination of STING signalling

doi: 10.1038/s41586-022-05354-0

Figure Lengend Snippet: a , Three-dimensional (3D) reconstructions of the complex in two different orientations. The atomic models of dimeric human LBD-STING (Protein Data Bank (PDB) code: 4KSY) and the AP-1 complex (PDB 6DFF) were fitted into the maps (grey) through rigid-body docking. b , High-resolution 3D reconstruction from focused refinement on the AP-1 core and pSTING tail at 2.34-Å resolution contoured at 3 σ . c , Ribbon representation of the AP-1 pSTING complex structure. d – f , Detailed views of the binding interface. e , Potentialhydrophobic interactions around the EXXXLI motif are indicated with dotted lines. The density map (grey mesh) of pSTING tail is contoured at 3 σ . f , Potential hydrogen bonds around pS366 indicated with dotted lines. The density map (grey mesh) of the pSTING tail is contoured at 3 σ . g , HEK293T cells transfected with Flag-tagged STING and HA-tagged wild-type AP-1σ or AP-1σ mutants σ KR (K60A/R61A), σ(I103S) and σ(V88D) were stimulated with diABZI for 2 h. Cell lysates were extracted, immunoprecipitated with anti-Flag antibody and analysed by western blot. h , WCL and CCV fractions from untreated and diABZI-treated HeLa STING KO cells reconstituted with Flag-tagged wild-type STING and the indicated STING mutants STING LI , STING LR (L374A/R375A) or STING(S358A) were analysed by western blot. CHC and GAPDH were used as loading controls. i , HeLa STING KO cells reconstituted with Flag-tagged STING and the indicated STING mutants STING(S358A), STING(S366A) or STING(S358/366A) were stimulated with diABZI for 2 h or left untreated. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. j , Schematic diagram of the function of AP-1 in the termination of STING signalling. One representative example of at least three ( g – i ) independent experiments is shown.

Article Snippet: The next day, the cells were stimulated by adding diABZI at 1 μM (MedChemExpress, HY-103665) for 2.5 h. They were then fixed and stained for pSTING as described for the AiryScan microscopy samples.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot