anti sting1  (R&D Systems)

Journal: bioRxiv

Article Title: Histone variant H3.3 mediates cGAS-STING pathway activation via telomere deprotection

doi: 10.1101/2024.08.07.606966

Figure Lengend Snippet: Expression of TRF2ΔBΔM triggers cGAS-STING pathway activation and cGAS-mediated growth inhibition. BJ t cells were separated into untreated and treatment groups, and 25 ng/ml doxycycline (DOX) was mixed into the culture medium of the treatment group to induce expression of TRF2ΔBΔM. siRNAs were added twice, i.e., at 60 h and 12 h before inducing expression via 2.5 ng/ml doxycycline (DOX). ( A ) Protein levels of phosphorylated STING S366 (pSTING), STING, cGAS, myc, and actin were determined by Western blotting. ( B ) The expression level of IFNβ mRNA was determined by RT-qPCR. qPCR experiments represent the mean ±SD of three technical repeats. ( C ) Numbers of surviving cells under the indicated conditions were determined by counting the DAPI signals in the culture dishes. Number of cells was normalized to day-1 after treatment in each experiment. Data represent the mean ±SD of three biological repeats. ( D ) Two sets of BJ t cells—inducibly expressing GFP or TRF2ΔBΔM—were treated with or without 25 ng/ml DOX. Surviving cells were counted 4 days after later.

Article Snippet: Primary antibodies including phosphorylated STING (19781, Cell Signaling), STING (MAB7169, R&D Systems), cGAS (15102, Abcam), H3.3 (09-838, Merkmillpore; ab176840, Abcam), myc (SI-M4439, Sigma), LIG4 (12695-1-AP, Proteintech), XRCC4 (15817-1-AP, Proteintech), ATRX (A301-045A, Bethyl), DAXX (A301-353A, Bethyl), HIRA (04-1488, Sigma-Aldrich), and ASF1 (sc-53171, Santa Cruz Biotechnology) were used at 1:1000 dilution.

Techniques: Expressing, Activation Assay, Inhibition, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Histone variant H3.3 mediates cGAS-STING pathway activation via telomere deprotection

doi: 10.1101/2024.08.07.606966

Figure Lengend Snippet: Depletion of H3.3 impairs cGAS-STING pathway activation and rescues growth defects due to expression of TRF2ΔBΔM. ( A ) BJ t cells stably expressing GFP or myc-tagged TRF2ΔBΔM were established. siRNAs were added twice, i.e., at 60 h and 12 h before inducing expression with doxycycline (DOX). Samples were cultured for 2.5 days to investigate the cGAS-STING signaling response. Protein levels of phosphorylated STING (pSTING), STING, H3.3, myc, and GAPDH were determined by Western blotting. ( B ) mRNA expression levels of IFNβ were determined by RT-qPCR. qPCR experiments represent the mean ±SD of three technical repeats. ( C ) Numbers of cells in the DOX+ or DOX-treatment groups were counted to evaluate cell growth 4 days later. Data represent the mean ±SD of three biological repeats. ( D ) A siRNA-insensitive H3.3 construct was introduced into cells via viral vectors and ectopically expressed. Protein levels of phosphorylated STING (pSTING), STING, H3.3, myc, and GAPDH were then determined by Western blotting.

Article Snippet: Primary antibodies including phosphorylated STING (19781, Cell Signaling), STING (MAB7169, R&D Systems), cGAS (15102, Abcam), H3.3 (09-838, Merkmillpore; ab176840, Abcam), myc (SI-M4439, Sigma), LIG4 (12695-1-AP, Proteintech), XRCC4 (15817-1-AP, Proteintech), ATRX (A301-045A, Bethyl), DAXX (A301-353A, Bethyl), HIRA (04-1488, Sigma-Aldrich), and ASF1 (sc-53171, Santa Cruz Biotechnology) were used at 1:1000 dilution.

Techniques: Activation Assay, Expressing, Stable Transfection, Cell Culture, Western Blot, Quantitative RT-PCR, Construct

Journal: bioRxiv

Article Title: Histone variant H3.3 mediates cGAS-STING pathway activation via telomere deprotection

doi: 10.1101/2024.08.07.606966

Figure Lengend Snippet: Depletion of LIG4 and XRCC4 disrupts the activation of the cGAS-STING response. BJ t cells inducibly expressing myc-tagged TRF2ΔBΔM were treated with siRNAs at 60 h, and 12 h before doxycycline (DOX) treatment. To correlate telomere fusion events and nuclear abnormalities, treated cell were lysed on day 2 after DOX treatment. ( A ) Protein levels of phosphorylated STING (pSTING), STING, H3.3, cGAS, LIG4, XRCC4, myc, and GAPDH were determined by Western blotting. ( B ) mRNA expression levels of IFNβ were determined by RT-qPCR. qPCR experiments represent the mean ±SD of three technical repeats.

Article Snippet: Primary antibodies including phosphorylated STING (19781, Cell Signaling), STING (MAB7169, R&D Systems), cGAS (15102, Abcam), H3.3 (09-838, Merkmillpore; ab176840, Abcam), myc (SI-M4439, Sigma), LIG4 (12695-1-AP, Proteintech), XRCC4 (15817-1-AP, Proteintech), ATRX (A301-045A, Bethyl), DAXX (A301-353A, Bethyl), HIRA (04-1488, Sigma-Aldrich), and ASF1 (sc-53171, Santa Cruz Biotechnology) were used at 1:1000 dilution.

Techniques: Activation Assay, Expressing, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Histone variant H3.3 mediates cGAS-STING pathway activation via telomere deprotection

doi: 10.1101/2024.08.07.606966

Figure Lengend Snippet: Depletion of H3.3 chaperones impairs cGAS-STING pathway activation. BJ t cells were treated with siRNAs at 60 h and 12 h before inducing expression of myc-tagged TRF2ΔBΔM with doxycycline (DOX). Samples were cultured for 2.5 days to investigate the cGAS-STING signaling response. ( A , B ) Protein levels of phosphorylated STING (pSTING), STING, ASF1, HIRA, ATRX, DAXX, H3.3, myc, and GAPDH were determined by Western blotting. ( C ) mRNA expression levels of IFNβ were determined by RT-qPCR. qPCR experiments represent the mean ±SD of three technical repeats. Another set of BJ t cells were treated with siRNAs including siCtrl, siH3.3, siATRX, siDAXX, siHIRA, and siLIG4 at 60 h and 12 h before DOX treatment to induce expression of TRF2ΔBΔM for 24 h. Cells were arrested using 0.1 μg/ml Demecolcine for another 24 h, and then spread on a slide to visualize telomere-to-telomere fusion events by staining with a TelC probe. ( D ) Quantitative results for telomere-to-telomere fusion events calculated from >2000 chromosomes for each sample. Data represent the mean ±SD of two set of experiments.

Article Snippet: Primary antibodies including phosphorylated STING (19781, Cell Signaling), STING (MAB7169, R&D Systems), cGAS (15102, Abcam), H3.3 (09-838, Merkmillpore; ab176840, Abcam), myc (SI-M4439, Sigma), LIG4 (12695-1-AP, Proteintech), XRCC4 (15817-1-AP, Proteintech), ATRX (A301-045A, Bethyl), DAXX (A301-353A, Bethyl), HIRA (04-1488, Sigma-Aldrich), and ASF1 (sc-53171, Santa Cruz Biotechnology) were used at 1:1000 dilution.

Techniques: Activation Assay, Expressing, Cell Culture, Western Blot, Quantitative RT-PCR, Staining

Journal: Nature communications

Article Title: ARF1 prevents aberrant type I interferon induction by regulating STING activation and recycling.

doi: 10.1038/s41467-023-42150-4

Figure Lengend Snippet: Fig. 2 | Expression of ARF1 R99C induces a STING-dependent type I IFN response. a ISRE promoter activity by SEAP reporter in 293-Dual-hSTING-R232 cells transiently expressing FLAG-tagged ARF1 WT, R99C, Q71L or T31N on (32 h post transfection and normalised to cell viability). Dots represent mean of n = 3 ± SEM (biological replicates). Lower panel: Corresponding immunoblots of whole-cell lysates (WCLs) stained with anti-FLAG. b Area under the curve analysis of the data in (a). c ISRE promoter activity by Firefly luciferase (Fluc) quantification in HEK293T cells transiently expressing STING (+STING) or empty vector (-STING) and FLAG-tagged ARF1 WT and R99C (32 h post transfection and normalised to GAPDH- Renilla luciferase). Dots represent mean of n = 3 ± SEM (biological replicates). Lower panel: Corresponding immunoblots of WCLs stained by anti-FLAG. d, e ISRE (d) or NF-κB (e) promoter activity in A549 Dual cells transduced with ARF1 WT and R99C. IFN-β (1000 U/mL, 16 h) and cGAMP (10 µg/ml, 16 h) served as positive controls. Bars represent mean of n = 3 ± SEM (biological replicates). Lower panels: Corresponding immunoblots of WCLs stained by anti-FLAG, anti-STING and anti- GAPDH. f Exemplary immunoblot of HEK293T WCLs transiently expressing

Article Snippet: After stripping, membranes were reblotted with anti-STING antibodies (mouse anti-STING, 1:1000, R&D Systems, MAB7169; rabbit anti-IRF3, 1:1000, Cell Signalling, 4302) in 2.5% non-fat milk in TBS buffer supplemented with 0.1% Tween.

Techniques: Expressing, Activity Assay, Transfection, Western Blot, Staining, Luciferase, Plasmid Preparation, Transduction

Journal: Nature communications

Article Title: ARF1 prevents aberrant type I interferon induction by regulating STING activation and recycling.

doi: 10.1038/s41467-023-42150-4

Figure Lengend Snippet: Fig. 4 | GTPase activity of ARF1 R99C is reduced. a Model of ARF1 (PDB: 2J59) ATP- bound (orange/yellow). R99 and D26 are highlighted. b, c Interaction of ARF1 WT (b) or R99C (c) with GTP and protein stability analysed by fluorescence thermal shift assay. Data are representative of two biological replicates. d GTPase activity of indicated ARF1 mutants purified from HEK293T cells expressing FLAG-ARF1. Lower panel: Corresponding immunoblot stained with anti-FLAG. Bars represent mean of n = 4 ± SEM (biological replicates). e ISRE promoter activity in 293-Dual-hSTING- R232 cells quantified by SEAP transiently expressing ARF1 WT or R99C with indi- cated GEF or GAP normalized to cell viability. Corresponding immunoblots of WCLs stained with anti-HA, anti-turboGFP (tGFP), anti-FLAG, anti-STING and anti-GAPDH. Bars represent mean of n = 3 ± SEM (biological replicates). f Protein abundance in ARF1 WT versus R99C large scale purification from HEK293T cells as assessed by

Article Snippet: After stripping, membranes were reblotted with anti-STING antibodies (mouse anti-STING, 1:1000, R&D Systems, MAB7169; rabbit anti-IRF3, 1:1000, Cell Signalling, 4302) in 2.5% non-fat milk in TBS buffer supplemented with 0.1% Tween.

Techniques: Activity Assay, Thermal Shift Assay, Expressing, Western Blot, Staining, Quantitative Proteomics