Journal: iScience

Article Title: Single-cell RNA-seq analysis reveals dual sensing of HIV-1 in blood Axl + dendritic cells

doi: 10.1016/j.isci.2023.106019

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Article Snippet: H-151 STING antagonist , Probechem , Cat# HY-112693; CAS: 941987-60-6.

Techniques: Virus, Infection, Recombinant, Transfection, Electron Microscopy, Staining, Cell Isolation, Purification, Reverse Transcription, SYBR Green Assay, Sequencing, Software, FCAP Assay

Journal: Molecular Medicine

Article Title: SHP-1 knockdown suppresses mitochondrial biogenesis and aggravates mitochondria-dependent apoptosis induced by all trans retinal through the STING/AMPK pathways

doi: 10.1186/s10020-022-00554-w

Figure Lengend Snippet: SHP-1 interacts with STING and suppresses its activation through posttranslational modifications. A – C ARPE-19 cells were exposed to atRAL (7.5 μM, 0, 3, 6, 12 h). Immunofluorescent images of cells costained for SHP-1 and calnexin ( A ) or for SHP-1 and STING ( B ) were shown. Scale bar = 50 μm. C Immunoprecipitation and western blotting of cell lysates. D , E Scramble-rLV- and shRNA-SHP-1-rLV-transfected ARPE-19 cells were treated with atRAL (7.5 μM, 6 h), and subject to immunofluorescent staining for STING ( D ) or immunoprecipitation analysis ( E ). Scale bar = 50 μm. F ARPE-19 cells were incubated with atRAL (7.5 μM, 0–12 h), and the ubiquitination of STING was detected by immunoprecipitation. G the K63- ubiquitination of STING in the scramble-rLV- and shRNA-SHP-1-rLV-transfected ARPE-19 cells exposed to atRAL (7.5 μM, 6 h)

Article Snippet: ARPE-19 cells transfected with scramble-rLV were used as control. atRAL(Sigma, St.Louis, MO, USA), a STING covalent antagonist (H151; Selleck Chemicals, Houston, TX, USA) and an AMPK inhibitor (Compound C; Selleck Chemicals), were dissolved and stored in the dark at -80 °C until use.

Techniques: Activation Assay, Immunoprecipitation, Western Blot, shRNA, Transfection, Staining, Incubation, Ubiquitin Proteomics

Journal: Molecular Medicine

Article Title: SHP-1 knockdown suppresses mitochondrial biogenesis and aggravates mitochondria-dependent apoptosis induced by all trans retinal through the STING/AMPK pathways

doi: 10.1186/s10020-022-00554-w

Figure Lengend Snippet: Effects of SHP-1 knockdown on cell apoptosis are attenuated by the inhibition of STING. Scramble-rLV- and shRNA-SHP-1-rLV-transfected ARPE-19 cells were treated with atRAL (7.5 μM), with or without pretreatment with the STING antagonist, H151 (1 μM), for the indicated time. A Viability of ARPE-19 cells (atRAL, 24 h) was examined using a CCK-8 assay. B Light microscopic images of the morphological features of ARPE-19 cells (atRAL, 24 h). Scale bar = 100 μm. C , D Representative images of ARPE-19 cells (atRAL, 12 h) stained with Annexin V-FITC/ PI ( C ), and quantitative analysis of the apoptosis rates ( D ). E , F ARPE-19 cells (atRAL, 12 h) were stained with JC-1 dyes ( E ), and the fluorescence intensity ratios (red/green) were calculated ( F ). Scale bar = 100 μm. G , H Western blotting analysis of Bcl-xL expression levels in ARPE-19 cells (atRAL, 24 h). * P < 0.05 and ** P < 0.01

Article Snippet: ARPE-19 cells transfected with scramble-rLV were used as control. atRAL(Sigma, St.Louis, MO, USA), a STING covalent antagonist (H151; Selleck Chemicals, Houston, TX, USA) and an AMPK inhibitor (Compound C; Selleck Chemicals), were dissolved and stored in the dark at -80 °C until use.

Techniques: Knockdown, Inhibition, shRNA, Transfection, CCK-8 Assay, Staining, Fluorescence, Western Blot, Expressing

Journal: Molecular Medicine

Article Title: SHP-1 knockdown suppresses mitochondrial biogenesis and aggravates mitochondria-dependent apoptosis induced by all trans retinal through the STING/AMPK pathways

doi: 10.1186/s10020-022-00554-w

Figure Lengend Snippet: Inhibition of STING reverses mitochondrial biogenesis suppressed by SHP-1 knockdown. Scramble-rLV- and shRNA-SHP-1-rLV- transfected ARPE-19 cells were incubated with atRAL (7.5 μM, 24 h), with or without H151 (1 μM). A The relative ATP levels in different groups were measured. mtDNA content, represented by mt -ND1 ( B ), and mt-CO3 ( C ) was assessed by qPCR. D – H Western blotting analysis of TOMM20 ( D , E ), PGC-1α ( F , G ), and nrf2 ( F , H ). * P < 0.05 and ** P < 0.01

Article Snippet: ARPE-19 cells transfected with scramble-rLV were used as control. atRAL(Sigma, St.Louis, MO, USA), a STING covalent antagonist (H151; Selleck Chemicals, Houston, TX, USA) and an AMPK inhibitor (Compound C; Selleck Chemicals), were dissolved and stored in the dark at -80 °C until use.

Techniques: Inhibition, Knockdown, shRNA, Transfection, Incubation, Western Blot

Journal: Molecular Medicine

Article Title: SHP-1 knockdown suppresses mitochondrial biogenesis and aggravates mitochondria-dependent apoptosis induced by all trans retinal through the STING/AMPK pathways

doi: 10.1186/s10020-022-00554-w

Figure Lengend Snippet: Inhibition of AMPK partially abrogates the effects of STING inhibition. A – C Western blotting of phosphorylated TBK1/TBK1 ( B ) and phosphorylated AMPKα/AMPKα ( C ) in ARPE-19 cells exposed to atRAL (7.5 μM, 24 h), with or without H151(1 μM). D Viability of scramble-rLV- and shRNA-SHP-1-rLV-transfected ARPE-19 cells exposed to atRAL (7.5 μM, 24 h), after pretreatment with Compound C (5 μM) and H151 (1 μM) as indicated. E-I Western blotting analysis of PGC-1α ( F ), nrf2 ( G ), Bcl-xL ( H ) and TOMM20 ( I ). * P < 0.05 and ** P < 0.01

Article Snippet: ARPE-19 cells transfected with scramble-rLV were used as control. atRAL(Sigma, St.Louis, MO, USA), a STING covalent antagonist (H151; Selleck Chemicals, Houston, TX, USA) and an AMPK inhibitor (Compound C; Selleck Chemicals), were dissolved and stored in the dark at -80 °C until use.

Techniques: Inhibition, Western Blot, shRNA, Transfection

Journal: Molecular Medicine

Article Title: SHP-1 knockdown suppresses mitochondrial biogenesis and aggravates mitochondria-dependent apoptosis induced by all trans retinal through the STING/AMPK pathways

doi: 10.1186/s10020-022-00554-w

Figure Lengend Snippet: Schematic diagram of the proposed model for atRAL-elicited responses within RPE cells regulated by SHP-1. atRAL causes mitochondrial damage, contributes to the production of ROS and leads to energy insufficiency in RPE cells. The energy stress-induced AMPK activation elicits adaptive protective responses to counterbalance mitochondrial dysfunction. However, the damaged mitochondria trigger K63-linked ubiquitination, aggregation and activation of STING in the ER, which phosphorylates TBK1, and subsequently suppresses AMPK activity. SHP-1 interacts with STING and restrains its activity. SHP-1 loss results in the overactivation of STING, dampening of mitochondrial biogenesis and antioxidant defense, and finally aggravation of oxidative stress-induced death in RPE cells

Article Snippet: ARPE-19 cells transfected with scramble-rLV were used as control. atRAL(Sigma, St.Louis, MO, USA), a STING covalent antagonist (H151; Selleck Chemicals, Houston, TX, USA) and an AMPK inhibitor (Compound C; Selleck Chemicals), were dissolved and stored in the dark at -80 °C until use.

Techniques: Activation Assay, Ubiquitin Proteomics, Activity Assay