Journal: EBioMedicine

Article Title: A STING antagonist modulating the interaction with STIM1 blocks ER-to-Golgi trafficking and inhibits lupus pathology

doi: 10.1016/j.ebiom.2021.103314

Figure Lengend Snippet: ISD017 associates with STING and STIM1 and prevents ER-to-Golgi Trafficking . (a) PMA-differentiated THP1 cells were treated with ISD017 (200 µg/ml). One h later, the cells were transfected with dsDNA (2 µg/ml). Cells were lysed at the indicated time points post-stimulation and immunoblotted by antibodies against pTBKs (S172), pSTING (S366), pIRF3 (S396), and Vinculin. ( n = 3). (b) PMA-differentiated THP1 cells were pretreated with ISD017 (200 µg/ml) for 1 h. The cells were transfected with dsDNA (6 µg/ml) and fixed 2 h later for imaging analysis after staining with anti-STING, anti-Calreticulin (ER), and anti-GM130 (Golgi) ( n = 3). Scale bar, 10 µm. (c) Lysates from PMA-differentiated THP1 cells were incubated with biotinylated ISD017, washed, and precipitated with streptavidin beads. Precipitates were immunoblotted with antibodies binding the indicated proteins ( n = 3). The light blue color of the bands for STEEP and vinculin in the input lane illustrates saturation of the signal. (d) Lysates from PMA-differentiated Wt, STING KO, and STIM1 KO THP1 cells were incubated with biotinylated ISD017, washed, and precipitated with streptavidin beads. Precipitates were immunoblotted with antibodies against STIM1, STING, and vinculin ( n = 3). (e) PMA-differentiated THP1 cells were treated with ISD017 (200 µg/ml) for 1 h, followed by stimulation with dsDNA for 2 hs. Cell lysates were subjected to immunoprecipitation with anti-STING and immunoblotting with anti-STIM1, anti-STING, and anti-vinculin ( n = 3). (f,g) Wt and STIM1 KO THP1 macrophages were treated with dsDNA in the presence or absence of ISD017 (200 µg/ml, pre-treatment). Supernatants were harvested 16 h post-stimulation, and IFN-I activity and TNFα levels were measured ( n = 3). The data in panel f and g are presented as means of biological triplicates +/- st.dev. [Statistical analysis of the data in f, g were performed using two-tailed one-way ANOVA test].

Article Snippet: The following antibodies and dilutions were used for immunoblotting anti‐STING (Cell Signaling, D2P2F/#13,647, 1:1000), sheep anti‐STING (R&D Systems, AF6516, 1:500), rabbit anti‐phosphoSTING (S366) (Cell Signaling Technology, #85,735, 1:1000), rabbit anti‐TBK1 (Cell Signaling, 3504, 1:1000), rabbit anti‐phospho-TBK1 (Ser172) (Cell Signaling, D52C2/#5483, 1:1000), IRF3 (Cell signaling, 11,904, 1:1000), phospho-IRF3 (S396) (Cell Signalling, 4947,1:1000, mouse anti‐p62 (Cell Signaling, # 88,588, 1:1000), anti-STEEP (Proteintech, 24,021–1-AP, 1:1000), anti-STIM1 (D88E10, Cell Signaling, #5668, 1:1000), rabbit anti‐LC3 (Cell Signaling, #3868, 1:1000), rabbit anti-cleaved caspase3 (Cell Signaling, #9664,1:1000), and mouse anti‐vinculin (Sigma, #V9131, 1:10,000).

Techniques: Transfection, Imaging, Staining, Incubation, Binding Assay, Immunoprecipitation, Western Blot, Activity Assay, Two Tailed Test

Journal: Journal of Virology

Article Title: Genotype 2 Strains of Porcine Reproductive and Respiratory Syndrome Virus Dysregulate Alveolar Macrophage Cytokine Production via the Unfolded Protein Response

doi: 10.1128/JVI.01251-17

Figure Lengend Snippet: Kinetics of IFN-α and TNF-α responses of porcine AMϕ. (A and C) ZMAC cells were stimulated with poly(I·C) (25 μg/ml) (A) or LPS (100 ng/ml) (C), and the amount of IFN-α (A) or TNF-α (C) present in cell-free culture supernatant at the indicated time after stimulation was determined by ELISA. The results represent the means ± standard deviations of three independent experiments for each agonist. (B) ZMAC cells were either mock treated or exposed to poly(I·C) (25 μg/ml) for 1 h, and their whole-cell lysates were analyzed by Western blotting to detect p-IRF3, total IRF3, and β-actin. (D) ZMAC cells were either mock treated or exposed to LPS (100 ng/ml), and their whole-cell lysates were harvested at the time points indicated and analyzed by Western blotting to detect p-NF-κB, total NF-κB, and β-actin. The results shown are representative of two independent experiments.

Article Snippet: The membranes were incubated in blocking buffer (2% fish gelatin in TBST solution [50 mM Tris, pH 7.5, 500 mM NaCl, and 0.5% Tween 20]) at RT for 1 h. Afterward, the membranes were incubated at 4°C overnight with one of the following unconjugated primary Abs (a 1:1,000 dilution of the manufacturer's original concentration in TBST with 5% BSA): anti-IRF3 (clone D83B9; Cell Signaling, Danvers, MA, USA), anti-phospho-IRF3 (Ser396) (clone 4D4G; Cell Signaling), anti-NF-κB-p65 (3034; Cell Signaling), anti-NF-κB-p65 (Sc109; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho NF-κB-p65 (Ser536) (clone 93H1; Cell Signaling), anti-STAT1 (sc346), anti-phospho STAT1 (Tyr701) (SC-7988; Santa Cruz Biotechnology), anti-eIF2 (9722; Cell Signaling), anti-phospho-eIF2 (9721; Cell Signaling), anti-CHOP (clone L63F7; Cell Signaling), or anti-β-actin (4967; Cell Signaling).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Journal of Virology

Article Title: Genotype 2 Strains of Porcine Reproductive and Respiratory Syndrome Virus Dysregulate Alveolar Macrophage Cytokine Production via the Unfolded Protein Response

doi: 10.1128/JVI.01251-17

Figure Lengend Snippet: Infection of AMϕ with PRRSV inhibits neither the activation of IRF3 or STAT1 nor the transcription of IFNB1, IRF7, and IFNA1 genes induced by stimulation with poly(I·C). (A and B) ZMAC cells were infected with either PRRSV strain FL12 or NADC20 (MOI = 5) and stimulated with poly(I·C) (25 μg/ml) at the indicated times postinfection. At 1 h after stimulation, whole-cell lysates were obtained and analyzed by Western blotting to sequentially detect p-IRF3, IRF3, and β-actin (A) or p-STAT1, STAT1, and β-actin (B). As a control, replicate cell sets were mock infected and cultured for 5 h before an additional 1-h incubation in the presence or absence of poly(I·C) and then harvested. (C, D, and E) ZMAC cells were mock infected or infected with PRRSV strain FL12 or NADC20 and stimulated with poly(I·C) (25 μg/ml) at 2 hpi. After 1, 4, or 7 h of stimulation, total RNA was obtained from each sample and subjected to real-time PCR analysis to detect IFNB1, IRF7, and IFNA1 gene transcripts. As a control, a replicate cell set was mock treated and cultured for 3 h before harvest. The fold changes in the amounts of these RNAs present in the virus-infected and poly(I·C)-stimulated AMϕ, as well as the mock-infected cells exposed to poly(I·C) for 4 or 7 h, relative to that in the mock-infected cells incubated with poly(I·C) for 1 h were determined by using the formula 2−ΔΔCt. The RPL32 gene was used as the reference housekeeping gene. RNA fold increases for IFNB1 and IFNA1 gene transcripts in mock-treated control cell samples cultured for 3 h were undetectable relative to those observed in cells exposed to poly(I·C) for 1 h, while the IRF7 gene transcript levels were approximately 2-fold greater. The asterisk indicates a statistically significant difference (*, P < 0.05).

Article Snippet: The membranes were incubated in blocking buffer (2% fish gelatin in TBST solution [50 mM Tris, pH 7.5, 500 mM NaCl, and 0.5% Tween 20]) at RT for 1 h. Afterward, the membranes were incubated at 4°C overnight with one of the following unconjugated primary Abs (a 1:1,000 dilution of the manufacturer's original concentration in TBST with 5% BSA): anti-IRF3 (clone D83B9; Cell Signaling, Danvers, MA, USA), anti-phospho-IRF3 (Ser396) (clone 4D4G; Cell Signaling), anti-NF-κB-p65 (3034; Cell Signaling), anti-NF-κB-p65 (Sc109; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho NF-κB-p65 (Ser536) (clone 93H1; Cell Signaling), anti-STAT1 (sc346), anti-phospho STAT1 (Tyr701) (SC-7988; Santa Cruz Biotechnology), anti-eIF2 (9722; Cell Signaling), anti-phospho-eIF2 (9721; Cell Signaling), anti-CHOP (clone L63F7; Cell Signaling), or anti-β-actin (4967; Cell Signaling).

Techniques: Infection, Activation Assay, Western Blot, Cell Culture, Incubation, Real-time Polymerase Chain Reaction

Journal: Journal of Virology

Article Title: Genotype 2 Strains of Porcine Reproductive and Respiratory Syndrome Virus Dysregulate Alveolar Macrophage Cytokine Production via the Unfolded Protein Response

doi: 10.1128/JVI.01251-17

Figure Lengend Snippet: Infection of AMϕ with PRRSV does not inhibit the activation of IRF3 induced by transfection with poly(I·C) despite the inhibition of IFN-α production. (A) ZMAC cells were mock infected or infected with PRRSV strain NADC20 or FL12 (MOI = 5). At 2 hpi, the cells were exposed to poly(I·C) either by transfection (400 ng/ml) or in free form (25 μg/ml). At 2 h after poly(I·C) treatment, whole-cell lysates were analyzed by Western blotting for the presence of p-IRF3 and total IRF3. Identical cultures were treated with the transfection reagent without poly(I·C) (mock transfection). (B) Duplicate cultures of ZMAC cells were mock infected or infected with PRRSV strain FL12 or NADC20 (MOI = 5). At 2 hpi, one member of each pair was exposed to either poly(I·C) (400 ng/ml) complexed with transfection reagent or mock transfected [exposed to transfection reagent without poly(I·C)]. After 8 h of culture, the amounts of IFN-α present in cell-free supernatants were determined by ELISA. The data represent the means ± standard deviations of two independent experiments. Statistical comparisons were made between the amounts of the cytokine present in the supernatants of infected versus mock-infected cultures stimulated with poly(I·C). The asterisks indicate statistical significance (**, P < 0.01).

Article Snippet: The membranes were incubated in blocking buffer (2% fish gelatin in TBST solution [50 mM Tris, pH 7.5, 500 mM NaCl, and 0.5% Tween 20]) at RT for 1 h. Afterward, the membranes were incubated at 4°C overnight with one of the following unconjugated primary Abs (a 1:1,000 dilution of the manufacturer's original concentration in TBST with 5% BSA): anti-IRF3 (clone D83B9; Cell Signaling, Danvers, MA, USA), anti-phospho-IRF3 (Ser396) (clone 4D4G; Cell Signaling), anti-NF-κB-p65 (3034; Cell Signaling), anti-NF-κB-p65 (Sc109; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho NF-κB-p65 (Ser536) (clone 93H1; Cell Signaling), anti-STAT1 (sc346), anti-phospho STAT1 (Tyr701) (SC-7988; Santa Cruz Biotechnology), anti-eIF2 (9722; Cell Signaling), anti-phospho-eIF2 (9721; Cell Signaling), anti-CHOP (clone L63F7; Cell Signaling), or anti-β-actin (4967; Cell Signaling).

Techniques: Infection, Activation Assay, Transfection, Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: MITOCHONDRIAL ANTIVIRAL PATHWAYS CONTROL ANTI-HIV RESPONSES AND ISCHEMIC STROKE OUTCOMES VIA THE RIG-1 SIGNALING AND INNATE IMMUNITY MECHANISMS

doi: 10.1101/2024.06.07.598027

Figure Lengend Snippet: (A) A diagram of antiviral RIG-1 pathway. Activated RIG-I and MDA5 molecules translocate to the mitochondria and interact with the MAVS adaptor. MAVS activates the downstream molecules TBK1/IKKε kinases, IRF3 and IRF7 inducing the transcriptional expression IFN-α/β. Pericytes were transfected with ocln-siRNA, ZO-1 siRNA, or PCMV3-OCLN vector as in , and the expression of RIG-1 (B) , IFIH1 (D) , TRAF3 (F) , MAVS (H) , IRF3 and p-IRF3 (J) , and TBK1 and p-TBK1 (K) was evaluated by q-PCR and Western Blotting. (L) Pericytes were transfected with RIG-1-siRNA, and p24 levels were assessed as in . (M-N) Pericytes were transfected with the PCMV3-OCLN vector and HIV-1 or mock-infected as in . IRF3 and IRF7 mRNA levels were measured by q-PCR. n= 4-8 per group. (C, E, G, I) RNA was extracted from isolated microvessels of age- and sex-matched ocln −/− , ocln +/− , and ocln +/+ mice (n=5-11 animals per group) as in . RIG-1 (C) , IFIH1 (E) , TRAF3 (G) , and MAVS (I) mRNA expression levels were measured by q-PCR. Graphs indicate the mean ± SD from three independent experiments. Individual animal data points are marked by blue (ocln +/+ ), green (ocln +/− ), and red (ocln −/− ) dots. ****p < 0.0001, ***p = 0.0002, **p = 0.003, *p < 0.0449.

Article Snippet: Blots were blocked with bovine serum album (BSA) at 5% in TBS-0.05% Tween20 for 1 h. Next, they were incubated overnight at 4°C with the following primary antibodies (all at 1:1000 in 5% BSA-TBS): anti-occludin (Thermo Fisher Scientific, Cat# 71-1500), anti-ISG15 (Thermo Fisher Scientist, Cat# 703131), anti-MX1 (Cell Signaling, Danvers, MA, USA, Cat# 37849), anti-MX2 (Cell Signaling, Cat# 43924), anti-IFIT1 (Abcam, Cambridge, UK, Cat# ab70023), anti-USP18 (Thermo Fisher Scientific, Cat# PA5-110555), anti-HERC5 (Thermo Fisher Scientific, Cat# PA5-114382), anti-DRP1 (Cell Signaling, Cat#14647), D-RP1 (616) (Cell Signaling, Cat# 44945), anti-RIG-1 (Thermo Fisher Scientific, Cat# 700366), anti-MDA5 (Abcam, Cat# ab283311), anti-MAVS (Thermo Fisher Scientific, Cat# 14341-1-AP), anti-TRAF3 (Abcam, Cat# ab239357), anti-IRF3 (Thermo Fisher Scientific, Cat# 703682), anti-p-IRF3 (Ser 396) (Thermo Fisher Scientific, Cat# B5-3195R), anti-TBK (Cell Signaling, Cat# 51872S), anti-p-TBK (Ser 172) (Cell Signaling, Cat# 5483S), anti-FIS1 (Abcam, Cat# ab156865), anti-MFN2 (Cell Signaling, Cat# 11925S), anti-OPA1 (Cell Signaling, Cat# 80471S), anti-MFF (Cell Signaling, Cat# 845805), anti-TOMM20 (Thermo Fisher Scientific, Cat# PA5-52843), anti-LC3BI/II (Cell Signaling, Cat# 4108), anti-P62 (Cell Signaling, Cat# 16177), anti-NDP52 (Cell Signaling, Cat# 60732), anti-PGC1α (Cell Signaling, Cat# 2178), anti-BAX (Cell Signaling, Cat# 2772), anti-BCL2 (Cell Signaling, Cat# 3498).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Infection, Isolation