Journal: PLoS ONE

Article Title: Chronic Oral Infection with Porphyromonas gingivalis Accelerates Atheroma Formation by Shifting the Lipid Profile

doi: 10.1371/journal.pone.0020240

Figure Lengend Snippet: Cells were stimulated with 1.0 µg/ml of P. gingivalis LPS, 0.1 µg/ml of E. coli LPS or 0.1 µg/ml of Pam3CSK4 for 12 hrs. Phosphorylation of IRF3 cellular extracts was analyzed by western blotting. Blot is a representative of three independent experiments.

Article Snippet: Rabbit anti-mouse IRF3 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse phospho-IRF3 (Cell Signaling Technology, Danvers, MA), rabbit anti-mouse GAPDH (Santa Cruz Biotechnology), peroxidase labeled anti-rabbit antibody (GE Healthcare, Munich, Germany) and ECL Plus Western Blotting Reagent Pack (Amersham Biosciences, Buckinghamshire, UK) were used for western blotting experiments.

Techniques: Phospho-proteomics, Western Blot

Journal: Molecular cell

Article Title: A Quantitative Genetic Interaction Map of HIV Infection

doi: 10.1016/j.molcel.2020.02.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-IRF7 , Cell Signaling , 13014S, RRID:AB_2737060.

Techniques: Virus, Luciferase, Mutagenesis, Recombinant, Purification, esiRNA, Cloning, Synthesized, Software

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 immunology (Baltimore, Md. : 1950)

Article Title: A Vitamin D-RelB/NF-κB Pathway Limits Chandipura Virus Multiplication by Rewiring the Homeostatic State of Autoregulatory Type 1 IFN-IRF7 Signaling.

doi: 10.4049/jimmunol.2101054

Figure Lengend Snippet: FIGURE 4. RelB deficiency triggers elevated basal expressions of T1-IFNs involving IRF7-mediated positive autoregulation. (A) GSEA comparing WT and Relb−/−

Article Snippet: Anti-IRF7 Ab (AHP-1180) was from Bio-Rad Laboratories.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Vitamin D-RelB/NF-κB Pathway Limits Chandipura Virus Multiplication by Rewiring the Homeostatic State of Autoregulatory Type 1 IFN-IRF7 Signaling.

doi: 10.4049/jimmunol.2101054

Figure Lengend Snippet: FIGURE 5. T1-IFNs and IRF7 cooperatively restrict viral multiplication in Relb−/−cells. Plaque assay reveal- ing the titer of progeny virus particles produced by WT and Relb−/−cells, including primary MEFs (A) or BMDMs (C, left panel) or immortalized MEFs (C, right panel), upon infection for 12 h with the indicated MOI of CHPV. (B) RT-qPCR showing the relative abundance of viral genomic RNA and mRNAs encoding viral nucleo- capsid protein and phosphoprotein in WT and Relb−/−

Article Snippet: Anti-IRF7 Ab (AHP-1180) was from Bio-Rad Laboratories.

Techniques: Plaque Assay, Virus, Produced, Infection, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Vitamin D-RelB/NF-κB Pathway Limits Chandipura Virus Multiplication by Rewiring the Homeostatic State of Autoregulatory Type 1 IFN-IRF7 Signaling.

doi: 10.4049/jimmunol.2101054

Figure Lengend Snippet: FIGURE 6. Vitamin D modulates the T1-IFNIRF7 pathway through RelB to exert antiviral effects. Vitamin D naive or vitamin Dconditioned WT or Relb−/−primary MEFs were examined for the relative abundance of IRF7 mRNA (A) and protein (B). Similarly, vitamin Dinduced expressions of IFN-b mRNA were scored in Relb−/−MEFs (C) and immortalized IRF7DCRISPR MEFs (E). Virus propaga- tion was scored in vitamin D naive and vitamin Dconditioned Relb−/−MEFs (D) or immortalized IRF7DCRISPR MEFs (F) subsequent to cell infection with 0.1 or 2 MOI of CHPV. The RT-qPCR data represent mean ± SEM of three indepen- dent experiments. The immunoblots represent three experi- mental replicates. The plaque assay data represent mean ± SEM of four biological replicates. *p# 0.05, **p# 0.01.

Article Snippet: Anti-IRF7 Ab (AHP-1180) was from Bio-Rad Laboratories.

Techniques: Virus, Infection, Quantitative RT-PCR, Western Blot, Plaque Assay

Journal: Cancer Research

Article Title: The m 6 A Reader YTHDF2 Promotes Bladder Cancer Progression by Suppressing RIG-I–Mediated Immune Response

doi: 10.1158/0008-5472.CAN-22-2485

Figure Lengend Snippet: RIG-I depletion can restore the phenotype of YTHDF2 deficiency in bladder carcinoma cells. A–D, Effects of knockdown ( A , C, and D ) or knockout ( B ) of either YTHDF2 or DDX58 , or both YTHDF2 and RIG-I in SW780 cells on cell viability measured by CCK-8 assays ( A ), cell proliferation analyzed by colony formation assays ( B ), the percentage of cells in S-phase determined by EdU assays ( C ), and relative cell death calculated by LDH release ( D ). E–G, Tumor size and weight of subcutaneous tumors from the indicated groups. H, H&E staining and sizes of tumors generated by Ddx58 KO and control MB49 cells implanted in mouse bladder lumen. I, A schematic model of signaling mediated by YTHDF2 in tumorigenesis of bladder carcinoma cells. YTHDF2 high-expressing cells fail to activate the type I IFN response and to suppress cell death. RIG-I detects and binds cytosolic double-stranded RNA (dsRNA) by its DExD/H-box domain, then opens its auto-repressed conformation, and recruits to the CARD domain of the MAVS, which induces phosphorylation of TBK1, IRF3, and IRF7, leading to the production of type I IFNs and cell death. In YTHDF2 high-expressing bladder carcinoma cells, excessive YTHDF2 binds to m 6 A modified RIG-I mRNA, which promotes RIG-I mRNA degradation, therefore, causing a decrease in RIGI-I protein expression level in the cell. Cytosolic dsRNA cannot be effectively recognized by the low-level RIG-I protein, thus weakening the activation of IFNI signaling and promoting bladder carcinoma tumorigenesis. Data in A–D are presented as the means ± SEM. Data in F and G are presented as the means ± SD of the experiment with 5 mice per group. P values in A–D, F, and G were calculated using the Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The following primary antibodies were used in this study: YTHDF2 (1:2,000, 24744–1-AP, Proteintech; RRID: AB_2687435), RIG-I (1:2,000, D261405; Sangon Biotech, RRID: AB_2537772), p-IRF3 (1:1,000, 37829; Cell Signaling Technology, RRID: AB_2799121), p-TBK1 (1:1,000, 5483; Cell Signaling Technology, RRID: AB_10693472), Flag (1:4,000, 80010–1-RR, Proteintech, RRID: AB_2882940), β-actin (1:10,000, RM2001, RayBiotech, RRID: AB_2756462), and GAPDH (1:20,000, 60004–1-Ig, Proteintech, RRID: AB_2107436).

Techniques: Knockdown, Knock-Out, CCK-8 Assay, Staining, Generated, Control, Expressing, Phospho-proteomics, Modification, Activation Assay

Journal: Journal of Inflammation Research

Article Title: LPS-Induced Activation of the cGAS-STING Pathway is Regulated by Mitochondrial Dysfunction and Mitochondrial DNA Leakage in Endometritis

doi: 10.2147/JIR.S374318

Figure Lengend Snippet: LPS induces STING undergoing perinuclear transfer and IRF3 transferring from the cytoplasm to the nucleus. ( A ) Representative images of immunofluorescence of STING in indicated groups of HESCs. ( B ) Representative images of immunofluorescence of IRF3 in indicated groups of HESCs. ( C ) The protein levels of IRF3 in cytoplasm and nucleus of HESCs in indicated groups by Western blot. ( D ) The protein levels of IRF3 in cytoplasm and nucleus of HESCs in indicated groups by Western blot. ( E ) Quantitative analysis of ( C and D ). (*p < 0.05).

Article Snippet: The antibodies used for this assay were as follows: anti-GAPDH (1:10000, 60004-1-Ig, Proteintech, USA); anti-tubulin (1:10000, 66031-1-Ig, Proteintech, USA); anti-Lamin B1 (1:10000, 66095-1-Ig, Proteintech, USA); anti-cGAS (1:1000, 83623, Cell Signaling Technology, USA); anti-STING (1:1000, 66680-1-Ig, Proteintech, USA; 1:1000, 13647, Cell Signaling Technology, USA), anti-TBK1 (1:1000, 38066, Cell Signaling Technology, USA); anti-Pho-TBK1 (1:1000, 5483, Cell Signaling Technology, USA); anti-IRF3 (1:1000, ab68481, Abcam, UK); anti-Pho-IRF3 antibody (1:1000, 11904, Cell Signaling Technology, USA); anti-Pho-IRF-3 (1:1000, 29047, Cell Signaling Technology, USA); horseradish enzyme-conjugated goat anti-rabbit IgG (H+L) (1:5000, ZB-2301, ZSGB-BIO, China); horseradish enzyme-conjugated goat anti-mouse IgG (H+L) (1:5000, ZB-2305, ZSGB-BIO, China).

Techniques: Transferring, Immunofluorescence, Western Blot