Journal: Frontiers in Immunology

Article Title: Characterization of a Novel Compound That Stimulates STING-Mediated Innate Immune Activity in an Allele-Specific Manner

doi: 10.3389/fimmu.2020.01430

Figure Lengend Snippet: M04 induces canonical activation of IRF3 which is essential to reporter signal generated by the compound. (A) Immunoblot showing phosphorylation status of TBK1 Ser172 and IRF3 Ser386 as well as corresponding total protein levels in MM6 cells (left) and THF (right) exposed for 4 h to 1% DMSO, 50 μM M04, or 1,000 HAU/mL SeV as indicated; (B) Indirect immunofluorescence showing subcellular localization of IRF3 in THF exposed for 4 h to 1% DMSO, transfected 2'3'cGAMP (10 μg/mL), 100 ng/mL TNFα, or 50 μM M04; (C) Reporter assay illustrating IFN-dependent LUC induction following overnight treatment with 1% DMSO, 1,000 U/mL IFNβ, 1,000 HAU/mL SeV, or 50 μM M04 in parental cells as well as those from which IRF3 was deleted as indicated. Data presented are mean ± SD relative luminescence units (RLU) using signal from DMSO-treated cells based on quadruplicate measurements. Student's T -test was used to compare RLU in the parental and ΔIRF3 cells ** P < 0.01; *** P < 0.001.

Article Snippet: Sources and concentrations of antibodies used against the following antigens are indicated in parentheses: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SC-51906; Santa Cruz) (1:10,000), IRF3 (4302; Cell Signaling), human phospho-IRF3 (76493; Abcam), mouse IRF3 (SC-9082; Santa Cruz), mouse phospho-S379 IRF3 (79945S; Cell Signaling), mouse phospho-S396 IRF3 (29047S; Cell Signaling), STING (13647S; Cell Signaling), phospho-S366 STING (19781S; Cell Signaling), TBK1 (3504S; Cell Signaling), phospho-TBK1 (5483S; Cell Signaling), NF-kB P65 (SC372; Santa Cruz), NF-kB P50 (3035; Cell Signaling), GM-130 (610823; BD Biosciences).

Techniques: Activation Assay, Generated, Western Blot, Phospho-proteomics, Immunofluorescence, Transfection, Reporter Assay

Journal: Frontiers in Immunology

Article Title: Characterization of a Novel Compound That Stimulates STING-Mediated Innate Immune Activity in an Allele-Specific Manner

doi: 10.3389/fimmu.2020.01430

Figure Lengend Snippet: Innate Activation by M04 requires STING but not MAVS, TRIF, or cytosolic DNA PRRs. (A) Reporter assay illustrating IFN-dependent LUC induction in THF-ISRE-ΔMAVS/TRIF following overnight treatment with 1% DMSO, transfected cGAMP (10 μg/mL), or 75 μM M04. Data presented are mean ± SD relative luminescence units (RLU) using signal from DMSO-treated cells as the basis ( n = 4 treatments); (B) Immunoblot showing phosphorylation status of IRF3 Ser386, total IRF3, and GAPDH in THF-ISRE-ΔMAVS/TRIF following 8 h treatment with 1% DMSO, 75 μM M04, 1,000 HAU/mL SeV or 25 μM ABZI as indicated; (C) Reporter assay illustrating IFN-dependent LUC induction in THF-ISRE-ΔSTING following overnight treatment with 1% DMSO, 1,000 U/mL IFNβ, 1,000 HAU/mL SeV, or 75 μM M04. Data presented are mean RLU ± SD as described above; Student's T -test was used to compare RLU *** p < 0.001; (D) Immunoblot showing phosphorylation status of IRF3 Ser386, total IRF3 in THF-ISRE-ΔSTING following 4 h treatment with 1% DMSO, 50 μM M04, 1,000 HAU/mL SeV or 25 μM ABZI as indicated; (E) Secretion of bioactive type I IFN from parental THF as well as THF-ISRE-ΔMAVS/TRIF and THF-ISRE-ΔSTING treated in triplicate overnight with 1% DMSO, 1,000 HAU/mL SeV, transfected cGAMP (10 μg/mL), or 75 μM M04. Data are expressed as mean concentrations ± SD for IFNβ equivalent units. Statistical significance between treated and untreated cells of similar genetic background was calculated using Student's T -test. **** p < 0.0001; (F) Reporter assay from WT parental THF-ISRE cells as well as from cells from which indicated dsDNA-specific PRRs were deleted. Values presented are mean fold changes ± SD for duplicates relative to the value for DMSO-treated cells.

Article Snippet: Sources and concentrations of antibodies used against the following antigens are indicated in parentheses: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SC-51906; Santa Cruz) (1:10,000), IRF3 (4302; Cell Signaling), human phospho-IRF3 (76493; Abcam), mouse IRF3 (SC-9082; Santa Cruz), mouse phospho-S379 IRF3 (79945S; Cell Signaling), mouse phospho-S396 IRF3 (29047S; Cell Signaling), STING (13647S; Cell Signaling), phospho-S366 STING (19781S; Cell Signaling), TBK1 (3504S; Cell Signaling), phospho-TBK1 (5483S; Cell Signaling), NF-kB P65 (SC372; Santa Cruz), NF-kB P50 (3035; Cell Signaling), GM-130 (610823; BD Biosciences).

Techniques: Activation Assay, Reporter Assay, Transfection, Western Blot, Phospho-proteomics

Journal: Frontiers in Immunology

Article Title: Characterization of a Novel Compound That Stimulates STING-Mediated Innate Immune Activity in an Allele-Specific Manner

doi: 10.3389/fimmu.2020.01430

Figure Lengend Snippet: Responsiveness to M04 can be Conferred through Introduction of WT STING Allelic Variant. (A) Reporter assay illustrating IFN-dependent LUC induction in THP-1-ISG-Lucia following overnight treatment with 1% DMSO, 1,000 HAU/mL SeV, 1,000 U/mL IFNβ, 75 μM TRIF agonist AV-C, 25 μM ABZI, or 75 μM M04. Data presented are mean ± SD relative luminescence units (RLU) using signal from DMSO-treated cells as the basis ( n = 4 treatments). Student's T -test was used to compare RLU *** p < 0.001, **** p < 0.0001; (B) lmmunoblot showing phosphorylation status of IRF3 Ser386 and total IRF3 in THP-1 whole cell lysates following 4 h treatment with 1% DMSO, 50 μM M04, 1,000 HAU/mL SeV, 25 μM ABZI, or 10 μg/mL cGAMP as indicated; (C) lmmunoblot showing expression of endogenous or ectopically expressed WT hSTING in THP-1 as indicated. (D) Immunoblot showing phosphorylation status of IRF3 Ser386 in THP-1 cells from which endogenous STING was deleted and WT STING stably introduced following indicated treatment as described above.

Article Snippet: Sources and concentrations of antibodies used against the following antigens are indicated in parentheses: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SC-51906; Santa Cruz) (1:10,000), IRF3 (4302; Cell Signaling), human phospho-IRF3 (76493; Abcam), mouse IRF3 (SC-9082; Santa Cruz), mouse phospho-S379 IRF3 (79945S; Cell Signaling), mouse phospho-S396 IRF3 (29047S; Cell Signaling), STING (13647S; Cell Signaling), phospho-S366 STING (19781S; Cell Signaling), TBK1 (3504S; Cell Signaling), phospho-TBK1 (5483S; Cell Signaling), NF-kB P65 (SC372; Santa Cruz), NF-kB P50 (3035; Cell Signaling), GM-130 (610823; BD Biosciences).

Techniques: Variant Assay, Reporter Assay, Phospho-proteomics, Expressing, Western Blot, Stable Transfection

Journal: Frontiers in Immunology

Article Title: Characterization of a Novel Compound That Stimulates STING-Mediated Innate Immune Activity in an Allele-Specific Manner

doi: 10.3389/fimmu.2020.01430

Figure Lengend Snippet: Transient transfection of vectors encoding WT and R232H but not HAQ hSTING confer responsiveness to M04. (A) lmmunoblot from HEK293T whole cell lysates showing expression of indicated STING variants following transient transfection, S386 phosphorylation status of IRF3 and total IRF3. Cells were left untreated or exposed to 75 μM M04, 100 nM diABZI, or 10 μg/mL cGAMP as indicated; (B) Reporter assay using cells ( n = 4) treated as described in (A) . Values displayed are mean fold changes ± SD relative to cells transfected with empty vector; (C) Expression of IFIT1 and Viperin mRNA as determined by qPCR in parental A549 cells as well as those transduced with hSTING following treatment with 1% DMSO or 75 μM M04. Data are mean fodl changes ± SD relative to DMSO-treated cells based on duplicates; (D) Synthesis of cGAMP by A549-hSTING cells as determined by ELISA following overnight treatment with 1% DMSO, HCMV, or M04. Data presented are mean pg/mL ± SD based on duplicate samples. Student's T -test was used to compare RLU and mRNA levels *** p < 0.001, **** p < 0.0001.

Article Snippet: Sources and concentrations of antibodies used against the following antigens are indicated in parentheses: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SC-51906; Santa Cruz) (1:10,000), IRF3 (4302; Cell Signaling), human phospho-IRF3 (76493; Abcam), mouse IRF3 (SC-9082; Santa Cruz), mouse phospho-S379 IRF3 (79945S; Cell Signaling), mouse phospho-S396 IRF3 (29047S; Cell Signaling), STING (13647S; Cell Signaling), phospho-S366 STING (19781S; Cell Signaling), TBK1 (3504S; Cell Signaling), phospho-TBK1 (5483S; Cell Signaling), NF-kB P65 (SC372; Santa Cruz), NF-kB P50 (3035; Cell Signaling), GM-130 (610823; BD Biosciences).

Techniques: Transfection, Expressing, Phospho-proteomics, Reporter Assay, Plasmid Preparation, Transduction, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Characterization of a Novel Compound That Stimulates STING-Mediated Innate Immune Activity in an Allele-Specific Manner

doi: 10.3389/fimmu.2020.01430

Figure Lengend Snippet: Responsiveness to M04 can be conferred to murine cells by ectopic expression of human STING-WT variant. (A) Reporter assay illustrating IFN-dependent LUC induction in RAW264.7-ISG-Lucia cells followni g overnight treatment with 1% DMSO, 160 HAU/mL SeV, 25 μM DMXAA, or 75 μM M04.Data presented are mean ± SD relative luminescence units (RLU) using signal from DMSO-treated cells as the basis ( n = 4 treatments); (B) qPCR examining in vivo ISG induction following IP injection of DMXAA or M04; (C) lmmunoblot showing expression of endogenous or ectopically expressed hSTING-WT in RAW264.7 cells as indicated; (D) lmmunoblot showing phosphorylation status of IRF3 Ser379 and Ser396 as well as total IRF3 in RAW264.7-hSTING cells following 4 h treatment with 1% DMSO, 75 μM M04, 160 HAU/mL SeV, or transfection of cGAMP as indicated; (E) qPCR examining transcription of IFIT1 or Viperin following overnight treatment of parental RAW264.7 and RAW264.7-hSTING cells with 75 μM M04 ( n = 3). Data presented are mean fold changes ± SD of mRNA relative to cells treated with 1% DMSO.

Article Snippet: Sources and concentrations of antibodies used against the following antigens are indicated in parentheses: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SC-51906; Santa Cruz) (1:10,000), IRF3 (4302; Cell Signaling), human phospho-IRF3 (76493; Abcam), mouse IRF3 (SC-9082; Santa Cruz), mouse phospho-S379 IRF3 (79945S; Cell Signaling), mouse phospho-S396 IRF3 (29047S; Cell Signaling), STING (13647S; Cell Signaling), phospho-S366 STING (19781S; Cell Signaling), TBK1 (3504S; Cell Signaling), phospho-TBK1 (5483S; Cell Signaling), NF-kB P65 (SC372; Santa Cruz), NF-kB P50 (3035; Cell Signaling), GM-130 (610823; BD Biosciences).

Techniques: Expressing, Variant Assay, Reporter Assay, In Vivo, Injection, Phospho-proteomics, Transfection

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: bioRxiv

Article Title: Long noncoding RNA AL109754.1 Regulates Myeloid Dendritic Cell Differentiation and Potentiates TLR signaling

doi: 10.1101/2023.03.01.530613

Figure Lengend Snippet: In vitro human monocyte-derived DC were transfected with scramble or si-AL109754.1. After 48h, cells were challenged with TLR2, TLR4, TLR5, TLR7 or TLR9 agonists. ( A ) Effect of lncRNA AL109754.1 knockdown on NFkB phosphorylation, IRF3, and IRF7 using phosphoflow. The half overlaid histograms in left panel were generated in Flowjo_v10.8.1 to elucidate the comparison phosphorylated species in control vs si-AL109754.1 transfected cells. After 48h post transfections all the cells were fixed with Cytofix Buffer (BD Biosciences), permeabilized with methanol and stained with various phospho-antibodies for TFs-associated with the TLR signaling. ( B ) Representative scatter plots show the phosphorylated percentage of downstream TLR molecules generated in Flowjo_v10.8.1. ( C ) Bar graph showing average percentage of cells containing phosohorylated TLR specific downstream signaling molecules. Each bar shows mean ±SD. Students t-test were used to calculate p-values and p <0.05 was considered significant. *p<0.05, **p<0.01, ***p<0.001 .

Article Snippet: Following antibodies were used to detect the downstream phosphorylation: NF-κB (BV421 conjugated NF-κB [p65], clone: K10-895.12.50, BD Biosciences), IRF7 (Alexa Fluor® 647 conjugated Mouse anti-IRF-7 [pS477/pS479], clone: K47-671, BD Biosciences), IRF3 (PE conjugated Rabbit mAb Phospho-IRF-3 [Ser396], clone: D6O1M, Cell Signaling).

Techniques: In Vitro, Derivative Assay, Transfection, Knockdown, Phospho-proteomics, Generated, Comparison, Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: Heat Shock Protein 60 Restricts Release of Mitochondrial dsRNA to Suppress Hepatic Inflammation and Ameliorate Non-Alcoholic Fatty Liver Disease in Mice

doi: 10.3390/ijms23010577

Figure Lengend Snippet: Analyses of the protein of TLR3 ( A ), MDA5 ( B ), and p-IRF3 ( C ), the mRNA of the il-6 ( D ) and il1-il1β ( E ) expressions, and the ( F ) mitochondria DNA copy number per cell in the WT and HSP60Tg mice livers following HFD or CD for 26 weeks. Data calculated from five to eight samples per group are expressed as the mean ± SE. * p < 0.05, ** p < 0.01 and *** p < 0.001 between the indicated groups. WT, wild-type mice; HFD, high-fat diet; HSP60Tg, mice harboring overexpression of heat shock protein 60; CD, chow diet; TLR3, toll-like receptor 3; MDA5, melanoma differentiation-associated gene 5; p-IRF3, phosphorylated-interferon regulatory factor 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Following electrophoresis, it was transferred to a PVDF membrane (Millipore, MA), and the blots were incubated with primary antibodies at 4 °C overnight against HSP60 (sc-1052, Santa Cruz, CA, USA), TLR3 (ab62566, abcam, Cambridge, UK), MDA5 (#5321, Cell Signaling, MA, USA), p-IRF3 (orb6225, Biorbyt, Cambridge, UK), IRF3 (ab68481, abcam, Cambridge, UK) and GAPDH (60004-1-lg, PROTEINTECH, IL, USA) for protein control.

Techniques: Over Expression

Journal: International Journal of Molecular Sciences

Article Title: Heat Shock Protein 60 Restricts Release of Mitochondrial dsRNA to Suppress Hepatic Inflammation and Ameliorate Non-Alcoholic Fatty Liver Disease in Mice

doi: 10.3390/ijms23010577

Figure Lengend Snippet: Analyses of HSP60 ( A ), TLR3 ( B ), MDA5 ( C ), and p-IRF3 ( D ) on the 10th culture day of HepG2 after treatment with a HSP60 overexpression vector for 24 h. Data are expressed as the mean ± SE of the four to six samples per group. ** p < 0.01 and *** p < 0.001 between the indicated groups. TLR3, toll-like receptor 3; MDA5, melanoma differentiation-associated gene 5; P-IRF3, phosphorylated-interferon regulatory factor 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Following electrophoresis, it was transferred to a PVDF membrane (Millipore, MA), and the blots were incubated with primary antibodies at 4 °C overnight against HSP60 (sc-1052, Santa Cruz, CA, USA), TLR3 (ab62566, abcam, Cambridge, UK), MDA5 (#5321, Cell Signaling, MA, USA), p-IRF3 (orb6225, Biorbyt, Cambridge, UK), IRF3 (ab68481, abcam, Cambridge, UK) and GAPDH (60004-1-lg, PROTEINTECH, IL, USA) for protein control.

Techniques: Over Expression, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Heat Shock Protein 60 Restricts Release of Mitochondrial dsRNA to Suppress Hepatic Inflammation and Ameliorate Non-Alcoholic Fatty Liver Disease in Mice

doi: 10.3390/ijms23010577

Figure Lengend Snippet: Proposed model of heat shock protein 60 restricts release of mitochondrial dsRNA to suppress hepatic inflammation and ameliorate non-alcoholic steatohepatitis in mice. High-fat diet (HFD) composed of 60% calories from fat can induce an obesity animal model that is characterized by steatohepatitis, glucose intolerance, and insulin resistance. Gain of heat shock protein 60 (HSP60) alleviated not only HFD-induced body weight gain, fat accumulation, and hepatocellular steatosis, but also glucose tolerance and insulin resistance. Furthermore, overexpression of HSP60 in the HFD group resulted in inhibited release of mitochondrial dsRNA (mt-dsRNA) compared to wild-type mice. In addition, overexpression of HSP60 also inhibited the activation of toll-like receptor 3 (TLR3), melanoma differentiation-associated gene 5 (MDA5), and phosphorylated-interferon regulatory factor 3 (p-IRF3), as well as inflammatory biomarkers such as mRNA of il-1β and il-6 expression in the liver in response to HFD.

Article Snippet: Following electrophoresis, it was transferred to a PVDF membrane (Millipore, MA), and the blots were incubated with primary antibodies at 4 °C overnight against HSP60 (sc-1052, Santa Cruz, CA, USA), TLR3 (ab62566, abcam, Cambridge, UK), MDA5 (#5321, Cell Signaling, MA, USA), p-IRF3 (orb6225, Biorbyt, Cambridge, UK), IRF3 (ab68481, abcam, Cambridge, UK) and GAPDH (60004-1-lg, PROTEINTECH, IL, USA) for protein control.

Techniques: Animal Model, Over Expression, Activation Assay, Expressing