Journal: Frontiers in Immunology

Article Title: CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX

doi: 10.3389/fimmu.2022.1050250

Figure Lengend Snippet: STINGPOX induces IFN signaling in a STING-dependent manner. (A) Primary murine BMDCs were infected with STINGPOX or OncoVACV for 24 h. Whole cell lysates were analyzed by WB for phospho-STING (p-STING S365), STING, phospho-TBK1 (p-TBK1 S172), TBK1, phospho-IRF3 (p-IRF3 S396), IRF3, IFIT1, cGAS and FLAG (for disA expression). β-actin levels were used as loading control. Data are representative of three independent experiments. (B) THP1-Dual cells were infected with STINGPOX or OncoVACV (at a range of MOIs) and relative luciferase activity was assessed as a measure of IFN signaling (n = 3). (C) Supernatants IFN-β levels in THP1-Dual infected with STINGPOX or OncoVACV (MOI of 0.3 or 1) for 24 h were measured by ELISA. (D) Heatmap of relative fold change in expression (to mock) in primary human macrophages infected with OncoVACV or STINGPOX (MOI = 3) for 18 hours, and analyzed by RNA-seq. Log2 transformed data is shown for genes associated Jak/STAT or IFN αβ signaling pathways that were activated at least 4-fold in STINGPOX infected cells relative to OncoVACV infection conditions. (E) IFN signaling measured as described in (B) using WT, cGAS KO, and STING KO THP1-Dual cells. (F) Immunoblot analyses of primary murine WT and cGAS -/- BMDCs infected with STINGPOX or OncoVACV, as described in (A) . Data are shown as mean ± SEM.

Article Snippet: The primary antibodies used for Western blotting were from the following sources: Abcam: IFIT1 (ab11821); Cell Signaling Technology: phospho-TBK1 (S172, #5483), TBK1 (3504), phospho-IRF3 (S396, #4947), IRF3 (#4302), phospho-STING (S365, #72971), STING (#3337), FLAG (#14793), cGAS (#31659); Sigma: β-actin antibody (A5441).

Techniques: Infection, Expressing, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing Assay, Transformation Assay, Western Blot

Journal: Cell Death Discovery

Article Title: Transglutaminase type 2-dependent crosslinking of IRF3 in dying melanoma cells

doi: 10.1038/s41420-022-01278-w

Figure Lengend Snippet: A , B Western blot analysis of H2AX and γH2AX expression in A375 cells treated with doxorubicin for 6, 16, and 24 h. GAPDH was used as loading control. ( n = 3; means ± SEM; *** p < 0,001). C , D Western blot analysis of TBK1 and STING protein expression in A375 treated with doxorubicin. Actin was used as loading control. ( n = 3; means ± SEM; p = ns). E Western blot and densitometric analysis of IRF3 levels in A375 treated with doxorubicin. Tubulin was used as loading control. ( n = 3; means ± SEM; *** p < 0.001).

Article Snippet: Anti-IRF3 (D83B9) Cat#4302 (Cell Signaling); anti-TG2CUB7402 Cat#MS-224-P (Neomarkers), anti-Actin Cat#A2066 (Sigma); anti-GAPDH Cat#G9545 (Sigma); anti- STING (D2P2F) Cat# 13647 (Cell Singaling); anti-TBK1/NAK (D1B4) Cat#3504 (Cell Signaling), anti-TBP Cat#22006-1-AP (Proteintech), anti- H2AX Cat# PA5-28778 (ThermoFisher), anti-γH2AX Cat#ab81299 (Abcam), anti-β Tubulin Cat#T4026.

Techniques: Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: cGAS deficiency enhances inflammasome activation in macrophages and inflammatory pathology in pristane-induced lupus

doi: 10.3389/fimmu.2022.1010764

Figure Lengend Snippet: Inhibition of cGAS increases the expression of Caspase 11 but not phosphorylation TBK1 in the lungs of pristane-induced lupus mice. (A–L) The relative RNA expression (normalized to actin) of (A) Tlr7 , (B) Tlr9 , (C) Rig1 , (D) Mda5 , (E) Sting , (F) Ifi16 , (G) Dai , (H) Ddx41 , (I) Irf3 , (J) Ifnb , (K) Cxcl10 , (L) Ifng , (M) Aim2 , (N) Nlrp3 , (O) Casp1 , (P) Casp11 , (Q) Il1b , (R) Il1a , (S) Il18, and (T) Il6 from the lungs of WT and Cgas -/- mice at 8 months after pristane injection (n = 4-6 per group). (U–W) Western blot analysis of (U) STING, (V) phosphorylation of TBK1 (Ser172), and (W) caspase-11 and gasdermin D from the lungs of WT and Cgas -/- mice at 8 months after pristane injection (representative blot, n = 3). Data are shown as the mean ± SEM. *p < 0.05, **p < 0.01, *** p < 0.001.

Article Snippet: Proteins were separated by SDS-PAGE, then proteins were transferred to nitrocellulose membranes and probed with STING antibody (clone: GTN-01; 1:2000) (CUSB-in house, BKK, TH), Phospho-TBK1 antibody (Ser172) (clone D52C2, cat: 5483; 1:1000) (Cell Signaling, MA, USA), TBK1 antibody (clone D1B4, cat: 3504; 1:1000) (Cell Signaling, MA, USA), Caspase11 antibody (clone 17D9, cat: 14340; 1:1000) (Cell Signaling, MA, USA), and Gasdermin D antibody (clone E9S1X, cat: 39754; 1:1000) (Cell Signaling, MA, USA).

Techniques: Inhibition, Expressing, RNA Expression, Injection, Western Blot

Journal: Journal for Immunotherapy of Cancer

Article Title: CD4 T cell-intrinsic STING signaling controls the differentiation and effector functions of T H 1 and T H 9 cells

doi: 10.1136/jitc-2021-003459

Figure Lengend Snippet: Transcriptional regulation of T H 1 and T H 9 cell differentiation following STING activation. (A) Heatmap representing Ifng , Tbx21 , Irf1 , Gata3 , Il9 , Irf4 , Irf8 , Batf , Rorc , and Foxp3 mRNA expression (Z-score) from naive CD4 T cells stimulated with cGAMP or control and polarized into T H 1 or T H 9 cells for 72 hours. (B–D) WT naive CD4 T cells stimulated with cGAMP or control for 4(B) or 6(C, D) hours. (B) STING, p-STING, TBK1 and p-TBK1 protein levels. (C) Cyt and Nuc localization of IRF3, p65 NF-κB and their phosphorylated forms. (D) Heatmap representing Ifna , Ifnb1 , Il6 , Tnfa , Ifit1 , Ifit2 , Mx2 and Cxcl10 mRNA expression (Log2FC) between CD4 T cells stimulated with cGAMP and control. Mean of replicates from three independent experiments. P values (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) determined by two-way analysis of variance (A) or unpaired t test (D). (E, F) RNA sequencing analysis from WT naive CD4 T cells stimulated with cGAMP or control and polarized into T H 1 or T H 9 cells for 16 hours. Biological replicates from three independent experiments. (E) Gene set enrichment analysis comparing expression in cGAMP-stimulated cells to control cells. Enrichment plot and score, Nom. P value and FDR q value shown for the three gene sets. (F) Heatmaps illustrating the hierarchical clustering of expression levels (rld values) of ISGs. cGAMP, 2′3′-cyclic guanosine monophosphate–adenosine monophosphate; Cyt, cytosolic; FDR, false discovery rate; ISG, interferon-stimulated gene; Nom, nominal; Nuc, nuclear; STING, stimulator of interferon genes; WT, wild type.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (CST) and diluted 1:1000 in TBS-T 5% BSA: Phospho-STING (Ser 366, 19781), TBK1/NAK (clone D1B4, 3504) Phopho-TBK1 (Ser 172, clone D52C2, 5483), Phospho-IRF-3 (Ser 396, clone 4D4G, 4947), IRF3 (clone D83B9, 4302), Phospho-NF kappa B P65 (Ser 536, clone 93H1, 3033), NF kappa B P65 (clone D14E12, 8242), Phospho-p70S6K (Thr389, clone 108D2, 9234), and Phospho-S6 (Ser235/236, clone D57.2.2E, 4858).

Techniques: Cell Differentiation, Activation Assay, Expressing, RNA Sequencing Assay

Journal: Theranostics

Article Title: IKKε phosphorylates kindlin-2 to induce invadopodia formation and promote colorectal cancer metastasis

doi: 10.7150/thno.40397

Figure Lengend Snippet: IKKε directly phosphorylates kindlin-2 at S159. (A) Slot blot analysis of the anti-phospho-kindlin-2-S159 antibody. The antibody was probed against an un-modified kindlin-2 peptide and phosphorylated peptides. (B) HCT116 cells were transfected with the indicated plasmids and whole cell extracts were immunoprecipitated (IP) with FLAG-conjugated M2 beads and then analyzed by immunoblotting (IB). (C)HCT116 cells were treated with the inhibitor IKK-3 at the indicated concentration for 24h and analyzed by immunoblotting. (D and E) HCT116 cells were transfected with the indicated siRNAs targeting IKKα, IKKβ, IKKε or TBK1. Cell lysates were analyzed by immunoblotting. (F) HCT116 cells were transfected with FLAG-tagged kindlin-2 and GFP-tagged IKKε for 48 h. Total cell lysates were immunoprecipitated using FLAG-conjugated M2 beads and analyzed by immunoblotting. (G) IKKε was immunoprecipitated using anti-IKKε antibody or the control antibody IgG from lysates of HCT116 cells. (H) HCT116 cells were transfected with the indicated plasmids and the whole cell extracts were prepared and analyzed by Co-IP assay flowed by immunoblotting with the indicated antibodies. (I) GST-tagged wild-type kindlin-2 or kindlin-2 (S159A) were incubated without IKKε (-), or with purified wild-type IKKε (WT) or mutant IKKε (K38A) in kinase buffer and resolved by SDS-PAGE, followed by immunoblotting analysis with the indicated antibodies.

Article Snippet: The following antibodies were used in this study: anti-kindlin-2-S159 (CTM-152, PTM Biolabs) ; anti-kindlin-2 (K3269) and anti-FLAG (F3165) (Sigma-Aldrich); anti-IKKα (2682), anti-IKKβ (2678), anti-IKKε (2905), anti-TBK1 (3504) (Cell Signaling Technology); anti-GAPDH (sc-3223), anti-FISH (sc-376211) and anti-cortactin (sc-55579) (Santa Cruz); anti-IKKε (ab7891) (Abcam); anti-SH3PXD2A (18976-1-AP) (Proteintech); anti-MIG2/Kindlin-2 (NBP1-47745) (NOVUS).

Techniques: Dot Blot, Modification, Transfection, Immunoprecipitation, Western Blot, Concentration Assay, Co-Immunoprecipitation Assay, Incubation, Purification, Mutagenesis, SDS Page