Journal: Theranostics

Article Title: Hepatoma cell-intrinsic TLR9 activation induces immune escape through PD-L1 upregulation in hepatocellular carcinoma

doi: 10.7150/thno.44417

Figure Lengend Snippet: TLR9 activation upregulated PD-L1 expression by promoting STAT3 Tyr705 phosphorylation in HCC cells. ( A ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 5, 10, or 20μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( B ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, or 10μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( C ) PD-L1 protein expression after treatment with ODN1585 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, and 10 μM; times: 0, 6, 12, 24, and 36 hours in 5μM) in Hepa1-6 cells. PD-L1 protein levels were analyzed by Western blotting. ( D ) PD-L1 + tumor cells were detected by flow cytometry after TLR9 agonist (Hep3B and Huh7 cells with ODN2216; Hepa1-6 cells with ODN1585) treatment with indicated concentration. (values are mean ± SD, *p < 0.05, **p < 0.01, ***p<0.001, NS indicates no significance). ( E ) PD-L1 expression after TLR9 overexpression in Huh7 cells. PD-L1 expression levels were analyzed by immunofluorescence. ( F ) PD-L1 protein expression after TLR9 overexpression in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( G ) PD-L1 protein expression in TLR9 knockdown or TLR9 rescue Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( H and I ) mRNA levels of PD-L1 in Hep3B ( H ) and Huh7 ( I ) cells measured by qRT-PCR after stimulation with different concentrations of ODN2216. (values are mean ± SD, *p < 0.05, **p < 0.01, NS indicates no significance) ( J ) TLR9 overexpression-induced PD-L1 expression after MYC, JUN, IRF1, IRF3, STAT1 or STAT3 silencing. PD-L1 mRNA expression in Huh7 cells was analyzed after TLR9 overexpression alone or in the presence of MYC-, JUN-, IRF1-, IRF3-, STAT1- or STAT3-specific siRNA or siRNA-NC. (values are mean ± SD, ***p<0.001). (K) p-STAT3 (Tyr705) levels in Hep3B cells after treatment with different concentrations of ODN2216 (a TLR9 agonist; ODN2216: 0, 2.5, 5, or 10μM) analyzed by Western blotting. ( L ) p-STAT3 (Tyr705) levels after TLR9 overexpression in Huh7 cells analyzed by Western blotting. ( M ) TLR9 overexpression-induced p-STAT3 (Tyr705) levels after TLR9 inhibition. p-STAT3 (Tyr705) levels were analyzed after TLR9 overexpression alone or in the presence of the TLR9 antagonist chloroquine diphosphate. ( N ) p-STAT3-induced PD-L1 levels after STAT3 inhibition. PD-L1 levels were analyzed after TLR9 overexpression alone or in the presence of the STAT3-specific small molecular inhibitor BP-1-102.

Article Snippet: The antibodies listed below were used in Western blotting, immunohistochemical and flow cytometry analyses: anti-TLR9 (ab37154, Abcam, Cambridge, UK; NBP2-24729, Novus Biologicals, Minneapolis, USA), anti-PARP1 (#9532; Cell Signaling Technology, Danvers, MA, USA; ab227244, Abcam), anti-PAR (#83732; Cell Signaling Technology, Danvers, MA, USA), anti-Ubiquitin (#3936; Cell Signaling Technology, Danvers, MA, USA); anti-STAT3 (#9139; Cell Signaling Technology, Danvers, MA, USA), anti-p-STAT3 (Tyr705) (#9145; Cell Signaling Technology, Danvers, MA, USA); anti-PD-L1 (#13684T, Cell Signaling Technology, Danvers, MA, USA; 329702, BioLegend, San Diego, CA, USA; ab205921, Abcam, Cambridge, UK), anti-Jak2 (#3230; Cell Signaling Technology, Danvers, MA, USA), anti-p-Jak2 (#3774; Cell Signaling Technology, Danvers, MA, USA), anti-STAT1(#14994, Cell Signaling Technology, Danvers, MA, USA), anti-JUN(#9165; Cell Signaling Technology, Danvers, MA, USA), anti-IRF1(#8478; Cell Signaling Technology, Danvers, MA, USA), anti-IRF3(#11904; Cell Signaling Technology, Danvers, MA, USA); anti-Myc (Sigma-Aldrich, St. Louis, MO, USA), anti-granzyme B (ab4059; Abcam), anti-CD8 (ab22378; Abcam; 560776; BD Biosciences), anti-JNK (AF6318; Affinity Biosciences, USA), anti-p-JNK (AF3318; Affinity Biosciences, USA), anti-ERK (AF0155; Affinity Biosciences, USA), anti-p-ERK (AF1015; Affinity Biosciences, USA), anti-p-p38 (AF4001; Affinity Biosciences, USA), anti-p38 (AF6456; Affinity Biosciences, USA), anti-PARG (#27808; Proteintech, Wuhan), anti-RNF146 (#ARP43340; Aviva Systems Biology Co., Ltd. USA; #bs-11669R; Bioss Antibodies Inc. USA), anti-CD4 (550954; BD Biosciences), anti-PD-1 (551892; BD Biosciences), anti-CD11b (557395; BD Biosciences), anti-NK1.1 (557391; BD Biosciences), anti-CD25 (553071; BD Biosciences), anti-Foxp3 (560402; BD Biosciences), anti-F4/80 (565612;BD Bioscience), anti-CD11c (553800; BD Biosciences), anti-CD317 (566431; BD Biosciences), PDD-00017273 (#5952; Tocris Bioscience, USA), ODN2216 (#tlrl-2216; Invivogen, USA), ODN2243(ODN2216 Control, #tlrl-2243; Invivogen, USA) and SP600125 (#T3109), U0126(#T6223), SB203580(#T1764), BP-1-102 (#T3708), Chloroquine diphosphate (#T0194) were purchased from Topscience (Topscience Co., Ltd. Shanghai).

Techniques: Activation Assay, Expressing, Western Blot, Flow Cytometry, Concentration Assay, Over Expression, Immunofluorescence, Quantitative RT-PCR, Inhibition

Journal: Journal of Immunology Research

Article Title: Mechanism of Nucleic Acid Sensing in Retinal Pigment Epithelium (RPE): RIG-I Mediates Type I Interferon Response in Human RPE

doi: 10.1155/2021/9975628

Figure Lengend Snippet: Identification of RIG-I as the key sensor in type I IFN response in RPE. (a, b) RNA but not DNA could be sensed by RPE to induce type I IFN response, suggesting the lack of DNA sensors in unstimulated RPE. THP-1 and THP-1 cGAS KO cells were used as controls (a). Cells were treated with indicated inducers at 0.25 μ g/mL, with or without the Lipofectamine transfection reagent, and intracellular ISG15 was measured by ELISA 24 h after stimulation. Each bar represents biological replicates ( n = 3) and is indicated as mean ± SD. Note that ARPE-19 without transfection did not respond to most tested inducers except for poly(I:C). ∗ p < 0.05 compared with corresponded vehicle-treated groups. # p < 0.05 compared with same induces in parental cells. (c) Type I IFN response was induced via the RIG-I–MAVS–IRF3 axis in ARPE-19 cells. ARPE-19 cells were cultured 10 d after reaching confluence for screening purposes. Different key nodes for the activation of the IFN pathway were knocked down using siRNAs (at least 3 different siRNAs for each gene). Cells were then treated with indicated nucleic acids, and the release of secreted IFN- β was measured by ELISA 24 h after stimulation. Similar results were observed in 3 independent experiments with different inducers (indicated in different lines) or different siRNAs (shown as different column); a single representative experiment is shown as an example. A heatmap was used to better visualize the percentage (%) change in the results. The efficiency of the mRNA knockdown was validated by qPCR and shown at the top in the form of heatmap. About ~70% inhibition was observed in most tested siRNAs.

Article Snippet: Following three washes with TBST, the membrane was incubated overnight with primary antibodies purchased from Cell Signaling Technology, including cGAS (CST#15102), IRF3 (CST#11904), RIG-I (CST#3743), Histone H3 (CST#4499), β -actin (CST#5125), and GAPDH (CST#8884), at 1 : 1000 dilution.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay, Inhibition

Journal: Theranostics

Article Title: Arginine starvation elicits chromatin leakage and cGAS-STING activation via epigenetic silencing of metabolic and DNA-repair genes

doi: 10.7150/thno.54695

Figure Lengend Snippet: cGAS-STING pathway was activated by arginine starvation. (A) Cells overexpressing cGAS-V5 were deprived for 72 h and stained with anti-V5 antibodies. Scale bars, 10 μm. (B) CWR22Rv1 cells were deprived for indicate timepoints, and endogenous IRF3 was immunoprecipitated. IRF3 phosphorylation and TBK1 association were determined by immunoblots. Fold changes are listed below each blot. (C) IRF3 phosphorylation level was quantified and normalized with the IRF3. (n = 3, **p < 0.01). (D) RT-qPCR analysis of type I interferon expression in CWR22Rv1 cells starved for 72 h. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) (E) IFNβ secretion by CWR22Rv1 cells after arginine starvation. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) (F) IFNβ promoter activity in CWR22Rv1 cells deprived for 72 h. (n = 3, *p < 0.05)

Article Snippet: IRF3 was immunoprecipitated with the IRF3 antibody (Cell Signaling, 11904) or control rabbit IgG (Millipore, pp64), and the precipitated immune complex was detected with IRF3, p-IRF3 (S396), TBK1 and p-TBK1 (S172) antibodies.

Techniques: Staining, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Expressing, Activity Assay

Journal: Theranostics

Article Title: Arginine starvation elicits chromatin leakage and cGAS-STING activation via epigenetic silencing of metabolic and DNA-repair genes

doi: 10.7150/thno.54695

Figure Lengend Snippet: Translocation of the cGAS downstream effectors. (A) Nuclear translocation of p65 was determined by subcellular fractionation in arginine-deprived CWR22Rv1 cells. (B) Nuclear p65 was quantified and normalized to lamin A/C. (n = 3, ***p < 0.001) (C) Immunostaining of p65 in CWR22Rv1 cells. Scale bars, 10 μm. (D) ChIP-qPCR analysis of IRF3 binding on the promoter regions of IFNA14 and IFNB1 in CWR22Rv1. (n = 3, *p < 0.05) (E) CWR22Rv1 cells overexpressing STING-V5 were subjected to arginine deprivation and stained with V5, calnexin and RCAS1 antibodies. Scale bars, 10 μm.

Article Snippet: IRF3 was immunoprecipitated with the IRF3 antibody (Cell Signaling, 11904) or control rabbit IgG (Millipore, pp64), and the precipitated immune complex was detected with IRF3, p-IRF3 (S396), TBK1 and p-TBK1 (S172) antibodies.

Techniques: Translocation Assay, Fractionation, Immunostaining, Binding Assay, Staining

Journal: PLoS ONE

Article Title: Structural and Functional Analyses of DNA-Sensing and Immune Activation by Human cGAS

doi: 10.1371/journal.pone.0076983

Figure Lengend Snippet: (A) Human cGAS mutants used for functional analyses. Human cGAS is shown as a ribbon model, with the same coloring as in . Mutated residues are shown as red ball-and-stick models. (B) cGAS-induced phosphorylation of TBK1, IRF3, and STING. cGAS WT or mutants were expressed in HEK293T cells stably expressing human STING. Cell lysates were analyzed by western blotting, using the indicated antibodies. The asterisk indicates phosphorylated STING. (C) Reporter assays for IFN-β (left panel) and NF-κB (right panel). The cell lysates are the same as in (B), except for the co-expression with reporter plasmids, and were measured for luciferase activities. Luciferase activities are shown as mean ± s.d. (n = 3). (D) Induction of IFN-β and A20 by human cGAS and its mutants. The relative mRNA expression levels of IFN-β (left) and A20 (right) were analyzed by Real-Time PCR using total RNAs isolated from the cells shown in (B). Relative expression levels are shown as mean ± s.d. (n = 3). (E) Immunoblotting for cGAS-induced phosphorylation (left panel), reporter assays for IFN-β (upper panel) and NF-κB (lower panel), the same as (B) and (C), respectively. (F) Pull-down experiment between biotinylated-ISD and human cGAS mutants. Human cGAS mutants were expressed and purified from E. coli , and mixed with Streptavidin beads in the presence or absence of biotinylated-ISD. Bound proteins were eluted with SDS sample buffer and analyzed by SDS-PAGE.

Article Snippet: Anti-STING (#3337), anti-phospho-TBK1 (#5483), anti-TBK1 (#3504), anti-phospho-IRF3 (#4947), and anti-IRF3 (#11904) antibodies were purchased from Cell Signaling.

Techniques: Functional Assay, Stable Transfection, Expressing, Western Blot, Luciferase, Real-time Polymerase Chain Reaction, Isolation, Purification, SDS Page

Journal: Nature

Article Title: Clathrin-associated AP-1 controls termination of STING signalling

doi: 10.1038/s41586-022-05354-0

Figure Lengend Snippet: a , Schematic diagram of the CTT of human STING (hSTING) and sequence logo of the CTT as indicated from 50 species. b , HeLa STING-knockout (KO) cells transfected with Flag-tagged STING ΔCΤΤ (Δ1–341), wild-type (WT) STING, STING E (E360A), STING LI (L364A/I365A) or STING ELI (E360A/L364A/I365A) were treated with diABZI for 2 h. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. c , WCL and extracted CCV fractions from HeLa STING KO cells reconstituted with Flag-tagged wild-type STING or STING LI (L364A/I365A) and treated or not with diABZI were analysed by western blot. CHC was used as a loading control. d , Glutathione sepharose pull-down assays of wild-type LBD-STING or LBD-STING ELI by glutathione S -transferase (GST)-tagged AP-1 core with or without ARF1. e , HeLa STING cells transfected with NC siRNA or siRNAs against TBK1 or IRF3 were treated with or without diABZI. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. GAPDH was used as a loading control. f , HeLa wild-type cells, HeLa TBK1 KO cells and HeLa IRF3 KO cells stimulated with diABZI for 0, 1, 2 or 4 h were analysed by western blot. Ratios of target proteins versus loading control normalized to the 0-h time point of each condition. Vinculin was used as a loading control. g , Bio-layer interferometry binding studies of LBD-STING (top) or TBK1-phosphorylated LBD-STING (pSTING) (bottom) with AP-1 ΔμCTD. The right graphs show the binding affinity of STING (top) and pSTING (bottom). One representative example of at least three ( b , d , f ) or two ( c , e , g ) independent experiments is shown.

Article Snippet: Primary antibodies used: mouse monoclonal anti-vinculin (hVIN-1) (Sigma-Aldrich, V9264, immunoblot 1:5,000), rabbit monoclonal anti-GAPDH (14C10) (Cell Signaling Technology, 2118, immunoblot 1:3,000), mouse monoclonal anti-Flag (M2) (Sigma-Aldrich, F1804, immunoblot 1:3,000), rabbit monoclonal anti-human phospho-STING (Ser366) (D7C3S) (Cell Signaling Technology, 19781, immunoblot 1:3,000), rabbit monoclonal anti-phospho-TBK1/NAK (Ser172) (D52C2) (Cell Signaling Technology, 5483, immunoblot 1:1,000), rabbit monoclonal anti-phospho-IRF3 (Ser386) (EPR2346) (Abcam, ab76493, immunoblot 1:1,000), rabbit monoclonal anti-TBK1/NAK (D1B4) (Cell Signaling Technology, 3504, immunoblot 1:1,000), rabbit polyclonal anti-TMEM173/STING (Proteintech, 19851-1-AP, immunoblot 1:1,000), rabbit monoclonal anti-IRF3 (D6I4C) (Cell Signaling Technology, 11904, immunoblot 1:1,000), rabbit monoclonal anti-clathrin heavy chain (P1663) (Cell Signaling Technology, 2410, immunoblot 1:500), mouse anti-clathrin heavy chain monoclonal antibody (X22) (Thermo Fisher Scientific, MA1-065, immunofluorescence (IF) 1:100), rabbit polyclonal anti-AP1S1 (Thermo Fisher Scientific, PA5-63913, immunoblot 1:1,000), rabbit polyclonal anti-AP1G1 (Thermo Fisher Scientific, PA5-65290, immunoblot 1:1,000), rabbit polyclonal anti-AP1B1 (Sigma-Aldrich, HPA065226, immunoblot 1:1,000), rabbit polyclonal anti-AP1M1 (Proteintech, 12112-1-AP, immunoblot 1:1,000), mouse monoclonal anti-HSV-1 ICP0 (11060) (Santa Cruz, sc-53090, immunoblot 1:500), mouse monoclonal anti-HA.11 epitope tag (16B12) (Biolegend, MMS-101R, immunoblot 1:2,000). mouse monoclonal γ-adaptin (AP1G1) (100/3) (Sigma-Aldrich, A4200, IF 1:100), mouse monoclonal EEA1 (E9Q6G) (Cell Signaling, 48453, IF 1:100), mouse monoclonal LAMP1 (H4A3) (Abcam, ab25630, IF 1:100), rabbit monoclonal anti-human phospho-STING (Ser366) (D8K6H) (Cell Signaling Technology, 40818, IF 1:100, STED 1:50), mouse monoclonal RAB7 (E9O7E) (Cell Signaling Technology, 95746, IF 1:100) and sheep polyclonal human TGN46 (Bio-Rad, AHP500G, IF 1:200).

Techniques: Sequencing, Knock-Out, Transfection, Immunoprecipitation, Western Blot, Binding Assay