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: International Journal of Biological Sciences

Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

doi: 10.7150/ijbs.70742

Figure Lengend Snippet: Aberrant TBK1 expression in CRC. (A) Differential TBK1 gene expression between CRC and normal samples from the TCGA database and three published microarray datasets (GSE117606, GSE68468, GSE37182). (B) WB analysis of TBK1 protein in tissue lysates from six randomly selected paired specimens. CRC tumors (T); normal tissues (N). (C) HE staining of normal tissue and CRC tissues. (D) IHC staining of TBK1 in representative normal and CRC tissues; scale bar = 50 µm. (E) Analysis of TBK1 IHC scores in normal tissues and CRC tissues and TBK1 IHC scores in CRC tissues with or without lymph node metastasis (LNM). * P< 0.05, *** P< 0.001, **** P< 0.0001.

Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

Techniques: Expressing, Microarray, Staining, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

doi: 10.7150/ijbs.70742

Figure Lengend Snippet: Relationship between TBK1 expression and clinicopathological factors in CRC patients (* P < 0.05)

Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

Techniques: Expressing

Journal: International Journal of Biological Sciences

Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

doi: 10.7150/ijbs.70742

Figure Lengend Snippet: TBK1 restrained the mTORC1 signaling activation in CRC. (A) Correlation analysis of TBK1 and mTOR in normal tissues and CRC tissues based on the TCGA database. (B) GESA analyses of gene sets for mTORC1 signaling. NES: normalized enrichment score; FDR: false discovery rate. Negative NES indicates lower expression in TBK1-WT to TBK1-KO. (C) The expression of TBK1 and mTOR in CRC cells revealed by IF. Green: mTOR; red: TBK1; blue: DAPI; scale bar: 50 µm. (D) The expression of TBK1 in the five CRC cell lines (HCT116, HT-29, SW480, SW620, LOVO). TBK1 expression was quantified by the gray-scale value of straps. (E) HCT116 cells and SW480 were transfected with NC-siRNA, TBK1-si207 and TBK1-si1953, TBK1, T-mTOR, p-mTOR, AKT, p-AKT, T-S6K1, p-S6K1, 4E-BP1, p-4E-BP1 and GAPDH were analyzed by WB.

Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

Techniques: Activation Assay, Expressing, Transfection

Journal: International Journal of Biological Sciences

Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

doi: 10.7150/ijbs.70742

Figure Lengend Snippet: TBK1 depletion suppressed cell migration, proliferation and drug resistance in CRC. (A) Representative photographs of scratch wound assay of HCT116 and SW480 cells transfected with NC-siRNA or TBK1-siRNA. (scale bar = 50 µm). (B) The quantification analysis of the relative scratch area, mean ± SD (n=3). (C) Representative photographs of transwell assay of HCT116 and SW480 cells transfected with NC-siRNA or TBK1-siRNA. (scale bar = 50 µm). (D) The quantification of the migratory cell rate, mean ± SD (n=3). The relative cell viabilities of HCT116 (E) and SW480 (F) cells were tested with a CCK-8 assay, mean ± SD (n=3). CTL: transfected with NC-siRNA, 50 nM; DMSO: treated with DMSO; si-TBK1: transfected with TBK1-siRNA, 50 nM; 5-FU: treated with 5-FU; si-TBK1+5-FU: transfected with TBK1-siRNA+5-FU.

Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

Techniques: Migration, Scratch Wound Assay Assay, Transfection, Transwell Assay, CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

doi: 10.7150/ijbs.70742

Figure Lengend Snippet: The inhibition of mTORC1 signaling increased the GLUT1 expression in CRC. (A) Correlation analysis of TBK1 and GLUT1 in normal colorectal and CRC tissues based on the TCGA database. (B) The lysates of HCT116 and SW480 transfected with two TBK1 siRNAs were blotted for GLUT1 and GAPDH. (C) HCT116 and SW480 cells transfected with NC-siRNA, TBK1-siRNA, vector plasmid and TBK1 WT plasmid as indicated, the cell lysis was immunoblotted. (D) The expression of TBK1, p-mTOR, p-S6K1, GLUT1and p-4E-BP1 were quantified. Data are mean ± SD (n=3) NC: negative control; TBK1-KD: TBK1 knockdown; TBK1-OE: TBK1 overexpression. (E) HCT116 and SW480 were treated with rapamycin (5 µM) for 12 h, the lysis was blotted and the GLUT1 expression was quantified. * P< 0.05, ** P< 0.01.

Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

Techniques: Inhibition, Expressing, Transfection, Plasmid Preparation, Lysis, Negative Control, Over Expression

Journal: International Journal of Biological Sciences

Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

doi: 10.7150/ijbs.70742

Figure Lengend Snippet: TBK1-induced autophagy inhibited GLUT1 degradation in CRC. (A) The mRNA levels of GLUT1, mTOR, Raptor and S6K1 in HCT116 cells transfected with NC-siRNA or TBK1-si207 for 24 h. (B) HCT116 cells were treated with cycloheximide (CHX) for 12 h after being transfected with NC-siRNA or TBK1-si207 for 24 h. GLUT1 and GAPDH of the cell lysates were blotted. The degradation curve is according to the relative GLIT1 grayscale value of each time point, and the bands were quantified and presented as the mean ± SD (n=3). (C) WB analysis of P62, GLUT1, LC3 II/I and GAPDH from whole-cell lysates. HCT116 cells were transfected with NC-siRNA, TBK1-si207, TBK1-si1953, vector plasmid and TBK1 WT plasmid as indicated. (D) WB analysis of TBK1, P62, GLUT1 and GAPDH in HCT116 cells with stable TBK1 knockdown or negative control. (E) Negative control (NC) and stable TBK1-knocked down HCT116 cells were separately transfected with NC-siRNA, ATG7-siRNA and TBC1D5-siRNA, and the whole cell lysates were immunoblotted for the indicated proteins.

Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

Techniques: Transfection, Plasmid Preparation, Negative Control

Journal: International Journal of Biological Sciences

Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

doi: 10.7150/ijbs.70742

Figure Lengend Snippet: TBK1 facilitated the cell membrane localization of GLTU1 in CRC. (A) Representative IHC staining of TBK1 and GLUT1 in normal, dysplasia and CRC tissues (scale bar = 50 µm). Insets: Magnification of the boxed regions. (B) IF staining for GLUT1 in HCT116 cell with GFP-GLUT1 expression with indicated treatment. Scale bars: 8 µm. Insets: Magnification of the boxed regions. (C) Fluorescence images of HCT116 cells treated with 2-NBDG (100 µM) for 3h after NC-siRNA, TBK1-siRNA, Vector plasmid or TBK1 WT plasmid. The mean fluorescence intensities were quantified with Image J. Mean±SD, n=3, * P< 0.05.

Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

Techniques: Immunohistochemistry, Staining, Expressing, Fluorescence, Plasmid Preparation

Journal: International Journal of Biological Sciences

Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

doi: 10.7150/ijbs.70742

Figure Lengend Snippet: TBK1 is a promising target for CRC treatment. (A) HCT116 and SW480 cells were treated with amlexanox (100 µM) or poly (I:C) (2 µg/ml) for 24 h, and the lysates were blotted for GLUT1. (B) The GLTU1 expression was quantified (mean±SD, n=3, * P< 0.05). (C) Representative image of tumors derived from NC-shRNA or TBK1-shRNA transfected HCT116 cells in nude mice (5/group). (D) Quantification of tumor volume and weight of NC and TBK1-KD groups (mean ± SD, n=5.) CCK-8 assay of HCT116 (E) and SW480 (F) cells treated as indicated. 5-FU, 5 µM; amlexanox (100 µM) for 24, 48 and 72 h. Data are mean ± SD (n=3). * P< 0.05, ** P< 0.01, *** P< 0.001.

Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

Techniques: Expressing, Derivative Assay, shRNA, Transfection, CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

doi: 10.7150/ijbs.70742

Figure Lengend Snippet: TBK1 expression in colorectal tumor tissues and paracancer tissues

Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

Techniques: Expressing

Journal: Theranostics

Article Title: Plasma-derived DNA containing-extracellular vesicles induce STING-mediated proinflammatory responses in dermatomyositis

doi: 10.7150/thno.59152

Figure Lengend Snippet: Inhibition of STING impaired immunostimulatory effects of DM plasma derived small extracellular vesicles in PBMCs. (A) sEVs-stimulated PBMCs secreted less IFNβ release when STING antagonist H-151 (1μM) was present ((21.58 ± 5.45 vs. 28.34 ± 4.25) pg/mL; n = 6). (B) sEVs-stimulated PBMCs secreted less TNFα release when STING antagonist H-151 was present (434.8 ± 231.5 vs. 919.1 ± 325.7) pg/mL; n = 6). (C) sEVs-stimulated PBMCs secreted less IL6 release when STING antagonist H-151 was present ((611.5 ± 132.8 vs. 844.2 ± 180.3) pg/mL; n = 6). (D) STING antagonist H-151 suppressed DM plasma-derived sEV-induced STING phosphorylation and its downstream signaling pathway TBK1, IRF3, and NFκB phosphorylation in PBMCs. (E) Relative intensity of phosphorylated STING, phosphorylated TBK1, phosphorylated IRF3, and phosphorylated NFκB in PBMCs stimulated with/without DM derived sEVs in the presence/absence of STING antagonist H-151 (n = 3). Data in (A,B,C,E) represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 between groups as indicated. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test. Comparison between two groups was analyzed by the Student t test.

Article Snippet: Phospho-STING (Ser366) (D7C3S) rabbit antibody, Phospho-STING (Ser366) (D8K6H) rabbit antibody, Phospho-STING (Ser365) (D8F4W) rabbit antibody, STING (D2P2F) rabbit antibody, phospho-TBK1/NAK (Ser 172) (D52C2) rabbit antibody, TBK1/NAK (D1B4) rabbit antibody, phospho-IRF-3 (Ser396) (D601M) rabbit antibody, IRF-3 (D83B9) rabbit antibody, and β-Actin (8H10D10) mouse antibody were obtained from Cell Signaling Technology Company (Danvers, MA).

Techniques: Inhibition, Derivative Assay

Journal: Theranostics

Article Title: Plasma-derived DNA containing-extracellular vesicles induce STING-mediated proinflammatory responses in dermatomyositis

doi: 10.7150/thno.59152

Figure Lengend Snippet: Inhibition of TBK1 decreased DM plasma derived small extracellular vesicles' immunostimulatory effects in PBMCs. (A) Representative immunofluorescent staining images showing that sEVs derived from DM plasma induced phosphorylation of TBK1 in PBMCs. (Scale bar 100 µm) (B) Bar graphic depicting the relative intensity of phosphorylated TBK1 immunofluorescent staining in PBMCs with/without DM plasma-derived sEVs stimulation (n = 5). (C) TBK1 inhibitors suppressed DM plasma-derived sEVs induced TBK1 and IRF3 phosphorylation in PBMCs. (D) DM plasma-derived sEVs induced IFNβ release in PBMCs (11.40 ± 4.669 pg/mL, n = 5) when compared with untreated PBMCs (2.000 ± 0.7674 pg/mL, n = 5). 2.5 µM of Amlexanox (TBK1 inhibitor) pretreatment impaired sEVs-triggered IFNβ release in PBMCs (3.933 ± 2.002 pg/mL, n = 5); 2.5 µM of MRT67307 (TBK1 inhibitor) pretreatment impaired DM sEVs-triggered IFNβ release in PBMCs (4.067 ± 1.511 pg/mL, n = 5). Data in B represent median. Data in D represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 between groups as indicated. Comparison between two groups was analyzed by the Student t test. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test.

Article Snippet: Phospho-STING (Ser366) (D7C3S) rabbit antibody, Phospho-STING (Ser366) (D8K6H) rabbit antibody, Phospho-STING (Ser365) (D8F4W) rabbit antibody, STING (D2P2F) rabbit antibody, phospho-TBK1/NAK (Ser 172) (D52C2) rabbit antibody, TBK1/NAK (D1B4) rabbit antibody, phospho-IRF-3 (Ser396) (D601M) rabbit antibody, IRF-3 (D83B9) rabbit antibody, and β-Actin (8H10D10) mouse antibody were obtained from Cell Signaling Technology Company (Danvers, MA).

Techniques: Inhibition, Derivative Assay, Staining

Journal: Theranostics

Article Title: Plasma-derived DNA containing-extracellular vesicles induce STING-mediated proinflammatory responses in dermatomyositis

doi: 10.7150/thno.59152

Figure Lengend Snippet: Digestion of DM plasma-derived small extracellular vesicles-captured DNA impaired their triggered STING signaling pathway activation in PBMCs. (A) The effects of DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without DNase I on STING phosphorylation and its downstream signaling pathway TBK1, and IRF3 phosphorylation in PBMCs. (B) The effects of DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without dsDNase on STING phosphorylation and its downstream signaling pathway TBK1, and IRF3 phosphorylation in PBMCs. Relative intensity of phosphorylated STING (C), phosphorylated TBK1(D), and phosphorylated IRF3 (E) in PBMCs stimulated by DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without DNase I (n = 3). Relative intensity of phosphorylated STING (F), phosphorylated TBK1(G), and phosphorylated IRF3 (H) in PBMCs stimulated by DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without dsDNase (n = 3). Data were represent mean ± SD. * P < 0.05,** P < 0.01 between groups as indicated. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test.

Article Snippet: Phospho-STING (Ser366) (D7C3S) rabbit antibody, Phospho-STING (Ser366) (D8K6H) rabbit antibody, Phospho-STING (Ser365) (D8F4W) rabbit antibody, STING (D2P2F) rabbit antibody, phospho-TBK1/NAK (Ser 172) (D52C2) rabbit antibody, TBK1/NAK (D1B4) rabbit antibody, phospho-IRF-3 (Ser396) (D601M) rabbit antibody, IRF-3 (D83B9) rabbit antibody, and β-Actin (8H10D10) mouse antibody were obtained from Cell Signaling Technology Company (Danvers, MA).

Techniques: Derivative Assay, Activation Assay

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

Journal: Nature

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

doi: 10.1038/s41586-022-05354-0

Figure Lengend Snippet: a , HeLa STING KO cells transfected with FLAG-tagged STING WT or STING LR(L374A/I375A) were treated with 2.5 µM diABZI for 0, 1 or 2 h, immunoprecipitated with anti-FLAG antibody, and analysed by western blot. b , HeLa TBK1 KO cells reconstituted with an empty plasmid or with plasmids expressing TBK1 WT or enzyme-dead TBK1 S172A were treated with 2.5 µM diABZI for 2 h and analysed by western blot. GAPDH was used as a processing control. c , HeLa cells pretreated with DMSO or 2 µM BX795 for 24 h were stimulated with 2.5 µM diABZI or not (2 h) and analysed by western blot. Vinculin was used as a loading control. One representative of at least two ( a – c ) independent experiments is shown. Ratios of target proteins versus loading control normalized to the untreated sample of each condition ( b , c ). d , Mass spectrometry detected molecular weight of SUMO LBD-STING and TBK1-phosphorylated LBD-STING (pSTING). e , Bio-layer interferometry binding studies of LBD-STING ELI(E360A/L364A/I365A) with AP-1 ΔμCTD. f , Bio-layer interferometry binding studies of LBD-STING 3S(S355D/S358D/S366D) with AP-1 ΔμCTD. One representative of at least two ( e , f ) 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: Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Expressing, Mass Spectrometry, Molecular Weight, Binding Assay

Journal: mBio

Article Title: Caspase-Mediated Regulation and Cellular Heterogeneity of the cGAS/STING Pathway in Kaposi’s Sarcoma-Associated Herpesvirus Infection

doi: 10.1128/mbio.02446-22

Figure Lengend Snippet: Caspase activity prevents cGAS activation during KSHV lytic replication to block IFN-β induction. (A to C) iSLK.219 cells were transfected with a negative-control siRNA or siRNAs targeting the indicated proteins. For cGAS and RIG-I, the transfection was carried out twice, 2 days prior to and on the day of lytic cycle induction; for STING, MAVS, and IFI16, one transfection was carried out 2 days prior to induction. The cells were then lytically reactivated with doxycycline (1 μg/mL) and treated with either dimethyl sulfoxide (DMSO, vehicle) or IDN-6556 (10 μM) as indicated. (A and B) Total RNA was extracted at day 5 postreactivation, and the levels of IFN-β (A) and ORF37 (B) mRNA were measured by RT-qPCR and normalized to levels of 18S rRNA ( n ≥ 3). (C) Cell lysates were harvested at day 4 postreactivation and subjected to Western blotting for cGAS and β-tubulin as a loading control. The asterisk indicates a nonspecific band. Blots are representative of 3 replicates. (D to F) iSLK.219 cells were lytically reactivated with doxycycline (1 μg/mL) and treated with either DMSO (vehicle) or IDN-6556 (10 μM) and the cGAS inhibitor RU.521 (cGASi; 24.1 μM), where indicated. (D) Total RNA was extracted from iSLK.219 cells 3 days after reactivation. Levels of IFN-β mRNA were measured by RT-qPCR and normalized to 18S rRNA ( n = 3). (E) Cell lysates were harvested at day 4 postreactivation and subjected to Western blotting for p-TBK-1, TBK-1, p-IRF-3, IRF-3, p-STING, STING, and β-tubulin (as a loading control) as indicated. The asterisk indicates a nonspecific band. Blots are representative of 3 replicates. (F) Levels of 2′,3′-cGAMP in lysate collected from iSLK.219 cells at day 4 postreactivation were measured by ELISA ( n = 4). ns, not significant ( P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Tukey’s multiple-comparison test after two-way ANOVA).

Article Snippet: The following Cell Signaling Technologies antibodies were used with PVDF at a 1:1,000 dilution in 5% bovine serum albumin (BSA) in TBST: anti-caspase-8 (no. 9746), anti-cleaved caspase-8 (no. 9496), anti-IRF3 (D83B9) (no. 4302), anti-phospho-IRF3 Ser396 (4D4G) (no. 4947), anti-TBK1 (D1B4) (no. 3504), anti-phospho-TBK1 Ser172 (D52C2) (no. 5483), anti-STING (D2P2F) (no. 13647), anti-phospho-STING Ser366 (D7C3S) (no. 19781), anti-RIG-I (D14G6) (no. 3743), anti-NF-κB p65/RelA (D14E12) (no. 8242S), anti-phospho-NF-κB p65 Ser536/RelA (93H1) (no. 3033), and anti-β-tubulin (9F3) (no. 2128).

Techniques: Activity Assay, Activation Assay, Blocking Assay, Transfection, Negative Control, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Biological Research

Article Title: Intravitreal injection of mitochondrial DNA induces cell damage and retinal dysfunction in rats

doi: 10.1186/s40659-022-00390-6

Figure Lengend Snippet: A1 Western blotting was used to evaluate the changes in proteins in retinas at 1, 3, 5, and 7 days after mtDNA injection (0.02 µg/µl, 2 µl). A2 – A6 Western blotting analysis of cGAS, STING, phospho-TBK1, phospho-IRF3, and INF-β. B1 – B3 Real-time PCR was used to evaluate the transcription of cGAS, STING, and IFNB1. *P < 0.05, **P < 0.01, n = 3 biological replicates in each group

Article Snippet: The following primary antibodies were used: rabbit anti-BAX (#2772, Cell Signaling Technology), rabbit anti-BAK (#12105, Cell Signaling Technology), rabbit anti-caspase 9 (AF6348, Affinity Biosciences Ltd), rabbit anti-cleaved caspase 9 (AF5240, affbiotech), rabbit anti-caspase 3 (ab44976, Abcam), rabbit anti-cleaved caspase 3 (ab49822, Abcam), anti-β-actin (ab8227, Abcam), anti-cGAS (ab179785, Abcam), rabbit anti-STING (D1V5L) (#50,494, Cell Signaling Technology), rabbit anti-TBK1/NAK (D1B4) (#3504, Cell Signaling Technology), rabbit anti-phospho-TBK1/NAK (Ser172) (D52C2) XP ® (#5483, Cell Signaling Technology), rabbit anti-IRF3 (D83B9) (#4302, Cell Signaling Technology), rabbit anti-phospho-IRF3 (Ser396) (D6O1M) (#29047, Cell Signaling Technology), anti-interferon β (ab140211, Abcam), rabbit anti-cytochrome c (10993-1-AP, Proteintech), and rabbit anti-VDAC1 (ab154856; Abcam).

Techniques: Western Blot, Injection, Real-time Polymerase Chain Reaction