Journal: Journal of Virology

Article Title: The Interferon Stimulator Mitochondrial Antiviral Signaling Protein Facilitates Cell Death by Disrupting the Mitochondrial Membrane Potential and by Activating Caspases

doi: 10.1128/JVI.02174-09

Figure Lengend Snippet: Caspase-cleaved MAVS loses the ability to activate the IFN promoter. N18 cells were cotransfected with p125-Luc (0.3 μg), IRF-3/pCR3.1 (0.3 μg), pRL-TK (0.1 μg), and the indicated MAVS constructs (0.6 μg) for 24 h, and the cell lysates were harvested and analyzed by dual-luciferase assay and by Western blotting, using antibodies against Flag, Ser396-phosphorylated IRF-3, IRF-3, and actin, as indicated at the left. Firefly luciferase activity was normalized to that of Renilla luciferase, and the change in induction relative to that of the green fluorescent protein (GFP) control was determined. The results are expressed as averages and standard deviations (n = 3 per group).

Article Snippet: The nonspecific antibody binding sites were blocked with skim milk in PBS-T and then incubated with the following primary antibodies: anti-MAVS (1:1,000; Axxora), anti-phospho-IRF-3 (Ser396), anti-IRF-3, anti-caspase-3 (1:1,000; Cell Signaling), anti-actin (1:10,000; Chemicon), anti-His (1:1,000; Novagen), and anti-Flag M2 (1:5,000; Sigma-Aldrich).

Techniques: Construct, Luciferase, Western Blot, Activity Assay

Journal: Journal of Virology

Article Title: The Interferon Stimulator Mitochondrial Antiviral Signaling Protein Facilitates Cell Death by Disrupting the Mitochondrial Membrane Potential and by Activating Caspases

doi: 10.1128/JVI.02174-09

Figure Lengend Snippet: Bcl-xL but not dnIRF-3 blocks MAVS-induced caspase-3 activation. N18 cells were transfected with plasmids encoding dominant-negative IRF-3 fused with GFP, Flag-tagged Bcl-xL, GFP control (1 μg) plus Flag-MAVS, or Flag-GFP control (1 μg), as indicated at the top, for 24 h. Cell lysates were harvested for Western blot analyses with antibodies against GFP, the Flag tag, caspase-3, and actin, as indicated at the left.

Article Snippet: The nonspecific antibody binding sites were blocked with skim milk in PBS-T and then incubated with the following primary antibodies: anti-MAVS (1:1,000; Axxora), anti-phospho-IRF-3 (Ser396), anti-IRF-3, anti-caspase-3 (1:1,000; Cell Signaling), anti-actin (1:10,000; Chemicon), anti-His (1:1,000; Novagen), and anti-Flag M2 (1:5,000; Sigma-Aldrich).

Techniques: Activation Assay, Transfection, Dominant Negative Mutation, Western Blot, FLAG-tag

Journal: Journal of Virology

Article Title: The Interferon Stimulator Mitochondrial Antiviral Signaling Protein Facilitates Cell Death by Disrupting the Mitochondrial Membrane Potential and by Activating Caspases

doi: 10.1128/JVI.02174-09

Figure Lengend Snippet: MAVS- and virus-induced IFN signaling is attenuated in MAVS knockdown cells. (A) Lentivirus-transduced A549 cells with (+) or without (−) MAVS-targeting shRNA were transfected with p125-Luc, IRF-3/pCR3.1, pRL-TK plus GFP, or MAVS as described in the legend to Fig. ​Fig.3.3. For DEN-2-induced IFN-β promoter analysis, the cells were adsorbed with DEN-2 (MOI = 5) for 2 h and then transfected with p125-Luc (0.6 μg) and pRL-TK (0.15 μg) for 24 h. The change in IFN-β promoter activation was determined as described in the legend to Fig. ​Fig.3.3. The IFN-β induction of the indicated groups was compared by two-tailed Student's t test. (B and C) Cells were prepared as described for panel A, and the cell lysates were analyzed by Western blotting with the antibodies indicated at the left.

Article Snippet: The nonspecific antibody binding sites were blocked with skim milk in PBS-T and then incubated with the following primary antibodies: anti-MAVS (1:1,000; Axxora), anti-phospho-IRF-3 (Ser396), anti-IRF-3, anti-caspase-3 (1:1,000; Cell Signaling), anti-actin (1:10,000; Chemicon), anti-His (1:1,000; Novagen), and anti-Flag M2 (1:5,000; Sigma-Aldrich).

Techniques: shRNA, Transfection, Activation Assay, Two Tailed Test, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: WDR5 is essential for assembly of the VISA-associated signaling complex and virus-triggered IRF3 and NF-?B activation

doi: 10.1073/pnas.0908967107

Figure Lengend Snippet: WDR5 is associated with VISA, TRAF3, and TRAF6. (A) WDR5 interacts with VISA, TRAF3, and TRAF6 but not RIG-I, MITA, TBK1, or IRF3 in overexpression system; 293 cells (2 × 106) were transfected with the indicated plasmids (5 μg each). Coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies (Upper). Expression of the transfected proteins were analyzed by immunoblots with anti-HA or anti-Flag (Lower). (B) Domain mapping of the VISA-WDR5 interaction. 293 cells (2 × 106) were transfected with the indicated plasmids (5 μg each). Coimmunoprecipitation was performed with anti-Flag or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-HA (Top). Expression of the transfected proteins were analyzed by immunoblots with anti-HA (Middle) or anti-Flag (Bottom). (C) WDR5 interacts with VISA, TRAF3, and TRAF6 in untransfected cells; 293 cells (5 × 107) were infected with SeV for the indicated time or left uninfected. Cell lysates were immunoprecipitated with the indicated antisera or control serum. The immunoprecipitates were analyzed by immunoblots with anti-VISA or anti-WDR5 as indicated (Upper). The levels of the proteins were analyzed by immunoblots with the indicated antibodies (Lower). Ig, control mouse IgG; αF, anti-Flag; Pre, preimmune serum; αT3, anti-TRAF3; αT6, anti-TRAF6.

Article Snippet: IL-1 (R&D Systems); MitoTracker Red (Molecular Probes); mouse monoclonal antibodies against Flag, HA and β-actin (Sigma), cIAP1 (R&D), AIF, KDEL, and H2B (Santa Cruz Biotechnology); rabbit polyclonal antibodies against TRAF3, TRAF6, and IRF3 (Santa Cruz Biotechnology), and phospho-IRF3 (Ser396) (Upstate) were purchased from the indicated manufacturers.

Techniques: Over Expression, Transfection, Western Blot, Expressing, Infection, Immunoprecipitation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: WDR5 is essential for assembly of the VISA-associated signaling complex and virus-triggered IRF3 and NF-?B activation

doi: 10.1073/pnas.0908967107

Figure Lengend Snippet: Knockdown of WDR5 inhibits SeV-induced activation of IRF3, NF-κB and the IFN-β promoter (A) Effects of WDR5-RNAi plasmids on the expression of WDR5. 293 cells (2 × 105) were transfected with expression plasmids for Flag-WDR5 and Flag-V59 (0.1 μg each), and the control or indicated WDR5 RNAi plasmids (1 μg). Twenty-four hours after transfection, cell lysates were analyzed by immunoblot with anti-Flag. The WDR5 bands from three independent experiments were quantitated using the Bio-Rad Quantity One Program and normalized by levels of the control protein V-59. The average levels of WDR5 from the three experiments are shown at the bottom of the blot. **, P < 0.01, n = 3. (B, D, and F) Effects of WDR5-RNAi plasmids on SeV-induced activation of the IFN-β promoter (B), ISRE (D), and NF-κB (F); 293 cells (2 × 105) were transfected with the indicated RNAi plasmids together with the indicated reporter plasmids. Cells were left uninfected or infected with SeV for 8 h before luciferase assays were performed. Graphs show mean ± SD, n = 3. **, P < 0.01. (C) Effects of WDR5 knockdown on transcription of endogenous IFNB1 gene. 293 cells (2 × 105) were transfected with a control or the indicated WDR5-RNAi plasmids. Twenty-four hours after transfection, cells were infected with SeV for 6 h or left uninfected before RT-PCRs were performed with the indicated primers. (E) Knockdown of WDR5 inhibits SeV-induced IRF3 phosphorylation; 293 cells (2 × 105) were transfected with a control or the indicated WDR5-RNAi plasmids. Twelve hours after transfection, cells were selected with puromycin (1 μg/mL) for 24 h, then infected with SeV for 6 h or left uninfected. Cell lysates were analyzed by immunoblots with the indicated antibodies. (G) WDR5-RNAi does not inhibit IL-1-induced NF-κB activation. 293 cells (2 × 105) were transfected with NF-κB reporter plasmid together with a control or WDR5-RNAi plasmid. Twenty-four hours after transfection, cells were left untreated or treated with IL-1 (20 ng/mL) for 8 h before luciferase assays were performed. Graphs show mean ± SD, n = 3. (H) Knockdown of WDR5 inhibits RIG-I-, RIG-I-CARD, VISA-, MITA- but not TBK1-mediated ISRE activation. 293 cells (2 × 105) were firstly transfected with a WDR5-RNAi or control plasmid (1 μg). Twenty-four hours later, cells were selected with puromycin (1 μg/mL) for 24 h and then retransfected with ISRE reporter and the indicated expression plasmids (0.1 μg each). Luciferase assays were performed 24 h after the second transfection. Graphs show mean ± SD, n = 3. *, P < 0.05; **, P < 0.01.

Article Snippet: IL-1 (R&D Systems); MitoTracker Red (Molecular Probes); mouse monoclonal antibodies against Flag, HA and β-actin (Sigma), cIAP1 (R&D), AIF, KDEL, and H2B (Santa Cruz Biotechnology); rabbit polyclonal antibodies against TRAF3, TRAF6, and IRF3 (Santa Cruz Biotechnology), and phospho-IRF3 (Ser396) (Upstate) were purchased from the indicated manufacturers.

Techniques: Activation Assay, Expressing, Transfection, Western Blot, Infection, Luciferase, Plasmid Preparation

Journal: Cancer research

Article Title: STING promotes homeostasis via regulation of cell proliferation and chromosomal stability

doi: 10.1158/0008-5472.CAN-18-1972

Figure Lengend Snippet: (A) Western analyses of lysates from WT and STING−/− primary MEFs 48 hours post-exposure to increasing doses of IR. The membranes were probed for STING, TBK1, phospho-TBK1 Ser172, IRF3, and phosphor-IRF3 Ser396. Β-actin antibody was used for loading control. (B) IFN-β protein level in WT and STING−/− MEFs supernatant 48 hours following exposure to increasing doses of IR. (C) IFN- β protein secretion in MC-38 with stable shSTING knockdown 48 hours following exposure to increasing doses of IR. (D) Overexpression of STING in HEK293 cells led to a higher IFN-β promoter-driven induction of luciferase activity following exposure to increasing IR dose. (E) Transcriptional profiling of C57BL/6 wild-type (WT) and STINGko primary MEFs 48 hours following 0 or 6 Gy. Venn diagram displays the number of overlapping differentially expressed genes (DEGs) between the basal differences in WT vs. STINGko MEFs and the effects of irradiation in WT MEFs. (F) Top-ranked cellular pathway classification of the 265 DEGs that are activated by IR in WT and STINGko MEFs analyzed using Ingenuity Pathway Analysis (IPA). (G) CDKN1A was predicted as a top upstream regulator that potentially affects most of the 265 DEGs analyzed using IPA Upstream Analysis. (H-K) Western analyses of STING and p21 expression in lysates from primary WT and STINGko MEFs (H), SV40-immortalized WT and STINGko MEFs (I), shSTING HCT116 (J), and shSTING SCC61 (K) harvested 48 hours post-exposure to increasing doses of IR. (L) Kinetic analysis of siCDKN1A HCT116 proliferation in vitro were measured over time in response to 6 Gy IR using the IncuCyte live cell imaging system. In vitro growth curve data are representative of at least two experiments, each with n = 3 per group. P-values were determined using unpaired Student’s t-test. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.005.

Article Snippet: Western blotting antibodies For confirmation of targeted knockdown experiments as well as transient transfection/reconstitution experiments in both murine and human cell lines, the following primary antibodies were used: anti-STING (clone D2P2F; #13647; Cell Signaling Technology), anti-cGAS (#15102 and #31659; Cell Signaling Technology), anti-p21 (ab109199; Abcam), anti-TBK1 (sc-9910; Santa Cruz Biotechnology), anti-phospho-TBK1 Ser172 (clone D52C2; #5483S; Cell Signaling Technology), anti-IRF-3 (clone FL-425; sc-9082; Santa Cruz Biotechnology), anti-phospho IRF3 Ser396 (70R-35220; Fitzgerald Antibodies), anti-STAT1 p84/p91 (clone C-136; sc-464; Santa Cruz Biotechnology), anti- NF-κB p65 (clone D14E12; #8242; Cell Signaling Technology), anti-phospho-NF-κB p65 Ser536 (clone 93H1; #3033; Cell Signaling Technology), anti-FLAG (M2 clone; Sigma), anti-CDC2 (#77055S; Cell Signaling Technology), anti-phospho-CDC2 Tyr15 (clone 10A11; #4539; Cell Signaling Technology), anti-Rb (#9313S; Cell Signaling Technology) anti-phospho-Rb Ser807/811 (clone D20B12; #8516; Cell Signaling Technology), and anti-Actin-HRP (sc-47778; Santa Cruz Biotechnology).

Techniques: Western Blot, Over Expression, Luciferase, Activity Assay, Irradiation, Expressing, In Vitro, Live Cell Imaging