Journal: bioRxiv

Article Title: Microsecond pulse electrical stimulation modulates cell migration

doi: 10.1101/2022.10.23.513372

Figure Lengend Snippet: (a) A graphical illustration showed the effects of μsPEF (pulse width:20 μs, frequency: 10 Hz, duration: 5 s) on the skin wound, promoting cell migration and extracellular matrix remodeling. (b) Representative time-lapse images showing the fibroblasts morphology after μsPEF (i.e., 750 and 1500 V/cm) treatment of different intensity at different time points (i.e., 0, 1 and 2 h). Detached cells are marked by yellow arrow. Scale bar, 50 μm. (c) The line graph representing the cell migration average speed per 1 h from 0 to 12 h after different intensity μsPEF (i.e., 750 and 1500 V/cm) treatment (blue circle: control; orange square: 750 V/cm; pink triangle: 1500 V/cm). Results are presented as mean ± standard deviation with 95% CI (n CTRL =42 cells, n 750 v/cm =48 cells, n 1500 v/cm =47 cells); * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 versus control by one-way ANOVA for multiple comparisons. (d) Box plot showing the average cell migration speed with different intensity μsPEF (i.e., 750 and 1500 V/cm) and control treatments in 24 h. Results are presented as mean ± standard deviation with 95% CI (n CTRL =470 cells, n 750 V/cm =979 cells, n 1500 V/cm =535 cells). (e) Effects of μsPEF on the secretion of COLA2. The level of collagen type I α2 in cellular supernatants was measured after 48 h. The concentration of COLA2 was 0.109 ± 0.018 ng/mL in the control group, 0.257 ± 0.058 ng/mL in the 750 V/cm group and 0.363 ± 0.034 ng/mL in the 1500 V/cm group. Results are presented as mean ± standard deviation with 95% CI (n=5); * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 versus control by one-way ANOVA for multiple comparisons. (f) Effects of μsPEF on the expression of FGF2. The level of FGF2 in cellular supernatants was measured after 48 h. The concentration of FGF2 was 44.139 ± 0.360 ng/mL in the control group, 48.012 ± 1.488 ng/mL in the 750 V/cm group and 48.523 ± 1.944 ng/mL in the 1500 V/cm group. Results are presented as mean ± standard deviation with 95% CI (n=3); ns=0.7329, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 versus control by one-way ANOVA for multiple comparisons.

Article Snippet: Then, the cells were cultured in serum-free medium for 48 h. The content of type I α collagen and basic fibroblast growth factor (FGF-2) in the supernatant were measured using commercially available Human COL1A2 (Collagen Type I Alpha 2) ELISA Kit (Elabscience, Wuhan, China) and Human bFGF/FGF2 (Basic Fibroblast Growth Factor) ELISA Kit (Elabscience, Wuhan, China) according to the manufacturer’s protocol respectively.

Techniques: Migration, Control, Standard Deviation, Concentration Assay, Expressing

Journal: JCI Insight

Article Title: An IKBKE variant conferring functional cGAS/STING pathway deficiency and susceptibility to recurrent HSV-2 meningitis

doi: 10.1172/jci.insight.173066

Figure Lengend Snippet: ( A – H ) iPSC derived microglia ( A – D ) and neurons ( E – H ) were transfected with IKBKE or control siRNA. ( A and E ) The efficiency of transfection was examined by measuring IKBKE mRNA levels. Cells were infected with HSV-2 for 6 hours, and IFNA2 , IFNB1 , and TNFA mRNA levels were quantified. ( I – L ) iPSC-derived microglia were transfected with siRNA targeting cGAS ( I and J ) or TLR3 ( K and L ). Microglia were infected with HSV-2, and IFNB1 mRNA was quantified ( J and L ). Unpaired t test was used for statistical analysis. ( M ) Graphical illustration of experimental setup for microglia-neuron crosstalk experiments. Created with BioRender.com. ( N ) iPSC-derived neurons were treated with Human Type 1 IFN Neutralizing Antibody Mixture or control IgG (both 1:100) 30 minutes before addition of supernatants from HSV-2–infected microglia or treatment with IFN-β (10 ng/mL). Six hours later, the medium was removed, and the neurons were infected with HSV-2. Supernatants were collected after 16 hours for plaque assay ( O ). Microglia subjected to IKBKE or control knockdown with siRNA were infected with HSV-2. The cells were washed, and the medium was replaced after 1 hour. Supernatants were collected from the microglia after 24 hours and added to the neurons. Six hours later, the medium was removed, and the neurons were infected with HSV-2. Supernatants were collected 16 hours later for plaque assay. Data presented are pooled from 2 independently performed experiments. ( P ) Cells were treated as in panel N , and culture supernatants were analyzed for cell viability. Data presented are from 1 of 2 experiments performed. ( N – P ) Groups were compared with Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. Error bars represent SEM ( A – H ) and SD ( N – P ), and *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: To neutralize type I IFN bioactivity in supernatants from microglia, neurons were cultured with Human Type 1 IFN Neutralizing Antibody Mixture (PBL) 30 minutes before addition of culture supernatants from microglia (1:100).

Techniques: Derivative Assay, Transfection, Control, Infection, Plaque Assay, Knockdown

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: HSV1 recombinant viruses expressing cGAS and/or STING exhibit restricted replication in human cancer cells (A) Schematic diagram of rHSV1 constructs. (B–N) 2 × 10 5 293T, hTERT, HT29, and SW48 cells infected with HSV1-Δγ34.5, HSV1-STING, HSV1-cGAS, and HSV1-STING-P2A-cGAS (HSV1-2A) at the MOI indicated. (B) Immunoblot analysis of cGAS, STING, phospho-STING, phospho-TBK1, phospho-IRF3, and β-actin 6 h post infection, (C) percentage of viable cells ( n = 4 biological replicates), and (D) virus titers ( n = 2 biological replicates). (E) Measurement by ELISA of the quantity of 2′3′ cGAMP in 5 × 10 5 293T cells 24 h post infection ( n = 3 biological replicates). (F) IFN-β-luciferase activity in 293T cells 24 h after plasmid transfection followed by 6 h of infection ( n = 3 technical replicates). (G, I, and K) Percentage of viable cells ( n = 3 biological replicates) and (H, J, and L) virus titers ( n = 3 biological replicates) on infected hTERT, HT29, and SW48 cells at MOI 1. (M) qPCR of Cxcl10 ( n = 2 biological replicates) and (N) ELISA analysis of human IFNβ production in hTERT, HT29, and SW48 cells 24 h after infection ( n = 6 biological replicates). Error bars indicate mean ± SEM; Student’s t test ∗ p < 0.05.

Article Snippet: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

Techniques: Recombinant, Expressing, Construct, Infection, Western Blot, Virus, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Plasmid Preparation, Transfection

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Recombinant HSV1 exhibits diminutive oncolytic activity yet retains in vivo anti-tumor properties dependent on extrinsic STING signaling (A) Immunoblot analysis of cGAS, STING, and β-actin in B16-OVA, B16-OVA cGAS KO (CKO), B16-OVA STING KO (SKO), and B16-OVA STING/cGAS KO (S/CKO) cells. (B and C) 2 × 10 5 B16 cells were infected with HSV1-Δγ34.5, HSV1-STING, HSV1-cGAS, and HSV1-STING-P2A-cGAS (HSV1-2A) at MOI 5 for 24 h. (B) Virus titers ( n = 2 biological replicates) and (C) percentage of viable cells were measured ( n = 2 biological replicates). (D–F) 2 × 10 5 293T and B16-OVA cells were infected at MOI 0.1 or 5 with HSV1- Δγ34.5-GFP. (D) The virus titer was determined by plaque assay ( n = 3 biological replicates), (E) the percentage of GFP + cells were measured by cytometry ( n = 3 biological replicates), and (F) the quantification of HSV1- Δγ34.5 genome was done by qPCR at 3 or 6 h, 24 h, and 48 h post infection ( n = 3 biological replicates). (G) B16 cells were infected with HSV1- Δγ34.5, HSV1-STING, HSV1-cGAS, or HSV1-2A at MOI 5 for 6 h. B16-OVA cells were treated with 3 μg/mL dsDNA90 as a control, and Cxcl10 was analyzed by qPCR ( n = 4 technical replicates). (H–Q) Wild-type (H–J: n = 6–8 mice per group; L–N: n = 4 mice per group), STING KO C57BL/6J ( n = 11–12 mice per group on 2 independent experiments), and BALB/c nude mice ( n = 7 mice by groups) were subcutaneously injected as indicated with B16-OVA, B16-OVA CKO, or B16-OVA S/CKO cells on the flank (5 × 10 5 cells/mouse). 5 × 10 6 PFU of replicating HSV1- Δγ34.5, HSV1-STING, HSV1-cGAS, or HSV1-2A was injected intratumorally (black arrows) three times. (H–L and O) The tumor volume was measured on the indicated days and calculated with the formula V = (length × width 2 )/2. At 16 or 17 days, the spleen and the tumors were extracted. (M and P) Digital photograph of tumors and (N and Q) ELISpot to measure IFNg release from CD8 + T cells. (R and S) Phagocytosis of B16-OVA and B16-OVA S/CKO cells by murine WT and STING KO macrophages. 1 × 10 6 cells were infected with HSV1-Δγ34.5 for 40 h at MOI 20 then irradiated by UV (120 mJ/cm) and incubated for 24 h. The irradiated cells were fed to macrophages (MØ) (2 × 10 5 cells). (R) Schematic representation and (S) ELISA analysis of IFN-β at 24 h in macrophages following engulfment of B16 ( n = 5 [WT macrophages] and 3 [STING macrophages] technical replicates). Error bars indicate mean ± SEM; Student’s t test and (H–L and O) ordinary one-way ANOVA test with Tukey’s multiple comparisons test ∗ p < 0.05.

Article Snippet: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

Techniques: Recombinant, Activity Assay, In Vivo, Western Blot, Infection, Virus, Plaque Assay, Cytometry, Control, Injection, Enzyme-linked Immunospot, Irradiation, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Nano-STAVs are readily opsonized by macrophages to stimulate STING signaling (A) Transmission electron microscopy image of nano-empty and nano-STAVs. (B and C) Western blot analysis of phosphorylated and total cGAS, STING, TBK1, and IRF3 proteins in WT or SKO murine macrophages (bone marrow-derived macrophages, BMDMs) and B16-OVA (2 × 10 5 cells) treated with nano-empty, nano-STAVs, or lipofectamine +/− STAVs at 1 μg/ml for 6 h. (D) macrophages and B16-OVA (5 × 10 4 cells) treated with nano-STAVs-Cy5 (red) (3 μg/mL) for 16 h, fixed and stained with DAPI (blue), and analyzed by microscopy confocal. (E and F) B16-OVA and macrophages (5 × 10 4 cells) were treated with nano-STAVs-Cy5 (red) (3 μg/mL) for 16 h, fixed and stained with EEA1-FITC, RAB7-FITC, CD63-FITC, LAMP1-FITC (green), and DAPI (blue), and analyzed by confocal microscopy. (G–N) (G–I and K–M) qPCR analysis of IFNb1, CXCL10, and CCL5 and (J and N) IFNb ELISA in B16-OVA, WT, and SKO macrophages (2 × 10 5 cells), treated with nano-empty, nano-STAVs, or lipofectamine +/− STAVs at 1 μg/ml for 6 h (qPCR) or 24 h (ELISA) ( n = 2 biological replicates). Error bars indicate mean ± SEM; Student’s t test ∗ p < 0.05.

Article Snippet: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Derivative Assay, Staining, Microscopy, Confocal Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Murine and human tumor cells exposed to nano-STAVs activate APCs in trans in an STING-dependent manner, augmenting checkpoint therapeutic activity in vivo (A) Schematic representation of the phagocytosis of mouse and human cells by macrophages. 1 × 10 6 cells were treated with 1 μg/mL of nano-empty or nano-STAVs or transfected with lipofectamine + STAVs and irradiated by UV (120 mJ/cm). The irradiated cells were fed to murine or human macrophages (MØ) (2 × 10 5 cells) 24 h after UV irradiation. (B) Confocal microscopy analysis with nano-STAVs-cy5 (red) in CD11b + FITC murine macrophages (green). Cells were treated for 6 h with nanoparticles, and the phagocytosis was evaluated at 6 h. (C–H) (C, E, and G) RT-qPCR analysis of Cxcl10 at 6 h and (D, F, and H) IFN-β ELISA at 24 h in human and murine WT macrophages following engulfment of B16, SK-MEL-31, and SK-MEL-5 cells in presence or absence of nano-STAVs for 24 h ( n = 4 [mouse cell] and 3 [human cells] biological replicates). (I–L) Mice were subcutaneously injected with B16-OVA cells (5 × 10 5 cells/mouse) ( n = 13–17 mice per group on 2 independent experiments) on the right flank. On days 7, 10, and 13, after tumor inoculation, the mice were intratumorally injected with PBS, STAVs, nano-empty, or nano-STAVs (0.1 μg/mouse) and/or intraperitoneally with PD1 (50 μg/mouse) (black arrows). At day 19, the spleen was extracted to measure IFNg release from CD8 + T cells. (I) Schematic representation of experimental design. (J) The tumor volume was measured and calculated with the formula V = (length × width 2 )/2. (K) Digital photographs of tumors. (L) IFNg ELISpot. (C–H) Error bars indicate mean ± SEM; Student’s t test and (J and L) two-way ANOVA test with Tukey’s multiple comparisons test ∗ p < 0.05.

Article Snippet: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

Techniques: Activity Assay, In Vivo, Transfection, Irradiation, Confocal Microscopy, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Injection, Enzyme-linked Immunospot

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Nano-STAVs activity is augmented by type I IFN (A–D) Mice were subcutaneously injected with B16 OVA cells (5 × 10 5 cells/mouse) ( n = 17–18 mice per group on 2 independent experiments) on the right flank. On days 7, 10, and 13, after tumor inoculation, the mice were intratumorally injected with PBS, STAVs, nano-empty, or nano-STAVs (0.1 μg/mouse) and/or intraperitoneally with PD1 (50 μg/mouse) and/or IFNa (10,000 U/mouse) (black arrows). At day 17, the spleen was extracted to measure IFNg release from CD8 + T cells. (A) Schematic representation of experimental design. (B) The tumor volume was measured and calculated with the formula V = (length × width 2 )/2. (C) IFNg ELISpot. (D) Digital photographs of tumors. (E–J) qPCR analysis of CXCL10 and CCL5 in B16-OVA, B16-OVA-cGAS KO (CKO), and WT and SKO murine macrophages (2 × 10 5 cells), treated with IFNa at 1000 U/ml for 6 h ( n = 4 biological replicates). (K and L) Flow cytometry for H-2Kb and CD86 on macrophages (2 × 10 5 cells) following IFNa treatment at 1000 U/ml for 24 h ( n = 2 biological replicates). (M) Schematic representation of the phagocytosis of B16-OVA cells by macrophages. 1 × 10 6 B16-OVA cells were treated with IFNa at 1000 U/ml for 24 h and irradiated by UV (120 mJ/cm). The irradiated cells were fed to murine macrophages (MØ) (2 × 10 5 cells) previously treated or not with IFNa at 1000 U/ml for 24 h. (N) RT-qPCR analysis of Cxcl10 at 6 h ( n = 2 biological replicates). (O) IFN-β ELISA at 24 h of murine WT macrophages following engulfment of B16-OVA treated with IFNa ( n = 3 biological replicates). (P) Schematic representation of the phagocytosis of untreated B16-OVA cGAS KO cells (B16 CKO) by macrophages previously treated with IFNa at 1000 U/ml for 24 h. The conditions applied were the same as in (M). (Q) Flow cytometry for H-2Kb-SIINFEKL (OVA) on macrophages at 24 h following phagocytosis of B16-OVA cGAS KO ( n = 2 biological replicates). Error bars indicate mean ± SEM; Student’s t test and (B and C) two-way ANOVA test with Tukey’s multiple comparisons test ∗ p < 0.05.

Article Snippet: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

Techniques: Activity Assay, Injection, Enzyme-linked Immunospot, Flow Cytometry, Irradiation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Nano-STAVs can facilitate the immunotherapeutic effects of radiation treatment in a head and neck tumor model (A–H) 2 × 10 5 MOC2 and Cal27 head and neck cell lines were treated with 1 μg/ml nano-empty, nano-STAVs, or lipofectamine +/− STAVs. (A and B) Western blot analysis of total and phosphorylated cGAS, STING, TBK1, IRF3 proteins, (C, D, F, and G) qPCR analysis of IFNb1 and CXCL10 after 6 h, and (E and H) IFNb ELISA 24 h after treatment ( n = 2 biological replicates). (I and J) MOC2 cells were irradiated with 10 Gy X-ray and then incubated for 24 h followed by treatment with nano-STAVs (1 μg/ml) for another 24 h. (I) For checking protein expression, MOC2 cells were collected and lysed with RIPA buffer and then analyzed by immunoblotting with indicated antibodies. (J) For phagocytosis assay, 2 × 10 6 MOC2 cells were phagocytosed with 2 × 10 6 dendritic cells (bone marrow-derived dendritic cells, BMDCs) for 6 h followed by isolating CD11c+ dendritic cells. Cxcl10 expression in dendritic cells was evaluated by qPCR analysis ( n = 2 biological replicates). (K and L) Immunocompetent C57BL/6J mice were subcutaneously injected with 5 × 10 5 MOC2 cells per mouse on the right flank and 2.5 × 10 5 MOC2 cells per mouse on left side. Right-side tumors were irradiated at days 4, 5 and 6 after tumor inoculation at 8 Gy (red arrows). On days 7, 10 and 13, after tumor inoculation, the mice were intratumorally injected on right side only with PBS, STAVs, nano-empty, or nano-STAVs (0.1 μg/mouse) and/or with PD1 (100 μg/mouse) (black arrows). The tumor volume was measured and calculated with the formula V = (length × width 2 )/2. (K) Schematic representation of experimental design. (L and M) (L) Immunocompetent WT ( n = 6 mice per group) or (M) STING KO C57BL/6J mice ( n = 6 mice per group). The tumor volumes are represented from day 21 or 24. (N and O) (N) Primary (right flank) and (O) abscopal effects (left flank) of the treatments ( n = 6 mice per group). Error bars indicate mean ± SEM; (C–J and O) Student’s t test and (N) two-way ANOVA test with Tukey’s multiple comparisons test ∗ p < 0.05.

Article Snippet: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Irradiation, Incubation, Expressing, Phagocytosis Assay, Derivative Assay, Injection

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet:

Article Snippet: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

Techniques: Control, Staining, Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Knock-Out, Plasmid Preparation, Expressing, Software, Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: A novel highly selective allosteric inhibitor of tyrosine kinase 2 (TYK2) can block inflammation- and autoimmune-related pathways

doi: 10.1186/s12964-023-01299-7

Figure Lengend Snippet: QL-1200186 blocks the TYK2 signaling pathway. A Effect of QL-1200186 on the JAK/STAT/ISGF3 pathway. B Human PBMCs were stimulated with human IFNα and pSTAT5 levels were determined. C Th17 cells were stimulated with IL-23 and pSTAT3 levels were determined. D IL-12 induced IFNγ production in NK92 cells. Data are the mean ± SD. At least three independent experiments were carried out

Article Snippet: With regard to IFNα-stimulated phosphorylation of STAT5 in human peripheral blood mononuclear cells (PBMCs), after incubation with QL-1200186 for 1 h, human PBMCs were stimulated with recombinant human IFNα (1000 U/mL; 11200–2; PBL Assay Science) for 30 min. With respect to GM-CSF-stimulated JAK2 activation in U937 cells (Cobioer Biosciences, CBP60277), after 1 h of treatment with QL-1200186, U937 cells were stimulated with GM-CSF (20 ng/mL; C003; Novoprotein, Suzhou, China) for 15 min. With regard to IL-2-stimulated JAK1/3 activation in human PBMCs, after 1 h of incubation with QL-1200186, human PBMCs were stimulated with human IL-2 (20 ng/mL; C013; Novoprotein) for 20 min. With respect to IL-2-stimulated JAK1/3 activation in human whole blood cells, whole blood (50 μL) was preincubated with compounds for 30 min and then stimulated with IL-2 (20 ng/mL) for 15 min.

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: A novel highly selective allosteric inhibitor of tyrosine kinase 2 (TYK2) can block inflammation- and autoimmune-related pathways

doi: 10.1186/s12964-023-01299-7

Figure Lengend Snippet: Inhibitory function and selectivity of QL-1200186 compared with other TYK2 inhibitors. A Effect of TYK2 inhibitors on IFNα-induced pTYK2 expression. B , C Effects of TYK2 inhibitors on pSTAT1 expression in CD3 + T and CD19 + B cells ( n = 3). D Effects of TYK2 inhibitors on IL-12-induced IFNγ production in human whole blood cells ( n = 3). E Effects of TYK2 inhibitors on the differentiation of monocytes into dendritic cells ( n = 2). F Effect of a TYK2 inhibitor and tofacitinib on the activity of JAK 1/3 was detected by IL-2-induced pSTAT5 expression ( n = 3). G , H Effect of compounds on JAK 2/2 activity was determined by thrombopoietin-induced pSTAT3/5 expression ( n = 3)

Article Snippet: With regard to IFNα-stimulated phosphorylation of STAT5 in human peripheral blood mononuclear cells (PBMCs), after incubation with QL-1200186 for 1 h, human PBMCs were stimulated with recombinant human IFNα (1000 U/mL; 11200–2; PBL Assay Science) for 30 min. With respect to GM-CSF-stimulated JAK2 activation in U937 cells (Cobioer Biosciences, CBP60277), after 1 h of treatment with QL-1200186, U937 cells were stimulated with GM-CSF (20 ng/mL; C003; Novoprotein, Suzhou, China) for 15 min. With regard to IL-2-stimulated JAK1/3 activation in human PBMCs, after 1 h of incubation with QL-1200186, human PBMCs were stimulated with human IL-2 (20 ng/mL; C013; Novoprotein) for 20 min. With respect to IL-2-stimulated JAK1/3 activation in human whole blood cells, whole blood (50 μL) was preincubated with compounds for 30 min and then stimulated with IL-2 (20 ng/mL) for 15 min.

Techniques: Expressing, Activity Assay

Journal: FEBS letters

Article Title: Rac1 and PAK1 are upstream of IKK-epsilon and TBK-1 in the viral activation of interferon regulatory factor-3.

doi: 10.1016/j.febslet.2004.04.069

Figure Lengend Snippet: Fig. 6. Inhibition of Rac1 activity results in enhanced influenza A virus or Sendai virus propagation. MDCK cells (A), A549 (B) or HUVEC (C) were treated with toxin B-10436 (10 ng/ml) before and during infection with different influenza A viruses (MOI ¼ 1). (D–F) Alternatively, MDCK cells were transfected with vector or plasmids expressing Rac1wt (E), Rac1N17 (D,E) or dominant negative IKK-e (IKK-e K38A) (E) prior to infection with influenza virus (MOI ¼ 1) or Sendai virus (MOI ¼ 0,1). (G) A549 cells were incubated with 20 lg/ml of a type I IFN receptor blocking antibody and infected with different influenza A viruses (MOI ¼ 1). Supernatants were assayed for progeny virus yields 13 h (A–C), 8 h (D) or 24 h (E,G) post- infection in standard plaque titrations. Virus yields of mock-treated cells were arbitrarily set as 100%.

Article Snippet: A receptor-neutralizing monoclonal antibody (mab) against the type I IFN receptor was purchased from PBL Biomedical Laboratories.

Techniques: Inhibition, Activity Assay, Virus, Infection, Transfection, Plasmid Preparation, Expressing, Dominant Negative Mutation, Incubation, Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: Influenza A Virus Induces Interleukin-27 through Cyclooxygenase-2 and Protein Kinase A Signaling *

doi: 10.1074/jbc.M111.308064

Figure Lengend Snippet: IL-27 treatment stimulates STAT1, STAT2, STAT3, and PKR phosphorylation. A549 cells were treated with recombinant IL-27 (50 ng/ml) for 12 h. Cells were harvested for the detection of: A, STAT1 and phosphorylated STAT1; B, STAT2 and phosphorylated STAT2; C, STAT3 and phosphorylated STAT3; or D, PKR and phosphorylated PKR expression. Densitometric analysis was normalized to the corresponding control in each experiment (*, p < 0.05). E, IFN-α expression level in IL-27-treated PBMCs. For IFN-α neutralization and antiviral assays, freshly isolated PBMCs were treated with recombinant IL-27 (100 ng/ml) for 2 h, then the culture medium supernatant was harvested and incubated with IFN-α neutralization antibody (5 μl/ml) or control IgG (5 μl/ml) at 37 °C for 1 h. The IFN-α Ab-neutralized culture medium was then used for the following antiviral assays (*, p < 0.05). F, A549 cells were incubated with IFN-α Ab-neutralized or control IgG-neutralized culture medium for 12 h before IAV A/Hong Kong/498/97 (H3N2) infection (m.o.i. = 1). Cells were collected 3 h post-infection and total RNA was isolated for detection of NP-specific mRNA, cRNA, and vRNA. The values of IAV-infected A549 cells for three RNAs were designated as 1. Data are expressed as fold-induction relative to the values of IAV-infected A549 cells for three RNAs (*, p < 0.05). G and H, A549 cells were treated as described in F for 12 h, then phosphorylated STAT1 (G) and phosphorylated PKR (H) were detected. The blot is representative of three experiments with similar results. Densitometric analysis relative to STAT1 or PKR levels was expressed as fold-change (*, p < 0.05).

Article Snippet: IFN-α neutralization antibody (21100-2) was purchased from PBL Interferon Source (Piscataway, NJ).

Techniques: Phospho-proteomics, Recombinant, Expressing, Control, Neutralization, Isolation, Incubation, Infection