Journal: Chemical science

Article Title: Dual-targeting biomimetic delivery for anti-glioma activity via remodeling the tumor microenvironment and directing macrophage-mediated immunotherapy.

doi: 10.1039/c7sc04853j

Figure Lengend Snippet: Fig. 4 (A) The cytotoxicity test (IC50) in the U87 and GL261 cells. (B) The cytotoxicity test in the U87 cells cultured with M1-CM or M2-CM. (C) The expression of MRs in TAM1 and TAM2 and the polarization modulation of the drugs. (D) The re-education of TAM2 treated with drugs and the expression of MRs and B7-H4. The ELISA analysis of the expression of TNF-a (E) and TGF-b1 (F) in TAM1 and TAM2 after drug treatment. The ELISA analysis of the expression of TNF-a (G) and TGF-b1 (H) in TAM1 and TAM2 cocultured with U87 cells after treatment.

Article Snippet: The mouse IFN-g Elisa Kit was purchased from R&D Systems (USA).

Techniques: Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Clinical and Experimental Immunology

Article Title: Splenectomy modulates the immune response but does not prevent joint inflammation in a mouse model of RA

doi: 10.1093/cei/uxac052

Figure Lengend Snippet: Comparison of the intracellular Ca 2+ signaling in B and T cells from inguinal and mesenteric LNs of the arthritic control ( n = 3) and -splenectomized ( n = 3) mice. Cells of the inguinal ( A , C , E ) and mesenteric ( B , D , F ) LNs were isolated and loaded with the Ca 2+ -specific indicator Fluo-3, then stimulated with selective activators for B or T cells. The intracellular Ca 2+ change was detected as time-dependent FL-1 fluorescence change for 5 min using a flow cytometer. The baseline was measured for 1 min, and then the cells were stimulated and measured for 5 min. B cells were activated using anti-IgM- ( A , B ) or anti-IgG ( C , D ) antibodies, while T cells ( E , F ), were activated using anti-CD3 antibody cross-linking. Values are presented as mean ± SEM.

Article Snippet: Levels of the sera rheumatoid factor (RF) IgG and IgM levels were measured using the FineTest Mouse RF-IgG or -IgM ELISA Kits (FineTest, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Comparison, Control, Isolation, Fluorescence, Flow Cytometry

Journal: Cell reports

Article Title: SPHK2-Generated S1P in CD11b + Macrophages Blocks STING to Suppress the Inflammatory Function of Alveolar Macrophages

doi: 10.1016/j.celrep.2020.02.112

Figure Lengend Snippet: (A–C) At indicated times lungs were harvested, homogenized and supernatant were used to quantify IL-6 (A), IL1β (B), and IFN-β levels (C) using an ELISA kit (n = 4 mice/group). (D) Neutrophil count was performed (per field) on hematoxylin and eosin-stained bronchoalveolar lavage at indicated times (n = 3 mice/group). (E–G) 30 min before sacrificing the mice at indicated times, (E) Evans blue-labeled albumin was injected retro-orbitally into macrophages (Mφ) or macrophage dep mice. (F) albumin influx (n = 4 mice/group) and (G) the lung wet/dry ratio (n = 5 mice/group) were determined. A representative image of Evans blue accumulation in the lung is shown in E from experiments that were repetated four times. (H) Macrophages were counted in hematoxylin and eosin-stained bronchoalveolar lavage at indicated times (n = 3 mice/group). (I) BrdU (5-bromo-2’-deoxyuridine) was injected i.t. 4 h before sacrificing the mice. Lungs were digested with collagenase and stained with CD11c and BrdU antibodies (n = 3 mice/group). (J) Lung cells were stained with CD11c, CD45, and Siglec-F antibodies and cytokine expression in alveolar macrophages (CD11c + /CD45 + /Siglec-F + ) were determined using qPCR, taking GAPDH as control in (n = 4 mice/group). Alveolar macrophages were sorted twice independently. (A–D) and (F–J) show mean ± SD. *p < 0.05, ** p < 0.01, *** p < 0.001 relative to macrophages or macrophage dep mice receiving vehicle alone; # p < 0.05, ## p < 0.01, ### p < 0.001 relative to macrophage group after receiving LPS at the indicated time. Analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: IFN-β ELISA Kit, Mouse , Signosis , Cat# CUS-EA-2001.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Labeling, Injection, Expressing

Journal: Cell reports

Article Title: SPHK2-Generated S1P in CD11b + Macrophages Blocks STING to Suppress the Inflammatory Function of Alveolar Macrophages

doi: 10.1016/j.celrep.2020.02.112

Figure Lengend Snippet: (A–C) Lungs receiving 1 × 10 4 CFU of P. aeruginosa (PA) i.t. were harvested at the indicated times and homogenized, and supernatants were used to quantify IL-6 (A), IL-1β (B), and IFN-β (C) levels using an ELISA kit (n = 4 mice/group). (D) Lung edema determined as described in . Plot shows individual values (n = 5 mice/group). (E) Mouse survival was assessed every 6–12 h after 1 × 10 6 CFU of PA instillation (n = 10 mice/group). (A–E) are shown as mean ± SD from experiments that were repeated three times independently. *p < 0.05, *** p < 0.001 relative to unexposed macrophages (Mφ) or macrophage dep mice; ### p < 0.001 relative to macrophage group after PA challenge at indicated times. Analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: IFN-β ELISA Kit, Mouse , Signosis , Cat# CUS-EA-2001.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: SPHK2-Generated S1P in CD11b + Macrophages Blocks STING to Suppress the Inflammatory Function of Alveolar Macrophages

doi: 10.1016/j.celrep.2020.02.112

Figure Lengend Snippet: (A) Phosphorylation of TBK1, IRF3, and p65 subunit of NF-κB in lung lysates of mice after LPS challenge was determined using indicated antibodies. Actin was used as a loading control. Experiments were repeated three times independently. (B) cGAMP was extracted from lungs and quantified by liquid chromatography-mass spectrometry (LC-MS). Data are represented as mean ± SD from experiments that were repeated twice independently (n = 4 mice/group). *p < 0.05, *** p < 0.001 relative to untreated macrophage (MF) and macrophage dep mice; ## p < 0.01 relative to macrophage mice after a 16-h LPS challenge. (C) WT and Sting −/− BMDMs were exposed to LPS, and IRF3 phosphorylation was determined as in (A). A representative immunoblot is shown from experiments that were independently repeated three times. (D) Wet/dry ratio in naive or LPS-exposed WT and Sting −/− lungs was determined as in . n = 5 mice/group. Data are represented as mean ± SD from experiments that were repeated two times independently (n = 4 mice/group). *p < 0.05, *** p < 0.001 relative to WT and Sting −/− untreated mice; ## p < 0.01 relative to WT mice after a 4-h LPS challenge. (E) Protocol shows timing of adoptive transfer of bone marrow CD11b monocytes (CD11b Mo) i.v. in macrophage dep mice after LPS challenge and DT injection. Lung injury was determined at 24 h as in . Macrophage dep mice receiving PBS in lieu of cells and macrophage dep mice challenged with or without LPS served as controls. (F) Phosphorylation of indicated proteins was determined without or with adoptive transfer of cells and LPS challenge as in (A). A representative immunoblot is shown from experiments that were independently repeated three times. (G and H) GM-CSF (G) and IFN-β (H) RNA expression without or with adoptive transfer of cells and LPS challenge using qPCR (n = 4 mice/group). (I and J) A representative immunoblot shows alteration in IFN-β expression after LPS challenge at indicated times. Actin was used as a loading control. Plot in J shows individual pixel intensities of IFN-β expressed as arbitrary units (a.u.) along with mean and SD (n = 3 mice/group). (K) Lung wet/dry weight ratio without or with adoptive transfer of cells and LPS challenge (n = 5 mice/group). Data in (G), (H), (J), and (K) are represented as mean ± SD from experiments that were repeated two to three times independently. *p < 0.05, ** p < 0.01, *** p < 0.001 relative to untreated macrophages and macrophage dep mice; ## p < 0.01, ### p < 0.001 relative to macrophage dep mice receiving LPS for 24 h or PBS after a 24-h LPS challenge. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: IFN-β ELISA Kit, Mouse , Signosis , Cat# CUS-EA-2001.

Techniques: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Adoptive Transfer Assay, Injection, RNA Expression, Expressing

Journal: Cell reports

Article Title: SPHK2-Generated S1P in CD11b + Macrophages Blocks STING to Suppress the Inflammatory Function of Alveolar Macrophages

doi: 10.1016/j.celrep.2020.02.112

Figure Lengend Snippet: (A) BMDMs isolated from indicated mice were exposed to LPS and IFN-β level was determined as in . Data are represented as mean ± SD from three independent experiments. ** p < 0.01, *** p < 0.001 relative to unstimulated WT or SPHK2-null BMDMs; ### p < 0.001 relative to WT BMDMs after an 8-h LPS challenge. (B) Phosphorylation of TBK1, IRF3, and p65 subunit of NF-κB in indicated BMDMs as in . A representative immunoblot is shown from experiments that were independently repeated three times. (C) BMDM lysates containing equal amount of protein were incubated with ATP and sphingosine at 37°C. After 1 h, the reaction was stopped and S1P was extracted. S1P levels were determined using an ELISA kit. SPHK activity was calculated as pmol/min/μg of protein. Experiments were performed twice independently using duplicate samples. Data are represented as mean ± SD from two independent experiments. *p < 0.05, *** p < 0.001 relative to unstimulated WT or SPHK2-null BMDMs; # p < 0.05 relative to LPS-exposed WT BMDMs. (D) BMDMs were pretreated with vehicle or SPHK2 inhibitor ABC294640 (5 μM) for 1 h followed by stimulation with LPS for the indicated times. Phosphorylation of indicated proteins was determined. A representative immunoblot is shown from experiments that were independently repeated three times. (E) U937 human monocytic cells were differentiated into macrophages by exposing them to phorbol myristate acetate (PMA) (100 ng/mL) for 48 h, deprived of serum for 1 h, and followed by the addition of 1 μg/mL LPS for determining phosphorylation of indicated proteins. A representative immunoblot is shown from experiments that were independently repeated three times. (F) BMDMs stimulated with or without LPS were stained with anti-STING antibody followed by FITC-labeled secondary antibody and DAPI. A representative confocal image is shown from experiments that were repeated two times. Scale bars, 10 μm. (G and H) BMDMs permeabilized with digitonin for 30 min were stimulated with 10 μM cGAMP for the indicated times and lysed. Phosphorylation was determined as in (G) while IFN-β levels were quantified as in (H). A representative immunoblot is shown from experiments that were independently repeated three times. Data in (H) are represented as mean ± SD from two independent experiments. ** p < 0.01, *** p < 0.001 relative to unstimulated WT or SPHK2-null BMDMs; ## p < 0.01 relative to WT BMDMs after cGAMP challenge. (I) BMDMs were transfected with WT SPHK2 or control vector. After 48 h, cells were stimulated with LPS to determine the phosphorylation of NF-κB-p65, TBK1, and IRF3. The western blot was then stripped and immunoblotted with GFP to assess SPHK2 expression. A representative immunoblot is shown from experiments that were independently repeated at least three times. (J) BMDM-DNA fragments were purified and immunoprecipitated (IP) with immunoglobulin G (IgG) or antibody against NF-κB, and the resulting chromatin fragments were subjected to PCR amplification using primers spanning SPHK2 promoter. PCR gel in right shows NF-κB enrichment on the SPHK2 promoter using ChIP. qPCR was performed using primers specific for SPHK2. Data are represented as mean ± SD from three independent experiments. *p < 0.05 relative to unstimulated WT BMDMs. Data were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: IFN-β ELISA Kit, Mouse , Signosis , Cat# CUS-EA-2001.

Techniques: Isolation, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Activity Assay, Staining, Labeling, Transfection, Plasmid Preparation, Expressing, Purification, Immunoprecipitation, Amplification

Journal: Cell reports

Article Title: SPHK2-Generated S1P in CD11b + Macrophages Blocks STING to Suppress the Inflammatory Function of Alveolar Macrophages

doi: 10.1016/j.celrep.2020.02.112

Figure Lengend Snippet: (A–C) At indicated times, lungs were digested with collagenase and stained with CD11c, CD11b, CD45, CD64, and Ly6G antibodies. Alveolar macrophages (CD11c + /CD45 + /CD64 + /CD11b/LY6G) and recruited macrophages (CD11b + /CD45 + /CD64 + /CD11c/LY6G) were sorted and lysed, and the expression of SPHK1 and SPHK2 was determined (A) using antibodies. Plots in B and C show individual pixel intensities of indicated proteins (expressed as a.u.) with mean ± SD (n = 4 mice/group). *** p < 0.05 relative to WT CD11b + macrophages. (D) BAL macrophages (MF) were isolated from indicated mice and times after LPS challenge and incubated with ATP and sphingosine at 37°C. SPHK activity was determined as in . Experiments were performed twice independently using duplicate samples. (E) BAL macrophages were lysed and S1P levels were determined using an ELISA kit. Each experiment was performed twice independently using duplicate samples (n = 2). Data in (D) and (E) are represented as mean ± SD. ** p < 0.01, *** p < 0.001 relative to unstimulated WT or SPHK2-null BMDMs; ### p < 0.001 relative to LPS-exposedWT BMDMs. (F and G) CD11b-DTR mice were first exposed to (F) LPS by inhalation or (G) i.t. PA by instillation followed by DT as described in . Thereafter, 2 × 10 6 CD11b + monocytes isolated from bone marrow of WT or Sphk2 −/− mice were injected i.v. into CD11b-DTR mice and the lung wet/dry weight ratio was determined. Mice receiving PBS served as controls. Shown are individual data with mean ± SD (n = 5 mice/group). *** p < 0.001 relative to unexposed macrophages or macrophage dep mice or 24-h LPS-/72-h PA exposed macrophage dep mice; ### p < 0.001 relative to macrophage dep mice receiving 24-h LPS/72-h PA and WT CD11b monocytes. (H) Lung lysates without or with LPS challenge and CD11b monocyte adoptive transfer were processed for determining phosphorylation of indicated proteins as in . A representative immunoblot is shown from experiments that were independently repeated three times. (I and J) Lungs from indicated mice were homogenized at the indicated times without or with LPS challenge or adoptive transfer, and expression of (I) IFN-β and (J) GM-CSF was determined using qPCR (n = 4). GAPDH was used as internal control. (K) Lung lysates from indicated mice without or with PA challenge and monocyte adoptive transfer were processed for determining phosphorylation as described in . A representative immunoblot is shown from experiments that were independently repeated three times. (L–N) Lungs lysates without or with PA challenge or adoptive transfer from indicated mice were processed for determining IFN-β expression at the level of protein (L) (n = 3 mice/group) or mRNA (N) (n = 4 mice/group). Plot in M shows individual pixel intensity for IFN-β protein expressed as arbitrary units (a.u.) along with mean and SD. Data in (I), (J), (M), and (N) are expressed as mean ± SD from two or three independent experiments. *p < 0.05, ** p < 0.01, *** p < 0.001 relative to unexposed macrophages or macrophage dep mice or 24-h LPS-/72-h PA exposed macrophage dep mice; # p < 0.05, ## p < 0.01, ### p < 0.001 relative to 24-h LPS-/72-h PA exposed macrophage dep mice receiving WT CD11b monocytes. All data were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test and an unpaired t test with Welch’s correction.

Article Snippet: IFN-β ELISA Kit, Mouse , Signosis , Cat# CUS-EA-2001.

Techniques: Staining, Expressing, Isolation, Incubation, Activity Assay, Enzyme-linked Immunosorbent Assay, Injection, Adoptive Transfer Assay, Western Blot

Journal: Cell reports

Article Title: SPHK2-Generated S1P in CD11b + Macrophages Blocks STING to Suppress the Inflammatory Function of Alveolar Macrophages

doi: 10.1016/j.celrep.2020.02.112

Figure Lengend Snippet: (A) WT BMDM lysates were immunoprecipitated with streptavidin or S1P-conjugated streptavidin beads. Immunocomplexes and total lysates were then immunoblotted with anti-STING antibody. (B) Expression of IFN-β following stimulation with 10 μM cGAMP as described in . GAPDH expression was used as an internal control. n = 3 mice/group. (C) Lysates from HEK cells transducing vector, HA-tagged full-length STING, or HA-tagged STING-CTD were immunoprecipitated using S1P streptavidin beads. Lysates and immunocomplexes were immunoblotted with HA antibody to determine the S1P interaction with STING. (D) Binding poses of S1P to STING-CTD. (i), (ii), and (iii) indicate overplayed binding poses of S1P (PyMOL native atom colors) with cGAMP (blue) with STING-CTD (light pick) (PDB: 4KSY) in resting, closed (PDB: 4LOJ), and open conformation (PDB:4EF4), respectively. In (A) and (C), a representative immunoblot is shown from experiments that were independently repeated three times. The data in (B) are represented as mean ± SD from two to three independent experiments. *p < 0.05, ***p < 0.001 relative to unstimulated WT or SPHK2-null BMDMs; ### p < 0.001 relative to S1P plus cGAMP-stimulated BMDMs. Data were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: IFN-β ELISA Kit, Mouse , Signosis , Cat# CUS-EA-2001.

Techniques: Immunoprecipitation, Expressing, Plasmid Preparation, Binding Assay, Western Blot

Journal: Cell reports

Article Title: SPHK2-Generated S1P in CD11b + Macrophages Blocks STING to Suppress the Inflammatory Function of Alveolar Macrophages

doi: 10.1016/j.celrep.2020.02.112

Figure Lengend Snippet: KEY RESOURCE TABLE

Article Snippet: IFN-β ELISA Kit, Mouse , Signosis , Cat# CUS-EA-2001.

Techniques: Recombinant, Cell Isolation, Enzyme-linked Immunosorbent Assay, Software