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  • 99
    ATCC thp 1 cells
    Phagosomal copper concentration differs among primary macrophages and macrophage cell lines. Common macrophage cell lines (P388D1, RAW264.7, J774.1, and PMA-differentiated <t>THP-1</t> cells) and unactivated primary cells (bone marrow derived macrophages (BMDMs), peritoneal macrophages (PMs), and alveolar macrophages (AMs)) were infected with H . capsulatum yeasts (MOI 1:2) with TEF1 or CTR3 promoter- gfp fusions for 48 hours. Macrophages were lysed, intracellular yeasts recovered, and the GFP fluorescence of individual yeast was quantified by microscopy (n > 100 yeasts for each sample). CTR3 promoter activity as indicated by the CTR3 promoter- gfp fusion was normalized to the average fluorescence of the TEF1 promoter- gfp fusion. Horizontal bars represent the average CTR3 promoter activity.
    Thp 1 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 6417 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore thp 1 cell line
    lefA mutant fails to grow in human monocytic <t>THP-1</t> cells. Infected THP-1 cells were cultured for 2, 6, 12, and 24 h. Data are the averages of triplicate samples from three identical experiments, and error bars represent standard deviations. Statistically significant differences compared to Phi-1 are indicated by asterisks (* P
    Thp 1 Cell Line, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher thp 1 cells
    TNFα increases intracellular production of S100 proteins in <t>THP-1</t> cells and S100A8 and S100A9 is co-localized in the vesicles along the endocytic pathway. THP-1 cells were cultured for 48h in presence or absence of 10 ng/ml TNFα. S100A8 and S100A9 mRNA expression was determined using qRT-PCR and was normalized to β-actin mRNA level. Data indicate mean ± SD of triplicate samples. Differences between treated and untreated groups are significant at ***P
    Thp 1 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5538 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Amaxa thp 1 cells
    Identification of the 40-kDa protein as pleckstrin by mass spectrometry. The membrane fraction of <t>THP-1</t> cells stimulated with 200 nM PMA for 20 min was harvested. Proteins in the membrane fraction were then separated on a 10% SDS-PAGE gel. The band corresponding to 40 kDa (arrow in A ) was excised from the SDS-PAGE gel stained with Coomassie blue for analysis. B , The results of MS analysis. Five tryptic peptides (A-E) mapping to pleckstrin protein sequence were found in MS. Three (A-C) are statistically significant as demonstrated by the Xcorr value > 2.0. The A-E peptide sequences are shown along with the total protein coverage of pleckstrin (bold type).
    Thp 1 Cells, supplied by Amaxa, used in various techniques. Bioz Stars score: 92/100, based on 587 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    InvivoGen thp1 xblue cells
    Full-length and truncated needle proteins activate NF-κB/AP-1 in <t>THP1-XBlue</t> cells in an MyD88-dependent manner. THP-1 (A) and THP-1 defMyd88 (B) cells were seeded in wells and treated with PBS, 1 μg/ml of HKLM (Heat Killed Listeria monocytogenes
    Thp1 Xblue Cells, supplied by InvivoGen, used in various techniques. Bioz Stars score: 89/100, based on 179 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher cellsensor nfκb bla thp 1 cell line
    Full-length and truncated needle proteins activate NF-κB/AP-1 in <t>THP1-XBlue</t> cells in an MyD88-dependent manner. THP-1 (A) and THP-1 defMyd88 (B) cells were seeded in wells and treated with PBS, 1 μg/ml of HKLM (Heat Killed Listeria monocytogenes
    Cellsensor Nfκb Bla Thp 1 Cell Line, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam thp 1 cells
    SAMHD1 knockdown and Vpx enhance the dNTP pool in PMA-treated <t>THP-1</t> cells. (a) THP-1, engineered to stably express shRNA scrambled (control) or shRNA specifically targeting SAMHD1, were differentiated overnight with PMA. Where indicated, the cells were incubated for 2 h with Vpx-containing and control VLP (+/−Vpx). Cell extracts were analyzed by western blot with the indicated antibodies. (b) dNTP concentrations were determined by the single nucleotide incorporation assay described in Diamond et al . The experiment was performed in duplicate and one representative result is shown. (c) dNTP triphosphohydrolase activity of recombinant SAMHD1. dNTPase activity assay was performed as described in the methods section in the presence of the indicated dNTP, 1 μCi of the corresponding γ- 32 P-dNTP and 1 μM of wild type SAMHD1. Where indicated 200 μM of unlabeled dGTP was added. The non-labeled standards were visualized by UV shadowing and γ- 32 P-labeled nucleotides were visualized using a phosphorimager (Fluorescent Image Analyzer FLA3000 (Fuji).
    Thp 1 Cells, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Beyotime thp 1 cells
    MBL blocks PGN-induced phosphorylation of I κ B α and p65 nuclear translocation in PMA-activated <t>THP-1</t> cells. (a) PMA-activated THP-1 cells were stimulated with PGN (200 μ g/ml), MBL (10 μ g/ml) mixed with PGN, or anti-MBL pAb (10 μ g/ml) mixed with MBL and PGN; then the cells were incubated at 37°C in a 5% ( v / v ) CO 2 environment for 8 h. All of the mixture was generated by preincubation for 2 h at room temperature. Cells were harvested, and Western blots were performed using I κ B α and p-I κ B α or antibodies (upper panels). β -Actin was used as loading control. The protein levels were quantitatively analyzed based on densitometry analysis (lower panel). (b) MBL inhibits p65 nuclear translocation. Cells were stimulated as described in (a). Nuclear extracts were prepared as described in Materials and Methods, and Western blots were performed using p65 antibody (upper panel). Histone H1 was used as loading control. The protein levels were quantitatively analyzed based on densitometry analysis (lower panel). Images are representative of three experiments. ∗ p
    Thp 1 Cells, supplied by Beyotime, used in various techniques. Bioz Stars score: 97/100, based on 199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Sumitomo Dainippon thp 1 cells
    Fluorescence intensity (arbitrary unit, a.u.) related to TMRM from <t>THP-1</t> cells applied with or without RF current plotted against the time after the RF application. Open square: control (n=2321–2641). Open circle: CRF (n=2321–2679). Closed circle (red): PRF (n=2578–2697). Data are expressed as mean±SD.
    Thp 1 Cells, supplied by Sumitomo Dainippon, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    InvivoGen thp1 blue cells
    Fluorescence intensity (arbitrary unit, a.u.) related to TMRM from <t>THP-1</t> cells applied with or without RF current plotted against the time after the RF application. Open square: control (n=2321–2641). Open circle: CRF (n=2321–2679). Closed circle (red): PRF (n=2578–2697). Data are expressed as mean±SD.
    Thp1 Blue Cells, supplied by InvivoGen, used in various techniques. Bioz Stars score: 96/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Phagosomal copper concentration differs among primary macrophages and macrophage cell lines. Common macrophage cell lines (P388D1, RAW264.7, J774.1, and PMA-differentiated THP-1 cells) and unactivated primary cells (bone marrow derived macrophages (BMDMs), peritoneal macrophages (PMs), and alveolar macrophages (AMs)) were infected with H . capsulatum yeasts (MOI 1:2) with TEF1 or CTR3 promoter- gfp fusions for 48 hours. Macrophages were lysed, intracellular yeasts recovered, and the GFP fluorescence of individual yeast was quantified by microscopy (n > 100 yeasts for each sample). CTR3 promoter activity as indicated by the CTR3 promoter- gfp fusion was normalized to the average fluorescence of the TEF1 promoter- gfp fusion. Horizontal bars represent the average CTR3 promoter activity.

    Journal: PLoS Pathogens

    Article Title: Macrophage activation by IFN-γ triggers restriction of phagosomal copper from intracellular pathogens

    doi: 10.1371/journal.ppat.1007444

    Figure Lengend Snippet: Phagosomal copper concentration differs among primary macrophages and macrophage cell lines. Common macrophage cell lines (P388D1, RAW264.7, J774.1, and PMA-differentiated THP-1 cells) and unactivated primary cells (bone marrow derived macrophages (BMDMs), peritoneal macrophages (PMs), and alveolar macrophages (AMs)) were infected with H . capsulatum yeasts (MOI 1:2) with TEF1 or CTR3 promoter- gfp fusions for 48 hours. Macrophages were lysed, intracellular yeasts recovered, and the GFP fluorescence of individual yeast was quantified by microscopy (n > 100 yeasts for each sample). CTR3 promoter activity as indicated by the CTR3 promoter- gfp fusion was normalized to the average fluorescence of the TEF1 promoter- gfp fusion. Horizontal bars represent the average CTR3 promoter activity.

    Article Snippet: THP-1 cells (ATCC TIB-202) were maintained in RPMI-1640 medium supplemented with 10% (FBS) and were differentiated in 10 ng/ml phorbol 12-myristate 13-acetate (PMA) for 48 h before use.

    Techniques: Concentration Assay, Derivative Assay, Affinity Magnetic Separation, Infection, Fluorescence, Microscopy, Activity Assay

    ZCCHC3 positively regulates dsDNA-triggered signaling. a ZCCHC3 activates the IFN-β promoter in a dose-dependent manner. HEK293 cells were transfected with the IFN-β reporter and increased amounts of ZCCHC3 plasmid for 18 h, and then left un-infected or infected with HSV-1 for 12 h before luciferase assays. b Effects of ZCCHC3 on transcription of downstream genes induced by HSV-1. Control HFF cells and HFFs stably expressing ZCCHC3 were infected with HSV-1 for the indicated times before qPCR analysis. c Effects of ZCCHC3 on transcription of downstream genes induced by cytosolic dsDNA. Control and HFFs stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. d Effects of ZCCHC3-deficiency on transcription of downstream genes induced by HSV-1. ZCCHC3- KO HFFs were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HFFs were left un-infected or infected with HSV-1 for the indicated times before qPCR analysis. ZCCHC3-deficiency in the KO cells was confirmed by immunoblotting analysis (right blots). e Effects of ZCCHC3 on transcription of downstream genes induced by cytosolic dsDNA. ZCCHC3-KO and control HFFs were transfected with the indicated nucleic acids (3 μg/ml) for 4 h before qPCR analysis. f Effects of ZCCHC3-deficiency on HSV-1-induced phosphorylation of TBK1 and IRF3. ZCCHC3-KO and control HFFs were left un-infected or infected with HSV-1 for the indicated times before immunoblotting analysis. g Effects of ZCCHC3-deficiency on IFN-β-induced transcription of downstream genes. Control and ZCCHC3- KO THP1 cells were generated by the CRISPR-Cas9 method. The cells were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. h Effects of ZCCHC3-deficiency on IFN-β-induced phosphorylation of STAT1. ZCCHC3-KO and control THP1 cells were left un-treated or treated with IFN-β (100 ng/ml) for the indicated times before immunoblotting analysis. * P

    Journal: Nature Communications

    Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

    doi: 10.1038/s41467-018-05559-w

    Figure Lengend Snippet: ZCCHC3 positively regulates dsDNA-triggered signaling. a ZCCHC3 activates the IFN-β promoter in a dose-dependent manner. HEK293 cells were transfected with the IFN-β reporter and increased amounts of ZCCHC3 plasmid for 18 h, and then left un-infected or infected with HSV-1 for 12 h before luciferase assays. b Effects of ZCCHC3 on transcription of downstream genes induced by HSV-1. Control HFF cells and HFFs stably expressing ZCCHC3 were infected with HSV-1 for the indicated times before qPCR analysis. c Effects of ZCCHC3 on transcription of downstream genes induced by cytosolic dsDNA. Control and HFFs stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. d Effects of ZCCHC3-deficiency on transcription of downstream genes induced by HSV-1. ZCCHC3- KO HFFs were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HFFs were left un-infected or infected with HSV-1 for the indicated times before qPCR analysis. ZCCHC3-deficiency in the KO cells was confirmed by immunoblotting analysis (right blots). e Effects of ZCCHC3 on transcription of downstream genes induced by cytosolic dsDNA. ZCCHC3-KO and control HFFs were transfected with the indicated nucleic acids (3 μg/ml) for 4 h before qPCR analysis. f Effects of ZCCHC3-deficiency on HSV-1-induced phosphorylation of TBK1 and IRF3. ZCCHC3-KO and control HFFs were left un-infected or infected with HSV-1 for the indicated times before immunoblotting analysis. g Effects of ZCCHC3-deficiency on IFN-β-induced transcription of downstream genes. Control and ZCCHC3- KO THP1 cells were generated by the CRISPR-Cas9 method. The cells were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. h Effects of ZCCHC3-deficiency on IFN-β-induced phosphorylation of STAT1. ZCCHC3-KO and control THP1 cells were left un-treated or treated with IFN-β (100 ng/ml) for the indicated times before immunoblotting analysis. * P

    Article Snippet: HEK293 cells (Cat # CRL-11268) and THP1 cells (Cat # TIB-202) were obtained from ATCC.

    Techniques: Transfection, Plasmid Preparation, Infection, Luciferase, Stable Transfection, Expressing, Real-time Polymerase Chain Reaction, Generated, CRISPR

    CX3CL1-Fc reduces monocyte cell adhesion to endothelial monolayers. (A) In vitro THP-1 adhesion assay under flow conditions. THP-1 cells pre-treated with CX3CL1-Fc are significantly less adherent to a HUVEC monolayer when under a shear stress of 1 dyne/cm 2 compared to VEH-treated controls. Representative images are shown (n = 3). (B–C) Static in vitro THP-1 adhesion assays. CX3CL1-Fc pre-treatment of THP-1 cells leads to reduced adhesion to a HUVEC monolayer under normal (B) and LPS-stimulated conditions (C). Representative images are shown (n = 3). (D–E) Static in vitro peripheral blood monocyte adhesion assays. Peripheral blood monocytes pre-treated with CX3CL1-Fc are less adherent to a HUVEC monolayer under normal (D) and LPS-stimulated (E) conditions. Representative images are shown (n = 3). Monocytes are shown in green and nuclei are labeled with DAPI. Scale bar = 100 μm. Data in both panels are presented as mean ± SD. *p

    Journal: Molecular Metabolism

    Article Title: CX3CL1-Fc treatment prevents atherosclerosis in Ldlr KO mice

    doi: 10.1016/j.molmet.2018.11.011

    Figure Lengend Snippet: CX3CL1-Fc reduces monocyte cell adhesion to endothelial monolayers. (A) In vitro THP-1 adhesion assay under flow conditions. THP-1 cells pre-treated with CX3CL1-Fc are significantly less adherent to a HUVEC monolayer when under a shear stress of 1 dyne/cm 2 compared to VEH-treated controls. Representative images are shown (n = 3). (B–C) Static in vitro THP-1 adhesion assays. CX3CL1-Fc pre-treatment of THP-1 cells leads to reduced adhesion to a HUVEC monolayer under normal (B) and LPS-stimulated conditions (C). Representative images are shown (n = 3). (D–E) Static in vitro peripheral blood monocyte adhesion assays. Peripheral blood monocytes pre-treated with CX3CL1-Fc are less adherent to a HUVEC monolayer under normal (D) and LPS-stimulated (E) conditions. Representative images are shown (n = 3). Monocytes are shown in green and nuclei are labeled with DAPI. Scale bar = 100 μm. Data in both panels are presented as mean ± SD. *p

    Article Snippet: Monocytes or human THP-1 cells (ATCC, catalog# TIB-202) were stained green with PKH67 (Sigma, catalog# PKH67GL-1 KT, St. Louis, MO, USA), pre-incubated in CX3CL1-Fc (1.4 nmol/L) or saline (VEH)-containing medium for 30 min and then added to washed human umbilical vein endothelial cell (HUVECs) (ATCC, catalog# PCS-100-010) monolayers for 1 h. For LPS stimulation (Sigma, catalog# LPS25), HUVEC monolayers were pre-treated with 100 ng/ml LPS for 30 min, while monocytes or human THP-1 cells were pre-treated with CX3CL1-Fc (1.4 nmol/L) or saline for 30 min before layering on confluent LPS-stimulated HUVECs.

    Techniques: In Vitro, Cell Adhesion Assay, Flow Cytometry, Labeling

    A schematic summary of the IVD degeneration in vitro model and effects of PBM on ECM-modifying enzymes in human NP cells. In this IVD degeneration in vitro model, macrophage THP-1 like cells express proinflammatory cytokines such as IL-1β and TNF-α. These molecules activate NF-κB downstream signaling, which control expression of inflammatory catabolic genes encoding including MMP1 and MMP3 via nucleus translocation of NF-κB (p65 and p50 subunits). Possible effect sites of PBM observed in this study are indicated by red lines. Abbreviations: IκB, inhibitor of nuclear factor κB; IL-1R1, IL-1 receptor 1; IL-1RAcP, IL-1 receptor accessory protein; NFκB, nuclear factor κB; TNF-α, tumor necrosis factor- alpha; IL-1β, interleukin-1beta; TNFR, TNF receptor 1; MMP, matrix metalloproteinase; TIMP, a tissue inhibitor of metalloproteinases.

    Journal: Scientific Reports

    Article Title: Photobiomodulation of extracellular matrix enzymes in human nucleus pulposus cells as a potential treatment for intervertebral disk degeneration

    doi: 10.1038/s41598-018-30185-3

    Figure Lengend Snippet: A schematic summary of the IVD degeneration in vitro model and effects of PBM on ECM-modifying enzymes in human NP cells. In this IVD degeneration in vitro model, macrophage THP-1 like cells express proinflammatory cytokines such as IL-1β and TNF-α. These molecules activate NF-κB downstream signaling, which control expression of inflammatory catabolic genes encoding including MMP1 and MMP3 via nucleus translocation of NF-κB (p65 and p50 subunits). Possible effect sites of PBM observed in this study are indicated by red lines. Abbreviations: IκB, inhibitor of nuclear factor κB; IL-1R1, IL-1 receptor 1; IL-1RAcP, IL-1 receptor accessory protein; NFκB, nuclear factor κB; TNF-α, tumor necrosis factor- alpha; IL-1β, interleukin-1beta; TNFR, TNF receptor 1; MMP, matrix metalloproteinase; TIMP, a tissue inhibitor of metalloproteinases.

    Article Snippet: Differentiation of human monocytic leukemia THP-1 cells into activated macrophage-like cells and generation of macrophage-conditioned medium (MCM) The human monocytic leukemia THP-1 cell line (ATCC TIB202; ATCC, Manassas, VA, USA) was seeded into 75-cm2 cell culture flasks containing Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 160 nM phorbol myristate acetate (PMA), 1% FBS, and 1% P/S.

    Techniques: In Vitro, Expressing, Translocation Assay

    A Δ pldA mutant has impaired growth in THP-1 macrophages. a Growth of C. burnetii wild type, Δ pldA, and Δ pldA comp strains in ACCM-2 (left panel) and THP-1 macrophages (right panel). Data represent fold increases in genome equivalents (GE) after 6 days of growth (early SCV) for 3 independent experiments performed in triplicate. Asterisks indicate a statistically significant difference (* = P

    Journal: BMC Microbiology

    Article Title: A Coxiella burnetii phospholipase A homolog pldA is required for optimal growth in macrophages and developmental form lipid remodeling

    doi: 10.1186/s12866-018-1181-0

    Figure Lengend Snippet: A Δ pldA mutant has impaired growth in THP-1 macrophages. a Growth of C. burnetii wild type, Δ pldA, and Δ pldA comp strains in ACCM-2 (left panel) and THP-1 macrophages (right panel). Data represent fold increases in genome equivalents (GE) after 6 days of growth (early SCV) for 3 independent experiments performed in triplicate. Asterisks indicate a statistically significant difference (* = P

    Article Snippet: The human acute monocytic leukemia cell line THP-1 (TIB-202; American Type Culture Collection) was grown at 37 °C and 5% CO2 in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone).

    Techniques: Mutagenesis

    Contribution of extracellular mycobacteria to OCR and ECAR of infected macrophages and percentage of Mtb -infected macrophages. OCR ( A, B ) and ECAR ( C, D ) of the remaining extracellular Mtb ( A, C ) and BCG ( B,D ) in the final wash of infected THP-1 macrophages that were adhered to wells of an XF96 culture plate using Cell-Tak TM prior to a mitochondrial respiration assay. The percentage of THP-1 ( E ) and hMDM cells ( F ) that were infected with Mtb GFP-reporter strain at MOI 1, 2.5 or 5 after 16 h. Data shown are the mean ± SD (n = 5 biological replicates) Student’s t test, ϕ, p

    Journal: eLife

    Article Title: Mycobacterium tuberculosis induces decelerated bioenergetic metabolism in human macrophages

    doi: 10.7554/eLife.39169

    Figure Lengend Snippet: Contribution of extracellular mycobacteria to OCR and ECAR of infected macrophages and percentage of Mtb -infected macrophages. OCR ( A, B ) and ECAR ( C, D ) of the remaining extracellular Mtb ( A, C ) and BCG ( B,D ) in the final wash of infected THP-1 macrophages that were adhered to wells of an XF96 culture plate using Cell-Tak TM prior to a mitochondrial respiration assay. The percentage of THP-1 ( E ) and hMDM cells ( F ) that were infected with Mtb GFP-reporter strain at MOI 1, 2.5 or 5 after 16 h. Data shown are the mean ± SD (n = 5 biological replicates) Student’s t test, ϕ, p

    Article Snippet: Cell culture THP-1 cells (ATCC TIB-202) were cultured in RPMI1640 [final concentrations: 4.5 g/L glucose, 2 mM L-GlutaMAXTM , 10 mM HEPES and 1 mM sodium pyruvate] containing 10% (v/v) FBS and 0.05 mM 2-mercaptoethanol.

    Techniques: Infection, Respiration Assay

    Mtb at a MOI of 2.5 decelerates flux through glycolysis in THP-1 cells and induces breaks in the TCA cycle at citrate and succinate. 13 C-tracing of extracted metabolites of THP-1 cells infected with Mtb , BCG and ∆Dead Mtb at a MOI of 2.5 for 10 h followed by incubation with [U- 13 C] glucose for 8 h. The stacked mass isotopomer distributions of the intracellular metabolites depict the contribution of glucose to the TCA cycle and the PPP and some amino acids associated with the TCA cycle.

    Journal: eLife

    Article Title: Mycobacterium tuberculosis induces decelerated bioenergetic metabolism in human macrophages

    doi: 10.7554/eLife.39169

    Figure Lengend Snippet: Mtb at a MOI of 2.5 decelerates flux through glycolysis in THP-1 cells and induces breaks in the TCA cycle at citrate and succinate. 13 C-tracing of extracted metabolites of THP-1 cells infected with Mtb , BCG and ∆Dead Mtb at a MOI of 2.5 for 10 h followed by incubation with [U- 13 C] glucose for 8 h. The stacked mass isotopomer distributions of the intracellular metabolites depict the contribution of glucose to the TCA cycle and the PPP and some amino acids associated with the TCA cycle.

    Article Snippet: Cell culture THP-1 cells (ATCC TIB-202) were cultured in RPMI1640 [final concentrations: 4.5 g/L glucose, 2 mM L-GlutaMAXTM , 10 mM HEPES and 1 mM sodium pyruvate] containing 10% (v/v) FBS and 0.05 mM 2-mercaptoethanol.

    Techniques: Infection, Incubation

    Mtb infection alters the mitochondrial substrate preference of macrophages. ( A–C ) UK5099, etomoxir and BPTES were used to assess the mitochondrial flexibility and dependency on glucose (Glc), fatty acids (FA) and glutamine (Gln) in ( B ) THP-1 and ( C ) hMDM cells infected with Mtb , BCG and ∆Dead Mtb at MOIs of 1 and 5, respectively, for 18 h. Data shown are the mean ±SEM of five independent experiments. Student’s t test relative to uninfected cells (UI); #, p

    Journal: eLife

    Article Title: Mycobacterium tuberculosis induces decelerated bioenergetic metabolism in human macrophages

    doi: 10.7554/eLife.39169

    Figure Lengend Snippet: Mtb infection alters the mitochondrial substrate preference of macrophages. ( A–C ) UK5099, etomoxir and BPTES were used to assess the mitochondrial flexibility and dependency on glucose (Glc), fatty acids (FA) and glutamine (Gln) in ( B ) THP-1 and ( C ) hMDM cells infected with Mtb , BCG and ∆Dead Mtb at MOIs of 1 and 5, respectively, for 18 h. Data shown are the mean ±SEM of five independent experiments. Student’s t test relative to uninfected cells (UI); #, p

    Article Snippet: Cell culture THP-1 cells (ATCC TIB-202) were cultured in RPMI1640 [final concentrations: 4.5 g/L glucose, 2 mM L-GlutaMAXTM , 10 mM HEPES and 1 mM sodium pyruvate] containing 10% (v/v) FBS and 0.05 mM 2-mercaptoethanol.

    Techniques: Infection, Gas Chromatography

    Mtb infection reduces the glycolytic proton efflux rate of macrophages. ( A–D ) Profiles of total proton efflux rate (PER) in red and the glycolytic proton efflux rate (glycoPER) in blue of hMDMs infected with Mtb , BCG and dead Mtb at MOI of 1 for 18 h. The difference between the total PER and glycoPER will give the PER caused by mitochondrial respiration. ( E–L ) Basal and compensatory glycolytic PER of THP-1 cells ( E–H ) and hMDMs ( I–L ) infected with Mtb , BCG and dead- Mtb at MOIs of 1 and 2.5 for 18 h. ( M, N ) % Contribution of glycolysis and OXPHOS to the total rate of ATP production in ( M ) THP-1 cells and ( N ) hMDM cells infected with Mtb , BCG or dead Mtb at indicated MOI for 18 h. Profiles, PER and % contribution of glycolysis and OXPHOS to total ATP production are representative of two independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to the uninfected cells; #, p

    Journal: eLife

    Article Title: Mycobacterium tuberculosis induces decelerated bioenergetic metabolism in human macrophages

    doi: 10.7554/eLife.39169

    Figure Lengend Snippet: Mtb infection reduces the glycolytic proton efflux rate of macrophages. ( A–D ) Profiles of total proton efflux rate (PER) in red and the glycolytic proton efflux rate (glycoPER) in blue of hMDMs infected with Mtb , BCG and dead Mtb at MOI of 1 for 18 h. The difference between the total PER and glycoPER will give the PER caused by mitochondrial respiration. ( E–L ) Basal and compensatory glycolytic PER of THP-1 cells ( E–H ) and hMDMs ( I–L ) infected with Mtb , BCG and dead- Mtb at MOIs of 1 and 2.5 for 18 h. ( M, N ) % Contribution of glycolysis and OXPHOS to the total rate of ATP production in ( M ) THP-1 cells and ( N ) hMDM cells infected with Mtb , BCG or dead Mtb at indicated MOI for 18 h. Profiles, PER and % contribution of glycolysis and OXPHOS to total ATP production are representative of two independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to the uninfected cells; #, p

    Article Snippet: Cell culture THP-1 cells (ATCC TIB-202) were cultured in RPMI1640 [final concentrations: 4.5 g/L glucose, 2 mM L-GlutaMAXTM , 10 mM HEPES and 1 mM sodium pyruvate] containing 10% (v/v) FBS and 0.05 mM 2-mercaptoethanol.

    Techniques: Infection

    Mitochondrial respiration assays without FCCP. Mitochondrial respiratory profiles of THP-1 cells ( A–D ) and hMDMs ( E, F ) without the addition of FCCP were generated to measure the non-mitochondrial respiration. The non-mitochondrial respiration was then used to calculate the basal respiration and proton leak in scenarios when the addition of FCCP induces oxidative bursts in infected cells at a high MOI. Profiles and respiratory parameters are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p

    Journal: eLife

    Article Title: Mycobacterium tuberculosis induces decelerated bioenergetic metabolism in human macrophages

    doi: 10.7554/eLife.39169

    Figure Lengend Snippet: Mitochondrial respiration assays without FCCP. Mitochondrial respiratory profiles of THP-1 cells ( A–D ) and hMDMs ( E, F ) without the addition of FCCP were generated to measure the non-mitochondrial respiration. The non-mitochondrial respiration was then used to calculate the basal respiration and proton leak in scenarios when the addition of FCCP induces oxidative bursts in infected cells at a high MOI. Profiles and respiratory parameters are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p

    Article Snippet: Cell culture THP-1 cells (ATCC TIB-202) were cultured in RPMI1640 [final concentrations: 4.5 g/L glucose, 2 mM L-GlutaMAXTM , 10 mM HEPES and 1 mM sodium pyruvate] containing 10% (v/v) FBS and 0.05 mM 2-mercaptoethanol.

    Techniques: Generated, Infection

    Respiratory profiles and parameters of infected macrophages are dependent on cell type, mycobacterial strain and MOI. Respiratory profiles (OCR) and respiratory parameters of ( A–D ) PMA differentiated THP-1 macrophages, and ( E–H ) hMDMs infected with Mtb , BCG and dead Mtb at MOIs of 1 or 2.5 for 24 h. Profiles and respiratory parameters are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p

    Journal: eLife

    Article Title: Mycobacterium tuberculosis induces decelerated bioenergetic metabolism in human macrophages

    doi: 10.7554/eLife.39169

    Figure Lengend Snippet: Respiratory profiles and parameters of infected macrophages are dependent on cell type, mycobacterial strain and MOI. Respiratory profiles (OCR) and respiratory parameters of ( A–D ) PMA differentiated THP-1 macrophages, and ( E–H ) hMDMs infected with Mtb , BCG and dead Mtb at MOIs of 1 or 2.5 for 24 h. Profiles and respiratory parameters are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p

    Article Snippet: Cell culture THP-1 cells (ATCC TIB-202) were cultured in RPMI1640 [final concentrations: 4.5 g/L glucose, 2 mM L-GlutaMAXTM , 10 mM HEPES and 1 mM sodium pyruvate] containing 10% (v/v) FBS and 0.05 mM 2-mercaptoethanol.

    Techniques: Infection

    Phenograms demonstrate that increasing MOI of Mtb shifts macrophages towards quiescent energy phenotypes. Basal OCR and ECAR measurements from the respiratory assay ( Figure 2 ) before addition of oligomycin were plotted to generate phenograms of ( A–C ) PMA-differentiated THP-1 cells and ( D–F ) hMDMs infected with Mtb , BCG and ∆Dead Mtb at MOIs of 1, 2.5 and 5. Data are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p

    Journal: eLife

    Article Title: Mycobacterium tuberculosis induces decelerated bioenergetic metabolism in human macrophages

    doi: 10.7554/eLife.39169

    Figure Lengend Snippet: Phenograms demonstrate that increasing MOI of Mtb shifts macrophages towards quiescent energy phenotypes. Basal OCR and ECAR measurements from the respiratory assay ( Figure 2 ) before addition of oligomycin were plotted to generate phenograms of ( A–C ) PMA-differentiated THP-1 cells and ( D–F ) hMDMs infected with Mtb , BCG and ∆Dead Mtb at MOIs of 1, 2.5 and 5. Data are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p

    Article Snippet: Cell culture THP-1 cells (ATCC TIB-202) were cultured in RPMI1640 [final concentrations: 4.5 g/L glucose, 2 mM L-GlutaMAXTM , 10 mM HEPES and 1 mM sodium pyruvate] containing 10% (v/v) FBS and 0.05 mM 2-mercaptoethanol.

    Techniques: Infection

    Extracellular acidification profiles and glycolytic parameters of THP-1 and hMDMs are affected by macrophage type, mycobacterial strain and MOI. ECAR profiles and glycolytic parameters of ( A–D ) PMA differentiated THP-1 macrophages, and ( E–H ) hMDMs infected with Mtb , BCG and dead Mtb at MOIs of 1 or 2.5 for 24 h. Profiles and glycolytic parameters are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p

    Journal: eLife

    Article Title: Mycobacterium tuberculosis induces decelerated bioenergetic metabolism in human macrophages

    doi: 10.7554/eLife.39169

    Figure Lengend Snippet: Extracellular acidification profiles and glycolytic parameters of THP-1 and hMDMs are affected by macrophage type, mycobacterial strain and MOI. ECAR profiles and glycolytic parameters of ( A–D ) PMA differentiated THP-1 macrophages, and ( E–H ) hMDMs infected with Mtb , BCG and dead Mtb at MOIs of 1 or 2.5 for 24 h. Profiles and glycolytic parameters are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p

    Article Snippet: Cell culture THP-1 cells (ATCC TIB-202) were cultured in RPMI1640 [final concentrations: 4.5 g/L glucose, 2 mM L-GlutaMAXTM , 10 mM HEPES and 1 mM sodium pyruvate] containing 10% (v/v) FBS and 0.05 mM 2-mercaptoethanol.

    Techniques: Infection

    YB-1 was protective against cell death in vitro. ( A , B ) Representative flow cytometry dot plot analyses of THP-1 ( A ) and U937 cells ( B ). Cells were treated with or without TNF (20 ng/mL) and cycloheximide (10 ng/mL) alone or together overnight. Cells were harvested and stained for annexin V and 7AAD. ( C ) Graphs representing flow cytometry analysis of live and dead cells in THP-1 and U937 cells. Statistical significance was calculated by two-way ANOVA with Bonferroni post hoc test, n = 3. Data represent the mean ± SEM. ** p

    Journal: Cancers

    Article Title: YB-1 Mediates TNF-Induced Pro-Survival Signaling by Regulating NF-κB Activation

    doi: 10.3390/cancers12082188

    Figure Lengend Snippet: YB-1 was protective against cell death in vitro. ( A , B ) Representative flow cytometry dot plot analyses of THP-1 ( A ) and U937 cells ( B ). Cells were treated with or without TNF (20 ng/mL) and cycloheximide (10 ng/mL) alone or together overnight. Cells were harvested and stained for annexin V and 7AAD. ( C ) Graphs representing flow cytometry analysis of live and dead cells in THP-1 and U937 cells. Statistical significance was calculated by two-way ANOVA with Bonferroni post hoc test, n = 3. Data represent the mean ± SEM. ** p

    Article Snippet: Cell Culture and Stimulation The cell lines THP-1 (TIB-202) and U937 (CRL-1593) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

    Techniques: In Vitro, Flow Cytometry, Staining

    Cell death was rescued using the pan-caspase inhibitor zVAD-fmk (ZVAD). ( A ) THP-1 cells were pretreated with zVAD and/or necrostatin-1 (Nec-1) for 1 h and treated with TNF and CHX overnight. The cells were harvested, stained with propidium iodide (PI), and analyzed by flow cytometry. The percentage of dead cells following treatment is indicated. ( B ) Graphs representing flow cytometry analysis of THP-1 (left panel) and U937 (right panel). Error bars specify the standard error mean (SEM) of at least three experiments. Statistical significance was calculated by two-way ANOVA with Bonferroni post hoc test, n = 3. Data represent the mean ± SEM. *** p ≤ 0.001, **** p ≤ 0.0001.

    Journal: Cancers

    Article Title: YB-1 Mediates TNF-Induced Pro-Survival Signaling by Regulating NF-κB Activation

    doi: 10.3390/cancers12082188

    Figure Lengend Snippet: Cell death was rescued using the pan-caspase inhibitor zVAD-fmk (ZVAD). ( A ) THP-1 cells were pretreated with zVAD and/or necrostatin-1 (Nec-1) for 1 h and treated with TNF and CHX overnight. The cells were harvested, stained with propidium iodide (PI), and analyzed by flow cytometry. The percentage of dead cells following treatment is indicated. ( B ) Graphs representing flow cytometry analysis of THP-1 (left panel) and U937 (right panel). Error bars specify the standard error mean (SEM) of at least three experiments. Statistical significance was calculated by two-way ANOVA with Bonferroni post hoc test, n = 3. Data represent the mean ± SEM. *** p ≤ 0.001, **** p ≤ 0.0001.

    Article Snippet: Cell Culture and Stimulation The cell lines THP-1 (TIB-202) and U937 (CRL-1593) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

    Techniques: Staining, Flow Cytometry

    YB-1 was required for TNF-induced NF-κB activation. ( A ) Bone marrow-derived macrophages (BMDM, WT, and YB-1 KO) were stimulated with TNF (20 ng/mL) for the indication of time periods to activate the NF-κB signaling pathway. Protein expression was analyzed by immunoblotting using the indicated antibodies. Vinculin was used as the loading control. The relative band intensities are indicated. ( B ) Relative YB-1 expression is shown for three independent experiments. WT (white) and KO (black bars). ( C ) Control and YB-1 knockdown (KD) cells (THP-1 and U937) were stimulated with TNF (20 ng/mL) to activate tumor necrosis factor receptor 1 (TNFR1). Proteins were analyzed by immunoblotting for expression of the indicated proteins. Vinculin was used as loading control. Control (CTRL; white) and knockdown (KD; black bars). ( D ) Relative YB-1 expression in KD cells is shown for three independent experiments. ( E ) Relative pp65, phospho- inhibitor of NF-κB alpha (p-IκBα), phospho-IkB kinase α/β (pIKKα/β), and TNF receptor-associated factor 2 (TRAF2) expression in WT and KD THP-1 and U937 cells are shown for three independent experiments. Control (CTRL; white) and knockdown (KD; black bars). Error bars specify the standard error of the mean (SEM). Statistical significance was calculated using an unpaired t -test, n = 3. Data represent the mean ± SEM. * p

    Journal: Cancers

    Article Title: YB-1 Mediates TNF-Induced Pro-Survival Signaling by Regulating NF-κB Activation

    doi: 10.3390/cancers12082188

    Figure Lengend Snippet: YB-1 was required for TNF-induced NF-κB activation. ( A ) Bone marrow-derived macrophages (BMDM, WT, and YB-1 KO) were stimulated with TNF (20 ng/mL) for the indication of time periods to activate the NF-κB signaling pathway. Protein expression was analyzed by immunoblotting using the indicated antibodies. Vinculin was used as the loading control. The relative band intensities are indicated. ( B ) Relative YB-1 expression is shown for three independent experiments. WT (white) and KO (black bars). ( C ) Control and YB-1 knockdown (KD) cells (THP-1 and U937) were stimulated with TNF (20 ng/mL) to activate tumor necrosis factor receptor 1 (TNFR1). Proteins were analyzed by immunoblotting for expression of the indicated proteins. Vinculin was used as loading control. Control (CTRL; white) and knockdown (KD; black bars). ( D ) Relative YB-1 expression in KD cells is shown for three independent experiments. ( E ) Relative pp65, phospho- inhibitor of NF-κB alpha (p-IκBα), phospho-IkB kinase α/β (pIKKα/β), and TNF receptor-associated factor 2 (TRAF2) expression in WT and KD THP-1 and U937 cells are shown for three independent experiments. Control (CTRL; white) and knockdown (KD; black bars). Error bars specify the standard error of the mean (SEM). Statistical significance was calculated using an unpaired t -test, n = 3. Data represent the mean ± SEM. * p

    Article Snippet: Cell Culture and Stimulation The cell lines THP-1 (TIB-202) and U937 (CRL-1593) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

    Techniques: Activation Assay, Derivative Assay, Expressing

    Depletion of YB-1 led to effector caspase activation. ( A , B ) Flow cytometry analyses of caspase-3/7 activation in THP-1 ( A ) and U937 cells ( B ). Cells were treated with or without TNF (20 ng/mL) alone or together with cycloheximide (10 ng/mL) for 4 h. Cells were harvested and stained for caspase-3/7 activation. Error bars specify the standard error mean (SEM) of at least three experiments. Statistical significance was calculated by unpaired t -test, n = 3. Data represent the mean ± SEM. ** p

    Journal: Cancers

    Article Title: YB-1 Mediates TNF-Induced Pro-Survival Signaling by Regulating NF-κB Activation

    doi: 10.3390/cancers12082188

    Figure Lengend Snippet: Depletion of YB-1 led to effector caspase activation. ( A , B ) Flow cytometry analyses of caspase-3/7 activation in THP-1 ( A ) and U937 cells ( B ). Cells were treated with or without TNF (20 ng/mL) alone or together with cycloheximide (10 ng/mL) for 4 h. Cells were harvested and stained for caspase-3/7 activation. Error bars specify the standard error mean (SEM) of at least three experiments. Statistical significance was calculated by unpaired t -test, n = 3. Data represent the mean ± SEM. ** p

    Article Snippet: Cell Culture and Stimulation The cell lines THP-1 (TIB-202) and U937 (CRL-1593) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

    Techniques: Activation Assay, Flow Cytometry, Staining

    Effect of adiponectin on LPS+PA induced NLRP3 inflammasome activation in THP-1 cells. A. Expression of NLRP3 protein was detected by western blotting in different groups. Adiponectin treated group expressed lower level of NLRP3 protein than LPS+PA treated group. B and D. Level of pro-caspase-1 protein and caspase-1 activity were also lower in adiponectin treated group than LPS+PA treated group. C. Adiponectin has no effect on LPS+PA-induced ASC protein expression in THP-1 cells. All data represent the mean ± SD of three independent experiments. **P

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Adiponectin inhibits NLRP3 inflammasome by modulating the AMPK-ROS pathway

    doi:

    Figure Lengend Snippet: Effect of adiponectin on LPS+PA induced NLRP3 inflammasome activation in THP-1 cells. A. Expression of NLRP3 protein was detected by western blotting in different groups. Adiponectin treated group expressed lower level of NLRP3 protein than LPS+PA treated group. B and D. Level of pro-caspase-1 protein and caspase-1 activity were also lower in adiponectin treated group than LPS+PA treated group. C. Adiponectin has no effect on LPS+PA-induced ASC protein expression in THP-1 cells. All data represent the mean ± SD of three independent experiments. **P

    Article Snippet: Monocytic THP-1 cells obtained from the American Type Culture Collection (ATCC) were grown at 37°C, under 5% CO2 in RPMI 1640 medium (Hyclone) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 mg/ml) and L-Glutamine (2.05 mM).

    Techniques: Activation Assay, Expressing, Western Blot, Activity Assay

    Effect of adiponectin on the LPS+PA induced secretion of IL-18 and IL-1β from THP-1 cells. Adiponectin group was pretreated with 2 μg/ml adiponectin for 12 h, and then stimulated with 100 ng/ml LPS for 3 hours and 0.5 mM PA for another 8 hours. Release of IL-18 and IL-1β was determined by ELISA. All data represent the mean ± SD of three independent experiments. **P

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Adiponectin inhibits NLRP3 inflammasome by modulating the AMPK-ROS pathway

    doi:

    Figure Lengend Snippet: Effect of adiponectin on the LPS+PA induced secretion of IL-18 and IL-1β from THP-1 cells. Adiponectin group was pretreated with 2 μg/ml adiponectin for 12 h, and then stimulated with 100 ng/ml LPS for 3 hours and 0.5 mM PA for another 8 hours. Release of IL-18 and IL-1β was determined by ELISA. All data represent the mean ± SD of three independent experiments. **P

    Article Snippet: Monocytic THP-1 cells obtained from the American Type Culture Collection (ATCC) were grown at 37°C, under 5% CO2 in RPMI 1640 medium (Hyclone) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 mg/ml) and L-Glutamine (2.05 mM).

    Techniques: Enzyme-linked Immunosorbent Assay

    Effect of adiponectin on LPS+PA induced mitochondrial ROS production in Si-NC and Si-AMPKa THP-1 cells. A. Adiponectin inhibits mitochondrial ROS generation. The ROS inhibitor NAC (N-acetyl-cysteine) was added 1 hour before the addition of LPS as a positive control. B. THP-1 cells were transfected with scramble or AMPKα1 siRNA for 72 h, followed by stimulation with LPS (100 ng/mL) for 3 h and then treated with PA (0.5 mM) for 8 h with or without 2 μg/mL adiponectin. All data represent the mean ± SD of three independent experiments. **P

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Adiponectin inhibits NLRP3 inflammasome by modulating the AMPK-ROS pathway

    doi:

    Figure Lengend Snippet: Effect of adiponectin on LPS+PA induced mitochondrial ROS production in Si-NC and Si-AMPKa THP-1 cells. A. Adiponectin inhibits mitochondrial ROS generation. The ROS inhibitor NAC (N-acetyl-cysteine) was added 1 hour before the addition of LPS as a positive control. B. THP-1 cells were transfected with scramble or AMPKα1 siRNA for 72 h, followed by stimulation with LPS (100 ng/mL) for 3 h and then treated with PA (0.5 mM) for 8 h with or without 2 μg/mL adiponectin. All data represent the mean ± SD of three independent experiments. **P

    Article Snippet: Monocytic THP-1 cells obtained from the American Type Culture Collection (ATCC) were grown at 37°C, under 5% CO2 in RPMI 1640 medium (Hyclone) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 mg/ml) and L-Glutamine (2.05 mM).

    Techniques: Positive Control, Transfection

    siRNA knockdown of AMPKα1 affects adiponectin by modulating LPS+PA-induced NLRP3 inflammasome activation. A. p-AMPK level was elevated in adiponectin treated group. B. p-AMPK and AMPK protein levels in Si-NC and Si-AMPKa THP-1 cells. C and E. The inhibition of NLRP3 protein and caspase-1 activity by adiponectin were abolished in Si-AMPKa THP-1 cells. D. Adiponectin has no effect on ASC protein expression in Si-NC and Si-AMPKa THP-1 cells. All data represent the mean ± SD of three independent experiments. **P

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Adiponectin inhibits NLRP3 inflammasome by modulating the AMPK-ROS pathway

    doi:

    Figure Lengend Snippet: siRNA knockdown of AMPKα1 affects adiponectin by modulating LPS+PA-induced NLRP3 inflammasome activation. A. p-AMPK level was elevated in adiponectin treated group. B. p-AMPK and AMPK protein levels in Si-NC and Si-AMPKa THP-1 cells. C and E. The inhibition of NLRP3 protein and caspase-1 activity by adiponectin were abolished in Si-AMPKa THP-1 cells. D. Adiponectin has no effect on ASC protein expression in Si-NC and Si-AMPKa THP-1 cells. All data represent the mean ± SD of three independent experiments. **P

    Article Snippet: Monocytic THP-1 cells obtained from the American Type Culture Collection (ATCC) were grown at 37°C, under 5% CO2 in RPMI 1640 medium (Hyclone) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 mg/ml) and L-Glutamine (2.05 mM).

    Techniques: Activation Assay, Inhibition, Activity Assay, Expressing

    Silencing of early growth response gene-1 (EGR1) or c-FOS increased the expression of phagocytic receptors. THP-1 macrophages were transfected with siEGR1 (A,B) or siFOS (C,D) for 24 h, and then infected with Pseudomonas aeruginosa at multiplicity of infection 1 for 1 h. The mRNA levels of phagocytic receptors including mannose receptor (MR) (A,C) and scavenger receptor (SR) (B,D) were analyzed by real-time PCR. Data are shown as the mean ± SEM of three independent experiments (* p

    Journal: Frontiers in Immunology

    Article Title: Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS

    doi: 10.3389/fimmu.2018.00211

    Figure Lengend Snippet: Silencing of early growth response gene-1 (EGR1) or c-FOS increased the expression of phagocytic receptors. THP-1 macrophages were transfected with siEGR1 (A,B) or siFOS (C,D) for 24 h, and then infected with Pseudomonas aeruginosa at multiplicity of infection 1 for 1 h. The mRNA levels of phagocytic receptors including mannose receptor (MR) (A,C) and scavenger receptor (SR) (B,D) were analyzed by real-time PCR. Data are shown as the mean ± SEM of three independent experiments (* p

    Article Snippet: Cell Culture Human monocytic cell line THP-1 (ATCC, TIB-202, Rockville, MA, USA) were treated with PMA (80 nM) at 37°C for 16 h, aiming to obtain the PMA-differentiated THP-1 macrophages.

    Techniques: Expressing, Transfection, Infection, Real-time Polymerase Chain Reaction

    Early growth response gene-1 (EGR1) or c-FOS enhanced autophagy in macrophages. (A–H) THP-1 macrophages were transfected with specific siRNA or overexpression plasmid for EGR1 or c-FOS for 24 h, and then infected with Pseudomonas aeruginosa (PA) for 1 h. Knockdown and overexpression effects of EGR1 (A,E) and c-FOS (B,F) were determined by western blot. Protein levels of microtubule associated protein 1 light chain 3 (LC3)-II (C,D,G,H) were tested by western blot in THP-1 macrophages before or after PA infection.

    Journal: Frontiers in Immunology

    Article Title: Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS

    doi: 10.3389/fimmu.2018.00211

    Figure Lengend Snippet: Early growth response gene-1 (EGR1) or c-FOS enhanced autophagy in macrophages. (A–H) THP-1 macrophages were transfected with specific siRNA or overexpression plasmid for EGR1 or c-FOS for 24 h, and then infected with Pseudomonas aeruginosa (PA) for 1 h. Knockdown and overexpression effects of EGR1 (A,E) and c-FOS (B,F) were determined by western blot. Protein levels of microtubule associated protein 1 light chain 3 (LC3)-II (C,D,G,H) were tested by western blot in THP-1 macrophages before or after PA infection.

    Article Snippet: Cell Culture Human monocytic cell line THP-1 (ATCC, TIB-202, Rockville, MA, USA) were treated with PMA (80 nM) at 37°C for 16 h, aiming to obtain the PMA-differentiated THP-1 macrophages.

    Techniques: Transfection, Over Expression, Plasmid Preparation, Infection, Western Blot

    Overexpression of early growth response gene-1 (EGR1) or c-FOS reversed the role of beta-defensins 2 (BD2) and beta-defensins 3 (BD3) in macrophage-mediated Pseudomonas aeruginosa (PA) elimination. THP-1 cells were transfected with EGR1, c-FOS, BD2, or BD3 overexpression plasmid control vs pSG5 plasmid for 24 h, followed by PA infection at multiplicity of infection 25, and then analyzed by phagocytosis (A–D) and killing assay (E–H) using the plate count method. Data are shown as the mean ± SEM of three independent experiments (* p

    Journal: Frontiers in Immunology

    Article Title: Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS

    doi: 10.3389/fimmu.2018.00211

    Figure Lengend Snippet: Overexpression of early growth response gene-1 (EGR1) or c-FOS reversed the role of beta-defensins 2 (BD2) and beta-defensins 3 (BD3) in macrophage-mediated Pseudomonas aeruginosa (PA) elimination. THP-1 cells were transfected with EGR1, c-FOS, BD2, or BD3 overexpression plasmid control vs pSG5 plasmid for 24 h, followed by PA infection at multiplicity of infection 25, and then analyzed by phagocytosis (A–D) and killing assay (E–H) using the plate count method. Data are shown as the mean ± SEM of three independent experiments (* p

    Article Snippet: Cell Culture Human monocytic cell line THP-1 (ATCC, TIB-202, Rockville, MA, USA) were treated with PMA (80 nM) at 37°C for 16 h, aiming to obtain the PMA-differentiated THP-1 macrophages.

    Techniques: Over Expression, Transfection, Plasmid Preparation, Infection

    Beta-defensins 2 (BD2) and beta-defensins 3 (BD3) suppressed autophagy in macrophages. (A–C) THP-1 macrophages were treated with recombinant human BD2 or BD3 or both peptides (1 µg/ml) for 6 h, and infected with Pseudomonas aeruginosa (PA) at multiplicity of infection 1 for 6 h. (D–G) RAW264.7 cells were transiently transfected with siBD2, siBD3, or both vs siNC for 24 h, and then infected with PA. (A,F) Cells were fixed, stained with anti- microtubule associated protein 1 light chain 3 (LC3) Alexa Fluro 488 fluorescent Ab, and then detected by immune fluorescence microscopy. Arrows indicate the LC3 puncta in THP-1 macrophages (A) and RAW264.7 cells (F) . (B,G) Quantification of cells containing LC3 puncta in THP-1 macrophages (B) and RAW264.7 cells (G) . (C,E) Protein levels of LC3-II were tested by western blot in THP-1 macrophages (C) and RAW264.7 cells (E) . (D) The knockdown efficiency was tested by RT PCR. Data are shown as the mean ± SEM of three independent experiments (* p

    Journal: Frontiers in Immunology

    Article Title: Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS

    doi: 10.3389/fimmu.2018.00211

    Figure Lengend Snippet: Beta-defensins 2 (BD2) and beta-defensins 3 (BD3) suppressed autophagy in macrophages. (A–C) THP-1 macrophages were treated with recombinant human BD2 or BD3 or both peptides (1 µg/ml) for 6 h, and infected with Pseudomonas aeruginosa (PA) at multiplicity of infection 1 for 6 h. (D–G) RAW264.7 cells were transiently transfected with siBD2, siBD3, or both vs siNC for 24 h, and then infected with PA. (A,F) Cells were fixed, stained with anti- microtubule associated protein 1 light chain 3 (LC3) Alexa Fluro 488 fluorescent Ab, and then detected by immune fluorescence microscopy. Arrows indicate the LC3 puncta in THP-1 macrophages (A) and RAW264.7 cells (F) . (B,G) Quantification of cells containing LC3 puncta in THP-1 macrophages (B) and RAW264.7 cells (G) . (C,E) Protein levels of LC3-II were tested by western blot in THP-1 macrophages (C) and RAW264.7 cells (E) . (D) The knockdown efficiency was tested by RT PCR. Data are shown as the mean ± SEM of three independent experiments (* p

    Article Snippet: Cell Culture Human monocytic cell line THP-1 (ATCC, TIB-202, Rockville, MA, USA) were treated with PMA (80 nM) at 37°C for 16 h, aiming to obtain the PMA-differentiated THP-1 macrophages.

    Techniques: Recombinant, Infection, Transfection, Staining, Fluorescence, Microscopy, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Beta-defensins 2 (BD2) and beta-defensins 3 (BD3) enhanced macrophage-mediated phagocytosis and intracellular killing of Pseudomonas aeruginosa (PA), and internalization of Zymosan bioparticles. (A,B) THP-1 macrophages were treated with recombinant BD2 or BD3 or both peptide (1 µg/ml) for 6 h, and infected with PA at multiplicity of infection (MOI) 25, and then the efficiency of phagocytosis (A) and intracellular killing (B) was determined by colony forming unit assay. (C–E) THP-1 macrophages were treated with BD2 or BD3 peptides (1 µg/ml) for 24 h, and treated with DQ-Red bovine serum albumin (lysosome marker, Red) for 6 h, and then incubated with Zymosan Alexa Fluro 488 Fluorescent Bioparticles (MOI = 25) for 1 h. Cells were fixed, stained with DAPI (Blue) to visualize the nuclei, and then examined by confocal microscopy [ (C) scale bar = 5 µm]. Internalization of Zymosan bioparticles was calculated by the number of phagosomes containing fluorescent Zymosan bioparticles (green dots) per cell (D) . Formation of phagolysosomes was determined by the co-localization of DQ-red positive lysosomes (red) and phagosomes containing zymosan bioparticles (green) (E) . Data are shown as the mean ± SEM of three independent experiments (* p

    Journal: Frontiers in Immunology

    Article Title: Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS

    doi: 10.3389/fimmu.2018.00211

    Figure Lengend Snippet: Beta-defensins 2 (BD2) and beta-defensins 3 (BD3) enhanced macrophage-mediated phagocytosis and intracellular killing of Pseudomonas aeruginosa (PA), and internalization of Zymosan bioparticles. (A,B) THP-1 macrophages were treated with recombinant BD2 or BD3 or both peptide (1 µg/ml) for 6 h, and infected with PA at multiplicity of infection (MOI) 25, and then the efficiency of phagocytosis (A) and intracellular killing (B) was determined by colony forming unit assay. (C–E) THP-1 macrophages were treated with BD2 or BD3 peptides (1 µg/ml) for 24 h, and treated with DQ-Red bovine serum albumin (lysosome marker, Red) for 6 h, and then incubated with Zymosan Alexa Fluro 488 Fluorescent Bioparticles (MOI = 25) for 1 h. Cells were fixed, stained with DAPI (Blue) to visualize the nuclei, and then examined by confocal microscopy [ (C) scale bar = 5 µm]. Internalization of Zymosan bioparticles was calculated by the number of phagosomes containing fluorescent Zymosan bioparticles (green dots) per cell (D) . Formation of phagolysosomes was determined by the co-localization of DQ-red positive lysosomes (red) and phagosomes containing zymosan bioparticles (green) (E) . Data are shown as the mean ± SEM of three independent experiments (* p

    Article Snippet: Cell Culture Human monocytic cell line THP-1 (ATCC, TIB-202, Rockville, MA, USA) were treated with PMA (80 nM) at 37°C for 16 h, aiming to obtain the PMA-differentiated THP-1 macrophages.

    Techniques: Recombinant, Infection, Colony-forming Unit Assay, Marker, Incubation, Staining, Confocal Microscopy

    Beta-defensins 2 (BD2) and beta-defensins 3 (BD3) reduced early growth response gene-1 (EGR1) and c-FOS expression and nuclear translocation in macrophages. (A) Affymetrix GeneChip was used to detect mRNA expression profiles in THP-1 macrophages stimulated with recombinant BD2 or BD3 peptides. Cluster analysis of gene expression profile showed the upregulated (red) or downregulated (green) genes after BD2 and BD3 stimulation in comparison with unstimulated control. All genes with expression variation in the range of 1.15-fold of control were clustered. The mRNA levels of EGR1 (B) and c-FOS (C) were tested by RT PCR in THP-1 macrophages after treatment with recombinant human BD2 or BD3 peptides at the indicated concentrations. Protein levels of EGR1 and c-FOS in the whole cell lysate (D) or in the separated part of cell cytoplasm (left) or nuclei (right) (E) were examined by western blot. Target band of c-FOS (62kD) was labeled with asterisk (D). (F,G) THP-1 cells plated in coverslips were treated with BD2 or BD3 peptides, following by PA infection. Then, the coverslips were sequentially incubated with primary EGR1 or c-FOS antibody and secondary Alexa Fluor 488 goat anti-rabbit IgG Ab. Finally, the coverslips were observed under a fluorescence microscope to capture fluorescence images. Data are shown as the mean ± SEM of three independent experiments (* p

    Journal: Frontiers in Immunology

    Article Title: Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS

    doi: 10.3389/fimmu.2018.00211

    Figure Lengend Snippet: Beta-defensins 2 (BD2) and beta-defensins 3 (BD3) reduced early growth response gene-1 (EGR1) and c-FOS expression and nuclear translocation in macrophages. (A) Affymetrix GeneChip was used to detect mRNA expression profiles in THP-1 macrophages stimulated with recombinant BD2 or BD3 peptides. Cluster analysis of gene expression profile showed the upregulated (red) or downregulated (green) genes after BD2 and BD3 stimulation in comparison with unstimulated control. All genes with expression variation in the range of 1.15-fold of control were clustered. The mRNA levels of EGR1 (B) and c-FOS (C) were tested by RT PCR in THP-1 macrophages after treatment with recombinant human BD2 or BD3 peptides at the indicated concentrations. Protein levels of EGR1 and c-FOS in the whole cell lysate (D) or in the separated part of cell cytoplasm (left) or nuclei (right) (E) were examined by western blot. Target band of c-FOS (62kD) was labeled with asterisk (D). (F,G) THP-1 cells plated in coverslips were treated with BD2 or BD3 peptides, following by PA infection. Then, the coverslips were sequentially incubated with primary EGR1 or c-FOS antibody and secondary Alexa Fluor 488 goat anti-rabbit IgG Ab. Finally, the coverslips were observed under a fluorescence microscope to capture fluorescence images. Data are shown as the mean ± SEM of three independent experiments (* p

    Article Snippet: Cell Culture Human monocytic cell line THP-1 (ATCC, TIB-202, Rockville, MA, USA) were treated with PMA (80 nM) at 37°C for 16 h, aiming to obtain the PMA-differentiated THP-1 macrophages.

    Techniques: Expressing, Translocation Assay, Recombinant, Reverse Transcription Polymerase Chain Reaction, Western Blot, Labeling, Infection, Incubation, Fluorescence, Microscopy

    Pseudomonas aeruginosa (PA) infection increased beta-defensins 2 (BD2) and beta-defensins 3 (BD3) expression in macrophages. THP-1 macrophages (A,B) and RAW264.7 cells (C,D) were infected with PA at multiplicity of infection (MOI) 1. The mRNA levels of BD2 (A,C) and BD3 (B,D) were analyzed by real-time PCR. (E) THP-1 macrophages were infected with PA at MOI 1. Protein levels of BD2 and BD3 were tested by western blot. (F) THP-1 macrophages were incubated with Zymosan Bioparticles (MOI = 1) at indicated time. Protein levels of BD2 and BD3 were tested by western blot.

    Journal: Frontiers in Immunology

    Article Title: Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS

    doi: 10.3389/fimmu.2018.00211

    Figure Lengend Snippet: Pseudomonas aeruginosa (PA) infection increased beta-defensins 2 (BD2) and beta-defensins 3 (BD3) expression in macrophages. THP-1 macrophages (A,B) and RAW264.7 cells (C,D) were infected with PA at multiplicity of infection (MOI) 1. The mRNA levels of BD2 (A,C) and BD3 (B,D) were analyzed by real-time PCR. (E) THP-1 macrophages were infected with PA at MOI 1. Protein levels of BD2 and BD3 were tested by western blot. (F) THP-1 macrophages were incubated with Zymosan Bioparticles (MOI = 1) at indicated time. Protein levels of BD2 and BD3 were tested by western blot.

    Article Snippet: Cell Culture Human monocytic cell line THP-1 (ATCC, TIB-202, Rockville, MA, USA) were treated with PMA (80 nM) at 37°C for 16 h, aiming to obtain the PMA-differentiated THP-1 macrophages.

    Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Incubation

    lefA mutant fails to grow in human monocytic THP-1 cells. Infected THP-1 cells were cultured for 2, 6, 12, and 24 h. Data are the averages of triplicate samples from three identical experiments, and error bars represent standard deviations. Statistically significant differences compared to Phi-1 are indicated by asterisks (* P

    Journal: Scientific Reports

    Article Title: Ciliate Paramecium is a natural reservoir of Legionella pneumophila

    doi: 10.1038/srep24322

    Figure Lengend Snippet: lefA mutant fails to grow in human monocytic THP-1 cells. Infected THP-1 cells were cultured for 2, 6, 12, and 24 h. Data are the averages of triplicate samples from three identical experiments, and error bars represent standard deviations. Statistically significant differences compared to Phi-1 are indicated by asterisks (* P

    Article Snippet: THP-1 cells culture and infection assay Cells from the human monocytic cell line THP-1 were grown in RPMI 1640 medium (Sigma–Aldrich), supplemented with 10% heat-inactivated FBS at 37 °C under an atmosphere containing 5% CO2 .

    Techniques: Mutagenesis, Infection, Cell Culture

    TNFα increases intracellular production of S100 proteins in THP-1 cells and S100A8 and S100A9 is co-localized in the vesicles along the endocytic pathway. THP-1 cells were cultured for 48h in presence or absence of 10 ng/ml TNFα. S100A8 and S100A9 mRNA expression was determined using qRT-PCR and was normalized to β-actin mRNA level. Data indicate mean ± SD of triplicate samples. Differences between treated and untreated groups are significant at ***P

    Journal: PLoS ONE

    Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

    doi: 10.1371/journal.pone.0145217

    Figure Lengend Snippet: TNFα increases intracellular production of S100 proteins in THP-1 cells and S100A8 and S100A9 is co-localized in the vesicles along the endocytic pathway. THP-1 cells were cultured for 48h in presence or absence of 10 ng/ml TNFα. S100A8 and S100A9 mRNA expression was determined using qRT-PCR and was normalized to β-actin mRNA level. Data indicate mean ± SD of triplicate samples. Differences between treated and untreated groups are significant at ***P

    Article Snippet: LDH assay Release of lactate dehydrogenase was assayed from the culture supernatants of THP-1 cells to determine Cytotoxicity by using Pierce LDH cytotoxicity assay kit (Thermoscientific, Rockford, USA) following manufacturer’s protocol.

    Techniques: Cell Culture, Expressing, Quantitative RT-PCR

    TNFα stimulates secretion of S100 proteins without causing cell death. THP-1 cells were cultured in triplicate either in the presence or absence of 10 ng/ml TNFα and TNFα + IL-10 (10 ng/ml). Culture supernatant was collected after 48h of incubation and S100A8/S100A9 dimer concentration was determined with ELISA following manufacturer’s protocol (A). Percent cell death was determined using LDH assay (B). In some experiments, THP-1 cells were stimulated with TNFα for 48 h in presence of methylamine (30 μM). In other experiments, cells were stimulated 12 h with TNFα without methylamine, followed by 36 h of culture with the drug. Alternatively, THP-1 cells were stimulated with TNFα; after 36 h, brefeldin A (5 μg/ml) was added and cells were cultured for another 12 h. Culture supernatant was collected after completion of incubation and S100A8/A9 heterodimer (C) and S100A9 (D) concentration was determined with ELISA following manufacturer’s protocol. Values are means of triplicates ± SD. Differences between various treatment groups are significant at **P

    Journal: PLoS ONE

    Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

    doi: 10.1371/journal.pone.0145217

    Figure Lengend Snippet: TNFα stimulates secretion of S100 proteins without causing cell death. THP-1 cells were cultured in triplicate either in the presence or absence of 10 ng/ml TNFα and TNFα + IL-10 (10 ng/ml). Culture supernatant was collected after 48h of incubation and S100A8/S100A9 dimer concentration was determined with ELISA following manufacturer’s protocol (A). Percent cell death was determined using LDH assay (B). In some experiments, THP-1 cells were stimulated with TNFα for 48 h in presence of methylamine (30 μM). In other experiments, cells were stimulated 12 h with TNFα without methylamine, followed by 36 h of culture with the drug. Alternatively, THP-1 cells were stimulated with TNFα; after 36 h, brefeldin A (5 μg/ml) was added and cells were cultured for another 12 h. Culture supernatant was collected after completion of incubation and S100A8/A9 heterodimer (C) and S100A9 (D) concentration was determined with ELISA following manufacturer’s protocol. Values are means of triplicates ± SD. Differences between various treatment groups are significant at **P

    Article Snippet: LDH assay Release of lactate dehydrogenase was assayed from the culture supernatants of THP-1 cells to determine Cytotoxicity by using Pierce LDH cytotoxicity assay kit (Thermoscientific, Rockford, USA) following manufacturer’s protocol.

    Techniques: Cell Culture, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay

    SPR analysis of S100A9 in isolated plasma membranes from THP-1 cells. (A) Binding of anti-S100A8 (8-5C2), S100A9 (43/8) and S100A8/S100A9 (27E10) to SA captured biotinylated plasma membranes. Antibodies were injected at 6.25–50 nM for 2 min at 30 μL/min in assay buffer (HBS-P with 1 mM Ca ++ + 20 μM Zn ++ ). A 15 μL pulse of 10 mM glycine-HCl was used for regeneration. Capture level ~ 2.1 kRU. Kinetic evaluation was made using a 1:1 model for mAb 43/8 at 25 and 50 nM. An affinity of 7.0 nM ( k on 1.9 × 10 5 1/Ms; k off 1.3 × 10 −3 1/s) was calculated with maximal binding of 46 RU. (B) Injection of non-biotinylated THP-1 lysate over immobilized mAb 8-5C2, 43/8 and 27E10 (density ~ 3 kRU). Lysate, 0.45 mg/mL, diluted 5 to 40 times in assay buffer, injected for 3 min at 20 μL/min. Distinct binding was obtained when the THP-1 lysate was injected over the 27E10 surface whereas no binding was observed when lysate was passed over 8-5C2 and 43/8. (C) Binding of ten-fold diluted non-biotinylated THP-1 lysate to mAb 27E10 is inhibited after pre-incubation with 50 nM 27E10 but not with 8-5C2 or 43/8. Binding was expressed as % of response without antibody. (D) Sensorgrams obtained after injection of 50 nM mAb 8-5C2, 43/8 and 27E10 over SA captured (level ~ 1.1 kRU) solubilized membranes from surface biotinylated human monocytes. An affinity of ~ 6.1 nM (( k on 2.7 × 10 5 1/Ms; k off 1.6 × 10 −3 1/s) and maximal binding at ~ 12 RU was obtained after fit of sensorgram obtained with the 43/8 antibody to a 1:1 model. (E) Interaction of non-biotinylated lysate from human monocytes with 8-5C2, 43/8 and 27E10. Serially diluted lysate (5- to 40-fold in assay buffer) was injected over immobilized antibodies as in Fig 1B . Binding at high levels was demonstrated to mAb 27E10 (reaching steady state levels at ~ 1,000 RU) and was completely blocked when lysate (diluted 5-fold) was pre-incubated with 50 nM 27E10 but not with 8-5C2 or 43/8 (Fig 2C). Specific (i.e. displaceable by pre-incubation with anti-S100A9; data not shown) binding at much lower levels was also shown to 43/8 whereas non-specific binding was demonstrated to 8-5C2 (Fig 2E).

    Journal: PLoS ONE

    Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

    doi: 10.1371/journal.pone.0145217

    Figure Lengend Snippet: SPR analysis of S100A9 in isolated plasma membranes from THP-1 cells. (A) Binding of anti-S100A8 (8-5C2), S100A9 (43/8) and S100A8/S100A9 (27E10) to SA captured biotinylated plasma membranes. Antibodies were injected at 6.25–50 nM for 2 min at 30 μL/min in assay buffer (HBS-P with 1 mM Ca ++ + 20 μM Zn ++ ). A 15 μL pulse of 10 mM glycine-HCl was used for regeneration. Capture level ~ 2.1 kRU. Kinetic evaluation was made using a 1:1 model for mAb 43/8 at 25 and 50 nM. An affinity of 7.0 nM ( k on 1.9 × 10 5 1/Ms; k off 1.3 × 10 −3 1/s) was calculated with maximal binding of 46 RU. (B) Injection of non-biotinylated THP-1 lysate over immobilized mAb 8-5C2, 43/8 and 27E10 (density ~ 3 kRU). Lysate, 0.45 mg/mL, diluted 5 to 40 times in assay buffer, injected for 3 min at 20 μL/min. Distinct binding was obtained when the THP-1 lysate was injected over the 27E10 surface whereas no binding was observed when lysate was passed over 8-5C2 and 43/8. (C) Binding of ten-fold diluted non-biotinylated THP-1 lysate to mAb 27E10 is inhibited after pre-incubation with 50 nM 27E10 but not with 8-5C2 or 43/8. Binding was expressed as % of response without antibody. (D) Sensorgrams obtained after injection of 50 nM mAb 8-5C2, 43/8 and 27E10 over SA captured (level ~ 1.1 kRU) solubilized membranes from surface biotinylated human monocytes. An affinity of ~ 6.1 nM (( k on 2.7 × 10 5 1/Ms; k off 1.6 × 10 −3 1/s) and maximal binding at ~ 12 RU was obtained after fit of sensorgram obtained with the 43/8 antibody to a 1:1 model. (E) Interaction of non-biotinylated lysate from human monocytes with 8-5C2, 43/8 and 27E10. Serially diluted lysate (5- to 40-fold in assay buffer) was injected over immobilized antibodies as in Fig 1B . Binding at high levels was demonstrated to mAb 27E10 (reaching steady state levels at ~ 1,000 RU) and was completely blocked when lysate (diluted 5-fold) was pre-incubated with 50 nM 27E10 but not with 8-5C2 or 43/8 (Fig 2C). Specific (i.e. displaceable by pre-incubation with anti-S100A9; data not shown) binding at much lower levels was also shown to 43/8 whereas non-specific binding was demonstrated to 8-5C2 (Fig 2E).

    Article Snippet: LDH assay Release of lactate dehydrogenase was assayed from the culture supernatants of THP-1 cells to determine Cytotoxicity by using Pierce LDH cytotoxicity assay kit (Thermoscientific, Rockford, USA) following manufacturer’s protocol.

    Techniques: SPR Assay, Isolation, Binding Assay, Injection, Mass Spectrometry, Incubation

    Western blot analysis under reducing conditions (10% SDS) of plasma membrane fractions prepared from cell surface biotinylated THP-1 cells and human monocytes. (A) S100A9 in the biotinylated plasma membrane fraction of THP-1 cells: non-biotinylated membrane fraction (lane a) and biotinylated cell surface fraction (lane b). (B) S100A8 expression in biotinylated THP-1 cells (same sample fractions as in (A). (C) Expression of S100A9 (left) and S100A8 (right) in the non-biotinylated membrane fraction (lane a) and biotinylated cell surface fraction (lane b) of human monocytes. Antibodies used were; α-S100A9 (NOVUS clone 1C10) and α-S100A8 (BMA; T1030; clone 8-5C2). 4 ng recombinant hS100A9 (rA9) was loaded.

    Journal: PLoS ONE

    Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

    doi: 10.1371/journal.pone.0145217

    Figure Lengend Snippet: Western blot analysis under reducing conditions (10% SDS) of plasma membrane fractions prepared from cell surface biotinylated THP-1 cells and human monocytes. (A) S100A9 in the biotinylated plasma membrane fraction of THP-1 cells: non-biotinylated membrane fraction (lane a) and biotinylated cell surface fraction (lane b). (B) S100A8 expression in biotinylated THP-1 cells (same sample fractions as in (A). (C) Expression of S100A9 (left) and S100A8 (right) in the non-biotinylated membrane fraction (lane a) and biotinylated cell surface fraction (lane b) of human monocytes. Antibodies used were; α-S100A9 (NOVUS clone 1C10) and α-S100A8 (BMA; T1030; clone 8-5C2). 4 ng recombinant hS100A9 (rA9) was loaded.

    Article Snippet: LDH assay Release of lactate dehydrogenase was assayed from the culture supernatants of THP-1 cells to determine Cytotoxicity by using Pierce LDH cytotoxicity assay kit (Thermoscientific, Rockford, USA) following manufacturer’s protocol.

    Techniques: Western Blot, Expressing, Recombinant

    Distribution of hS100A8 and hS100A9 proteins in THP-1 cells by confocal and electron microscopy. THP1 cells were cultured for 48h in presence or absence of 10ng/ml TNFα and stained with antibodies against S100A9 (purple) (A). THP-1 cells were treated with either TNFα (B) or TNFα + IL10 (C); stained with anti-S100A8 (red), anti-S100A9 (purple) and anti-S100A8/S100A9 (green) antibodies. Arrows indicate the vesicles containing only hS100A9 or hS100A8. (D) Distribution of hS100A8 and hS100A9 proteins in THP-1 cells by Transmission Electron Microscopy. Black and white boxes represent magnification of areas where S100A8 and S100A9 hetero- or homodimers can be observed respectively. Further, black arrows highlight cytoplasmic vesicles or plasma membrane patches where hS100A9 (big dots) and hS100A8 (small dots) proteins co-localize, while red arrows point to vesicles containing only hS100A9 or hS100A8 homodimers.

    Journal: PLoS ONE

    Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

    doi: 10.1371/journal.pone.0145217

    Figure Lengend Snippet: Distribution of hS100A8 and hS100A9 proteins in THP-1 cells by confocal and electron microscopy. THP1 cells were cultured for 48h in presence or absence of 10ng/ml TNFα and stained with antibodies against S100A9 (purple) (A). THP-1 cells were treated with either TNFα (B) or TNFα + IL10 (C); stained with anti-S100A8 (red), anti-S100A9 (purple) and anti-S100A8/S100A9 (green) antibodies. Arrows indicate the vesicles containing only hS100A9 or hS100A8. (D) Distribution of hS100A8 and hS100A9 proteins in THP-1 cells by Transmission Electron Microscopy. Black and white boxes represent magnification of areas where S100A8 and S100A9 hetero- or homodimers can be observed respectively. Further, black arrows highlight cytoplasmic vesicles or plasma membrane patches where hS100A9 (big dots) and hS100A8 (small dots) proteins co-localize, while red arrows point to vesicles containing only hS100A9 or hS100A8 homodimers.

    Article Snippet: LDH assay Release of lactate dehydrogenase was assayed from the culture supernatants of THP-1 cells to determine Cytotoxicity by using Pierce LDH cytotoxicity assay kit (Thermoscientific, Rockford, USA) following manufacturer’s protocol.

    Techniques: Electron Microscopy, Cell Culture, Staining, Transmission Assay

    S100A8 and S100A9 are present in endo-lysosmal enriched fraction. THP-1 cells were grown for 48 h in presence or absence of TNFα. Cells were harvested after 48 h (A) or after 24h and 48h (B) and gently homogenized in homogenization buffer. Crude nuclear pellet was discarded by centrifugation. The post-nuclear supernatant (PNS) centrifuged at 50,000g for 20 min. The supernatant (S50; post endo-lysosomal fraction) was collected and pellet (P50) was solubilized in 1% Triton X-100 and aliquots from PNS, P50 fractions and S50 fractions were analyzed by Western blotting using the indicated antibodies.

    Journal: PLoS ONE

    Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

    doi: 10.1371/journal.pone.0145217

    Figure Lengend Snippet: S100A8 and S100A9 are present in endo-lysosmal enriched fraction. THP-1 cells were grown for 48 h in presence or absence of TNFα. Cells were harvested after 48 h (A) or after 24h and 48h (B) and gently homogenized in homogenization buffer. Crude nuclear pellet was discarded by centrifugation. The post-nuclear supernatant (PNS) centrifuged at 50,000g for 20 min. The supernatant (S50; post endo-lysosomal fraction) was collected and pellet (P50) was solubilized in 1% Triton X-100 and aliquots from PNS, P50 fractions and S50 fractions were analyzed by Western blotting using the indicated antibodies.

    Article Snippet: LDH assay Release of lactate dehydrogenase was assayed from the culture supernatants of THP-1 cells to determine Cytotoxicity by using Pierce LDH cytotoxicity assay kit (Thermoscientific, Rockford, USA) following manufacturer’s protocol.

    Techniques: Homogenization, Centrifugation, Western Blot

    Identification of the 40-kDa protein as pleckstrin by mass spectrometry. The membrane fraction of THP-1 cells stimulated with 200 nM PMA for 20 min was harvested. Proteins in the membrane fraction were then separated on a 10% SDS-PAGE gel. The band corresponding to 40 kDa (arrow in A ) was excised from the SDS-PAGE gel stained with Coomassie blue for analysis. B , The results of MS analysis. Five tryptic peptides (A-E) mapping to pleckstrin protein sequence were found in MS. Three (A-C) are statistically significant as demonstrated by the Xcorr value > 2.0. The A-E peptide sequences are shown along with the total protein coverage of pleckstrin (bold type).

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Phosphorylation of Pleckstrin Increases Proinflammatory Cytokine Secretion by Mononuclear Phagocytes in Diabetes Mellitus 1

    doi:

    Figure Lengend Snippet: Identification of the 40-kDa protein as pleckstrin by mass spectrometry. The membrane fraction of THP-1 cells stimulated with 200 nM PMA for 20 min was harvested. Proteins in the membrane fraction were then separated on a 10% SDS-PAGE gel. The band corresponding to 40 kDa (arrow in A ) was excised from the SDS-PAGE gel stained with Coomassie blue for analysis. B , The results of MS analysis. Five tryptic peptides (A-E) mapping to pleckstrin protein sequence were found in MS. Three (A-C) are statistically significant as demonstrated by the Xcorr value > 2.0. The A-E peptide sequences are shown along with the total protein coverage of pleckstrin (bold type).

    Article Snippet: THP-1 cells, which were within the 2-10h passages, were differentiated with VitD3 2 days before the transfection. siRNA was introduced by electroporation using a Nucleofector instrument and specific Nucleofector kit for THP-1 Cells (Amaxa Biosystems).

    Techniques: Mass Spectrometry, SDS Page, Staining, Sequencing

    siRNA inhibition of pleckstrin in THP-1 cells. A , THP-1 cells (1 × 10 6 ) were transfected with control siRNA or pleckstrin (PLEK) siRNA and cultured for 24 h at 37°C, 5% CO 2 . The efficiency of the reduction on total pleckstrin protein was evaluated with Western blotting with pleckstrin Ab. Total pleckstrin expression was reduced by 30% as demonstrated by densitometry. B , THP-1 cells (1 × 10 6 ) were transfected with control siRNA or pleckstrin (PLEK) siRNA. After 24 h incubation, cells were treated with 5 μg/ml S100B. Phosphorylation of PKC substrates was monitored with pPKC(ser) Ab with Western blotting. Pleckstrin phosphorylation was also reduced by 30% as demonstrated by densitometry. Results are representative of three independent experiments.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Phosphorylation of Pleckstrin Increases Proinflammatory Cytokine Secretion by Mononuclear Phagocytes in Diabetes Mellitus 1

    doi:

    Figure Lengend Snippet: siRNA inhibition of pleckstrin in THP-1 cells. A , THP-1 cells (1 × 10 6 ) were transfected with control siRNA or pleckstrin (PLEK) siRNA and cultured for 24 h at 37°C, 5% CO 2 . The efficiency of the reduction on total pleckstrin protein was evaluated with Western blotting with pleckstrin Ab. Total pleckstrin expression was reduced by 30% as demonstrated by densitometry. B , THP-1 cells (1 × 10 6 ) were transfected with control siRNA or pleckstrin (PLEK) siRNA. After 24 h incubation, cells were treated with 5 μg/ml S100B. Phosphorylation of PKC substrates was monitored with pPKC(ser) Ab with Western blotting. Pleckstrin phosphorylation was also reduced by 30% as demonstrated by densitometry. Results are representative of three independent experiments.

    Article Snippet: THP-1 cells, which were within the 2-10h passages, were differentiated with VitD3 2 days before the transfection. siRNA was introduced by electroporation using a Nucleofector instrument and specific Nucleofector kit for THP-1 Cells (Amaxa Biosystems).

    Techniques: Inhibition, Transfection, Cell Culture, Western Blot, Expressing, Incubation

    The role of pleckstrin in proinflammatory cytokine release induced by S100B. THP-1 cells (1 × 10 6 ) were transfected with control siRNA or pleckstrin siRNA and cultured for 24 h at 37°C, 5% CO 2 . Cells were stimulated with 1 μg/ml S100B for 24 h. The supernatants were collected for ELISA analysis to detect the concentration of TNF-α ( A ) and IL-1β ( B ). The concentration of cytokine is expressed as picograms per milligram per microgram of total protein. All values represent the average ± SD from four independent experiments. *, p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Phosphorylation of Pleckstrin Increases Proinflammatory Cytokine Secretion by Mononuclear Phagocytes in Diabetes Mellitus 1

    doi:

    Figure Lengend Snippet: The role of pleckstrin in proinflammatory cytokine release induced by S100B. THP-1 cells (1 × 10 6 ) were transfected with control siRNA or pleckstrin siRNA and cultured for 24 h at 37°C, 5% CO 2 . Cells were stimulated with 1 μg/ml S100B for 24 h. The supernatants were collected for ELISA analysis to detect the concentration of TNF-α ( A ) and IL-1β ( B ). The concentration of cytokine is expressed as picograms per milligram per microgram of total protein. All values represent the average ± SD from four independent experiments. *, p

    Article Snippet: THP-1 cells, which were within the 2-10h passages, were differentiated with VitD3 2 days before the transfection. siRNA was introduced by electroporation using a Nucleofector instrument and specific Nucleofector kit for THP-1 Cells (Amaxa Biosystems).

    Techniques: Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay

    PMA induces the translocation of pleckstrin to the membrane. THP-1 cells were treated with PBS containing 0.25% DMSO or with 200 nM PMA for 5 min, lysed with hypertonic buffer, and fractionated into membrane and cytosol fractions. The amount of pleckstrin in these two fractions was detected by Western blotting with an Ab against pleckstrin. Results are representative of three independent experiments. Because β-actin is not expressed in both cell fractions; equal loading in each lane was confirmed by Coomassie blue staining (data not shown).

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Phosphorylation of Pleckstrin Increases Proinflammatory Cytokine Secretion by Mononuclear Phagocytes in Diabetes Mellitus 1

    doi:

    Figure Lengend Snippet: PMA induces the translocation of pleckstrin to the membrane. THP-1 cells were treated with PBS containing 0.25% DMSO or with 200 nM PMA for 5 min, lysed with hypertonic buffer, and fractionated into membrane and cytosol fractions. The amount of pleckstrin in these two fractions was detected by Western blotting with an Ab against pleckstrin. Results are representative of three independent experiments. Because β-actin is not expressed in both cell fractions; equal loading in each lane was confirmed by Coomassie blue staining (data not shown).

    Article Snippet: THP-1 cells, which were within the 2-10h passages, were differentiated with VitD3 2 days before the transfection. siRNA was introduced by electroporation using a Nucleofector instrument and specific Nucleofector kit for THP-1 Cells (Amaxa Biosystems).

    Techniques: Translocation Assay, Western Blot, Staining

    IP confirms pleckstrin as the 40-kDa protein. THP-1 cells were treated with either PBS containing 0.25% DMSO or 200 nM PMA for 5 min at 37°C, 5% CO 2 . The first two lanes are whole cell lysates with and without PMA stimulation, showing the total pleckstrin in the cells under these conditions. The last three lanes are immunoprecipitates from the membrane fraction with either control IgG or pPKC(s) Ab. An Ab against human pleckstrin was used to detect pleckstrin in these samples. Immunoprecipitates with pPKC(s) Ab from PMA-stimulated THP-1 cells contained the phosphorylated PKC substrates on the membrane; the arrow indicates the presence of pleckstrin. The 50-kDa band is the IgG band, representing the contaminating fragments of the Ab in the eluted Ags. Results are representative of three independent experiments.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Phosphorylation of Pleckstrin Increases Proinflammatory Cytokine Secretion by Mononuclear Phagocytes in Diabetes Mellitus 1

    doi:

    Figure Lengend Snippet: IP confirms pleckstrin as the 40-kDa protein. THP-1 cells were treated with either PBS containing 0.25% DMSO or 200 nM PMA for 5 min at 37°C, 5% CO 2 . The first two lanes are whole cell lysates with and without PMA stimulation, showing the total pleckstrin in the cells under these conditions. The last three lanes are immunoprecipitates from the membrane fraction with either control IgG or pPKC(s) Ab. An Ab against human pleckstrin was used to detect pleckstrin in these samples. Immunoprecipitates with pPKC(s) Ab from PMA-stimulated THP-1 cells contained the phosphorylated PKC substrates on the membrane; the arrow indicates the presence of pleckstrin. The 50-kDa band is the IgG band, representing the contaminating fragments of the Ab in the eluted Ags. Results are representative of three independent experiments.

    Article Snippet: THP-1 cells, which were within the 2-10h passages, were differentiated with VitD3 2 days before the transfection. siRNA was introduced by electroporation using a Nucleofector instrument and specific Nucleofector kit for THP-1 Cells (Amaxa Biosystems).

    Techniques:

    PMA induces the phosphorylation of pleckstrin. A , Normal human mononuclear phagocytes and THP-1 cells were treated with PBS containing 0.25% (v/v) DMSO (resting) or 200 nM PMA for 5 min. Western blot analysis was performed to detect phosphorylation of PKC substrates in the whole cell lysates. The arrow indicates the increased phosphorylation of pleckstrin. B , THP-1 cells were stimulated with 200 nM PMA for 5 and 20 min. Cells were lysed with hypertonic buffer for cell fractionation. Both membrane and cytosolic fractions were analyzed by Western blotting to detect phosphorylation of cPKC substrates and their cellular distribution. The arrow indicates that the phosphorylated 40-kDa pleckstrin is located in the membrane fraction after PMA stimulation. The amount of total protein loaded in each lane was confirmed by Coomassie blue staining. Results are representative of three independent experiments.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Phosphorylation of Pleckstrin Increases Proinflammatory Cytokine Secretion by Mononuclear Phagocytes in Diabetes Mellitus 1

    doi:

    Figure Lengend Snippet: PMA induces the phosphorylation of pleckstrin. A , Normal human mononuclear phagocytes and THP-1 cells were treated with PBS containing 0.25% (v/v) DMSO (resting) or 200 nM PMA for 5 min. Western blot analysis was performed to detect phosphorylation of PKC substrates in the whole cell lysates. The arrow indicates the increased phosphorylation of pleckstrin. B , THP-1 cells were stimulated with 200 nM PMA for 5 and 20 min. Cells were lysed with hypertonic buffer for cell fractionation. Both membrane and cytosolic fractions were analyzed by Western blotting to detect phosphorylation of cPKC substrates and their cellular distribution. The arrow indicates that the phosphorylated 40-kDa pleckstrin is located in the membrane fraction after PMA stimulation. The amount of total protein loaded in each lane was confirmed by Coomassie blue staining. Results are representative of three independent experiments.

    Article Snippet: THP-1 cells, which were within the 2-10h passages, were differentiated with VitD3 2 days before the transfection. siRNA was introduced by electroporation using a Nucleofector instrument and specific Nucleofector kit for THP-1 Cells (Amaxa Biosystems).

    Techniques: Western Blot, Cell Fractionation, Staining

    S100B induces the phosphorylation and translocation of pleckstrin to the membrane. THP-1 cells were stimulated with 5 μg/ml S100B for the times shown at 37°C, 5% CO 2 . The membrane portion of the cell lysates was extracted and the phosphorylation and presence of pleckstrin was detected by Western blotting with pPKC(s) Ab. The arrow indicates phosphorylated pleckstrin. Results are representative of three independent experiments. The amount of total protein loaded in each lane was confirmed by Coomassie blue staining.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Phosphorylation of Pleckstrin Increases Proinflammatory Cytokine Secretion by Mononuclear Phagocytes in Diabetes Mellitus 1

    doi:

    Figure Lengend Snippet: S100B induces the phosphorylation and translocation of pleckstrin to the membrane. THP-1 cells were stimulated with 5 μg/ml S100B for the times shown at 37°C, 5% CO 2 . The membrane portion of the cell lysates was extracted and the phosphorylation and presence of pleckstrin was detected by Western blotting with pPKC(s) Ab. The arrow indicates phosphorylated pleckstrin. Results are representative of three independent experiments. The amount of total protein loaded in each lane was confirmed by Coomassie blue staining.

    Article Snippet: THP-1 cells, which were within the 2-10h passages, were differentiated with VitD3 2 days before the transfection. siRNA was introduced by electroporation using a Nucleofector instrument and specific Nucleofector kit for THP-1 Cells (Amaxa Biosystems).

    Techniques: Translocation Assay, Western Blot, Staining

    Full-length and truncated needle proteins activate NF-κB/AP-1 in THP1-XBlue cells in an MyD88-dependent manner. THP-1 (A) and THP-1 defMyd88 (B) cells were seeded in wells and treated with PBS, 1 μg/ml of HKLM (Heat Killed Listeria monocytogenes

    Journal: Infection and Immunity

    Article Title: The N Terminus of Type III Secretion Needle Protein YscF from Yersinia pestis Functions To Modulate Innate Immune Responses

    doi: 10.1128/IAI.02687-14

    Figure Lengend Snippet: Full-length and truncated needle proteins activate NF-κB/AP-1 in THP1-XBlue cells in an MyD88-dependent manner. THP-1 (A) and THP-1 defMyd88 (B) cells were seeded in wells and treated with PBS, 1 μg/ml of HKLM (Heat Killed Listeria monocytogenes

    Article Snippet: The human monocyte cell line THP-1 (ATCC TIB-202) and THP1-XBlue cells (InvivoGen, San Diego, CA) were maintained in RPMI 1640 (Corning Cellgro, Manassas, VA) containing 10% (vol/vol) heat-inactivated fetal calf serum (Invitrogen), 25 mM HEPES (Fisher Scientific, Pittsburgh, PA), 2 mM l -glutamine (Corning Cellgro), 1 mM sodium pyruvate (Corning Cellgro), and 50 μg/ml of Pen-Strep (Corning Cellgro) at 37°C with 5% CO2 .

    Techniques:

    YscF truncated at 15 amino acids has the highest NF-κB/AP-1 and cytokine activation. (A) THP1-XBlue cells were seeded in wells and treated with PBS, 1 μg/ml of LPS, or 1 μg/ml of recombinant needle protein dissolved in PBS. SEAP

    Journal: Infection and Immunity

    Article Title: The N Terminus of Type III Secretion Needle Protein YscF from Yersinia pestis Functions To Modulate Innate Immune Responses

    doi: 10.1128/IAI.02687-14

    Figure Lengend Snippet: YscF truncated at 15 amino acids has the highest NF-κB/AP-1 and cytokine activation. (A) THP1-XBlue cells were seeded in wells and treated with PBS, 1 μg/ml of LPS, or 1 μg/ml of recombinant needle protein dissolved in PBS. SEAP

    Article Snippet: The human monocyte cell line THP-1 (ATCC TIB-202) and THP1-XBlue cells (InvivoGen, San Diego, CA) were maintained in RPMI 1640 (Corning Cellgro, Manassas, VA) containing 10% (vol/vol) heat-inactivated fetal calf serum (Invitrogen), 25 mM HEPES (Fisher Scientific, Pittsburgh, PA), 2 mM l -glutamine (Corning Cellgro), 1 mM sodium pyruvate (Corning Cellgro), and 50 μg/ml of Pen-Strep (Corning Cellgro) at 37°C with 5% CO2 .

    Techniques: Activation Assay, Recombinant

    The N terminus of YscF reduces NF-κB/AP-1 and cytokine activation by SsaG. THP1-XBlue cells were seeded in wells and treated with PBS, 1 μg/ml of LPS, or 1 μg/ml of needle proteins: YscF, SsaG, and chimeric YscF-SsaG (A) or YscF,

    Journal: Infection and Immunity

    Article Title: The N Terminus of Type III Secretion Needle Protein YscF from Yersinia pestis Functions To Modulate Innate Immune Responses

    doi: 10.1128/IAI.02687-14

    Figure Lengend Snippet: The N terminus of YscF reduces NF-κB/AP-1 and cytokine activation by SsaG. THP1-XBlue cells were seeded in wells and treated with PBS, 1 μg/ml of LPS, or 1 μg/ml of needle proteins: YscF, SsaG, and chimeric YscF-SsaG (A) or YscF,

    Article Snippet: The human monocyte cell line THP-1 (ATCC TIB-202) and THP1-XBlue cells (InvivoGen, San Diego, CA) were maintained in RPMI 1640 (Corning Cellgro, Manassas, VA) containing 10% (vol/vol) heat-inactivated fetal calf serum (Invitrogen), 25 mM HEPES (Fisher Scientific, Pittsburgh, PA), 2 mM l -glutamine (Corning Cellgro), 1 mM sodium pyruvate (Corning Cellgro), and 50 μg/ml of Pen-Strep (Corning Cellgro) at 37°C with 5% CO2 .

    Techniques: Activation Assay

    Alanine-scanning mutants of YscFΔ(S2-D15). THP1-XBlue (A), HEK 293 TLR2 (B), and HEK 293 TLR2 (C) cells were seeded in wells and treated with PBS, 1 μg/ml of LPS, 1 μg/ml of HKLM, or 1 μg/ml of recombinant needle proteins

    Journal: Infection and Immunity

    Article Title: The N Terminus of Type III Secretion Needle Protein YscF from Yersinia pestis Functions To Modulate Innate Immune Responses

    doi: 10.1128/IAI.02687-14

    Figure Lengend Snippet: Alanine-scanning mutants of YscFΔ(S2-D15). THP1-XBlue (A), HEK 293 TLR2 (B), and HEK 293 TLR2 (C) cells were seeded in wells and treated with PBS, 1 μg/ml of LPS, 1 μg/ml of HKLM, or 1 μg/ml of recombinant needle proteins

    Article Snippet: The human monocyte cell line THP-1 (ATCC TIB-202) and THP1-XBlue cells (InvivoGen, San Diego, CA) were maintained in RPMI 1640 (Corning Cellgro, Manassas, VA) containing 10% (vol/vol) heat-inactivated fetal calf serum (Invitrogen), 25 mM HEPES (Fisher Scientific, Pittsburgh, PA), 2 mM l -glutamine (Corning Cellgro), 1 mM sodium pyruvate (Corning Cellgro), and 50 μg/ml of Pen-Strep (Corning Cellgro) at 37°C with 5% CO2 .

    Techniques: Recombinant

    Ysc needles lacking the N terminus of YscF increases NF-κB/AP-1 and cytokine activation. (A) THP1-XBlue cells were seeded in wells and treated with PBS, 1 μg/ml LPS, or 1 μg/ml of sheared needle dissolved in PBS. SEAP levels were

    Journal: Infection and Immunity

    Article Title: The N Terminus of Type III Secretion Needle Protein YscF from Yersinia pestis Functions To Modulate Innate Immune Responses

    doi: 10.1128/IAI.02687-14

    Figure Lengend Snippet: Ysc needles lacking the N terminus of YscF increases NF-κB/AP-1 and cytokine activation. (A) THP1-XBlue cells were seeded in wells and treated with PBS, 1 μg/ml LPS, or 1 μg/ml of sheared needle dissolved in PBS. SEAP levels were

    Article Snippet: The human monocyte cell line THP-1 (ATCC TIB-202) and THP1-XBlue cells (InvivoGen, San Diego, CA) were maintained in RPMI 1640 (Corning Cellgro, Manassas, VA) containing 10% (vol/vol) heat-inactivated fetal calf serum (Invitrogen), 25 mM HEPES (Fisher Scientific, Pittsburgh, PA), 2 mM l -glutamine (Corning Cellgro), 1 mM sodium pyruvate (Corning Cellgro), and 50 μg/ml of Pen-Strep (Corning Cellgro) at 37°C with 5% CO2 .

    Techniques: Activation Assay

    SAMHD1 knockdown and Vpx enhance the dNTP pool in PMA-treated THP-1 cells. (a) THP-1, engineered to stably express shRNA scrambled (control) or shRNA specifically targeting SAMHD1, were differentiated overnight with PMA. Where indicated, the cells were incubated for 2 h with Vpx-containing and control VLP (+/−Vpx). Cell extracts were analyzed by western blot with the indicated antibodies. (b) dNTP concentrations were determined by the single nucleotide incorporation assay described in Diamond et al . The experiment was performed in duplicate and one representative result is shown. (c) dNTP triphosphohydrolase activity of recombinant SAMHD1. dNTPase activity assay was performed as described in the methods section in the presence of the indicated dNTP, 1 μCi of the corresponding γ- 32 P-dNTP and 1 μM of wild type SAMHD1. Where indicated 200 μM of unlabeled dGTP was added. The non-labeled standards were visualized by UV shadowing and γ- 32 P-labeled nucleotides were visualized using a phosphorimager (Fluorescent Image Analyzer FLA3000 (Fuji).

    Journal: Nature immunology

    Article Title: SAMHD1 restricts HIV-1 by reducing the intracellular pool of deoxynucleotide triphosphates

    doi: 10.1038/ni.2236

    Figure Lengend Snippet: SAMHD1 knockdown and Vpx enhance the dNTP pool in PMA-treated THP-1 cells. (a) THP-1, engineered to stably express shRNA scrambled (control) or shRNA specifically targeting SAMHD1, were differentiated overnight with PMA. Where indicated, the cells were incubated for 2 h with Vpx-containing and control VLP (+/−Vpx). Cell extracts were analyzed by western blot with the indicated antibodies. (b) dNTP concentrations were determined by the single nucleotide incorporation assay described in Diamond et al . The experiment was performed in duplicate and one representative result is shown. (c) dNTP triphosphohydrolase activity of recombinant SAMHD1. dNTPase activity assay was performed as described in the methods section in the presence of the indicated dNTP, 1 μCi of the corresponding γ- 32 P-dNTP and 1 μM of wild type SAMHD1. Where indicated 200 μM of unlabeled dGTP was added. The non-labeled standards were visualized by UV shadowing and γ- 32 P-labeled nucleotides were visualized using a phosphorimager (Fluorescent Image Analyzer FLA3000 (Fuji).

    Article Snippet: SAMHD1 and alpha-tubulin expression in THP-1 cells was controlled using antibodies purchased respectively from Abcam (ab67820) and Sigma (T9026).

    Techniques: Stable Transfection, shRNA, Incubation, Western Blot, Activity Assay, Recombinant, Labeling

    MBL blocks PGN-induced phosphorylation of I κ B α and p65 nuclear translocation in PMA-activated THP-1 cells. (a) PMA-activated THP-1 cells were stimulated with PGN (200 μ g/ml), MBL (10 μ g/ml) mixed with PGN, or anti-MBL pAb (10 μ g/ml) mixed with MBL and PGN; then the cells were incubated at 37°C in a 5% ( v / v ) CO 2 environment for 8 h. All of the mixture was generated by preincubation for 2 h at room temperature. Cells were harvested, and Western blots were performed using I κ B α and p-I κ B α or antibodies (upper panels). β -Actin was used as loading control. The protein levels were quantitatively analyzed based on densitometry analysis (lower panel). (b) MBL inhibits p65 nuclear translocation. Cells were stimulated as described in (a). Nuclear extracts were prepared as described in Materials and Methods, and Western blots were performed using p65 antibody (upper panel). Histone H1 was used as loading control. The protein levels were quantitatively analyzed based on densitometry analysis (lower panel). Images are representative of three experiments. ∗ p

    Journal: Mediators of Inflammation

    Article Title: Mannan-Binding Lectin Suppresses Peptidoglycan-Induced TLR2 Activation and Inflammatory Responses

    doi: 10.1155/2019/1349784

    Figure Lengend Snippet: MBL blocks PGN-induced phosphorylation of I κ B α and p65 nuclear translocation in PMA-activated THP-1 cells. (a) PMA-activated THP-1 cells were stimulated with PGN (200 μ g/ml), MBL (10 μ g/ml) mixed with PGN, or anti-MBL pAb (10 μ g/ml) mixed with MBL and PGN; then the cells were incubated at 37°C in a 5% ( v / v ) CO 2 environment for 8 h. All of the mixture was generated by preincubation for 2 h at room temperature. Cells were harvested, and Western blots were performed using I κ B α and p-I κ B α or antibodies (upper panels). β -Actin was used as loading control. The protein levels were quantitatively analyzed based on densitometry analysis (lower panel). (b) MBL inhibits p65 nuclear translocation. Cells were stimulated as described in (a). Nuclear extracts were prepared as described in Materials and Methods, and Western blots were performed using p65 antibody (upper panel). Histone H1 was used as loading control. The protein levels were quantitatively analyzed based on densitometry analysis (lower panel). Images are representative of three experiments. ∗ p

    Article Snippet: Cytoplasmic and nuclear proteins were obtained from different treatments of THP-1 cells by Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotech) according to the manufacturer's instruction.

    Techniques: Translocation Assay, Incubation, Generated, Western Blot

    MBL decreases PGN-induced phosphorylation of MAPKs in PMA-activated THP-1 cells. (a) PMA-activated THP-1 cells were stimulated with PGN (200 μ g/ml), MBL (10 μ g/ml) mixed with PGN, or anti-MBL pAb (10 μ g/ml) mixed with MBL and PGN; then the cells were incubated at 37°C in a 5% ( v / v ) CO 2 environment for 8 h. All of the mixture was generated by preincubation for 2 h at room temperature. Cells were harvested, and Western blots were performed using p38 antibody (upper panel). β -Actin was used as loading control. The protein levels were quantitatively analyzed based on densitometry analysis (lower panel). (b) Cells were stimulated as described in (a). Western blots were performed using p-ERK (upper panel). The protein levels were quantitatively analyzed based on densitometry analysis (lower panel). (c) Western blots were performed using p-JNK (upper panel). The protein levels were quantitatively analyzed based on densitometry analysis (lower panel). Images are representative of three experiments. ∗ p

    Journal: Mediators of Inflammation

    Article Title: Mannan-Binding Lectin Suppresses Peptidoglycan-Induced TLR2 Activation and Inflammatory Responses

    doi: 10.1155/2019/1349784

    Figure Lengend Snippet: MBL decreases PGN-induced phosphorylation of MAPKs in PMA-activated THP-1 cells. (a) PMA-activated THP-1 cells were stimulated with PGN (200 μ g/ml), MBL (10 μ g/ml) mixed with PGN, or anti-MBL pAb (10 μ g/ml) mixed with MBL and PGN; then the cells were incubated at 37°C in a 5% ( v / v ) CO 2 environment for 8 h. All of the mixture was generated by preincubation for 2 h at room temperature. Cells were harvested, and Western blots were performed using p38 antibody (upper panel). β -Actin was used as loading control. The protein levels were quantitatively analyzed based on densitometry analysis (lower panel). (b) Cells were stimulated as described in (a). Western blots were performed using p-ERK (upper panel). The protein levels were quantitatively analyzed based on densitometry analysis (lower panel). (c) Western blots were performed using p-JNK (upper panel). The protein levels were quantitatively analyzed based on densitometry analysis (lower panel). Images are representative of three experiments. ∗ p

    Article Snippet: Cytoplasmic and nuclear proteins were obtained from different treatments of THP-1 cells by Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotech) according to the manufacturer's instruction.

    Techniques: Incubation, Generated, Western Blot

    MBL was added before or after treatment with PGN to determine the effect on cellular response. (a) The cytokine levels of IL-6. NC bar indicates a negative control; PGN bar indicates that PMA-activated THP-1 cells were stimulated with PGN (200 μ g/ml) alone for 24 h; (MBL + PGN) (6 h) bar indicates preincubation with PGN and MBL (10 μ g/ml) for 6 h in room temperature and then were added into PMA-activated THP-1 cells for 24 h; PGN (6 h) + MBL bar indicates that PMA-activated THP-1 cells were preincubated cells with PGN for 6 h and then were added MBL for 24 h; MBL (6 h) + PGN bar indicated that PMA-activated THP-1 cells were preincubated cells with MBL for 6 h and then were added PGN for 24 h. All the cells were incubated at 37°C in a 5% ( v / v ) CO 2 environment. Cell culture supernatants were collected, and IL-6 was measured by ELISA. (b) The cytokine levels of TNF- α . Cell culture supernatants from (a) were collected, and TNF- α was measured by ELISA. Data shown represent three independent experiments. ∗ p

    Journal: Mediators of Inflammation

    Article Title: Mannan-Binding Lectin Suppresses Peptidoglycan-Induced TLR2 Activation and Inflammatory Responses

    doi: 10.1155/2019/1349784

    Figure Lengend Snippet: MBL was added before or after treatment with PGN to determine the effect on cellular response. (a) The cytokine levels of IL-6. NC bar indicates a negative control; PGN bar indicates that PMA-activated THP-1 cells were stimulated with PGN (200 μ g/ml) alone for 24 h; (MBL + PGN) (6 h) bar indicates preincubation with PGN and MBL (10 μ g/ml) for 6 h in room temperature and then were added into PMA-activated THP-1 cells for 24 h; PGN (6 h) + MBL bar indicates that PMA-activated THP-1 cells were preincubated cells with PGN for 6 h and then were added MBL for 24 h; MBL (6 h) + PGN bar indicated that PMA-activated THP-1 cells were preincubated cells with MBL for 6 h and then were added PGN for 24 h. All the cells were incubated at 37°C in a 5% ( v / v ) CO 2 environment. Cell culture supernatants were collected, and IL-6 was measured by ELISA. (b) The cytokine levels of TNF- α . Cell culture supernatants from (a) were collected, and TNF- α was measured by ELISA. Data shown represent three independent experiments. ∗ p

    Article Snippet: Cytoplasmic and nuclear proteins were obtained from different treatments of THP-1 cells by Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotech) according to the manufacturer's instruction.

    Techniques: Negative Control, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

    MBL suppresses PGN-induced inflammatory cytokine production. (a) The mRNA levels of TNF- α and IL-6. PMA-activated THP-1 cells were stimulated with PGN (200 μ g/ml), MBL (10 μ g/ml) mixed with PGN, or anti-MBL pAb (10 μ g/ml) mixed with MBL and PGN; then the cells were incubated at 37°C in a 5% ( v / v ) CO 2 environment for 24 h. All of the mixture was generated by preincubation for 2 h at room temperature. Total RNA was extracted from the treated cells, and the relative mRNA level of TNF- α and IL-6 was analyzed by qRT-PCR by normalizing to internal β -actin. Value is mean ± SEM of three experiments. ∗ p

    Journal: Mediators of Inflammation

    Article Title: Mannan-Binding Lectin Suppresses Peptidoglycan-Induced TLR2 Activation and Inflammatory Responses

    doi: 10.1155/2019/1349784

    Figure Lengend Snippet: MBL suppresses PGN-induced inflammatory cytokine production. (a) The mRNA levels of TNF- α and IL-6. PMA-activated THP-1 cells were stimulated with PGN (200 μ g/ml), MBL (10 μ g/ml) mixed with PGN, or anti-MBL pAb (10 μ g/ml) mixed with MBL and PGN; then the cells were incubated at 37°C in a 5% ( v / v ) CO 2 environment for 24 h. All of the mixture was generated by preincubation for 2 h at room temperature. Total RNA was extracted from the treated cells, and the relative mRNA level of TNF- α and IL-6 was analyzed by qRT-PCR by normalizing to internal β -actin. Value is mean ± SEM of three experiments. ∗ p

    Article Snippet: Cytoplasmic and nuclear proteins were obtained from different treatments of THP-1 cells by Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotech) according to the manufacturer's instruction.

    Techniques: Incubation, Generated, Quantitative RT-PCR

    TLR2 is activated by PGN, and the upregulation of TLR2 is inhibited by MBL. (a) The mRNA levels of TLR2. PMA-activated THP-1 cells were stimulated with PGN (200 μ g/ml), MBL (10 μ g/ml) mixed with PGN, or anti-MBL pAb (10 μ g/ml) mixed with MBL and PGN; then the cells were incubated at 37°C in a 5% ( v / v ) CO 2 environment for 24 h. All of the mixture was generated by preincubation for 2 h at room temperature. Cells were harvested, and total RNA was isolated. The mRNA level of TLR2 was analyzed using RT-PCR (upper panel). β -Actin was used as loading control. The mRNA levels were quantitatively analyzed based on densitometry analysis (lower panel). (b) The protein level of TLR2. Cells were treated as described in (a), and the protein level of TLR2 was also analyzed using Western blots (upper panel). β -Actin was used as loading control. The protein levels were quantitatively analyzed based on densitometry analysis (lower panel). (c) Blocking of TLR2 significantly abolished the inhibitory effect of MBL on IL-6 production. PMA-activated THP-1 cells were pretreated with anti-TLR2 Ab (10 μ g/ml) or with the isotype control of antibody (10 μ g/ml), respectively, and stimulated with PGN (200 μ g/ml), MBL (10 μ g/ml) mixed with PGN, or anti-MBL pAb (10 μ g/ml) mixed with MBL and PGN. All of the mixture was generated by preincubation for 2 h at room temperature. Then the cells were incubated at 37°C in a 5% ( v / v ) CO 2 environment for another 24 h. Cell culture supernatants were collected and measured by ELISA. (d) Blocking of TLR2 significantly abolished the inhibitory effect of MBL on TNF- α production. Cells were treated as described in (c). Cell culture supernatants were collected and measured by ELISA. (e) The expression of TLR2 is not influenced by MBL in the mRNA level. PMA-activated THP-1 cells were stimulated with different concentrations of 0, 5, 10, and 20 ( μ g/ml) MBL for 24 h, and the mRNA level of TLR2 was detected by RT-PCR (upper panel). β -Actin was used as loading control. The mRNA levels were quantitatively analyzed based on densitometry analysis (lower panel). (f) The expression of TLR2 is not influenced by MBL in the protein level. PMA-activated THP-1 cells were stimulated with different concentrations of MBL (0, 5, 10, and 20 ( μ g/ml)) for 24 h, and the protein level of TLR2 was detected by Western blot (upper panel). β -Actin was used as loading control. The protein levels were quantitatively analyzed based on densitometry analysis (lower panel). Images and data are representative of three experiments. ∗ p

    Journal: Mediators of Inflammation

    Article Title: Mannan-Binding Lectin Suppresses Peptidoglycan-Induced TLR2 Activation and Inflammatory Responses

    doi: 10.1155/2019/1349784

    Figure Lengend Snippet: TLR2 is activated by PGN, and the upregulation of TLR2 is inhibited by MBL. (a) The mRNA levels of TLR2. PMA-activated THP-1 cells were stimulated with PGN (200 μ g/ml), MBL (10 μ g/ml) mixed with PGN, or anti-MBL pAb (10 μ g/ml) mixed with MBL and PGN; then the cells were incubated at 37°C in a 5% ( v / v ) CO 2 environment for 24 h. All of the mixture was generated by preincubation for 2 h at room temperature. Cells were harvested, and total RNA was isolated. The mRNA level of TLR2 was analyzed using RT-PCR (upper panel). β -Actin was used as loading control. The mRNA levels were quantitatively analyzed based on densitometry analysis (lower panel). (b) The protein level of TLR2. Cells were treated as described in (a), and the protein level of TLR2 was also analyzed using Western blots (upper panel). β -Actin was used as loading control. The protein levels were quantitatively analyzed based on densitometry analysis (lower panel). (c) Blocking of TLR2 significantly abolished the inhibitory effect of MBL on IL-6 production. PMA-activated THP-1 cells were pretreated with anti-TLR2 Ab (10 μ g/ml) or with the isotype control of antibody (10 μ g/ml), respectively, and stimulated with PGN (200 μ g/ml), MBL (10 μ g/ml) mixed with PGN, or anti-MBL pAb (10 μ g/ml) mixed with MBL and PGN. All of the mixture was generated by preincubation for 2 h at room temperature. Then the cells were incubated at 37°C in a 5% ( v / v ) CO 2 environment for another 24 h. Cell culture supernatants were collected and measured by ELISA. (d) Blocking of TLR2 significantly abolished the inhibitory effect of MBL on TNF- α production. Cells were treated as described in (c). Cell culture supernatants were collected and measured by ELISA. (e) The expression of TLR2 is not influenced by MBL in the mRNA level. PMA-activated THP-1 cells were stimulated with different concentrations of 0, 5, 10, and 20 ( μ g/ml) MBL for 24 h, and the mRNA level of TLR2 was detected by RT-PCR (upper panel). β -Actin was used as loading control. The mRNA levels were quantitatively analyzed based on densitometry analysis (lower panel). (f) The expression of TLR2 is not influenced by MBL in the protein level. PMA-activated THP-1 cells were stimulated with different concentrations of MBL (0, 5, 10, and 20 ( μ g/ml)) for 24 h, and the protein level of TLR2 was detected by Western blot (upper panel). β -Actin was used as loading control. The protein levels were quantitatively analyzed based on densitometry analysis (lower panel). Images and data are representative of three experiments. ∗ p

    Article Snippet: Cytoplasmic and nuclear proteins were obtained from different treatments of THP-1 cells by Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotech) according to the manufacturer's instruction.

    Techniques: Incubation, Generated, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Blocking Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing

    Fluorescence intensity (arbitrary unit, a.u.) related to TMRM from THP-1 cells applied with or without RF current plotted against the time after the RF application. Open square: control (n=2321–2641). Open circle: CRF (n=2321–2679). Closed circle (red): PRF (n=2578–2697). Data are expressed as mean±SD.

    Journal: Journal of Pain Research

    Article Title: Continuous But Not Pulsed Radiofrequency Current Generated by NeuroTherm NT500 Impairs Mitochondrial Membrane Potential in Human Monocytic Cells THP-1

    doi: 10.2147/JPR.S242204

    Figure Lengend Snippet: Fluorescence intensity (arbitrary unit, a.u.) related to TMRM from THP-1 cells applied with or without RF current plotted against the time after the RF application. Open square: control (n=2321–2641). Open circle: CRF (n=2321–2679). Closed circle (red): PRF (n=2578–2697). Data are expressed as mean±SD.

    Article Snippet: The relative fluorescence intensity from THP-1 cells tended to decrease according to the temperature of TMRM pre-incubation, but not significantly (without pre-incubation, 13.9%±1.7; with pre-incubation at 70 °C for 3 min, 10.9%±2.1; at 80 °C for 5 min, 9.3%±2.5,;n=3 each, P=0.087), indicating approximately 78% inhibition of activity as a fluorescence probe after pre-incubation at 70 °C for 3 min. By contrast, fluorescence intensity related to TMRM from THP-1 cells applied with CRF current at 70 °C for 3 min significantly decreased to less than 40% of that from cells without RF application ( and ), supporting the concept that CRF current significantly suppressed the mitochondrial membrane potential in THP-1 cells.

    Techniques: Fluorescence

    Suspension of THP-1 cells applied with or without RF current was analyzed using flow cytometry. THP-1 cells were gated by a circle in the FSC-A/SSC-H plot and dotted in blue. Fluorescence related to TMRM was measured by setting excitation with a 488-nm blue laser beam and detection through a 585/42-nm bandpass filter.

    Journal: Journal of Pain Research

    Article Title: Continuous But Not Pulsed Radiofrequency Current Generated by NeuroTherm NT500 Impairs Mitochondrial Membrane Potential in Human Monocytic Cells THP-1

    doi: 10.2147/JPR.S242204

    Figure Lengend Snippet: Suspension of THP-1 cells applied with or without RF current was analyzed using flow cytometry. THP-1 cells were gated by a circle in the FSC-A/SSC-H plot and dotted in blue. Fluorescence related to TMRM was measured by setting excitation with a 488-nm blue laser beam and detection through a 585/42-nm bandpass filter.

    Article Snippet: The relative fluorescence intensity from THP-1 cells tended to decrease according to the temperature of TMRM pre-incubation, but not significantly (without pre-incubation, 13.9%±1.7; with pre-incubation at 70 °C for 3 min, 10.9%±2.1; at 80 °C for 5 min, 9.3%±2.5,;n=3 each, P=0.087), indicating approximately 78% inhibition of activity as a fluorescence probe after pre-incubation at 70 °C for 3 min. By contrast, fluorescence intensity related to TMRM from THP-1 cells applied with CRF current at 70 °C for 3 min significantly decreased to less than 40% of that from cells without RF application ( and ), supporting the concept that CRF current significantly suppressed the mitochondrial membrane potential in THP-1 cells.

    Techniques: Flow Cytometry, Fluorescence