Journal: Cell Communication and Signaling : CCS

Article Title: Activation of C6 glioblastoma cell ceruloplasmin expression by neighboring human brain endothelia-derived interleukins in an in vitro blood–brain barrier model system

doi: 10.1186/s12964-014-0065-7

Figure Lengend Snippet: LPS-induced activation of hBMVEC basolateral iron efflux through modulation of hBMVEC interleukins with subsequent C6 glioma cell sCp gene activation. (A) LPS (100 μg/mL) was added to hBMVEC for the times indicated before total hBMVEC RNA was collected and assayed for IL-1β and IL-6 transcript abundance via qPCR. (B) Total RNA was isolated from C6 glioma cells seeded distal to hBMVEC incubated with or without the addition of LPS (100 μg/mL) to the apical chamber for 24 h. Soluble Cp transcript abundance was assessed via qPCR. (C) C6 glioma cells were incubated alone or distal to hBMVEC for 20 h prior to the addition of LPS (100 μg/mL) to the apical chamber and IRAP (1 μg/mL) and/or SC144 (20 μM) to the basal chamber for an additional 4 h as indicated. Total C6 glioma RNA was isolated and assayed for sCp transcript abundance via qPCR. Significance of the data was determined using either a paired t-test or one-way ANOVA statistical analyses. ***P

Article Snippet: IL-1β, IL-6, and IRAP were purchased from Peprotech (Rocky Hill, NJ), SB203580 (Santa Cruz Biotechnology), SC144 and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich.

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Isolation, Incubation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Th2/1 Hybrid Cells Occurring in Murine and Human Strongyloidiasis Share Effector Functions of Th1 Cells

doi: 10.3389/fcimb.2017.00261

Figure Lengend Snippet: Th2/1 cells are capable of supporting classical macrophage effector function. Murine Th1, Th2, and Th2/1 hybrid cells were generated from naïve CD4 + T cells in vitro and surveyed for (A) GATA-3 (left) and T-bet (right) expression. (B) IL-4 and IFN-γ expression by in vitro -generated Th1, Th2, and Th2/1 cells in response to PMA/ionomycin stimulation. (C) Geometric mean fluorescence intensity of IFN-γ in Th1, Th2, and Th2/1 hybrid cells. (D) Nitrite accumulation in supernatant of peritoneal macrophages left untreated (–), stimulated with recombinant IFN-γ or incubated with Th1, Th2, and Th2/1 cells in the depicted T cell:macrophage ratios for 24 h. (E) Nitrite concentrations in supernatant of macrophages left untreated (–), stimulated with LPS or co-cultured with T cells (1:1 ratio) in presence of LPS. Mean and SD of five cultures is shown. Data are representative for three independent experiments. * p

Article Snippet: 1 × 105 in vitro generated Th1, Th2 or Th2/1 cells were added and co-cultured in 200 μl for 24 h. Lipopolysaccheride (LPS, L5529, Sigma) was added for co-stimulation at 0.5 μg/ml to some wells.

Techniques: Generated, In Vitro, Expressing, Fluorescence, Recombinant, Incubation, Cell Culture

Journal: PLoS ONE

Article Title: Doxycycline Inhibits Inflammation-Induced Lymphangiogenesis in Mouse Cornea by Multiple Mechanisms

doi: 10.1371/journal.pone.0108931

Figure Lengend Snippet: Doxycycline suppresses LPS-induced macrophage activation by modulating the PI3K/Akt/NF-κB pathway. The levels of IL-1β (A) and TNF-α (B) in the supernatants were measured using ELISA. The expression of VEGF-C mRNA (C) was determined using real-time PCR. Doxycycline treatment significantly reduced PI3K activity (D). The levels of (phosphorylated) NF-κBp65 (E), IκB-α (F) and (phosphorylated) Akt (G) were determined using Western blotting. * p

Article Snippet: Antibodies and Reagents Doxycycline, hydroxypropyl-β-cyclodextrin, poloxamer 407, poloxamer 188, VEGF-C and lipopolysaccharides (LPS) were purchased from Sigma (St. Louis, MO, USA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Western Blot

Journal: EBioMedicine

Article Title: AMSC-derived exosomes alleviate lipopolysaccharide/d-galactosamine-induced acute liver failure by miR-17-mediated reduction of TXNIP/NLRP3 inflammasome activation in macrophages

doi: 10.1016/j.ebiom.2018.08.054

Figure Lengend Snippet: AMSC-Exo suppress inflammasome activation by miR-17-mediated TXNIP inhibition. (a) qPCR detection of miR-223 and miR-17 family miRNAs (miR-17, miR-20a/b, miR-93, miR-106a/b) relative expression levels in AMSC-Exo (a, compared with miR-223; b, compared with miR-17). (b and c) qPCR detection of miR-17 (b) and Western blot analysis of TXNIP (c) expression levels in RAW264.7 cells treated with AMSC-Exo or transfected with miR-17 or control miRNA (miR-ctrl). U.T, untreated. (d and e) IL-1β and IL-18 secretion levels of LPS-stimulated RAW264.7 cells were measured by ELISA ( ns , nonsense). (f–i) Protein levels of TXNIP, cleaved-Casp1, IL-1β, and IL-18 in LPS-stimulated RAW264.7 cells were measured by Western blot analysis (f and g) and then quantified by gray value assay (h and i). Data are presented as mean ± SD (Statistical analysis was performed by Student's t -test, *p

Article Snippet: 2.8 ALF mouse model and AMSC-Exo treatment Mice (C57BL/6J, aged 5–6 weeks) were intraperitoneally injected with LPS (10 μg/kg, Sigma-Aldrich, St Louis, MO, USA) and D-GalN (400 mg/kg, Sigma-Aldrich) to establish a mouse model of LPS/GalN-induced ALF.

Techniques: Activation Assay, Inhibition, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay

Journal: EBioMedicine

Article Title: AMSC-derived exosomes alleviate lipopolysaccharide/d-galactosamine-induced acute liver failure by miR-17-mediated reduction of TXNIP/NLRP3 inflammasome activation in macrophages

doi: 10.1016/j.ebiom.2018.08.054

Figure Lengend Snippet: AMSC-Exo reduce NLRP3 inflammasome activation in macrophages. (a–d) Western blot analysis and gray value assay on expression levels of inflammasome-associated protein in murine liver samples (a and b, n = 6, 3 from 6 samples were shown in a) and macrophages (c and d, n = 3). (e) IL-1β and IL-18 secretion levels from macrophages by LPS exposure were measured by ELISA. (f) PKH26-labeled AMSC-Exo were colocalized with CD68-positive Kupffer cells. Scale bar: 30 μm. Data are presented as mean ± SD (Statistical analysis was performed by Student's t -test, *p

Article Snippet: 2.8 ALF mouse model and AMSC-Exo treatment Mice (C57BL/6J, aged 5–6 weeks) were intraperitoneally injected with LPS (10 μg/kg, Sigma-Aldrich, St Louis, MO, USA) and D-GalN (400 mg/kg, Sigma-Aldrich) to establish a mouse model of LPS/GalN-induced ALF.

Techniques: Activation Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Labeling

Journal: EBioMedicine

Article Title: AMSC-derived exosomes alleviate lipopolysaccharide/d-galactosamine-induced acute liver failure by miR-17-mediated reduction of TXNIP/NLRP3 inflammasome activation in macrophages

doi: 10.1016/j.ebiom.2018.08.054

Figure Lengend Snippet: AMSC-Exo administration ameliorates LPS/D-GalN-induced liver failure. (a and b) Serum levels of ALT, AST, and inflammatory cytokines were elevated by LPS/D-GalN injection (Vehicle group) and significantly decreased by treatment with AMSC-Exo. Data are presented as mean ± SD (Statistical analysis was performed by Student's t -test, **p

Article Snippet: 2.8 ALF mouse model and AMSC-Exo treatment Mice (C57BL/6J, aged 5–6 weeks) were intraperitoneally injected with LPS (10 μg/kg, Sigma-Aldrich, St Louis, MO, USA) and D-GalN (400 mg/kg, Sigma-Aldrich) to establish a mouse model of LPS/GalN-induced ALF.

Techniques: AST Assay, Injection

Journal: Journal of the American Society of Nephrology : JASN

Article Title: The Ubiquitin-Like Protein FAT10 Mediates NF-?B Activation

doi: 10.1681/ASN.2009050479

Figure Lengend Snippet: FAT10 is necessary for TNF-α– and LPS-induced activation of the NF-κB reporter vector pGL2/NF-κB luciferase in FAT10 −/− RTECs. (A) FAT10 +/+ and FAT10 −/− RTECs were co-transfected with pGL2/NF-κB luciferase and renilla luciferase plasmids. After 24 h, TNF-α (10 ng/ml) or LPS (1 μg/ml) was added to the media and the cells were further incubated for 16 h. (B) FAT10 −/− RTECs were co-transfected with pGL2/NF-κB luciferase, renilla, and either pHR/FAT10-IRES-EGFP or pHR-IRES-EGFP. After 24 h, TNF-α (10 ng/ml) or LPS (1 μg/ml) was added and cells were further incubated for 16 h. Luciferase activity was measured using a luminometer. Results are normalized to renilla luciferase and expressed as fold induction relative to TNF-α or LPS untreated cells. Data are means ± SD ( n = 3). ** P

Article Snippet: An NF-κB reporter plasmid (pGL2/NF-κB luciferase) was transfected into FAT10 +/+ and FAT10 −/− RTECs for 24 h, and cells were assayed for luciferase activity after additional incubation with TNF-α or LPS (Invitrogen) for 16 h. A plasmid encoding renilla luciferase was co-transfected as an internal control for transfection efficiency.

Techniques: Activation Assay, Plasmid Preparation, Luciferase, Transfection, Incubation, Activity Assay

Journal: PLoS ONE

Article Title: ESAT-6 and HspX Improve the Effectiveness of BCG to Induce Human Dendritic Cells-Dependent Th1 and NK Cells Activation

doi: 10.1371/journal.pone.0075684

Figure Lengend Snippet: TLR2 is involved in IL-12-dependent IFN-γ secretion by CD4 + cells co-cultured with ESAT-6, HspX and BCG-treated DCs. DCs cultured in the absence (filled bars) or presence (open bars) of 5 µg/ml TLR2-blocking antibody were treated for 24 hrs with Mtb, BCG alone or combined with HspX and ESAT6, 10 µg/ml Pam3CSK4 (Pam3) or 100 ng/ml LPS. (A) Supernatants were collected and IL-12 release was analyzed by ELISA. Results are the mean value+SD of four experiments. Statistical analysis: antibody-treated vs antibody-untreated cells, ns P > 0.05, *P

Article Snippet: Ultrapure lipolysaccharide (LPS) from E. coli (0111: B4 strain) and palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4) were purchased from InvivoGen (San Diego, CA).

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

Journal: Cellular microbiology

Article Title: Galectin-3 binds Neisseria meningitidis and increases interaction with phagocytic cells

doi: 10.1111/j.1462-5822.2012.01838.x

Figure Lengend Snippet: Galectin-3 binds to meningococci expressing LPS with a terminal LacNAc

Article Snippet: For labelling of bacteria the cells were incubated for 1 hr with an anti-lipoplysaccharide (LPS) antibody (α-L3,7,9) and with an anti-galectin-3 antibody (Santa Cruz Biotechnology), after which the cells were washed twice in PBS then incubated for 30 min with an anti-mouse Alexa Fluor 488-conjugated antibody and anti-human Alexa Fluor 555-conjugated antibody (Invitrogen) to label bacteria and cells respectively.

Techniques: Expressing

Journal: Cellular microbiology

Article Title: Galectin-3 binds Neisseria meningitidis and increases interaction with phagocytic cells

doi: 10.1111/j.1462-5822.2012.01838.x

Figure Lengend Snippet: Full length lipopolysaccharide is required for galectin-3 binding to N. meningitidis

Article Snippet: For labelling of bacteria the cells were incubated for 1 hr with an anti-lipoplysaccharide (LPS) antibody (α-L3,7,9) and with an anti-galectin-3 antibody (Santa Cruz Biotechnology), after which the cells were washed twice in PBS then incubated for 30 min with an anti-mouse Alexa Fluor 488-conjugated antibody and anti-human Alexa Fluor 555-conjugated antibody (Invitrogen) to label bacteria and cells respectively.

Techniques: Binding Assay

Journal: Molecular microbiology

Article Title: Chemical activation of adenylyl cyclase Rv1625c inhibits growth of Mycobacterium tuberculosis on cholesterol and modulates intramacrophage signaling

doi: 10.1111/mmi.13701

Figure Lengend Snippet: V-58 stimulates intracellular Mtb to produce cAMP J774.16 murine macrophages were infected with live Mtb H37Rv or CDC1551, and heat-killed CDC1551 were treated with either 0.1 % DMSO (black bars) or 10 μM V-58 (gray bars). cAMP levels were quantified by RIA from the A) intracellular bacteria and B) macrophage lysates. The data shown are the means of two biologically independent experiments. Error bars represent the standard deviation between replicates. C) Macrophages were infected with Mtb CDC1551 at the indicated MOI and treated with 10 ng/mL LPS, 250 μM 8-Br-cAMP or V-58 at the indicated concentrations for 18 h and supernatant TNF-α levels were determined by ELISA. Data are means of four biologically independent experiments and the error bars represent the standard deviation between experiments. Statistical significance was determined with a two-tailed unpaired t-test, *p≤0.05, **p≤0.005 and ****p≤0.0001.

Article Snippet: Following 2 h of infection, the cultures were washed twice with warm supplemented DMEM, replenished with the same medium containing 8Br-cAMP (Sigma) at the indicated concentrations, LPS 10 ng/ml (List Biological Laboratories), V-58 at the indicated concentrations, or DMSO vehicle control.

Techniques: Infection, Standard Deviation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: PLoS Neglected Tropical Diseases

Article Title: Extracellular Vesicles from Leishmania-Infected Macrophages Confer an Anti-infection Cytokine-Production Profile to Naïve Macrophages

doi: 10.1371/journal.pntd.0003161

Figure Lengend Snippet: Effect of membrane vesicles from macrophages infected with L. amazonensis (EVLa) or not (EV) and stimulated with LPS or not on cytokine production by naïve macrophages. A and B, IL-12p70. C and D, IL-1β. E and F, TNF-α. G and H, IL-6. I and J, IL-10. A, C, E, G, and I, unstimulated macrophages; B, D, F, H, and J, macrophages stimulated with bacterium lipopolysacharide for 48 hours. The symbols and lines represent the averages of replicates of individual experiment. Data from six independent experiments are represented. Values obtained from cultures to which no EVs were added were subtracted from the data shown. *, P

Article Snippet: These peritoneal macrophages were incubated for 48 hours with EVs from uninfected bone marrow-derived macrophages (EVmedium) and with EVs from L. amazonensis -infected bone marrow-derived macrophages (EVLa) with or without 1 ng/mL bacterial lipopolysacharide (LPS; Escherichia coli 0127:B8, Sigma-Aldridge, St. Louis, MO, USA), at 37°C and 5% CO2 .

Techniques: Infection

Journal: American journal of obstetrics and gynecology

Article Title: Docosahexaenoic acid Confers Neuroprotection in a Rat Model of Perinatal Hypoxia-ischemia potentiated by E. coli lipopolysaccharide-induced systemic inflammation

doi: 10.1016/j.ajog.2009.01.020

Figure Lengend Snippet: DHA pretreatment attenuates forepaw placing deficits among hypoxic ischemic pups exposed to LPS pretreatment

Article Snippet: [ ] Rats received intraperitoneal Escherichia coli lipolysaccharide (LPS) 055:B5 (0.1 mg/kg) (Sigma, St. Louis) followed 2.5 hours later by right carotid artery ligation.

Techniques:

Journal: Journal of Inflammation (London, England)

Article Title: Stimulation of TLR4 by recombinant HSP70 requires structural integrity of the HSP70 protein itself

doi: 10.1186/1476-9255-9-11

Figure Lengend Snippet: Proteinase K-agarose digestion ablates HSP70 activity . (A) 200 μg rhHSP70 was incubated with digestion buffer (lane 1), Proteinase K-agarose beads (lane 2), or control agarose beads (lane 3) for 3 hr at 37°C, as described in Methods . Protein integrity and endotoxin content were determined by silver staining and the LAL assay, respectively. (B) 100 pg of ultrapure LPS, which is an approximation of the endotoxin content in 200 μg of rhHSP70 (see Results and Discussion ), was incubated with Proteinase K-agarose beads or control agarose beads, under the conditions described in A . The ultrapure LPS treated with Proteinase K-agarose beads (PK) or control agarose beads (CTRL) were then used to stimulate primary human macrophages for 16 hr at 100 pg/mL LPS final concentration in each culture. Cell culture supernatants were collected and analyzed for TNF-α. (C) The HSP70 preparations in A were used to stimulate primary human macrophages for 4 hr at 10 μg/mL final concentration. Cell culture supernatants were collected and analyzed for TNF-α. Endotoxin levels shown at bottom are the calculated amounts in the macrophage cultures based on the LAL determinations in A . Untreated macrophage cultures produced ≤ 100 pg/mL of TNF-α under these conditions. Results shown are representative of two independent digestion and stimulation experiments. *** p

Article Snippet: Reagents Recombinant low-endotoxin human HSP70 (cat. # ADI-ESP-555-F; lot # 05040922) and ultrapure LPSs (cat. # ALX-581-007-L002 and ALX-581-013-L002) were from Enzo Life Sciences (Plymouth Meeting, PA).

Techniques: Activity Assay, Incubation, Silver Staining, LAL Assay, Concentration Assay, Cell Culture, Produced

Journal: European Journal of Immunology

Article Title: TLR activation enhances C5a-induced pro-inflammatory responses by negatively modulating the second C5a receptor, C5L2

doi: 10.1002/eji.201041350

Figure Lengend Snippet: Sensitivity to C5a of PBMCs pre-exposed to TLR ligands. (A–C) Levels of IL-8 in culture supernatants of PBMCs (1.5×10 5 /well) (A) stimulated for 14 h with the indicated concentrations of C5a and (B) after washing and re-culture following pre-exposure (14 h) to LPS (100 pg/mL or as indicated), Pam 3 Cys (100 ng/mL), Zymosan (1 μg/mL), Flagellin (5 μg/mL), Imiquimod (3 μg/mL) or mock-pre-exposure (no TLR ligand). IL-8 concentrations were estimated by subtracting the background levels of IL-8 present in cultures not activated with C5a and pre-exposed or not to TLR ligands from the corresponding C5a-activated samples (IL-8 background levels (ng/mL): No ligand/No C5a, 1.6±0.7; +LPS, 2.3±1.2; +Pam 3 Cys, 1.5±0.6; +Zymosan, 1.3±0.9; +Flagellin, 6.9±2.5; +Imiquimod, 5.3±1.1; n ≥4). (C) IL-8 fold increases were determined by comparing IL-8 levels – after background subtraction – between C5a-stimulated (10 nM) cell samples pre-exposed and not pre-exposed to LPS. (D) Levels of IL-6 in culture supernatants of PBMCs stimulated for 14 h with the indicated concentrations of C5a, after washing and re-culture following pre-exposure to LPS. (E and F) Determination of (E) IL-6 and IL-8 mRNA levels in RNA samples and (F) NF-κB concentrations in the nuclear extracts of PBMCs pre-exposed or not to LPS and subsequently stimulated with C5a as described for A–D. (E) mRNA levels are relative to control (No LPS/No C5a). Results are from one experiment (+SD) representative of at least four for each ligand (A, B) or three (C–F). * p

Article Snippet: For cell activation experiments, triplicate cell aliquots (1.5×105 cells/well, unless stated otherwise) were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% heat-inactivated (56°C, 30 min) FCS (HyClone; < 0.06 U/mL endotoxin) and 2 mM glutamine (complete medium), and stimulated at 37°C for 14 h or the time indicated with optimal concentrations of ultra-pure LPS (E. coli O111:B4 strain), zymosan, flagellin, imiquimod – all from Invivogen – Pam3 -Cys-Ser-(Lys)4 HCl (Pam3 Cys; EMC microcollections GmbH) as indicated, or medium alone (mock stimulation).

Techniques:

Journal: Infection and Immunity

Article Title: The Oligosaccharide of Gonococcal Lipooligosaccharide Contains Several Epitopes That Are Recognized by Human Antibodies ▿

doi: 10.1128/IAI.01445-09

Figure Lengend Snippet: ). (B) OS structure of 15253 LOS. (C) Analysis of affinity purification by PAGE (silver staining). Lanes: IgG, purified human IgG (control); 1, bound to the LOS column; 2, unbound to the protein G column; 3, bound to the protein G column; 4, purified IgG after the inactivated matrix column. (D) Subclass analysis. The purified IgG was analyzed with a MAb IgG/IgA subclass typing kit. (E) ELISA analysis. Each well was coated with 15253 LOS, JW31R LOS, or S. minnesota Re 595 LPS (200 ng each). Anti-human IgG (AP conjugate) and p -nitrophosphate were used, and the absorbances (405 nm) at 70 min are shown.

Article Snippet: Escherichia coli LPS and the following Salmonella rough mutant LPSs were purchased from Sigma (St. Louis, MO): Ra ( S almonella enterica serovar Typhimurium TV119), Rc (“ S. minnesota ” R5), Rd ( S. minnesota R7), and Re ( S. minnesota Re 595).

Techniques: Affinity Purification, Polyacrylamide Gel Electrophoresis, Silver Staining, Purification, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Rapid Downregulation of DAB2 by Toll-Like Receptor Activation Contributes to a Pro-Inflammatory Switch in Activated Dendritic Cells

doi: 10.3389/fimmu.2019.00304

Figure Lengend Snippet: DAB2 is downregulated in human and murine dendritic cells after Toll-like receptor activation through MyD88 and TRIF pathways. (A) Bone-marrow derived dendritic cells (BMDC) differentiated from C57BL/6J mice, DC2.4 cells, and human monocytes-derived dendritic cells were incubated with 100 ng/mL LPS, and DAB2 expression was quantified using western blotting . (B) DAB2 expression in BMDCs treated with agonist for extracellular TLR or (C) agonist for intracellular TLR. (D,E) DAB2 expression after treatment with HKLM, HMW, or LPS in BMDC differentiated from WT, MyD88 −/− , and or TRIF −/− C57BL/6 mice. Bar graphs represent mean values of three independent experiments combined ( n = 3 mice, ** p

Article Snippet: BMDCs were also treated for a short period with 100 ng/mL LPS (InvivoGen) or pre-treated with 100 nM bafilomycin A1 (Sigma Aldrich) or 2 μM MG132 (Sigma Aldrich) for 30 min before treatment with 100 ng/mL LPS for 1 h. To study Dab2 protein and mRNA stability, BMDCs were treated with 5 μg/mL cycloheximide (Sigma Aldrich) or 10 μg/mL actinomycin D (Santa Cruz Biotechnology) for different durations.

Techniques: Activation Assay, Derivative Assay, Mouse Assay, Incubation, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: Rapid Downregulation of DAB2 by Toll-Like Receptor Activation Contributes to a Pro-Inflammatory Switch in Activated Dendritic Cells

doi: 10.3389/fimmu.2019.00304

Figure Lengend Snippet: TLR4 activation represses both DAB2 protein and gene expression with different dynamics. (A) BMDCs were incubated with 100 ng/mL LPS for up to 90 min and DAB2 protein expression was evaluated by Western blotting. (B) Dab2 mRNA expression was evaluated after incubation with 100 ng/mL LPS for up to 16 h. Dab2 and Tbp (as an internal control) gene expression was quantified using qRT-PCR. * p

Article Snippet: BMDCs were also treated for a short period with 100 ng/mL LPS (InvivoGen) or pre-treated with 100 nM bafilomycin A1 (Sigma Aldrich) or 2 μM MG132 (Sigma Aldrich) for 30 min before treatment with 100 ng/mL LPS for 1 h. To study Dab2 protein and mRNA stability, BMDCs were treated with 5 μg/mL cycloheximide (Sigma Aldrich) or 10 μg/mL actinomycin D (Santa Cruz Biotechnology) for different durations.

Techniques: Activation Assay, Expressing, Incubation, Western Blot, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: The Role of Human T-Lymphotropic Virus Type 1 (HTLV-1)-Infected Dendritic Cells in the Development of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis

doi:

Figure Lengend Snippet: Expression of various molecules on DCs pulsed with HTLV-1. Immature DCs were differentiated from normal healthy donor monocytes, pulsed with 20 CCID 50 of live HTLV-1 or equivalent dose of heat-inactivated virions, and matured by LPS (10 ng/ml). –––, control MAb; ———, indicated MAb. The number in each panel represents the mean fluorescence intensity.

Article Snippet: Maturation of DCs was performed by addition of 10 ng of lipopolysaccharide (LPS) from Escherichia coli O111:B4 (Difco Laboratories, Detroit, Mich.) per ml for 24 h, and matured DCs were assessed for APC functions after treatment with 50 μg of mitomycin C per ml.

Techniques: Expressing, Fluorescence

Journal: Journal of Virology

Article Title: The Role of Human T-Lymphotropic Virus Type 1 (HTLV-1)-Infected Dendritic Cells in the Development of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis

doi:

Figure Lengend Snippet: Comparison of T-cell-stimulating activities of HTLV-1-infected DCs and inactivated virion-pulsed DCs. (a) Immature DCs were either infected with various doses of HTLV-1 or pulsed with an equivalent dose of heat-inactivated virions, subsequently matured by LPS, and cocultured with autologous unseparated, CD4 + and CD8 + T cells (10 5 /well) for 5 days at a DC:T-cell ratio of 1:20. (b) LPS-matured DCs were exposed to live or inactivated virus for 4 days and cocultured with various T cells (10 5 /well) for 5 days at a DC:T-cell ratio of 1:20. A representative result of three independent experiments is shown. Each experiment was performed in triplicate.

Article Snippet: Maturation of DCs was performed by addition of 10 ng of lipopolysaccharide (LPS) from Escherichia coli O111:B4 (Difco Laboratories, Detroit, Mich.) per ml for 24 h, and matured DCs were assessed for APC functions after treatment with 50 μg of mitomycin C per ml.

Techniques: Infection