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

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 2. C/EBP, NF-B p50, STAT3, c-Rel, and TBP bind the endogenous CRP promoter. Agarose gel of a ChIP assay performed on Hep3B cells treated with cytokines IL-1 and IL-6 for 0–15 h, as described in Materials and Methods. Abs to C/EBP, NF-B p50, STAT3, c-Rel, and TBP were used in the assays with primers flanking the CRP proximal promoter (118 to 115). The mock is a no Ab control. Input is a 1/10 dilution of total chromatin after sonication and preclearing. C/EBP, NF-B p50, and input are shown (top row). STAT3, c-Rel, and mock are shown in the middle, and TBP is shown in the bottom row. Results are representative of four experiments.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Agarose Gel Electrophoresis, Control, Sonication

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

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 3. C/EBP binds the endogenous CRP promoter in response to cytokines. Real-time PCR of ChIP assays performed on Hep3B cells treated with cytokines IL-1 and IL-6. Zero time values were determined in each case. Subsequently three time courses were followed 30 min–6 h (n 3 assays) (a), 8–16 h (n 4) (b), and 12–36 h (n 4) (c). Abs were used in the assays with primers flanking the CRP proximal promoter (118 to 115), as described in Fig. 2. Data show C/EBP occupancy expressed as fold change after subtraction of mock and normalization to input signal (see Materials and Methods). Results are an average of three to four ex- periments, each done in duplicate. Error bar represents SD. Statistical sig- nificance of each time point compared with basal levels was determined by a one-way ANOVA and is defined. , p 0.5.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction

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

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 4. p50 occupancy of the CRP promoter changes modestly in the presence of cytokines. Real-time PCR of ChIP assays performed on Hep3B cells treated with cytokines IL-1 and IL-6. Zero time values were determined in each case. Subsequently three time courses were followed 30 min–6 h (n 3 assays) (a), 8–16 h (n 4) (b), and 12–36 h (n 4) (c). Abs were used in the assays with primers flanking the CRP proximal pro- moter (118 to 115), as described in Fig. 2. Data show NF-B p50 occupancy expressed as fold change after subtraction of mock and nor- malization to input signal (see Materials and Methods). Results are an average of three to four experiments, each done in duplicate. Error bar represents SD. Statistical significance of each time point compared with basal levels was determined by a one-way ANOVA at p 0.5, but the experiment had insufficient statistical power to reliably calculate p values.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction

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

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 5. STAT3 occupancy of the CRP promoter rises modestly in response to cytokines. Real-time PCR of ChIP assays performed on Hep3B cells treated with cytokines IL-1 and IL-6. Zero time values were deter- mined in each case. Subsequently three time courses were followed 30 min–6 h (n 3 assays) (a), 8–16 h (n 4) (b), and 12–36 h (n 4) (c). Abs were used in the assays with primers flanking the CRP proximal pro- moter (118 to 115), as described in Fig. 2. Data show STAT3 occu- pancy expressed as fold change after subtraction of mock and normaliza- tion to input signal (see Materials and Methods). Results are an average of three to four experiments, each done in duplicate. Error bar represents the SD. Statistical significance of each time point compared with basal levels was determined by a one-way ANOVA is defined. , p 0.5.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction

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

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 6. c-Rel and TBP occupy the CRP promoter in parallel. Real-time PCR of ChIP assays performed on Hep3B cells treated with cytokines IL-1 and IL-6. Zero time values were determined in each case. Subsequently three time courses were followed 30 min–6 h (n 3 assays) (a), 8–16 h (n 4) (b), and 12–36 h (n 4) (c). Abs were used in the assays with primers flanking the CRP proximal promoter (118 to 115), as described in Fig. 2. Data show c-Rel (solid line, Œ) and TBP (dashed line, f) occupancy expressed as fold change after subtraction of mock and normalization to input signal (see Materials and Methods). a–c, Average of three to four experiments, each done in duplicate. Error bar represents SD. d and e, Profiles from individual ChIP experiments of c-Rel and TBP promoter occupancy. Statistical significance of each time point compared with basal levels was determined by a one-way ANOVA. , p 0.5 is defined for c-Rel and , p 0.5 is indicated for TBP.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction

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

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 7. CRP mRNA accumulates in response to cytokines. a, Rep- resentative agarose gel of RT-PCR performed on Hep3B cells treated with cytokines IL-1 and IL-6 for the indicated times (hours). CRP mRNA levels are shown at top and -actin mRNA levels are shown at bottom. b, Average quantification of band intensity measured using ImageQuant of CRP mRNA normalized to -actin mRNA (n 4 measurements). Error bar represents SD.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

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

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 8. Composite graph of transcription factor occupancy on the CRP promoter and CRP mRNA ac- cumulation in response to cytokines. Common time points for transcription factor occupancy from the 12– 36-h data and CRP mRNA accumulation from the 3–24-h data (12, 18, and 24 h) were plotted together. The left y-axis is the fold change above mock for the transcription factor promoter occupancy. The right y- axis is average normalized band intensity for CRP mRNA accumulation. CRP mRNA (dotted dashed gray line F), C/EBP (black line ), STAT3 (light gray line ‚), p50 (light gray line E), c-Rel (short dashed line Œ), and TBP (black dashed line f) are shown.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques:

Journal: Nature immunology

Article Title: The encephalitogenicity of T H 17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF

doi: 10.1038/ni.2031

Figure Lengend Snippet: (a) Naive CD4+CD25−CD62LhiCD44lo T cells from spleens of C57BL/6 mice were sorted by flow cytometry and activated with anti-CD3 and anti-CD28 in the presence of TGF-β plus IL-6, anti-IFN-γ and anti-IL-4 for 72 h (first stimulation). Cells were rested 2 days in the presence of IL-2 and then reactivated with anti-CD3 and anti-CD28 (second stimulation) during 72 h in the presence of TGF-β plus IL-6, TGF-β, IL-6 and IL-23, IL-23 or without added cytokines. Cells were then stimulated with PMA and ionomycin in the presence of GolgiPlug for the final 4 h, stained and analyzed by flow cytometry. CD4+ cells are shown. (b) Percentage of GM-CSF+ cells among CD4+IL-17A+ cells after the second stimulation. (c) GM-CSF, IL-10 and IL-17A concentrations in cell culture supernatants after the second stimulation measured by ELISA. (d) TH17 cells restimulated in the presence of TGF-β and IL-6 for 72 h were treated with anti-IL-10 or isotype control (goat IgG) and analyzed by flow cytometry. (e) GM-CSF concentrations in culture supernatants of cells stimulated as in (d) measured by ELISA. **p< 0.001. Data are representative of three independent experiments. (error bars, s.e.m).

Article Snippet: Anti-GM-CSF monoclonal antibody (clone 22E9.11; ATCC) was kindly provided by KaloBios Pharmaceuticals, Inc. (South San Francisco).

Techniques: Flow Cytometry, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: Nature immunology

Article Title: The encephalitogenicity of T H 17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF

doi: 10.1038/ni.2031

Figure Lengend Snippet: (a) Naive CD4+CD25−CD62LhiCD44lo T cells from spleens of C57BL/6 mice were FACS sorted and differentiated into TH17 cells during the first stimulation. Cells were then reactivated with anti-CD3 and anti-CD28 for 72 h in the presence of either IL-23 or TGF-β plus IL-6, in the presence of IL-1β (10 ng/ml) and/or TNF (10 ng/ml). CD4+ cells are shown. (b) GM-CSF, IL-17A, IL-21 and IL-22 concentrations in cell culture supernatants after the second stimulation measured by ELISA. (c) Naive CD4+ T cells from spleens of C57BL/6 and RORγt-deficient mice were activated during 72 h with anti-CD3 and anti-CD28 in the presence of either TGF-β and IL-6 or IL-1β, IL-6 and IL-23. Cells were then stimulated with PMA and ionomycin in the presence of GolgiPlug, stained and analyzed by flow cytometry. *p<0.01, **p < 0.001. Data are representative of two independent experiments. (error bars, s.e.m).

Article Snippet: Anti-GM-CSF monoclonal antibody (clone 22E9.11; ATCC) was kindly provided by KaloBios Pharmaceuticals, Inc. (South San Francisco).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

Journal: Nature immunology

Article Title: The encephalitogenicity of T H 17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF

doi: 10.1038/ni.2031

Figure Lengend Snippet: (a) Splenocytes from MOG35-55-immuzed C57BL/6 mice were activated with MOG35-55 (20 μg/ml) in the presence of TGF-β plus IL-6, IL-23 or without added cytokines. Cells were then stimulated with PMA and ionomycin in the presence of GolgiPlug, stained and analyzed by flow cytometry. CD4+ cells are shown. (b) Percentage of GM-CSF+ cells among CD4+IL-17A+ cells after the second stimulation. (c) GM-CSF, IL-10, and IL-17A concentrations in cell culture supernatants measured by ELISA. (d) GM-CSF and IL-17A concentrations in supernatants of cell cultures stimulated as in (a) in the presence of TGF-β plus IL-6 and treated with anti-IL-10 or goat IgG. (e) GM-CSF and IL-17A concentrations in supernatants of cell cultures stimulated as in (a) without added cytokines and treated with anti-IL-23p19 or goat IgG (f) Clinical scores of irradiated wild-type recipient mice that received 5×106 CD4+ cells enriched from EAE splenocytes activated in the presence of IL-23. Mice were treated with either anti-GM-CSF or rat IgG from day 2 to day 35 post cell transfer. *p< 0.01; **p < 0.001. Data are representative of two (d, e and f) or four (a, b, and c) independent experiments (error bars, s.e.m).

Article Snippet: Anti-GM-CSF monoclonal antibody (clone 22E9.11; ATCC) was kindly provided by KaloBios Pharmaceuticals, Inc. (South San Francisco).

Techniques: Staining, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Irradiation

Journal: Nature immunology

Article Title: The encephalitogenicity of T H 17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF

doi: 10.1038/ni.2031

Figure Lengend Snippet: Splenocytes from wild-type- or Csf2−/−-MBPAc1-11 transgenic mice were activated with MBPAc1-11 peptide in the presence of IL-12 (TH1 conditions) or TGF-β plus IL-6, anti-IFN-γ and anti-IL-4 during 72 h (TH17 conditions). Cells were rested 2 days in the presence of IL-2 and then reactivated with MBPAc1-11 peptide in the presence of IL-12 (TH1 conditions) or IL-23 (TH17 conditions). After 72 h, cells were stimulated with PMA and ionomycin in the presence of GolgiPlug. (a) Flow cytometric analysis of IL-17A and IFN-γ expression in wild-type and Csf2−/− TH1 and TH17 cells after the second stimulation. CD4+ cells are shown. (b) IL-17A, IFN-γ and GM-CSF concentrations were measured by ELISA in the supernatants after the second stimulation. (c) RORγt and T-bet expression on gated CD4+IFN-γ+ (TH1) or CD4+IL-17A+ cells (TH17) analyzed by flow cytometry after the second stimulation. Filled histograms represent isotype controls. (d) Real time PCR analysis of Ahr, CCL20, CCR6, IL-23R and RORα expression in CD4+ T cells enriched by magnetic beads after the second stimulation from wild-type or Csf2−/− MBPAc1-11-splenocytes cultured in TH17 conditions. (e) Clinical scores of recipient mice that received 5×106 of either MBPAc1-11-specific wild-type or Csf2−/− TH1 or TH17 cells enriched by magnetic beads after the second stimulation. Pertussis toxin was injected i.p. on days 0 and 2 post transfer. *p< 0.01; **p < 0.001. Data are representative of two independent experiments. (error bars, s.e.m).

Article Snippet: Anti-GM-CSF monoclonal antibody (clone 22E9.11; ATCC) was kindly provided by KaloBios Pharmaceuticals, Inc. (South San Francisco).

Techniques: Transgenic Assay, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Real-time Polymerase Chain Reaction, Magnetic Beads, Cell Culture, Injection

Journal: Frontiers in Nutrition

Article Title: Ponciri Fructus Immaturus ethanol extract attenuates septic shock through inhibition of the STAT1 signaling pathway

doi: 10.3389/fnut.2022.988309

Figure Lengend Snippet: PFIE suppresses the production of inflammatory cytokines by LPS in J774 cells. Indicated PFIE concentrations were used for pretreatment 1 h prior to treatment with 1 μg/mL of LPS for 24 h. mRNA levels of panels (A) TNF-α, (B) IL-1β, and (C) IL-6 were determined using real-time PCR. Relative mRNA expression levels were normalized to those of GAPDH. (D–F) Secreted levels of panel (D) TNF-α, (E) IL-1β, and (F) IL-6 in the culture medium were measured using ELISA. * P < 0.05, ** P < 0.01, and # P < 0.001 (Student’s t -test). Data are representative of three experiments.

Article Snippet: The levels of TNF-α, IL-1β, and IL-6 were measured using a mouse TNF-α DuoSet ELISA kit (DY-410; R&D Systems), mouse IL-1β DuoSet ELISA kit (DY-401; R&D Systems), and mouse IL-6 DuoSet ELISA kit. (DY-406; R&D Systems), respectively.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Nutrition

Article Title: Ponciri Fructus Immaturus ethanol extract attenuates septic shock through inhibition of the STAT1 signaling pathway

doi: 10.3389/fnut.2022.988309

Figure Lengend Snippet: PFIE prevents septic shock and attenuates proinflammatory responses in an LPS-induced endotoxemia mouse model. (A) Mortality was monitored for 7 days after LPS injection in the groups ( n = 10): PBS (circle); LPS (triangle); LPS + 10 mg/kg of PFIE (inverted triangle); and LPS + 20 mg/kg of PFIE (diamond). (B–I) Mouse serum samples ( n = 5) were collected 12 h after LPS injection. Serum levels of AST (B) , ALT (C) , and CK (D) measured using an automatic chemical analyzer. (F) Serum levels of nitric oxide (NO) metabolites measured using Griess reagent. The levels of TNF-α (G) , IL-1β (H) , and IL-6 (I) measured using ELISA. * P < 0.05, ** P < 0.01, and # P < 0.001 (Student’s t -test).

Article Snippet: The levels of TNF-α, IL-1β, and IL-6 were measured using a mouse TNF-α DuoSet ELISA kit (DY-410; R&D Systems), mouse IL-1β DuoSet ELISA kit (DY-401; R&D Systems), and mouse IL-6 DuoSet ELISA kit. (DY-406; R&D Systems), respectively.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Nutrition

Article Title: Ponciri Fructus Immaturus ethanol extract attenuates septic shock through inhibition of the STAT1 signaling pathway

doi: 10.3389/fnut.2022.988309

Figure Lengend Snippet: PFIE improves the survival rate and attenuates proinflammatory responses in a CLP-induced septic mouse model. (A) Mortality was monitored for 7 days after the surgery ( n = 10): sham (circle); CLP (triangle); CLP + 10 mg/kg of PFIE (inverted triangle); CLP + 20 mg/kg of PFIE (diamond). (B–I) Mouse serum samples ( n = 5) were collected 12 h after CLP. Serum levels of AST (B) , ALT (C) , and CK (D) measured using an automatic chemical analyzer. (F) Serum levels of nitric oxide (NO) metabolites measured using Griess reagent. Serum levels of TNF-α (G) , IL-1β (H) , and IL-6 (I) measured using ELISA. * P < 0.05 and ** P < 0.01 (Student’s t -test).

Article Snippet: The levels of TNF-α, IL-1β, and IL-6 were measured using a mouse TNF-α DuoSet ELISA kit (DY-410; R&D Systems), mouse IL-1β DuoSet ELISA kit (DY-401; R&D Systems), and mouse IL-6 DuoSet ELISA kit. (DY-406; R&D Systems), respectively.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: Quinolinic acid potentially links kidney injury to brain toxicity

doi: 10.1172/jci.insight.180229

Figure Lengend Snippet: Kidney cortex RNA and protein lysates were prepared from Mdm2 -cKO vs. control mice ( n = 4 per group). ( A ) Transformation related protein 53 ( Trp53 ) mRNA and ( B ) p53 protein levels. ( C ) Cyclin-dependent kinase inhibitor 1A ( Cdkn1a ) mRNA and ( D ) p21 protein levels. ( E ) Cytochrome b-245, beta polypeptide ( Cybb ), mRNA and ( F ) CYBB protein levels. ( G ) CXC chemokine ligand 1 ( Cxcl1 ) mRNA level. ( H ) CXCL1 protein concentration. ( I ) Interleukin-1β ( Il1b ) mRNA level. ( J ) IL-1β protein concentration. ( K ) Neutrophil-to-lymphocyte ratio (VetScan Analyzer). ( L ) Plasma interleukin-6 (IL-6) protein concentration. ( M ) Plasma soluble urokinase plasminogen activator receptor (suPAR) concentration. Quantitative PCR (qPCR) results (normalized to Gapdh expression) and protein levels measured via Western blot (normalized to actin) are presented as fold-change relative to control. The proteins analyzed via ELISA are reported as concentrations. Graphs display means ± SEM. Two-tailed t tests: * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Quantitative protein levels in plasma and tissue lysates were assessed using ELISA kits following manufacturer’s protocols: mouse uPAR DuoSet ELISA (R&D Systems, Bio-Techne, DY531), mouse CXCL1/KC Quantikine ELISA Kit (R&D Systems, Bio-Techne, MKC00B-1), mouse IL-6 Quantikine ELISA Kit (R&D Systems, Bio-Techne, M6000B-1), and mouse IL-1β/IL-1F2 Quantikine ELISA Kit (R&D Systems, Bio-Techne, MLB00C-1).

Techniques: Control, Transformation Assay, Protein Concentration, Clinical Proteomics, Concentration Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: JCI Insight

Article Title: Quinolinic acid potentially links kidney injury to brain toxicity

doi: 10.1172/jci.insight.180229

Figure Lengend Snippet: Mice were fed regular chow (control) or 0.2% adenine-enriched chow for 1 month ( n = 4 per group). ( A and B ) Plasma suPAR and IL-6 levels measured by ELISA. Concentrations of Trp, KYN, 3HK, QA, and 5-HIAA in ( C – G ) plasma, kidney cortex ( H – L ), and brain cortex ( M – Q ). QA and 5-HIAA were measured using a different mass spectrometry method. Graphs display means ± SEM. Two-tailed t test; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Quantitative protein levels in plasma and tissue lysates were assessed using ELISA kits following manufacturer’s protocols: mouse uPAR DuoSet ELISA (R&D Systems, Bio-Techne, DY531), mouse CXCL1/KC Quantikine ELISA Kit (R&D Systems, Bio-Techne, MKC00B-1), mouse IL-6 Quantikine ELISA Kit (R&D Systems, Bio-Techne, M6000B-1), and mouse IL-1β/IL-1F2 Quantikine ELISA Kit (R&D Systems, Bio-Techne, MLB00C-1).

Techniques: Control, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Translation of IRF-1 Restricts Hepatic Interleukin-7 Production to Types I and II Interferons: Implications for Hepatic Immunity

doi: 10.3389/fimmu.2020.581352

Figure Lengend Snippet: Interferon-(IFN)-α and IFN-γ, but not IFN-λ, induce hepatic interleukin-7 (IL-7) expression and secretion. (A) Quantification of IL-7 mRNA levels in relation to housekeeping GAPDH mRNA in Huh-7.5 cells, which were stimulated for the indicated hours with IFN-α (left), IFN-γ (middle), or IFN-λ2 (right) at the indicated dosages. IL-7 expression is shown relative to untreated cells. (B) IFN-λ signaling is present in Huh-7.5 cells. Quantification of interferon-stimulated gene (ISG) mRNA levels relative to housekeeping GAPDH mRNA in Huh-7.5 cells, which were stimulated with IFN-α, IFN-γ, or IFN-λ2 at the indicated dosages for 6 h. ISG expression is shown relative to untreated cells. (C) Analysis of protein levels of IL- via ELISA in cell culture supernatant of Huh7.5 cells and (D) primary human hepatic sinusoidal endothelial cell (HLSEC), which were stimulated with IFN-α (500 i.E./ml), IFN-γ (5 ng/ml), or IFN-λ2 (50 ng/ml) for 6 or 24 h. In (A–D) , standard deviations of three independent experiments (n=3) performed with three replicates each are shown. * P < 0.05 for comparison of the indicated condition with mock. f.c., fold change. IU, international units.

Article Snippet: Levels of human IL-7, IFNγ, IL-6, and TNFα in human serum samples as well as cell culture supernatant were analyzed using the Human IL-7 High sensitivity, IFNγ, IL-6, and TNFα Quantikine ELISA Kits (R&D systems), respectively.

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

Journal: Frontiers in Immunology

Article Title: Translation of IRF-1 Restricts Hepatic Interleukin-7 Production to Types I and II Interferons: Implications for Hepatic Immunity

doi: 10.3389/fimmu.2020.581352

Figure Lengend Snippet: The IFN-IRF-1-IL-7 axis is present in patients with liver cirrhosis. (A) Presence of the proteins interferon-response factor-1 (IRF1), Glycogen synthase kinase-3 (GSK3), CD68 as a macrophage marker, and CK18 as part of the hepatocellular cytoskeleton was analyzed by immunofluorescence in paraffin-embedded tissue of a representative patient with liver cirrhosis. One out of two experiments (n=2) is shown as demonstration. (B) Interferon-stimulated gene (ISG)-15 relative to GAPDH mRNA concentration was quantified by quantitative PCR in (PBMCs of 11 randomly selected patients with liver cirrhosis from (C) and correlated with serum concentrations of interleukin-7 (IL-7). P value for Pearson correlation. (C) Flow cytometric analyses of the percentage of CD127 positive myeloid cells relative to all myeloid cells from patients with liver cirrhosis and available PBMCs or healthy controls. (D) Serum concentrations of IL-7 and interferon-(IFN)-γ in healthy controls and in patients with different liver cirrhosis stages or with acute-on-chronic liver failure (ACLF) were determined by ELISA. P for comparison between healthy controls and all patients with liver cirrhosis. (E) Monocytes from peripheral blood and ascites-derived macrophages of patients with ascites (n=4), compensated liver cirrhosis (n=4) and decompensated liver cirrhosis (n=4, all measured in duplicate) were stimulated ex vivo with 10 ng/ml IL-7 and/or 10 ng/ml LPS, as indicated, for 6 h. Then, quantitative analysis of mRNA of IL-1β, IL-6, and tumor necrosis factor α (TNFα) levels relative to housekeeping GAPDH mRNA was performed. * P < 0.05 for comparison of the indicated condition versus mock; other comparisons were not significant.

Article Snippet: Levels of human IL-7, IFNγ, IL-6, and TNFα in human serum samples as well as cell culture supernatant were analyzed using the Human IL-7 High sensitivity, IFNγ, IL-6, and TNFα Quantikine ELISA Kits (R&D systems), respectively.

Techniques: Marker, Immunofluorescence, Concentration Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Comparison, Derivative Assay, Ex Vivo

Journal: Frontiers in Immunology

Article Title: Translation of IRF-1 Restricts Hepatic Interleukin-7 Production to Types I and II Interferons: Implications for Hepatic Immunity

doi: 10.3389/fimmu.2020.581352

Figure Lengend Snippet: Interleukin-7 (IL-7) decreases intracellular ATP content of monocyte-derived macrophages but augments pro-inflammatory cytokine production in response to lipopolysaccharide (LPS). (A) Monocyte-derived macrophages (MDMs) underwent pre-incubation with or without 10 ng/ml LPS for 16 h before being stimulated with IL-7 or the Glycogen synthase kinase-3 (GSK3) Inhibitor CHIR99021 (5 µM) for the displayed time points. Then the intracellular ATP content relative to unstimulated controls was analyzed. Standard deviations of three experiments (n=3) performed with three replicates each are shown. * P < 0.05 and # P < 0.1 for comparison of the indicated condition at the indicated time point versus mock. (B) MDMs were pre-incubated with 10 ng/ml LPS for 16 h prior to stimulation with 10 ng/ml IL-7 in presence or absence of 5 µg/ml IL-7 antibody or the GSK3 Inhibitor CHIR99021 (5 µM) for the time points shown. Then the intracellular ATP content relative to unstimulated controls was analyzed by luciferase reaction. Standard deviations of three experiments (n=3) performed with three replicates each are shown. * P < 0.05 for comparison of the indicated condition versus mock. (C) Analysis of mRNA levels of IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) relative to housekeeping GAPDH mRNA in MDMs pre-stimulated with 10 ng/ml LPS as indicated, which were then treated with the indicated reagents for 6 h. Standard deviations of three experiments (n=3) performed with three replicates each are shown. * P < 0.05 for indicated comparisons. (D) MDMs underwent pre-incubation with 10 ng/ml LPS for 16 h prior to stimulation with 10 ng/ml IL-7 in presence or absence of 5 µg/ml IL-7 antibody or the GSK3 Inhibitor CHIR99021 (5 µM) for 24 h. The amount of IL-6 (top) and TNFα (bottom) in cell culture supernatant was quantified by ELISA. Standard deviations of three experiments (n=3) performed with three replicates each are shown. # P < 0.1 for comparison of the indicated condition versus mock. (E) Assessment of mRNA levels of IL-1β, IL-6, and TNFα relative to housekeeping GAPDH mRNA in MDMs pre-incubated with or without two sequential stimuli with 10 ng/ml LPS as shown, and which were co-stimulated with or without 10 ng/ml IL-7 30 min after the first LPS stimulus. Standard deviations of three experiments (n=3) performed with three replicates each are shown. * P < 0.05 for indicated comparisons.

Article Snippet: Levels of human IL-7, IFNγ, IL-6, and TNFα in human serum samples as well as cell culture supernatant were analyzed using the Human IL-7 High sensitivity, IFNγ, IL-6, and TNFα Quantikine ELISA Kits (R&D systems), respectively.

Techniques: Derivative Assay, Incubation, Comparison, Luciferase, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Clinical and Translational Science

Article Title: Good Manufacturing Practice‐Grade of Megakaryocytes Produced by a Novel Ex Vivo Culturing Platform

doi: 10.1111/cts.12788

Figure Lengend Snippet: Confirmation of MK differentiation effect from SMC‐expanded hematopoietic stem cells. ( a ) Expansion folds of total cells, CD34 + cells, and CD34 + /CD38 ‐ cells after 5 days of expansion with SMC combination. ( b ) Percentage of CD34 + cells, CD34 + /CD38 ‐ cells, and CD41a + cells after 5 days of expansion with SMC combination. ( c ) Purity of CD41a + /CD42b + MKs differentiated from unexpanded or SMC‐expanded CD34 + cells over time. ( d ) Absolute number of total cells cultured from unexpanded or SMC‐expanded CD34 + cells over time. ( e ) Absolute number of CD41a + /CD42b + MKs cultured from unexpanded or SMC‐expanded CD34 + cells over time. ( f ) Yield of CD41a + /CD42b + MKs per input cord blood hematopoietic stem cells. Data are shown as mean ± SD, n = 3. *** P < 0.001. MKs, megakaryocytes; SMC, small‐molecule/cytokine cocktail.

Article Snippet: SMC was composed of stem cell factor (SCF 100 ng/mL; Biopharmagen, Suzhou, China), Fms‐related tyrosine kinase 3 ligand (100 ng/mL; Biopharmagen), thrombopoietin (TPO 40 ng/mL; Biopharmagen), interleukin (IL) −6 (15 ng/mL; Biopharmagen), stemregenin1 (SR1 1 μM; Selleck Chemicals, Houston, TX), CAY10433 (C433, 0.1 μM; Cayman Chemical, Ann Arbor, MI), and valproic acid (VPA 0.2 mM; Sigma‐Aldrich, St. Louis, MO).

Techniques: Cell Culture

Journal: Clinical and Translational Science

Article Title: Good Manufacturing Practice‐Grade of Megakaryocytes Produced by a Novel Ex Vivo Culturing Platform

doi: 10.1111/cts.12788

Figure Lengend Snippet: Optimization of culture conditions for producing MKs from small‐molecule/cytokine cocktail‐expanded hematopoietic stem cells. ( a ) Absolute number of CD41a + /CD42b + MKs after 9 days of culture with the indicated treatments. ( b ) Percentage of CD41a + and CD42b + cells after 9 days of culture with the indicated treatments. ( c ) Percentage of CD41a + and CD42b + cells over time induced by CC. ( d ) Absolute number of CD41a + /CD42b + MKs from day 7 to day 9 induced by SCF + TPO or CC. Data are shown as mean ± SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001. CC, cytokine cocktail; MKs, megakaryocytes; SCF, stem cell factor; SR1, stemregenin1; TPO, thrombopoietin; VPA, valproic acid.

Article Snippet: SMC was composed of stem cell factor (SCF 100 ng/mL; Biopharmagen, Suzhou, China), Fms‐related tyrosine kinase 3 ligand (100 ng/mL; Biopharmagen), thrombopoietin (TPO 40 ng/mL; Biopharmagen), interleukin (IL) −6 (15 ng/mL; Biopharmagen), stemregenin1 (SR1 1 μM; Selleck Chemicals, Houston, TX), CAY10433 (C433, 0.1 μM; Cayman Chemical, Ann Arbor, MI), and valproic acid (VPA 0.2 mM; Sigma‐Aldrich, St. Louis, MO).

Techniques:

Journal: Clinical and Translational Science

Article Title: Good Manufacturing Practice‐Grade of Megakaryocytes Produced by a Novel Ex Vivo Culturing Platform

doi: 10.1111/cts.12788

Figure Lengend Snippet: Scalable production and characterization analysis of mature MKs. ( a ) Schematic diagram of the stepwise culture protocol from cord blood CD34 + cells to MKs in the roller‐bottle culture system. ( b ) Absolute number of CD41a + /CD42b + MKs at the end of culture. ( c ) Cell size distribution of MKs measured by microscopy. Digital images of cells were captured and cell diameters were measured by Image J software. ( d ) Representative cell morphology of MKs and pro‐platelet‐like structures (red arrows). Scale bar = 20 μm. ( e ) Number of extending shafts from one adherent MK with ≧ 30 cells counted per condition. ( f ) DNA ploidy distribution of MKs. ( g ) The mRNA levels of megakaryopoiesis‐related gene assessed by real‐time quantitative polymerase chain reaction. β‐actin was used as an internal control. ( h ) Yield of CD41a + /CD42b + MKs per input cord blood hematopoietic stem cell. All data are shown as means ± SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001. CB, cord blood; MKs, megakaryocytes; SMC, small‐molecule/cytokine cocktail.

Article Snippet: SMC was composed of stem cell factor (SCF 100 ng/mL; Biopharmagen, Suzhou, China), Fms‐related tyrosine kinase 3 ligand (100 ng/mL; Biopharmagen), thrombopoietin (TPO 40 ng/mL; Biopharmagen), interleukin (IL) −6 (15 ng/mL; Biopharmagen), stemregenin1 (SR1 1 μM; Selleck Chemicals, Houston, TX), CAY10433 (C433, 0.1 μM; Cayman Chemical, Ann Arbor, MI), and valproic acid (VPA 0.2 mM; Sigma‐Aldrich, St. Louis, MO).

Techniques: Microscopy, Software, Real-time Polymerase Chain Reaction, Control

Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: TWEAK regulates the functions of hair follicle stem cells via the Fn14-Wnt/β-catenin-CXCR4 signalling axis.

doi: 10.1111/wrr.70032

Figure Lengend Snippet: FIGURE 1 TWEAK/Fn14 signalling upregulates the expressions of HFSC markers. (A) Immunofluorescence analysis of Fn14 and CXCR4 expression in mouse hair follicles. The white arrows indicate the Fn14- or CXCR4-positive cells; however, these positive cells were significantly diminished and even became indistinct in the Fn14-deficient specimen. Scale bar = 20 μm. (B) Histochemical analysis of Fn14, K19 and CD34 expression in mouse hair follicles. Scale bar = 20 μm. (C) Primary human HFSCs cultured in vitro. (D) Immunofluorescence analysis of β1 integrin and K15 expression in HFSCs stimulated with TWEAK (10 ng/mL). Scale bar = 10 μm. (e, f) Western blotting of K19, integrin β1 and K15 proteins. Data represent mean ± SEM from three independent experiments. *p < 0.05, compared with the 0 ng/mL TWEAK group. CXCR4, chemokine (C X C motif) receptor 4; Fn14, fibroblast growth factor-inducible 14; HFSC, hair follicle stem cells; K15/K19, keratins 15/19; TWEAK, tumour necrosis factor- like weak inducer of apoptosis.

Article Snippet: HFSCs were stimulated for 48 hours with recombinant human TWEAK (0–250 ng/mL; R&D Systems, Minneapolis, MN).

Techniques: Immunofluorescence, Expressing, Cell Culture, In Vitro, Western Blot

Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: TWEAK regulates the functions of hair follicle stem cells via the Fn14-Wnt/β-catenin-CXCR4 signalling axis.

doi: 10.1111/wrr.70032

Figure Lengend Snippet: FIGURE 2 TWEAK induces proliferation, migration and cytokine production in HFSCs. Human HFSCs were cultured in vitro and stimulated with TWEAK (0–250 ng/mL). (A) Proliferation of HFSCs analysed via flow cytometry. n = 5 per group. (B) Cell migration assessed via scratch analysis. n = 5 per group. (C) mRNA expression levels of EGF, BFGF, TGF-β, NGF and VEGF measured via qRT-PCR. n = 3 per group. (D) Cytokine levels in the supernatants determined via ELISA. n = 3 per group. Data represent mean ± SEM from three to five independent experiments. *p < 0.05 compared with the 0 ng/mL group. #p < 0.05 compared with the 10 ng/mL group. HFSC, hair follicle stem cells; qRT-PCR, quantitative real-time PCR; SEM, standard error of the mean; TWEAK, tumour necrosis factor-like weak inducer of apoptosis.

Article Snippet: HFSCs were stimulated for 48 hours with recombinant human TWEAK (0–250 ng/mL; R&D Systems, Minneapolis, MN).

Techniques: Migration, Cell Culture, In Vitro, Flow Cytometry, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: TWEAK regulates the functions of hair follicle stem cells via the Fn14-Wnt/β-catenin-CXCR4 signalling axis.

doi: 10.1111/wrr.70032

Figure Lengend Snippet: FIGURE 3 TWEAK upregulates the expressions of HFSC phenotype markers. Human HFSCs were cultured in vitro and stimulated with TWEAK (0–250 ng/mL). (A) Fn14 and TNFR2 expressions detected via immunofluorescence. (B) mRNA expression levels of FN14, TNFR2, IGFR and CXCR4 analysed via qRT-PCR. (C, D) Protein expressions of these markers determined via Western blotting, with quantification using ImageJ software. Data represent mean ± SEM from three independent experiments. *p < 0.05 compared with the 0 ng/mL group. #p < 0.05 compared with the 10 ng/mL group. Δp < 0.05 compared with the 50 ng/mL group. HFSC, hair follicle stem cells; qRT-PCR, quantitative real-time PCR; SEM, standard error of the mean; TWEAK, tumour necrosis factor-like weak inducer of apoptosis.

Article Snippet: HFSCs were stimulated for 48 hours with recombinant human TWEAK (0–250 ng/mL; R&D Systems, Minneapolis, MN).

Techniques: Cell Culture, In Vitro, Immunofluorescence, Expressing, Quantitative RT-PCR, Western Blot, Software, Real-time Polymerase Chain Reaction

Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: TWEAK regulates the functions of hair follicle stem cells via the Fn14-Wnt/β-catenin-CXCR4 signalling axis.

doi: 10.1111/wrr.70032

Figure Lengend Snippet: FIGURE 4 TWEAK activates Wnt/β-catenin signalling in HFSCs. Human HFSCs were cultured in vitro and stimulated with TWEAK (10 ng/mL). (A) mRNA expression levels of WNT5A, CTNNB and GSK3B analysed via qRT-PCR. (B, C) Protein expressions of these markers detected via Western blotting, with quantification using ImageJ software. (D) mRNA expression levels of TNFR2, IGFR and CXCR4 analysed in cells pretreated with the β-catenin inhibitor XAV-939. (E, F) Protein expressions of these markers detected via Western blotting, with quantification using ImageJ software. Data represent mean ± SEM from three independent experiments. In (A, C), *p < 0.05 compared with the blank group. In (D, F), *p < 0.05 compared with the PBS alone group; #p < 0.05 compared with the XAV-939 group. Δp < 0.05 compared with the TWEAK alone group. HFSC, hair follicle stem cells; qRT-PCR, quantitative real-time PCR; TWEAK, tumour necrosis factor- like weak inducer of apoptosis.

Article Snippet: HFSCs were stimulated for 48 hours with recombinant human TWEAK (0–250 ng/mL; R&D Systems, Minneapolis, MN).

Techniques: Cell Culture, In Vitro, Expressing, Quantitative RT-PCR, Western Blot, Software, Real-time Polymerase Chain Reaction

Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: TWEAK regulates the functions of hair follicle stem cells via the Fn14-Wnt/β-catenin-CXCR4 signalling axis.

doi: 10.1111/wrr.70032

Figure Lengend Snippet: FIGURE 5 CXCR4 inhibitor suppresses the effect of TWEAK on HFSCs. Human HFSCs were cultured in vitro and treated with TWEAK (10 ng/mL) in the presence or absence of the CXCR4 inhibitor EPI-X4. (A) Proliferation of HFSCs assessed via flow cytometry. n = 5 per group. (B, C) Protein expressions of differentiation markers detected via Western blotting and quantitated using ImageJ software. n = 3 per group. (D). Cytokines synthesised by HFSCs and measured via ELISA in the culture supernatants. n = 3 per group. Data represent mean ± SEM from three to five independent experiments. *p < 0.05 compared with the blank group. #p < 0.05 compared with the TWEAK alone group. CXCR4, chemokine (C X C motif) receptor 4; HFSC, hair follicle stem cells; TWEAK, tumour necrosis factor-like weak inducer of apoptosis.

Article Snippet: HFSCs were stimulated for 48 hours with recombinant human TWEAK (0–250 ng/mL; R&D Systems, Minneapolis, MN).

Techniques: Cell Culture, In Vitro, Flow Cytometry, Western Blot, Software, Enzyme-linked Immunosorbent Assay

Journal: Mediators of Inflammation

Article Title: Pretransplant Immune- and Apoptosis-Related Gene Expression Is Associated with Kidney Allograft Function

doi: 10.1155/2016/8970291

Figure Lengend Snippet: Significant correlations between pretransplant clinical parameters and gene expression.

Article Snippet: The peripheral blood gene expression of caspase-3 ( CASP3 , Hs00263337_m1), Fas ( FAS , Hs00236330_m1), p53 ( TP53 , Hs00153349_m1), Foxp3 ( FOXP3 , Hs00203958_m1), IFN-gamma ( IFNG , Hs00174143_m1), interleukin-2 ( IL-2 , Hs00174114_m1), interleukin-6 ( IL-6 , Hs00174131_m1), interleukin-8 ( IL-8 , Hs00174103_m1), interleukin-10 ( IL-10 , Hs00174086_m1), interleukin-17 ( IL-17A , Hs99999082_m1), interleukin-18 ( IL-18 , Hs00155517_m1), NGAL ( LCN2 , Hs00194353_m1), TGF-beta ( TGFB1 , Hs99999918_m1), and TNF-alpha ( TNF , Hs00174128_m1) was assessed with real-time PCR on custom-designed low density arrays (TaqMan) with TaqMan PCR Master Mix.

Techniques: Expressing, Gene Expression

Journal: Mediators of Inflammation

Article Title: Pretransplant Immune- and Apoptosis-Related Gene Expression Is Associated with Kidney Allograft Function

doi: 10.1155/2016/8970291

Figure Lengend Snippet: Expression level of the studied genes.

Article Snippet: The peripheral blood gene expression of caspase-3 ( CASP3 , Hs00263337_m1), Fas ( FAS , Hs00236330_m1), p53 ( TP53 , Hs00153349_m1), Foxp3 ( FOXP3 , Hs00203958_m1), IFN-gamma ( IFNG , Hs00174143_m1), interleukin-2 ( IL-2 , Hs00174114_m1), interleukin-6 ( IL-6 , Hs00174131_m1), interleukin-8 ( IL-8 , Hs00174103_m1), interleukin-10 ( IL-10 , Hs00174086_m1), interleukin-17 ( IL-17A , Hs99999082_m1), interleukin-18 ( IL-18 , Hs00155517_m1), NGAL ( LCN2 , Hs00194353_m1), TGF-beta ( TGFB1 , Hs99999918_m1), and TNF-alpha ( TNF , Hs00174128_m1) was assessed with real-time PCR on custom-designed low density arrays (TaqMan) with TaqMan PCR Master Mix.

Techniques: Expressing