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: Cellular and Molecular Gastroenterology and Hepatology

Article Title: siRNA Library Screening Identifies a Druggable Immune-Signature Driving Esophageal Adenocarcinoma Cell Growth

doi: 10.1016/j.jcmgh.2018.01.012

Figure Lengend Snippet: LIF hierarchically regulates STAT3 phosphorylation, IL6 expression, and cell proliferation in EAC cells. SiRNA-mediated silencing of LIF expression results in reduced LIF mRNA ( A ) expression, ( B ) secretion, and ( C ) a recombinant-recoverable reduction in cell proliferation in CP-D, SKGT4, and OE-33 EAC cell lines. Treatment of LIF silenced cells with recombinant LIF protein (rLIF) results in ( D ) recovery of LIF mRNA in CP-D, SKGT4, and OE33 EAC cells and ( E ) protein expression levels in SKGT4 EAC cells. ( F ) STAT3-Y705 phosphorylation levels in LIF-silenced SKGT4 EAC cells are rescued by treatment with recombinant LIF protein as determined by Western blot. ( G ) Silencing LIF expression in SKGT4 EAC cells results in decreased IL6 mRNA expression that is recovered upon exposure to rLIF protein. ( H ) Neither silencing IL6 expression by siRNA-mediated silencing nor treatment with anti-IL6 antibody affects LIF mRNA levels or SKGT4 EAC cell growth. ( I ) Silencing LIF expression results in decreased expression of C1QA mRNA but not TREM2 in SKGT4 EAC cells. ∗ P < .05, ** P < .01, *** P < .001, and **** P < .0001 in either Student t test (proliferation) or Mann–Whitney testing (mRNA expression). siNT, nontargeting siRNA pool; siLIF, LIF-targeted siRNA pool; rLIF, recombinant LIF protein; rIL6, recombinant IL6 protein.

Article Snippet: Treatments with recombinant leukemia inhibitory factor (LIF) (30 ng/mL, ab57665; Abcam), IL6 (30 ng/mL; R&D Systems, Minneapolis, MN), native C1q (75 μg/mL; Sigma, Oakville, Ontario), and IL6 blocking antibody (AB206NA) experiments were performed in either 6- or 96-well cell culture plates for protein and viability experiments, respectively.

Techniques: Phospho-proteomics, Expressing, Recombinant, Western Blot, MANN-WHITNEY

Journal: Nature Communications

Article Title: The negative cofactor 2 complex is a key regulator of drug resistance in Aspergillus fumigatus

doi: 10.1038/s41467-019-14191-1

Figure Lengend Snippet: a , b A549 epithelial cells or macrophages were infected with the wild-type (WT), the nctA null mutant ( ∆nctA ) or the nctA reconstituted isolate ( nctA rec ) for 24 h. Cytotoxicity of each mutant was evaluated by measuring the release of lactate dehydrogenase (LDH) activity into the culture medium. The data represent five different infection challenges with triplicate LDH activity measurements. Data are shown in fold change of LDH activity relative to the wild-type-infected cells. The error bars mean the standard error of the mean (SEM), and P- values were calculated by Kruskal–Wallis test with Dunn’s correction: * P < 0.0180; ** P < 0.0056 (for a , A549 cells). ** P < 0.0032; *** P < 0.0007 (for b , macrophages). c – f Granulocyte macrophage colony-stimulating factor-induced bone marrow-derived dendritic cells (gm-csf BMDCs) were infected with the A. fumigatus strains with the multiplicity of infection (MOI) = 5:1. Proinflammatory cytokines were quantified by ELISA. Data represent three biological replicates ( c – e ) or five biological replicates ( f ) with ± SEM. P- values were calculated by ANOVA with Tukey’s correction: *** P < 0.002; **** P < 0.0001. g , h Activation of dendritic cells measured by CD40 and CD80 markers by flow cytometry. Data represent three biological replicates with ±SEM. P -values were calculated by ANOVA: *** P < 0.0002; **** P < 0.0001 (for g ). *** P < 0.0004; **** P < 0.0001 (for h ). Source data are provided as a Source Data file.

Article Snippet: The concentration of IL-8 and IL-6 were determined in A549 epithelial cells co-cultured with A. fumigatus strains by using the Human IL-8/CXCL8 and IL-6 DuoSet ELISA according to the manufacturer’s instructions (R&D systems).

Techniques: Infection, Mutagenesis, Activity Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Flow Cytometry