Journal: The Journal of Biological Chemistry

Article Title: Innate lymphoid cell type 3–derived interleukin-22 boosts lipocalin-2 production in intestinal epithelial cells via synergy between STAT3 and NF-κB

doi: 10.1074/jbc.RA118.007290

Figure Lengend Snippet: Cytokine-mediated antimicrobial gene and protein expression in human IECs. A, HT-29 cells were stimulated for 24 h with IL-17A (50 ng/ml), IL-22 (50 ng/ml), TNF (20 ng/ml), IFN-γ (10 ng/ml), or a combination of these cytokines after which gene expression for the indicated genes was determined by qPCR. Subsequent hierarchical clustering was performed based on the calculated Pearson distance between genes. Colors represent average fold change on log scale compared with untreated controls based on three independent experiments. B, HT-29 cells and primary human CD45-depleted intestinal cells were stimulated for 24 h with IL-17A (50 ng/ml), IL-22 (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) after which gene expression for 19 antimicrobial genes was determined by qPCR. For HT-29 cells, values represent average fold change compared with no stimulation based on three independent experiments. For primary IECs, values indicate average fold change compared with no stimulation for three individual patient samples. C, HT-29, SW480, Caco-2, T84, and HCT116 cells were stimulated for 24 h with IL-17A (50 ng/ml), IL-22 (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) after which gene expression for the indicated genes was determined by qPCR. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Dunnett's post hoc test as compared with the unstimulated cells (expression value of 1). D–F, HT-29 and T84 cells were stimulated for 24 h with IL-17A (50 ng/ml), IL-22 (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) after which hBD-2 and LCN-2 concentrations in supernatant were determined by ELISA, and nitrite concentrations were determined by Griess assay. Values indicate the mean with S.E. of three independent experiments. Statistical analysis performed with two-way ANOVA followed by Šidák's post hoc test. p < 0.005 = **, and p < 0.0005 = ***.

Article Snippet: Reagents The following cytokines were obtained from BioTechne (Minneapolis, MN) and used at the following concentrations unless stated otherwise: 50 ng/ml human IL-22 (782-IL-010); 50 ng/ml human IL-17A (317-ILB-050); 20 ng/ml human TNF (210-TA-005); 10 ng/ml human IFN-γ (285-IF-100); 50 ng/ml human IL-1β (201-LB-005); 50 ng/ml human IL-23; and 50 ng/ml human IL-6 (206-IL-010).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Griess Assay

Journal: The Journal of Biological Chemistry

Article Title: Innate lymphoid cell type 3–derived interleukin-22 boosts lipocalin-2 production in intestinal epithelial cells via synergy between STAT3 and NF-κB

doi: 10.1074/jbc.RA118.007290

Figure Lengend Snippet: Specific cytokine synergy regulates the expression and release of LCN-2, NO, and hBD-2. A–F, HT-29 cells were stimulated for 24 h with all possible combinations of IL-22 (50 ng/ml), IL-17A (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) after which qPCR was performed to determine LCN2 (A) DEFB4 (B), and NOS2 (C) expression. Protein levels for LCN-2 (D) and hBD-2 (E) were determined in supernatants by ELISA, whereas nitrite levels (F) were determined by Griess assay. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with one-way ANOVA followed by Dunnett's post hoc test as compared with unstimulated cells. G, Venn diagrams representing gene expression levels (left) and protein/nitrite levels (right) for different cytokine combinations. Color intensity corresponds to the values in A–F redistributed on a gray scale from 0 to 255. p < 0.05 = *, p < 0.005 = **, and p < 0.0005 = ***.

Article Snippet: Reagents The following cytokines were obtained from BioTechne (Minneapolis, MN) and used at the following concentrations unless stated otherwise: 50 ng/ml human IL-22 (782-IL-010); 50 ng/ml human IL-17A (317-ILB-050); 20 ng/ml human TNF (210-TA-005); 10 ng/ml human IFN-γ (285-IF-100); 50 ng/ml human IL-1β (201-LB-005); 50 ng/ml human IL-23; and 50 ng/ml human IL-6 (206-IL-010).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Griess Assay

Journal: The Journal of Biological Chemistry

Article Title: Innate lymphoid cell type 3–derived interleukin-22 boosts lipocalin-2 production in intestinal epithelial cells via synergy between STAT3 and NF-κB

doi: 10.1074/jbc.RA118.007290

Figure Lengend Snippet: Growth of K. pneumoniae in cell supernatants Hill slope values ± S.E. were calculated from four-parameter fit models of bacterial growth curves. Hill slopes represent the estimated maximum growth rate during log phase, with higher values representing more rapid growth. Values indicate the mean with S.E. of three to four independent experiments.

Article Snippet: Reagents The following cytokines were obtained from BioTechne (Minneapolis, MN) and used at the following concentrations unless stated otherwise: 50 ng/ml human IL-22 (782-IL-010); 50 ng/ml human IL-17A (317-ILB-050); 20 ng/ml human TNF (210-TA-005); 10 ng/ml human IFN-γ (285-IF-100); 50 ng/ml human IL-1β (201-LB-005); 50 ng/ml human IL-23; and 50 ng/ml human IL-6 (206-IL-010).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Innate lymphoid cell type 3–derived interleukin-22 boosts lipocalin-2 production in intestinal epithelial cells via synergy between STAT3 and NF-κB

doi: 10.1074/jbc.RA118.007290

Figure Lengend Snippet: Involvement of STAT3 in IL-22, TNF, and IL-17A–mediated LCN-2 production. A and B, HT-29 cells were stimulated for 30 min with the indicated cytokines after which protein lysate was used for Western blot analysis to determine levels of pSTAT3–Tyr-705, pSTAT3–Ser-727, ac-STAT3–Lys-685, STAT3, and total protein. A, images of Western blottings representative of three independent experiments. B, band intensity quantification. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. C and D, HT-29 cells were stimulated for 24 h with IL-22 (50 ng/ml), IL-17A (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) in combination with indicated concentrations of STATTIC or DMSO control after which LCN-2 concentrations were determined by ELISA (C) and HT-29 viability was determined by WST-1 assay (D). Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Dunnett's post hoc test. E, HT-29 cells were stimulated with indicated cytokine combinations for 30 min after which ChIP-qPCR was performed using anti-STAT3 or control IgG. Fold-increase in enrichment is shown compared with unstimulated cells. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. p < 0.05 = *, p < 0.005 = **, and p < 0.0005 = ***. Molecular masses indicate the location of the closest protein ladder marker on the blot.

Article Snippet: Reagents The following cytokines were obtained from BioTechne (Minneapolis, MN) and used at the following concentrations unless stated otherwise: 50 ng/ml human IL-22 (782-IL-010); 50 ng/ml human IL-17A (317-ILB-050); 20 ng/ml human TNF (210-TA-005); 10 ng/ml human IFN-γ (285-IF-100); 50 ng/ml human IL-1β (201-LB-005); 50 ng/ml human IL-23; and 50 ng/ml human IL-6 (206-IL-010).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, WST-1 Assay, Marker

Journal: The Journal of Biological Chemistry

Article Title: Innate lymphoid cell type 3–derived interleukin-22 boosts lipocalin-2 production in intestinal epithelial cells via synergy between STAT3 and NF-κB

doi: 10.1074/jbc.RA118.007290

Figure Lengend Snippet: Involvement of NF-κB in IL-22, TNF, and IL-17A-mediated LCN-2 production. A and B, HT-29 cells were stimulated for 30 min with the indicated cytokines after which protein lysate was used for Western blot analysis to determine levels of pNF-κB–Ser-536, ac-NF-κB–Lys-310, IκB, NF-κB, and total protein. A, images of Western blottings representative of three independent experiments. B, band intensity quantification. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. C and D, HT-29 cells were stimulated for 24 h with IL-22 (50 ng/ml), IL-17A (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) in combination with indicated concentrations of BMS-345541 or DMSO control after which LCN-2 concentrations were determined by ELISA (C) and HT-29 viability was determined by WST-1 assay (D). Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Dunnett's post hoc test. E, HT-29 cells were stimulated with indicated cytokine combinations for 30 min after which ChIP-qPCR was performed using anti-NF-κB or control IgG. Fold-increase in enrichment was compared with unstimulated cells. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. p < 0.05 = *, p < 0.005 = **, p < 0.0005 = ***. Molecular masses indicate the location of the closest protein ladder marker on the blot.

Article Snippet: Reagents The following cytokines were obtained from BioTechne (Minneapolis, MN) and used at the following concentrations unless stated otherwise: 50 ng/ml human IL-22 (782-IL-010); 50 ng/ml human IL-17A (317-ILB-050); 20 ng/ml human TNF (210-TA-005); 10 ng/ml human IFN-γ (285-IF-100); 50 ng/ml human IL-1β (201-LB-005); 50 ng/ml human IL-23; and 50 ng/ml human IL-6 (206-IL-010).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, WST-1 Assay, Marker

Journal: The Journal of Biological Chemistry

Article Title: Innate lymphoid cell type 3–derived interleukin-22 boosts lipocalin-2 production in intestinal epithelial cells via synergy between STAT3 and NF-κB

doi: 10.1074/jbc.RA118.007290

Figure Lengend Snippet: Synergy between STAT3 and NF-κB inducers regulates LCN-2 production. A and B, HT-29 cells were stimulated for 30 min with the indicated cytokines after which protein lysate was used for Western blot analysis to determine levels of pNF-κB–Ser-536, ac-NF-κB–Lys-310, IκB, NF-κB, and total protein. A, images of Western blottings representative of three independent experiments. B, band intensity quantification. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. C and D, HT-29 cells were stimulated for 30 min with the indicated cytokines after which protein lysate was used for Western blot analysis to determine total levels of pSTAT3–Tyr-705, pSTAT3–Ser-727, ac-STAT3–Lys-685, STAT3, and total protein. C, images of Western blottings representative of two independent experiments. D, band intensity quantification. Values indicate the mean with S.E. of two independent experiments. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. E, HT-29 cells were stimulated for 24 h with the indicated combinations of IL-22 (50 ng/ml), IL-17A (50 ng/ml) + TNF (20 ng/ml), IL-6 (50 ng/ml), and IL-1β (50 ng/ml), after which LCN-2 concentrations were determined in the cell supernatant by ELISA. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with one-way ANOVA followed by Šidák's post hoc test. F, HT-29 cells were stimulated for 24 h with all indicated combinations of IL-22 (50 ng/ml), IL-17A (50 ng/ml) + TNF (20 ng/ml), IL-6 (50 ng/ml), and IL-1β (50 ng/ml) in combination with 5 μm BMS-345541, 2.5 μm STATTIC, or DMSO control, after which LCN-2 concentrations were determined in cell supernatant by ELISA. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Dunnett's post hoc test as compared with unstimulated cells. p < 0.05 = *, p < 0.005 = **, p < 0.0005 = ***. Molecular masses indicate the location of the closest protein ladder marker on the blot.

Article Snippet: Reagents The following cytokines were obtained from BioTechne (Minneapolis, MN) and used at the following concentrations unless stated otherwise: 50 ng/ml human IL-22 (782-IL-010); 50 ng/ml human IL-17A (317-ILB-050); 20 ng/ml human TNF (210-TA-005); 10 ng/ml human IFN-γ (285-IF-100); 50 ng/ml human IL-1β (201-LB-005); 50 ng/ml human IL-23; and 50 ng/ml human IL-6 (206-IL-010).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Marker