Journal: Oncotarget

Article Title: Oncogenic features of neuromedin U in breast cancer are associated with NMUR2 expression involving crosstalk with members of the WNT signaling pathway

doi: 10.18632/oncotarget.16121

Figure Lengend Snippet: (A) Real-time PCR-based validation of candidate NMU downstream genes in independent stably transfected SKBR3 NMU (n=5) and mock clones (n=5). * P < 0.05; ns: not significant (Mann-Whitney-U test). (B) Representative western blots showing differential protein expression of candidate NMU downstream genes in independent stably transfected SKBR3 NMU and mock clones. Loading controls: β-actin, β-tubulin, total RAC1. All experiments were performed in triplicate. (C) Densitometrical evaluation of the western blot results shown in B depicted as box plots. Box plot showing RAC1-GTP in relation to total RAC1 amounts in SKBR3 NMU (n=4) and mock clones (n=4), combines data of three independent experiments. * P < 0.05; ns: not significant (Mann-Whitney-U test). (D) Hypothetical model of NMU's oncogenic role in dependency of NMUR2 in breast cancer: crosstalk of NMU signaling with WNT, TGFβ and ERK cascade results in decreased expression of the canonical WNT target MYC and enhanced activation of the non-canonical WNT/planar cell polarity (PCP) pathway effector RAC1 among others, contributing to growth inhibition and promotion of cell migration.

Article Snippet: The following primary antibodies were used: NMU (1:250, HPA025926, Atlas Antibodies, Stockholm, Sweden), β-actin (1:5000, A5441, Sigma-Aldrich, Deisenhofen, Germany), β-tubulin (1:500, ab6046, Abcam, Cambridge, UK), CD44 (1:1000, 156-3C11), MYC (1:1000, D84C12), DKK1 (1:1000, 4687), LRP6 (1:1000, C47E12), P-LRP6 (1:1000, 2568) (all Cell Signaling, Danvers, MA, USA).

Techniques: Real-time Polymerase Chain Reaction, Biomarker Discovery, Stable Transfection, Transfection, Clone Assay, MANN-WHITNEY, Western Blot, Expressing, Activation Assay, Inhibition, Migration

Journal: Aging (Albany NY)

Article Title: Identification and validation of eight estrogen-related genes for predicting prognosis of papillary thyroid cancer

doi: 10.18632/aging.204582

Figure Lengend Snippet: The top 6 significantly enriched pathways in the high-risk and low-risk groups. GSEA identified six pathways significantly enriched in the high-risk group, including E2F target ( A ), G2M checkpoint ( B ), epithelial-mesenchymal transition ( C ), IL6/JAK/STAT3 signaling ( D ), KRAS signaling ( E ), and IL2/STAT5 signaling ( F ) (All p < 0.01). ( G ) Western blots of KRAS signaling in TPC-1 and KTC-1 cells with and without NMU knockdown. GAPDH was used as the loading control, p-AKT and p-ERK levels were significantly lower with NMU knockdown.

Article Snippet: Protein samples were subjected to 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane, and then incubated with a specific antibody at 4°C overnight: NMU (24862-1-AP; Proteintech; Rosemont, IL, USA), protein kinase B (AKT; 4685S; Cell Signaling Technology [CST]; Danvers, MA, USA), phosphorylated AKT (p-AKT; 4060S; CST), extracellular signal-regulated kinase (ERK; 4695S; CST), and phosphorylated ERK (p-ERK; 4370S; CST).

Techniques: Western Blot, Knockdown, Control

Journal: Cell metabolism

Article Title: Neuromedin U Does Not Act as a Decretin in Rats.

doi: 10.1016/j.cmet.2018.10.008

Figure Lengend Snippet: Figure 4. NMU Expression Down the Rat Gastrointestinal Tract, NMUR1 Receptor Activation, and NMUR1 and NMUR2 Expression in Pancreas and Gut (A) Extractable NMU (total) concentrations down the gastrointestinal tract in the rat. (B) NMUR1 receptor activation by synthetic rat NMU-23 in COS-7 cells tran- siently expressing the rat NMUR1 (measured as IP3 formation). (C) NMUR1, NMUR2, and GLP-1R expression in human islets. (D) NMUR1, NMUR2, and GLP-1R expression in rat islets. (E) Expression of NMUR1, NMUR2, and GLP-1R in human a, b, and d cell islets (by single-cell RNA sequencing). (F) Representative NMUR1 and NMUR2 staining in rat pancreas and small intestine. Data in (A) and (B) are shown as means ± SEM. Dots in (A) indicate individual data points. ‘‘I’’ in (F) indicated pancreatic islets and indicated scale bar applies to all. Statistical significance was assessed by one-way ANOVA for repeated measurements followed by Bonferroni multiple comparisons test (A, C, and D);

Article Snippet: COS-7 cells were maintained as described above and transiently transfection with the rat NMUR1 (untagged, from Origene, cat. no. RN208112 , Rockville, MD, USA) was performed using the calcium phosphate precipitation method with the addition of chloroquine (Kissow et al., 2012).

Techniques: Expressing, Activation Assay, RNA Sequencing, Staining

Journal: Frontiers in Immunology

Article Title: Neuronal-Activated ILC2s Promote IL-17A Production in Lung γδ T Cells During Sepsis

doi: 10.3389/fimmu.2021.670676

Figure Lengend Snippet: Sepsis promotes NMU expression in the lung and NMUR1 expression in lung ILC2s. (A, B) Real-time PCR (A) and western blot (B) detection of lung NMU expression from CLP or sham mice at 24h (n = 3). (C) Real-time PCR detection of nmur1 mRNA in sorted ILC2s under the treatment of LPS + TNF-α for 24h (n = 6). (D) Real-time PCR detection of nmur1 mRNA in three cell populations sorted from lung at 24h after CLP surgery (n = 3). All data are mean ± SEM, with symbols representing the values of individual mice. * P < 0.05, **** P < 0.0001, u.d., undetected. One-way ANOVA in (D) ; two-tailed Student’s t-test in (A–C) . Densitometry of western blotting bands was quantified by ImageJ software (gray-scale band analysis) of three independent experiments, non-parametric Mann-Whitney U test.

Article Snippet: Quantitative PCR was conducted in triplicate on a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with TaqMan Gene Expression Master Mix (Applied Biosystems) using the following TaqMan Gene Expression Assays (Applied Biosystems): Nmu (Mm00479868_m1); Nmur1 (Mm00515885_m1); Il5 (Mm00439646_m1); Il9 (Mm00434305_m1); Il13 (Mm00434204_m1); Il17a (Mm00439618_m1).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Two Tailed Test, Software, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Neuronal-Activated ILC2s Promote IL-17A Production in Lung γδ T Cells During Sepsis

doi: 10.3389/fimmu.2021.670676

Figure Lengend Snippet: NMU promotes lung IL-17A-producing γδ T cell expansion. (A) Survival study of mice monitored for 72h after CLP or sham surgery. Mice received PBS or NMU (0.2 µg/g) at 6h before and after CLP (n = 5). (B, C) (B) Representative flow cytometry plots for ILC2 population within lung live CD45 + Lineage - populations; (C) The percentages of ILC2s within lung live CD45 + populations at 24h after CLP or sham surgery. Mice received a single dose of NMU (1 µg/g) at 6h before CLP (n = 6). (D, E) Representative flow cytometry plots (D) and percentages (E) of the IL-17A + cell population within lung live CD45 + populations at 24h after CLP or sham surgery. Mice received a single dose of NMU (1 µg/g) at 6h before CLP (n = 4). (F, G) Representative flow cytometry plots (F) and percentages (G) of γδ T cell population within lung live CD45 + populations at 24h after CLP or sham surgery. Mice received a single dose of NMU (1 µg/g) at 6h before CLP (n = 4). All data are mean ± SEM, with symbols representing the values of individual mice. * P < 0.05, ** P < 0.01, *** P < 0.001. Kaplan–Meier analysis in (A) One-way ANOVA in (C) two-tailed Student’s t-test in (E, G) .

Article Snippet: Quantitative PCR was conducted in triplicate on a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with TaqMan Gene Expression Master Mix (Applied Biosystems) using the following TaqMan Gene Expression Assays (Applied Biosystems): Nmu (Mm00479868_m1); Nmur1 (Mm00515885_m1); Il5 (Mm00439646_m1); Il9 (Mm00434305_m1); Il13 (Mm00434204_m1); Il17a (Mm00439618_m1).

Techniques: Flow Cytometry, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Neuronal-Activated ILC2s Promote IL-17A Production in Lung γδ T Cells During Sepsis

doi: 10.3389/fimmu.2021.670676

Figure Lengend Snippet: ILC2s mediate NMU-indued increase in lung γδ T cells. (A–D) Representative flow cytometry plots (A) , numbers (B) , percentages (C) of γδ T cell population, and ELISA analysis (D) of supernatant IL-17A in different groups. ILC2s and γδ T cells were co-cultured for 48h with or without NMU (10 μg/ml). IL-1β (100 ng/ml) and IL-23 (100 ng/ml) were added to polarize IL-17A-producing γδ T cells, LPS (1 μg/ml) plus TNF-α (20 ng/ml) were added to mimic sepsis stimulation (n = 4). (E, F) Numbers (E) of γδ T cell population and ELISA analysis (F) of supernatant IL-17A in groups co-cultured with different numbers of ILC2s. ILC2s and γδ T cells were co-cultured for 48 hours with NMU (10 μg/ml) (n = 4). (G, H) Representative flow cytometry plots (G) of γδ T cell population and ELISA analysis (H) of supernatant IL-17A in co-culture group with different concentrations of NMU (1 or 10 μg/ml). ILC2s and γδ T cells were co-cultured for 48h (n = 4). (I) Real-time PCR detection of nmur1 mRNA in ILC2s after nmur1 sgRNA transfection using CRISPR/Cas9 approach for 48h (n = 3). (J–M) Representative flow cytometry plots (J) , numbers (K) , percentages (L) of γδ T cell population, and ELISA analysis (M) of supernatant IL-17A in control and nmur1 knockdown groups. ILC2s and γδ T cells were co-cultured for 48h (n = 4). All data are mean ± SEM, with symbols representing the values of individual mice. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant. One-way ANOVA in (B–D) ; two-tailed Student’s t-test in (E, F, H, I, K–M) .

Article Snippet: Quantitative PCR was conducted in triplicate on a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with TaqMan Gene Expression Master Mix (Applied Biosystems) using the following TaqMan Gene Expression Assays (Applied Biosystems): Nmu (Mm00479868_m1); Nmur1 (Mm00515885_m1); Il5 (Mm00439646_m1); Il9 (Mm00434305_m1); Il13 (Mm00434204_m1); Il17a (Mm00439618_m1).

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Co-Culture Assay, Real-time Polymerase Chain Reaction, Transfection, CRISPR, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Neuronal-Activated ILC2s Promote IL-17A Production in Lung γδ T Cells During Sepsis

doi: 10.3389/fimmu.2021.670676

Figure Lengend Snippet: IL-9 mediates ILC2 regulation of γδ T cell expansion and IL-17A production. (A) ELISA analysis of supernatant IL-9 in different groups (n = 3). ILC2s and γδ T cells were co-cultured for 48h with or without NMU (10 μg/ml). (B) ELISA analysis of supernatant IL-9 in groups after nmur1 sgRNA transfection using CRISPR/Cas9 approach for 48h and then co-cultured with γδ T cells for 48h (n = 4). (C) ELISA analysis of supernatant IL-9 in groups after Il9 sgRNA transfection using CRISPR/Cas9 approach for 48h (n = 4). (D–G) Representative flow cytometry plots (D) , numbers (E) , percentages (F) of γδ T cell population, and ELISA analysis (G) of supernatant IL-17A in control and Il9 knockdown groups. ILC2s and γδ T cells were co-cultured for 48h (n = 4). All data are mean ± SEM, with symbols representing the values of individual mice. * P < 0.05, ** P < 0.01, *** P < 0.001. One-way ANOVA in (A) ; two-tailed Student’s t-test in (B, C, E, F, G) .

Article Snippet: Quantitative PCR was conducted in triplicate on a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with TaqMan Gene Expression Master Mix (Applied Biosystems) using the following TaqMan Gene Expression Assays (Applied Biosystems): Nmu (Mm00479868_m1); Nmur1 (Mm00515885_m1); Il5 (Mm00439646_m1); Il9 (Mm00434305_m1); Il13 (Mm00434204_m1); Il17a (Mm00439618_m1).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Transfection, CRISPR, Flow Cytometry, Two Tailed Test

Journal: Cell Reports

Article Title: ILC precursors differentiate into metabolically distinct ILC1-like cells during Mycobacterium tuberculosis infection

doi: 10.1016/j.celrep.2022.110715

Figure Lengend Snippet:

Article Snippet: For the cytokine-based plasticity model, C57BL/6 or Rag2 −/− mice were treated i.n. with different combinations of cytokines specified in figures legends at day 1, 3, 5, 8 and sacrificed at day 9: 100 ng of IL-12 (R&D), IL-18 (R&D), IL-33 (Biolegend) or 20 μg of NMU (US Biological) per mouse and per instillation.

Techniques: Software, Recombinant, Staining, Enzyme-linked Immunosorbent Assay