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: 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: Figure 8: NMU signaling modulates the WNT receptor pathway in NMUR2-positive SKBR3 breast cancer cells. (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