Journal: Nature cell biology

Article Title: Obesity alters the lung myeloid cell landscape to enhance breast cancer metastasis through IL5 and GM-CSF

doi: 10.1038/ncb3578

Figure Lengend Snippet: IL5 signalling supports lung neutrophilia. (a) qRT–PCR of Ccl25, Csf2, Igfbp2, Il5, Mmp3, Mmp9, Cd40lg and Il6 in visceral and subcutaneous fat from mice on HF diet. n = 4 mice per tissue; mean ± s.e.m. (b) Trial schematic for c: WT BL6 mice were treated daily with rIL5 or PBS (5 d), and immune cells in lung were quantified by flow cytometry. (c) Representative flow cytometry plots (left) and quantification (right) of lung neutrophils following the trial depicted in b. n = 5 mice per group; mean ± s.e.m., two-tailed unpaired Student’s t-test. (d) Representative flow plots showing gating strategy and population distribution for IL5Rα+ cells. (e) qRT-PCR of Csf2ra, Csf2rb and Il5ra in FACS-purified lung neutrophils, monocytes and eosinophils from the DIO model. n = 5 mice per group; mean ± s.e.m., two-tailed unpaired Student’s t-test. (f) Representative flow plots showing IL5Rα+ populations in human blood. Eosinophils (blue) were used as a positive gating control, n = 7 healthy donors. (g) Quantification of cell proliferation in vitro in response to rIL5 treatment via flow cytometry for Ki67+ cells. Cells isolated from n = 5 mice per group; Tukey boxplot, two-tailed unpaired Student’s t-test. (h) qRT–PCR of Csf2 expression in FACS-purified IL5Rα+ cells after treatment with rIL5 in vitro (100 ng ml−1, 4 h). Cells isolated from n = 5 mice per group; mean ± s.e.m., two-tailed unpaired Student’s t-test. (i) Flow cytometry analysis of IL5Rα+ monocytes from blood and lung in the DIO model. n = 5 mice per group; Tukey boxplot, two-tailed unpaired Student’s t-test. (j) qRT–PCR of Csf2 expression in FACS-purified IL5Rα+ cell types from blood and lung in the DIO model. n = 5 mice per group; Tukey boxplot, two-tailed unpaired Student’s t-test. NS, not significant. Box plots represent median and interquartile range while whiskers represent maximum and minimum values excluding outliers. Each symbol represents one mouse.

Article Snippet: WT and ob/ob neutrophils were mixed at an equal ratio (confirmed by flow cytometry), and then 3 × 10 6 cells were injected via tail vein into WT, ob/ob or ob/ob mice + neutralizing antibody against IL5 (1 mg kg −1 body weight, dosed 3 d and 1 d prior to adoptive transfer intravenously, clone TRFK5, rat IgG1 isotype control; R&D Systems) 67 .

Techniques: Quantitative RT-PCR, Flow Cytometry, Two Tailed Test, Purification, Control, In Vitro, Isolation, Expressing

Journal: Nature cell biology

Article Title: Obesity alters the lung myeloid cell landscape to enhance breast cancer metastasis through IL5 and GM-CSF

doi: 10.1038/ncb3578

Figure Lengend Snippet: Serum GM-CSF is elevated in obesity in association with CD11b+Gr1+ cells. (a) Left, flow cytometry of circulating CD11b+Gr1+ cells in the DIO model. Right, CD11b+Gr1+ populations are shown as a red overlay upon total CD11b+ cells, graphed on Ly6C (x axis) by Ly6G (y axis) dot plots. LF, n = 8 mice; HF, n = 10 mice; minimum–maximum boxplots, all data points shown, two-tailed unpaired Student’s t-test. (b) Left, in vitro myelopoiesis assay, demonstrating increased differentiation of mouse BM cells towards a CD11b+Gr1+ phenotype after treatment with HF serum compared with LF serum from the DIO model. n = 3 independent BM isolations; mean ± s.e.m., two-tailed unpaired Student’s t-test. Right, representative flow plots are shown. (c) Left, in vitro myelopoiesis assay, demonstrating increased differentiation of mouse BM cells towards a CD11b+Gr1+ phenotype after treatment with obese serum compared with lean serum from human donors. n = 3 independent BM isolations; mean ± s.e.m., two-tailed unpaired Student’s t-test. Right, representative flow plots are shown. (d) Venn diagram of results from cross-species cytokine array (Supplementary Table 2). Out of 103 factors, 30 were elevated in HF versus LF mouse serum, and 16 out of 103 factors were elevated in obese versus lean human serum. Eight overlapping factors were identified, including CCL25, CD40L, GM-CSF, IGFBP2, IL5, IL6, MMP3 and MMP9. (e) In vitro myelopoiesis assay, testing the capacity of the 8 factors identified in d to regulate BM differentiation towards a CD11b+Gr1+ phenotype. n = 3 independent mouse BM isolations; mean ± s.e.m., one-way ANOVA and Dunnett’s multiple comparisons test. (f) In vitro myelopoiesis assay, demonstrating that GM-CSF neutralization reverses the effects of HF serum on CD11b+Gr1+ differentiation. n = 6 independent mouse BM isolations; mean ± s.e.m., one-way ANOVA and Dunnett’s multiple comparisons test. (g) qRT-PCR of Csf2 (GM-CSF) across different tissues in HF-fed animals. n = 4 mice per tissue; mean ± s.e.m. (h) qRT-PCR of Csf2 in different FACS-purified cell types from HF lung tissues. n = 5 mice per cell type; mean ± s.e.m. Box plots represent median and interquartile range while whiskers represent maximum and minimum values excluding outliers. Each symbol represents one mouse.

Article Snippet: WT and ob/ob neutrophils were mixed at an equal ratio (confirmed by flow cytometry), and then 3 × 10 6 cells were injected via tail vein into WT, ob/ob or ob/ob mice + neutralizing antibody against IL5 (1 mg kg −1 body weight, dosed 3 d and 1 d prior to adoptive transfer intravenously, clone TRFK5, rat IgG1 isotype control; R&D Systems) 67 .

Techniques: Flow Cytometry, Two Tailed Test, In Vitro, Neutralization, Quantitative RT-PCR, Purification

Journal: Nature cell biology

Article Title: Obesity alters the lung myeloid cell landscape to enhance breast cancer metastasis through IL5 and GM-CSF

doi: 10.1038/ncb3578

Figure Lengend Snippet: Weight loss reduces obesity-associated lung neutrophilia and metastasis in mice and humans. (a–c) Flow cytometry quantification of IL5Rα+ neutrophils (a), IL5Rα+ eosinophils (b) and IL5Rα+ monocytes (c) from lung in LF, HF and diet-switch (HF–LF) mice. LF, n = 5 mice; HF, n = 5 mice; HF–LF, n = 4 mice; mean ± s.e.m., one-way ANOVA and Dunnett’s multiple comparisons test. (d) Left, BLI of the 48 h metastasis assay with 99LN cells injected via the tail vein in the diet-switch trial. Right, representative images are shown. LF, n = 9 mice; HF, n = 10 mice; HF–LF, n = 10 mice; mean ± s.e.m., Kruskal–Wallis and Dunn’s multiple comparisons test. (e) qRT-PCR analysis of Cxcr2, Cxcr4, S100a8 and S100a9 expression in FACS-purified lung neutrophils. LF, n = 5 mice; HF, n = 5 mice; HF–LF, n = 4 mice; mean ± s.e.m., one-way ANOVA and Dunnett’s multiple comparisons test. (f) ELISA analysis of serum IL5 (left) and GM-CSF (right). LF, n = 5 mice; HF, n = 5 mice; HF–LF, n = 4 mice; mean ± s.e.m., one-way ANOVA and Dunnett’s multiple comparisons test. (g) ELISA analysis of serum IL5 (left) and GM-CSF (right) from human weight loss study. n = 10 donors per group; matched pre- and post-weight loss concentrations within a given individual are connected with a line; two-tailed paired Student’s t-test. NS, not significant. (h) Schematic representation of the proposed mechanism underlying obesity-associated lung neutrophilia. Adipose tissue-derived IL5 signals to IL5rα+ cells, leading to their expansion and upregulation of Csf2. This contributes to an environment that supports neutrophilia in the circulation and in the lungs. Lung neutrophils are reprogrammed by obesity to adopt pro-tumorigenic transcriptional signatures, and ultimately support metastatic progression.

Article Snippet: WT and ob/ob neutrophils were mixed at an equal ratio (confirmed by flow cytometry), and then 3 × 10 6 cells were injected via tail vein into WT, ob/ob or ob/ob mice + neutralizing antibody against IL5 (1 mg kg −1 body weight, dosed 3 d and 1 d prior to adoptive transfer intravenously, clone TRFK5, rat IgG1 isotype control; R&D Systems) 67 .

Techniques: Flow Cytometry, Injection, Quantitative RT-PCR, Expressing, Purification, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Derivative Assay

Journal: Nature cell biology

Article Title: Obesity alters the lung myeloid cell landscape to enhance breast cancer metastasis through IL5 and GM-CSF

doi: 10.1038/ncb3578

Figure Lengend Snippet: Obesity enhances lung homing of neutrophils in an IL5-dependent manner. (a) Schematic representation of the adoptive cell transfer experiment. Neutrophils were isolated from BM of WT or ob/ob mice, labelled with fluorescent CellTrace dye (green and red, respectively), mixed 1:1, and then injected via the tail vein (3 × 106 cells) into WT or ob/ob mice ± IL5 neutralizing antibody. Lungs were isolated for flow cytometry analysis after 4 h and 8 h to assess kinetics of neutrophil trafficking. (b) Flow cytometry validation of an equal 1:1 mix of WT (green; 49.3%) and ob/ob (red; 49.7%) donor neutrophils immediately prior to adoptive cell transfer injections. (c) Flow cytometry analysis of lung at 4 h post-adoptive transfer. The vast majority of labelled neutrophils at this time point were from ob/ob donors. n = 5 mice per recipient group, mean ± s.e.m., one-way ANOVA and Dunnett’s multiple comparisons test. (d) Representative flow cytometry plots for the data presented in c. (e) Flow cytometry analysis of fluorescently labelled circulating neutrophils 4 h post-adoptive transfer, demonstrating balanced representation of both red (ob/ob donor) and green (WT donor) cells. n = 5 mice per recipient group; mean ± s.e.m. (f) Flow cytometry analysis of lung at 8 h post-adoptive transfer. Equivalent representation of red and green donor neutrophils was observed at this time point compared with 4 h as in c. n = 5 mice per recipient group, mean ± s.e.m., one-way ANOVA and Dunnett’s multiple comparisons test. (g) Representative flow cytometry plots for the data presented in f. NS, not significant.

Article Snippet: WT and ob/ob neutrophils were mixed at an equal ratio (confirmed by flow cytometry), and then 3 × 10 6 cells were injected via tail vein into WT, ob/ob or ob/ob mice + neutralizing antibody against IL5 (1 mg kg −1 body weight, dosed 3 d and 1 d prior to adoptive transfer intravenously, clone TRFK5, rat IgG1 isotype control; R&D Systems) 67 .

Techniques: Isolation, Injection, Flow Cytometry, Biomarker Discovery, Adoptive Transfer Assay

Journal: Neuroscience

Article Title: Simvastatin inhibits protein isoprenylation in the brain.

doi: 10.1016/j.neuroscience.2016.04.053

Figure Lengend Snippet: Fig. 1. High-dose simvastatin treatment leads to change in isoelec- tric point (pI) of RhoA and Cdc42. N2a neuroblastoma cells were incubated in the presence of vehicle or simvastatin (10 lM) for 24 h. Cells were then harvested and cellular lysates were processed for 2D gel electrophoresis, followed by Western blotting using antibody specific for (A) RhoA or (B) Cdc42. Left and right panels demonstrate 2D and 1D gel blots, respectively. Protein levels and isoelectric point shift of RhoA and Cdc42 bands were assessed by Western blots (probed with anti-GAPDH as a loading control). Blots are represen- tative of four separate experiments with 1–3 blots per condition per experiment. Averages given ± SD, and ** (p < 0.01) connote statis- tical difference from vehicle control by paired t-test.

Article Snippet: Focused strips (2D-SDS PAGE) or 10–20 lg of each protein sample (1D SDS PAGE) were subject to electrophoretic molecular weight separation in the second dimension on precast Criterion 12% gels (BioRad) and then transferred to polyvinylidene fluoride or nitrocellulose membranes.

Techniques: Incubation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot, Control

Journal: Neuroscience

Article Title: Simvastatin inhibits protein isoprenylation in the brain.

doi: 10.1016/j.neuroscience.2016.04.053

Figure Lengend Snippet: Fig. 3. Simvastatin treatment leads to changes in isoelectric point of RhoA and Cdc42 in a dose-dependent manner to doses as low as 50 nM. N2a cells were treated with vehicle or with varying concentrations (50–500 nM) of simvastatin (SIM) for 48 h. Cells were then harvested and cellular lysates were processed either for 2D or 1D gel electrophoresis, followed by Western blotting using antibody specific for (A) RhoA or (B) Cdc42, and GAPDH as a loading control. Data representative of three experiments and 3–4 blots per experiment are shown and quantitations are presented as average ± SD. * (p < 0.05) and ** (p < 0.01) connote statistical difference by paired t-test.

Article Snippet: Focused strips (2D-SDS PAGE) or 10–20 lg of each protein sample (1D SDS PAGE) were subject to electrophoretic molecular weight separation in the second dimension on precast Criterion 12% gels (BioRad) and then transferred to polyvinylidene fluoride or nitrocellulose membranes.

Techniques: Nucleic Acid Electrophoresis, Western Blot, Control