Journal: PLoS Genetics

Article Title: Congenital lipodystrophy induces severe osteosclerosis

doi: 10.1371/journal.pgen.1008244

Figure Lengend Snippet: A) Transplanted fat depots 3 months after surgery. B) Fat depot weight 3 months after transplantation. C) Serum leptin and adiponectin of FF mice 3 months after fat depot transplantation. WT and non-transplanted FF mice serve as control. D) μCT analysis of trabecular bone volume and bone mineral density of distal femurs of FF mice 3 months after sham operation or transplantation of fat derived from WT or adipokine-deficient mice. Data are presented as mean ± SD. **p<0.01; *** p<0.001 as determined by ANOVA with Holm-Sidak's post hoc analysis for multiple comparisons test. D) comparison with FF Sham except where detailed.

Article Snippet: The primary antibody cocktail contained rat anti-mouse CD45-BUV395 (BD Horizon, clone 30-F11, final dilution factor 1:200), rat anti-mouse TER-119-APC (BioLegend, clone TER-119, 1:200), rat anti-mouse CD41-BV421 (BioLegend, clone MWReg30, 1:300), rat anti-mouse/human CD11b (BioLegend, clone M1/70, 1:400), and rat-anti mouse Leptin receptor (LepR)-biotin (R&D Systems, polyclonal, 1:50) in Brilliant Stain Buffer (BD Biosciences) containing 10 μg/mL FcBlock.

Techniques: Transplantation Assay, Control, Derivative Assay, Comparison

Journal: Cell Stem Cell

Article Title: Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis

doi: 10.1016/j.stem.2020.11.004

Figure Lengend Snippet: MSCs are fibrosis-driving cells in patients characterized by upregulation of S100A8/A9 (A) Diagnostic BM images of the patients. Representative H&E and reticulin stainings. For additional images (all controls) and detailed patient characteristics, see Figure S5 . (B) UMAP of cells in 1 PMF patient (MF2, n = 243 cells) and two control patients (MF0, n = 255 cells). In the left panel, cells are color coded by their annotated cellular identity, and in the right panel, by their patient source. (C) Top marker genes. Wilcoxon rank-sum test, p < 0.01. (D) Ridgeline plot comparing PMF (blue) versus control (red) condition. Competitive gene set enrichment analysis was used. (E) PROGENy analysis. Sampling-based permutation (10,000 permutations). Pathway activity scores are given as Z scores. (F) Ridgeline plot of S100A8/A9 expression in PMF (blue) or control (red). Significance estimated by modeling the dropout rate as a binomial process with the observed dropout rate per condition as estimator of p for both conditions, respectively. (G) Network plot of ligand-receptor activity in PMF compared to control. (H) Bar plot of top 10 most abundant ligands in all inferred ligand-receptor interactions. (I) Sankey plot of top 20 deregulated TGFB1 -mediated ligand-receptor interactions. The absolute difference in mean LR expression was used as a metric for the extent of deregulation. (J) Sankey plot of top 20 deregulated ligand-receptor interactions mediated by PF4, PF4V1, or PPBP. The absolute difference in mean LR expression was used as a metric for the extent of deregulation. See also Figure S6 and , , , , and .

Article Snippet: Samples were diluted 1:50 - 1:150 and S100A8 concentration was quantified using the Human S100A8 DuoSet ELISA (R&D Systems, DY4570-05) according to the manufacturer’s instructions.

Techniques: Diagnostic Assay, Control, Marker, Sampling, Activity Assay, Expressing

Journal: Cell Stem Cell

Article Title: Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis

doi: 10.1016/j.stem.2020.11.004

Figure Lengend Snippet: Spatial kinetics of S100A8/S100A9 detects disease progression in MPN and their pharmacological targeting ameliorates the disease (A) ELISA of S100A8 (and S100A9) in MPN (blue) and controls (red) plasma. Two-tailed, two-sample Welch test was used. (B) ELISA of S100A8 (and S100A9) in MPN with different MF grades (blue) and controls (red) plasma. Mean ± SEM. One-way-ANOVA with post hoc Tukey’s was used. (C) Frequency of S100A8 + cells BM biopsies; n = 64 patients. One-way-ANOVA with post hoc Tukey’s HSD was used. (D and E) Grading of S100A8 in the non-hematopoietic compartment in BM biopsies. Scale bar, 100 μm. n = 64 patients. Kruskal-Wallis H test with post hoc Wilcoxon rank-sum test was used. p values were adjusted for multiple hypothesis testing by the Holm-Bonferroni method. (F) White blood cell counts of WT mice transplanted with either JAK2 V617F (blue) or JAK2 WT overexpressing HSPCs (red) each either treated with Tasquinimod 30 mg/kg/day or vehicle control. Two-way repeated ANOVA pairwise comparisons were analyzed by estimated marginal means. (G) Spleens at sacrifice as indicated. (H) Relative spleen weights. Mean ± SEM. One-way-ANOVA with post hoc Tukey’s HSD. (I) Reticulin (MF) grade. Kruskal-Wallis H test with post hoc Wilcoxon rank-sum test. See also Figure S6 .

Article Snippet: Samples were diluted 1:50 - 1:150 and S100A8 concentration was quantified using the Human S100A8 DuoSet ELISA (R&D Systems, DY4570-05) according to the manufacturer’s instructions.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Two Tailed Test, Control

Journal: Cell Stem Cell

Article Title: Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis

doi: 10.1016/j.stem.2020.11.004

Figure Lengend Snippet:

Article Snippet: Samples were diluted 1:50 - 1:150 and S100A8 concentration was quantified using the Human S100A8 DuoSet ELISA (R&D Systems, DY4570-05) according to the manufacturer’s instructions.

Techniques: Recombinant, Staining, Plasmid Preparation, Virus, Multiplex Assay, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Avidin-Biotin Assay, Blocking Assay, Retroviral, Software

Journal: Scientific Reports

Article Title: P-Selectin preserves immune tolerance in mice and is reduced in human cutaneous lupus

doi: 10.1038/srep41841

Figure Lengend Snippet: ( a) Relative frequency of peripheral blood leukocyte populations of 1.5–3 month-old WT and P-Sel −/− mice. ( b , d ) Percentage of IL-10 + conventional dendritic cells (cDC), plasmacytoid DC (pDC), monocytes, granulocytes and B cells; and frequency of IL-10 and IL-17 producing CD4 + and CD8 + T lymphocytes, in 1.5-month-old ( b ) and >18-month-old ( d ) WT and P-Sel −/− mice. ( c) Representative dot plots of IL-10 + cDCs and IL-10 and IL-17 producing CD4 + T cells in 1.5-months old WT and P-Sel −/− mice. ( e) Phenotyping of CD4 + and CD8 + splenic T lymphocytes according to the expression of the naïve/memory/effector markers CD62L and CD44 in 1.5–3 months-old (upper panels) and >18 month-old (lower panels) WT and P-Sel −/− mice. ( f) Representative dot plots showing the distribution of 1.5–3-month-old mice splenic populations according to the expression of L-Selectin and CD44. In all cases, n = 4 mice per group. In all cases, n = 4 mice per group. Bars represent the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.005, by Student’s two tailed t test.

Article Snippet: Consecutive tissue sections of sun-exposed skin biopsies underwent immunohistochemistry with the Dako REAL EnVision Detection System Peroxidase/DAB+kit (Dako), using as primary antibodies anti-human P-Selectin (10 μg/ml) (R&D Systems; Minneapolis, MN, USA) and anti-human CD31 (1/50) (Abcam).

Techniques: Expressing, Two Tailed Test

Journal: Scientific Reports

Article Title: P-Selectin preserves immune tolerance in mice and is reduced in human cutaneous lupus

doi: 10.1038/srep41841

Figure Lengend Snippet: ( a) Representative photomicrographs (20×) of anti-CD31 and anti-P-Selectin stained skin biopsies of healthy donors and SLE patients (upper panels). Black arrows point blood vessels. ( b) 200% magnification of representative blood vessels from the original images are represented (lower panels). ( c) Classification and quantification of CD31+ dermal blood vessels according to the expression level of P-Selectin (healthy controls, n = 4; SLE patients, n = 4); bars show the mean ± SD. **p < 0.01 by Student’s two tailed t test.

Article Snippet: Consecutive tissue sections of sun-exposed skin biopsies underwent immunohistochemistry with the Dako REAL EnVision Detection System Peroxidase/DAB+kit (Dako), using as primary antibodies anti-human P-Selectin (10 μg/ml) (R&D Systems; Minneapolis, MN, USA) and anti-human CD31 (1/50) (Abcam).

Techniques: Staining, Expressing, Two Tailed Test

Journal: Cell reports

Article Title: Neurokinin-1 Receptor Signaling Is Required for Efficient Ca 2+ Flux in T-Cell-Receptor-Activated T Cells

doi: 10.1016/j.celrep.2020.02.054

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse / rat / human Substance P (rabbit polyclonal), Cy3 , Bioss USA , Cat # bs-0064R-Cy3.

Techniques: Functional Assay, Recombinant, Staining, Avidin-Biotin Assay, Blocking Assay, Plasmid Preparation, Inhibition, Fluorescence, SYBR Green Assay, Activation Assay, Bicinchoninic Acid Protein Assay, In Situ, Enzyme-linked Immunosorbent Assay, Activity Assay, Labeling, Software

Journal: bioRxiv

Article Title: Treatment with anti-HER2 chimeric antigen receptor tumor-infiltrating lymphocytes (CAR-TILs) is safe and associated with antitumor efficacy in mice and companion dogs

doi: 10.1101/2022.09.11.507449

Figure Lengend Snippet: mRNA encoding anti-HER2 CAR was electroporated into TILs from five cutaneous melanomas (MM1, MM3, MM4, MM5, and MM6) and one uveal melanoma (UM22) to produce CAR-TILs. CAR expression was detected by HER2-biotin binding at 5-16 hours post-transfection (a). MM1 TILs and CAR-TILs were co-cultured with the parental or HER2 KO 92-1 uveal melanoma cell line for 4-6 hours, followed by flow cytometry to measure degranulation (CD107a expression) (b) or IFN-γ-secreting cells (c). TILs and CAR-TILs not co-cultured with 92-1 cells (TILs alone; TA) were used as controls in b-c. Alternatively, cells were co-cultured for 48 h, and the supernatant was collected for analysis of secreted IFN-γ by ELISA (d). 92-1 cells not co-cultured with TILs (no TILs) were used as a control. Data are presented as mean ± standard deviation (SD) of duplicates. The experiments were performed twice, and representative results are shown.

Article Snippet: For analysis of HER2 protein binding capacity of CAR-TILs, 100.000 cells were incubated with 1 μg biotinylated HER2 protein (Abcam) for 30 min at 4 °C, followed by incubation with an allophycocyanin-conjugated streptavidin antibody (Jackson Immuno Research) for 25 min at 4 °C, and flow cytometry analysis was performed using an Accuri C6 flow cytometer (BD) equipped with the BD Accuri C6 software.

Techniques: Expressing, Binding Assay, Transfection, Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: bioRxiv

Article Title: Treatment with anti-HER2 chimeric antigen receptor tumor-infiltrating lymphocytes (CAR-TILs) is safe and associated with antitumor efficacy in mice and companion dogs

doi: 10.1101/2022.09.11.507449

Figure Lengend Snippet: Flow cytometry data showing HER2 CAR expression after HER2 CAR mRNA electroporation by detecting bound biotinylated HER2 protein. Representative plots for samples MM1, MM3, MM4, MM5, MM6 and UM22.

Article Snippet: For analysis of HER2 protein binding capacity of CAR-TILs, 100.000 cells were incubated with 1 μg biotinylated HER2 protein (Abcam) for 30 min at 4 °C, followed by incubation with an allophycocyanin-conjugated streptavidin antibody (Jackson Immuno Research) for 25 min at 4 °C, and flow cytometry analysis was performed using an Accuri C6 flow cytometer (BD) equipped with the BD Accuri C6 software.

Techniques: Flow Cytometry, Expressing, Electroporation

Journal: bioRxiv

Article Title: Treatment with anti-HER2 chimeric antigen receptor tumor-infiltrating lymphocytes (CAR-TILs) is safe and associated with antitumor efficacy in mice and companion dogs

doi: 10.1101/2022.09.11.507449

Figure Lengend Snippet: (a-b) TILs and CAR-TILs from MM1 were co-cultured with the parental or HER2 KO 92-1 uveal melanoma cell line for 24 hours, followed by cleaved caspase 3 (CC3) detection in the tumor cells using flow cytometry (a). Additionally, TILs and CAR-TILs from MM5 were co-cultured with autologous cancer cells for 24 hours, followed by cleaved CC3 detection using flow cytometry (b). Cancer cells not treated with TILs were used as a control (no TILs). (c-g) Viability of autologous cancer cells was measured by luciferase signal detected after 48 hours co-culture with increasing doses of TILs and CAR-TILs from UM22 (c), MM3 (d), MM4 (e), MM5 (f) and MM6 (g) in the indicated ratios (TILs:cancer cells). Data is presented as mean with SD of triplicates. Asterisks represent p-values of difference between similar doses of TILs and CAR-TILs. The experiments were performed twice, and representative data from one experiment is shown.

Article Snippet: For analysis of HER2 protein binding capacity of CAR-TILs, 100.000 cells were incubated with 1 μg biotinylated HER2 protein (Abcam) for 30 min at 4 °C, followed by incubation with an allophycocyanin-conjugated streptavidin antibody (Jackson Immuno Research) for 25 min at 4 °C, and flow cytometry analysis was performed using an Accuri C6 flow cytometer (BD) equipped with the BD Accuri C6 software.

Techniques: Cell Culture, Flow Cytometry, Luciferase, Co-Culture Assay

Journal: bioRxiv

Article Title: Treatment with anti-HER2 chimeric antigen receptor tumor-infiltrating lymphocytes (CAR-TILs) is safe and associated with antitumor efficacy in mice and companion dogs

doi: 10.1101/2022.09.11.507449

Figure Lengend Snippet: HER2 expression detected by qPCR (a) and immunohistochemistry (b) in tumors from four companion dogs (dogs 1-4). Canine cell lines D17.os and CF41.mg and canine PBMC were used as controls in a. (c) Expression of human melanoma marker Melan-a in tumors from dogs 3 and 4 with melanoma. (d) Classification of canine tumor biopsies from dogs 3 and 4 based on gene expression relative to TCGA cohort of > 10000 human tumor samples from 32 cancer types. The similarities between canine melanoma and samples in TCGA were visualized using tSNE dimensionality reduction. SKCM: melanoma cases. (e-f) TILs and mRNA electroporated CAR-TILs from dog 3 were co-cultured with the canine cell lines CF41.mg (e) and D17.os (f) for 24 h. The experiment was performed twice and representative data (n=3, data ± SD) from one experiment are shown. (g) Photograph of Dog 3 with metastatic oral malignant melanoma enrolled in the FIDO trial evaluating the safety of CAR-TIL treatment in companion dogs.

Article Snippet: For analysis of HER2 protein binding capacity of CAR-TILs, 100.000 cells were incubated with 1 μg biotinylated HER2 protein (Abcam) for 30 min at 4 °C, followed by incubation with an allophycocyanin-conjugated streptavidin antibody (Jackson Immuno Research) for 25 min at 4 °C, and flow cytometry analysis was performed using an Accuri C6 flow cytometer (BD) equipped with the BD Accuri C6 software.

Techniques: Expressing, Immunohistochemistry, Marker, Cell Culture

Journal: Journal of Inflammation (London, England)

Article Title: The PPAR-γ antagonist GW9662 elicits differentiation of M2c-like cells and upregulation of the MerTK/Gas6 axis: a key role for PPAR-γ in human macrophage polarization

doi: 10.1186/s12950-015-0081-4

Figure Lengend Snippet: GW9662 induces M2c-like cells that upregulate MerTK and its ligand Gas6. (A-C) Healthy monocytes were cultured in serum-free medium in the absence of cytokines or growth factors (M0 differentiation), with or without the PPAR-γ antagonist GW9662 (2.5-10 μM), for 4 days; when specified, the PPAR-γ agonist rosiglitazone (1 μM) was added. Expression of MerTK, CD163 and CD16 was measured by flow cytometry. (D-E) Gas6 production levels were quantified by ELISA in culture medium, upon incubation with or without GW9662 (2.5-10 μM) of otherwise untreated cells (M0 conditions), LPS (50 ng/ml; M1 conditions) or IL-4 (20 ng/ml; M2a conditions) exposed cells. (A-E) Pooled data are represented as mean values ± SEM. Analysis was performed using one-way repeated measures ANOVA with Newman-Keuls multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. When not specified by additional graphic signs, statistical annotations (asterisks) refer to comparisons with respect to the relative GW9662 untreated control group. Each set of data is representative of three independent experiments.

Article Snippet: Biotinylated goat polyclonal anti-human Gas6 antibody (R&D Systems), followed by HRP-conjugated streptavidin (Biolegend), was used for detection.

Techniques: Cell Culture, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Incubation, Control