Journal: Life sciences

Article Title: High ErbB3 activating activity in human blood is not due to circulating neuregulin-1 beta

doi: 10.1016/j.lfs.2020.117634

Figure Lengend Snippet: A. Graphical representation of ErbB1-4 receptors expression in a human microvascular endothelial cell line (HMEC1, positive control), a human monocytic cell line (THP1, negative control) and a human breast cancer cell line (MCF-7). B. Flow cytometric analysis of cell surface expression of ErbB receptors in MCF-7 cells. The open histogram depicts the specific ErbB antibody and the gray histogram represents the isotype-matched control. The Y-axis shows cell count; the X-axis indicates fluorescence intensity. C. Effect of 10 ng/ml neuregulin-1(NRG-1) on phosphorylation of ErbB3 in MCF-7 cells. The horizontal lines on the left are the molecular weight ladder. The ErbB3 molecular weight is 180kD. D. Graphical representation of data from western blot analysis of NRG-1 induced phosphorylation of ErbB3. Data are shown as mean±SEM; n=3.

Article Snippet: NRG-1 positive extracellular vesicles were identified using anti-NRG-1 antibody (AF377, Bio-Techne/RD Systems) conjugated to CF™488A (excitation/emission: 490/515 nm) using Mix-n-Stain™CF™488A Antibody Labeling kit (MIX488AS100, Sigma).

Techniques: Expressing, Positive Control, Negative Control, Cell Counting, Fluorescence, Molecular Weight, Western Blot

Journal: Life sciences

Article Title: High ErbB3 activating activity in human blood is not due to circulating neuregulin-1 beta

doi: 10.1016/j.lfs.2020.117634

Figure Lengend Snippet: A. Effect of NRG-1 (closed circles, n=12) and epidermal growth factor (EGF, open circles, n=3) on phosphorylation of ErbB3 in MCF-7 cells. EC50 [NRG1] = 26 ng/ml. B. Effect of TAK-165 (open circles), AG 1478 (closed circles), AST1306 (open triangles) and AZD8931 (closed triangles) on ErbB3 phosphorylation induced by 50 ng/ml rhNRG-1. The data are mean ±SEM, n=3.

Article Snippet: NRG-1 positive extracellular vesicles were identified using anti-NRG-1 antibody (AF377, Bio-Techne/RD Systems) conjugated to CF™488A (excitation/emission: 490/515 nm) using Mix-n-Stain™CF™488A Antibody Labeling kit (MIX488AS100, Sigma).

Techniques:

Journal: Life sciences

Article Title: High ErbB3 activating activity in human blood is not due to circulating neuregulin-1 beta

doi: 10.1016/j.lfs.2020.117634

Figure Lengend Snippet: A. Graphical representation of data for circulating levels of NRG-1 protein (ELISA) and plasma ErbB3 activating activity (ErbB3 AA) in healthy donor plasma (n=10). Mann Whitney test. B. Effect of plasma dilutions (1:2.5, 1:5, 1:10, 1:20, and 1:40) on phosphorylation of ErbB3. Results become linear with dilutions between 1:10 and 1:40. Repeated measures one-way AN OVA, Tukey’s posttest, n=10. C-D. The association between levels of NRG-1 protein and ErbB3 activating activity at 1:2.5 (C) and 1:20 (D) dilutions.

Article Snippet: NRG-1 positive extracellular vesicles were identified using anti-NRG-1 antibody (AF377, Bio-Techne/RD Systems) conjugated to CF™488A (excitation/emission: 490/515 nm) using Mix-n-Stain™CF™488A Antibody Labeling kit (MIX488AS100, Sigma).

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay, MANN-WHITNEY

Journal: Life sciences

Article Title: High ErbB3 activating activity in human blood is not due to circulating neuregulin-1 beta

doi: 10.1016/j.lfs.2020.117634

Figure Lengend Snippet: A. Effect of anti-NRG-1 antibody (AF377, closed circles) or control IgG’s on ErbB3 phosphorylation induced by 10 ng/ml rhNRG-1. The data are mean±SEM, n=3. Mann Whitney test. B. Effect of anti-NRG-1 antibody (AF377, closed circles) on ErbB3 phosphorylation induced by 10 ng/ml NRG-1 (N), healthy donor plasma (P), combinations of NRG-1 and plasma (N + P), NRG-1, plasma and 1 μg/ml (N + P + Ab1), or 5 μg/ml (N + P + Ab5) of anti-NRG-1 antibody. The data are mean±SEM, n=3. One-way ANOVA, Tukey’s posttest. C. Graphical representation of data showing synergism between recombinant NRG-1 and plasma. Open bar – sum of NRG-1 and plasma added separately to MCF-7 cells; Closed bar – NRG-1 and plasma added to cells simultaneously. Mann Whitney test, n=3. D. Effect of 1 μg/ml anti-NRG-1 antibody and control IgG’s on ErbB3 phosphorylation induced by plasma. Each plasma sample was tested in three independent experiments performed in duplicates. Mann Whitney test. E. Effect of 100 ng/ml recombinant NRG-1 (open bar), 100 ng/ml betacellulin (BTC, grey-shaded) and 100ng/ml heparin-binding epidermal growth factor (FIB-EGF, open) on phosphorylation of ErbB3 in MCF-7 cells. The data are mean ±SEM, n=2.

Article Snippet: NRG-1 positive extracellular vesicles were identified using anti-NRG-1 antibody (AF377, Bio-Techne/RD Systems) conjugated to CF™488A (excitation/emission: 490/515 nm) using Mix-n-Stain™CF™488A Antibody Labeling kit (MIX488AS100, Sigma).

Techniques: MANN-WHITNEY, Recombinant, Binding Assay

Journal: Life sciences

Article Title: High ErbB3 activating activity in human blood is not due to circulating neuregulin-1 beta

doi: 10.1016/j.lfs.2020.117634

Figure Lengend Snippet: A Western blot analysis of plasma samples obtained from two individuals with the highest level of circulating NRG-1 (Sub1 - 20 ng/ml, and Sub2 - 24 ng/ml). Different concentrations of extracellular domain of recombinant human NRG-1 (rhNRG-1) were used to identify the active form of NRG-1. B. Immunoprecipitation of plasma NRG-1 using mouse IgG2a (IgG, control) or anti-NRG-1 antibody (a-NRG, clone: 7D5) followed by western blot analysis of circulating NRG-1 with goat anti-NRG-1 antibody. C. Representative flow cytometric plots showing NRG-1 positive vesicles (red gate) in plasma samples before (unfiltered) and after (filtered) filtration through filters with pore size of 0.1 μm. Microbeads (5 x 104) with size of 2 μm (blue gate) were used to determine number of NRG-1 positive extracellular vesicles. D. Association between NRG-1 positive vesicles, determined using flow cytometric analysis, and NRG-1 protein levels in plasma samples. The rs and p values are from Spearman correlation (n = 10). E. Levels of NRG-1 protein, determined by ELISA, in plasma samples before (unfiltered) and after (filtered) filtration through 0.1 μm filters. Wilcoxon test. P-value is indicated.

Article Snippet: NRG-1 positive extracellular vesicles were identified using anti-NRG-1 antibody (AF377, Bio-Techne/RD Systems) conjugated to CF™488A (excitation/emission: 490/515 nm) using Mix-n-Stain™CF™488A Antibody Labeling kit (MIX488AS100, Sigma).

Techniques: Western Blot, Recombinant, Immunoprecipitation, Filtration, Pore Size, Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: CD47 prevents the elimination of diseased fibroblasts in scleroderma

doi: 10.1172/jci.insight.140458

Figure Lengend Snippet: ( A ) ATAC-Seq analysis for JUN and the hedgehog genes Gli1 and Ptch1 in scleroderma fibroblasts (SSCL), scleroderma fibroblasts after JUN knockout (JUN-KO) or under vismodegib, and normal skin fibroblasts. The promoter regions are highlighted with red boxes. n = 2. ( B ) ATAC-Seq analysis for the immune checkpoints CD47 and PD-L1 and the interleukin IL-6 in scleroderma fibroblasts, scleroderma fibroblasts after JUN knockout or under vismodegib, and normal skin fibroblasts. The promoter regions are highlighted with red boxes. n = 2. ( C ) Heatmap of differential open chromatin regulatory elements characterized from ATAC-Seq. The color bar shows the relative ATAC-Seq signal ( Z score of normalized read counts) as indicated. Samples 1 and 2 in both groups are individual samples. n = 2. ( D ) Fibrosis-linked genes with a 5-fold decline in promoter accessibility after JUN knockout. n = 2. ( E ) p-JUN expression in pulmonary fibroblasts and scleroderma fibroblasts on a 70 kPa hydrogel or a regular polystyrene plastic dish. Two-sided t test. ** P < 0.01; *** P < 0.001. n = 4. Tukey’s multiple comparison test. Bars represent means with standard deviations.

Article Snippet: For immunohistochemistry/immunofluorescence we used adiponectin (Abcam, ab22554), CD3 (Abcam, ab5690), CD11b (Novus, NB110-89474), CD26 (Abcam, ab28340), CD26 (R&D Systems, Bio-Techne, AF954), CD31 (Dako, m0823), CD47 (Thermo Fisher Scientific, 14-0479-82, clone B6H12), CD47 (R&D Systems, Bio-Techne, AF1866), CD68 (Agilent, GA60691-2, clone KP1), cleaved caspase-3 (CST, 96645, clone 5A1E), FSP1 (MilliporeSigma, 07-2274), FSP1 (Abcam, ab58597), collagen 1 (Abcam, ab34710), FSP1 (MilliporeSigma, 07-2274), Ki67 (Abcam, ab15580), PD-1 (Cell Marque, 315M-96, clone NAT105), PD-1 (R&D Systems, Bio-Techne, AF1021), PD-L1 (R&D Systems, Bio-Techne, AF1019), and phospho–c-Jun (Ser73) (CST, 32705, clone D47G9).

Techniques: Knock-Out, Expressing, Comparison

Journal: JCI Insight

Article Title: CD47 prevents the elimination of diseased fibroblasts in scleroderma

doi: 10.1172/jci.insight.140458

Figure Lengend Snippet: ( A ) Immunofluorescence stains against CD47 and FSP1 with and without JUN induction. Scale bar: 25 μm. n = 5. ( B ) Histogram of CD47 expression in fibroblasts with and without JUN induction. n = 5. ( C ) Percentage of CD47 positivity in different fibroblast populations with and without JUN induction. Fisher’s multiple comparison test. ** P < 0.01; *** P < 0.01. n = 5. Bars represent means with standard deviations. ( D ) Representative optical images of ectopically transplanted JUN-inducible mouse dermal fibroblasts ± CD47 inhibition. n = 4. ( E ) Corresponding quantification of photon emissions. Values are normalized to day 0. Fisher’s multiple comparison test. ** P < 0.01. n = 4. Bars represent means with standard deviations. ( F ) Fluorescent graft visualization under the dissection microscope after 7 days of CD47 inhibition. Scale bar: 5 mm. n = 2. ( G ) FACS plot for PE/RFP + CD11b + macrophages ± JUN induction ± CD47 inhibition in an in vitro phagocytosis assay. n = 3. ( H ) Corresponding quantification of RFP + macrophages. Tukey’s multiple comparison test. * P < 0.05; *** P < 0.01. n = 3. Bars represent means with standard deviations. ( I ) Schema of a macrophage depletion trial with subsequent skin fibrosis induction. n = 5. ( J ) Corresponding trichrome stains. Scale bar: 500 μm. n = 5. ( K ) Corresponding hydroxyproline assay. Two-sided t test. *** P < 0.001. n = 5. Bars represent means with standard deviations.

Article Snippet: For immunohistochemistry/immunofluorescence we used adiponectin (Abcam, ab22554), CD3 (Abcam, ab5690), CD11b (Novus, NB110-89474), CD26 (Abcam, ab28340), CD26 (R&D Systems, Bio-Techne, AF954), CD31 (Dako, m0823), CD47 (Thermo Fisher Scientific, 14-0479-82, clone B6H12), CD47 (R&D Systems, Bio-Techne, AF1866), CD68 (Agilent, GA60691-2, clone KP1), cleaved caspase-3 (CST, 96645, clone 5A1E), FSP1 (MilliporeSigma, 07-2274), FSP1 (Abcam, ab58597), collagen 1 (Abcam, ab34710), FSP1 (MilliporeSigma, 07-2274), Ki67 (Abcam, ab15580), PD-1 (Cell Marque, 315M-96, clone NAT105), PD-1 (R&D Systems, Bio-Techne, AF1021), PD-L1 (R&D Systems, Bio-Techne, AF1019), and phospho–c-Jun (Ser73) (CST, 32705, clone D47G9).

Techniques: Immunofluorescence, Expressing, Comparison, Inhibition, Dissection, Microscopy, In Vitro, Phagocytosis Assay, Hydroxyproline Assay

Journal: JCI Insight

Article Title: CD47 prevents the elimination of diseased fibroblasts in scleroderma

doi: 10.1172/jci.insight.140458

Figure Lengend Snippet: ( A ) Schematic outline of the therapeutic trial. ( B ) Representative H&E and trichrome stains of the different groups. Scale bar: 500 μm. n = 4. ( C ) Hydroxyproline content of the skin. Tukey’s multiple comparison test. * P < 0.05; ** P < 0.01. n = 6. Graph bars represent means with standard deviations. ( D ) Amount of fat tissue. Values indicate μm 2 /μm skin width. Tukey’s multiple comparison test. * P < 0.05. n = 8. Graph bars represent means with standard deviations. ( E ) Representative optical images of ectopically transplanted GFP/luciferase-labeled human scleroderma fibroblasts ± CD47/IL-6 inhibition. ( F ) Optical imaging of explanted kidneys on day 5. ( G ) Quantification of photon emissions of explanted kidney grafts normalized to the values of the untreated mice. Two-sided t test. * P < 0.05. n = 3–4. Bars represent means with standard deviations. ( H ) Corresponding caspase-3 staining of kidney grafts. Scale bar: 25 μm. ( I ) Corresponding percentage of caspase-3 + GFP + fibroblasts. Two-sided t test. ** P < 0.01. n = 4–5. Bars represent means with standard deviations.

Article Snippet: For immunohistochemistry/immunofluorescence we used adiponectin (Abcam, ab22554), CD3 (Abcam, ab5690), CD11b (Novus, NB110-89474), CD26 (Abcam, ab28340), CD26 (R&D Systems, Bio-Techne, AF954), CD31 (Dako, m0823), CD47 (Thermo Fisher Scientific, 14-0479-82, clone B6H12), CD47 (R&D Systems, Bio-Techne, AF1866), CD68 (Agilent, GA60691-2, clone KP1), cleaved caspase-3 (CST, 96645, clone 5A1E), FSP1 (MilliporeSigma, 07-2274), FSP1 (Abcam, ab58597), collagen 1 (Abcam, ab34710), FSP1 (MilliporeSigma, 07-2274), Ki67 (Abcam, ab15580), PD-1 (Cell Marque, 315M-96, clone NAT105), PD-1 (R&D Systems, Bio-Techne, AF1021), PD-L1 (R&D Systems, Bio-Techne, AF1019), and phospho–c-Jun (Ser73) (CST, 32705, clone D47G9).

Techniques: Comparison, Luciferase, Labeling, Inhibition, Optical Imaging, Staining