Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Semaphorin 4A exerts a proangiogenic effect by enhancing vascular endothelial growth factor-A expression in macrophages.

doi: 10.4049/jimmunol.1101435

Figure Lengend Snippet: FIGURE 1. Sema4A expression is increased in activated human macrophages. (A) Real-time RT-PCR analysis using RNA from human macrophages exposed to IFN-g, LPS alone, or combined with IFN-g for 18 h. RQ values are compared with control untreated macrophages and are mean 6 SD of n = 3 donors for each condition. Sema4A enhanced macrophage migration via PlexinD1. (B) Human recombinant Sema4A (from 0 up to 100 nmol/l) increased the migration in Boyden’s chamber of human macrophages treated for 18 h with LPS and IFN-g in a dose-response curve with a peak at 50 nmol/l of Sema4A. Values are mean 6 SD of four independent experiments. (C) Human macrophages exposed for 18 h to LPS and IFN-g were incubated in the presence or not of 20 mg/ml of PlexinD1 or PlexinB2-blocking Abs or appropriate control Ig and allowed to migrate toward 50 nmol/l of recombinant Sema4A for 4 h. Values are mean 6 SD (n = 4 donors) of four independent experiments. (D) Human macrophages exposed for 18 h to LPS and IFN-g were incubated in the presence or not of 20 mg/ml of PlexinD1-blocking Ab or IgG and stimulated with 50 nmol/l of recombinant Sema3E and Sema4A for 4 h. Values are mean 6 SD (n = 4 donors) of four independent experiments. (E and F) Sema4A and Sema3E protein levels were analyzed by Western blot analysis of lysates of control or LPS- and IFN-g–treated human macrophages. The data shown are representative (n = 4 donors) of four independent experiments. (F) Relative levels of Sema4A and Sema3E protein expression normalized to b-tubulin. (G) Protein levels of Sema4A were measured by ELISA both in the supernatants and cell lysates of human macrophages exposed or not for 18 h to LPS and IFN-g (see Materials and Methods). Values are mean 6 SD (n = 4 donors) of four independent experiments. Ctrl, Control. **p , 0.01, ***p , 0.001.

Article Snippet: Upper wells were loaded with 50 ml cells alone or in the presence of 50 nmol/l recombinant Sema4A, 20 mg/ml rabbit antiPlexinD1 Ab (H-70, sc-67145; Santa Cruz Biotechnology), 20 mg/ml mouse anti-PlexinB2 Ab (MAB5329; R&D Systems), and 20 mg/ml IgG or IgG2A controls, respectively (DakoCytomation).

Techniques: Expressing, Quantitative RT-PCR, Control, Migration, Recombinant, Incubation, Blocking Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Semaphorin 4A exerts a proangiogenic effect by enhancing vascular endothelial growth factor-A expression in macrophages.

doi: 10.4049/jimmunol.1101435

Figure Lengend Snippet: FIGURE 3. Sema4A and its receptors are modulated in macrophages and circulating inflammatory monocytes during peritoneal inflammation. (A) Sema4A expression on peritoneal macrophages at different time points after thioglycollate challenge was detected using F4/80-PE– and Sema4A-FITC– conjugated Abs in FACS analysis. Data are representative of n = 8 mice. (B) Coexpression of Sema4A with PlexinD1, PlexinB2, and TIM-2 on F4/80- positive peritoneal macrophages at different time points after thioglycollate challenge was investigated in FACS analysis by means of anti–Sema4A-FITC, anti–PlexinD1-allophycocyanin, anti–PlexinB2-APC, and anti–TIM-2-647 Abs, respectively. Data are representative of n = 8 mice. (C) PlexinD1, PlexinB2, and TIM-2 expression in peritoneal macrophages is expressed as mean (means 6 SD of n = 8 mice). (D and E) Western blot analysis using protein lysates of control or thioglycollate-treated C57/Bl6 mice peritoneal cells. (E) Relative levels of Sema4A protein expression normalized to b-tubulin. The data are representative of n = 12 mice analyzed. (F) Ly6Chigh and Ly6Clow monocyte subsets from the peripheral blood of control or thioglycollate-treated mice were isolated by staining with anti–CD11b-allophycocyanin, anti–CD115-PE, and biotinylated anti-Ly6c Abs, and Sema4A expression was detected by FACS analysis by using anti-Sema4A FITC Ab. Ly6Chigh monocytes expressed high amount of Sema4A compared with the Ly6Clow both in control and treated mice. Sema4A expression in circulating monocytes is expressed as median (means 6 SD of n = 4 mice). **p , 0.01, ***p , 0.001.

Article Snippet: Upper wells were loaded with 50 ml cells alone or in the presence of 50 nmol/l recombinant Sema4A, 20 mg/ml rabbit antiPlexinD1 Ab (H-70, sc-67145; Santa Cruz Biotechnology), 20 mg/ml mouse anti-PlexinB2 Ab (MAB5329; R&D Systems), and 20 mg/ml IgG or IgG2A controls, respectively (DakoCytomation).

Techniques: Expressing, Western Blot, Control, Isolation, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Semaphorin 4A exerts a proangiogenic effect by enhancing vascular endothelial growth factor-A expression in macrophages.

doi: 10.4049/jimmunol.1101435

Figure Lengend Snippet: FIGURE 4. Sema4A induces a proangiogenic activity on human macrophages. (A) Real-time RT-PCR analysis using RNA from human macrophages treated for 18 h with human recombinant Sema4A (10, 50, and 100 nmol/l). RQ values are compared with control macrophages and are mean 6 SD of n = 4 donors for each condition. (B and C) VEGF-A protein levels were analyzed by Western blot analysis of lysates of human macrophages upon 18-h treatment with different concentrations of recombinant Sema4A (10, 50, and 100 nmol/l). The data shown are representative (n = 4 donors) of four independent experiments. (C) Relative levels of VEGF-A protein expression normalized to b-tubulin. (D) Protein levels of VEGF-A were measured by ELISA in the supernatants of human macrophages treated with 10, 50, and 100 nmol/l of human recombinant Sema4A. Values are mean 6 SD (n = 4 donors) of four independent experiments. (E and F) Protein levels of VEGF-A were measured by ELISA in the supernatants and lysates of human macrophages treated with 50 nmol/l of human recombinant Sema4A in the presence or not of PlexinD1 and PlexinB2-blocking Abs or LPS. Values are mean 6 SD (n = 4 donors) of four independent experiments. (G) Phospho–VEGFR-2 and phospho–VEGFR-1 protein levels were analyzed by Western blot analysis of lysates of human macrophages upon 18-h treatment with 50 nmol/l of recombinant Sema4A. The data shown are representative (n = 4 donors) of four independent experiments. **p , 0.01, ***p , 0.001.

Article Snippet: Upper wells were loaded with 50 ml cells alone or in the presence of 50 nmol/l recombinant Sema4A, 20 mg/ml rabbit antiPlexinD1 Ab (H-70, sc-67145; Santa Cruz Biotechnology), 20 mg/ml mouse anti-PlexinB2 Ab (MAB5329; R&D Systems), and 20 mg/ml IgG or IgG2A controls, respectively (DakoCytomation).

Techniques: Activity Assay, Quantitative RT-PCR, Recombinant, Control, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal: Nature Communications

Article Title: Semaphorin 4C Plexin-B2 signaling in peripheral sensory neurons is pronociceptive in a model of inflammatory pain

doi: 10.1038/s41467-017-00341-w

Figure Lengend Snippet: Morphological and functional analyses in mice with early onset loss of Plexin-B2 in peripheral sensory neurons. a Quantitative RT-PCR for plxnb2 expression in the DRG of adult SNS-PB2 −/− or PB2 fl/fl mice ( n = 4 for PB2 fl/fl mice and n = 6 for SNS-PB2 −/− mice). b Examples ( left ) and densitometric quantifications ( right ) of western blot analysis of PlexinB2 in lysates of L3-L4 DRGs from SNS-PB2 −/− mice or their control littermates PB2 fl/fl . Data represent fold changes of the ratio of Plexin-B2 over β-TubulinIII signal and shown as mean ± s.e.m. n = 5 mice/group, P < 0.05. c – e Behavioral analysis of sensitivity to somatosensory nociceptive and non-nociceptive stimuli tested in the hindpaw SNS-PB2 −/− mice or their control PB2 fl/fl littermates. c Basal mechanical sensitivity measured as frequency of withdrawal responses to five applications each of von Frey filaments of varying force ( n = 12 mice/group). d Analysis of latency of paw withdrawal to 50 °C heat ( n = 10 mice/group). e Analysis of nocifensive behavior evoked by intraplantar injection of the algogen, capsaicin ( n = 10 mice/group). f , g Typical examples f and quantitative summary g of immunohistochemical determination of the spinal targeting of CGRP-positive and IB 4 -binding nociceptors ( n = 14–18 sections/group taken from three different mice/group). Scale bar , 100 µm. ANOVA repeated measures was performed in panel c comparing entire curves with each other, followed by Tukey’s test. In a , b , d , e and g , Student’s t -test was performed. In all panels, P < 0.05 indicated by * as compared to the corresponding control groups. Error bars represent s.e.m

Article Snippet: DRGs were stained with anti-β-galactosidase antibodies (rabbit 1:700; MP Biomedicals, 559762 and chicken 1:800; Abcam, ab9361), anti-PlexinB2 (rabbit 1:25, CSB-PA347467, CusAb-Cusabio), anti-CGRP antibody (1:2000; 24112, Immunostar), biotinylated Isolectin B 4 (1:200; B-1205, Vector), and anti-NF200 antibody (1:300, N4142, Sigma).

Techniques: Functional Assay, Quantitative RT-PCR, Expressing, Western Blot, Control, Injection, Immunohistochemical staining, Binding Assay

Journal: Nature Communications

Article Title: Semaphorin 4C Plexin-B2 signaling in peripheral sensory neurons is pronociceptive in a model of inflammatory pain

doi: 10.1038/s41467-017-00341-w

Figure Lengend Snippet: Analysis of Plexin-B2 expression in sensory neurons of the dorsal root ganglia (DRG) in adult mice and its regulation in inflammatory pain. a , b Expression of plxnb2 via immunofluorescence analysis of β-galactosidase in adult DRG in respective LacZ reporter knock-in mice. Typical examples a and quantitative analysis b of the distribution of DRG cell types expressing plxnb2 via co-immunolabeling with marker proteins ( n = 10–20 sections/group taken from at least three different mice/group). Scale bar , 50 µm. c , d Typical examples c and quantitative summary d of LacZ staining demonstrating plxnb2 expression in adult Plexin-B2-LacZ +/− mice following intraplantar injection of either vehicle (control) or Complete Freund´s Adjuvant (CFA) stimulating inflammatory pain ( n = 20–30 sections/group taken from at least 3 different mice/group). Scale bars represent 50 µm. In d , Student’s t -test (two sides) was performed. P < 0.05 indicated by † as compared to basal. In all diagrams data are represented as mean, whereas error bars represent s.e.m. e , f Examples ( left ) and densitometric quantifications ( right ) of western blot analysis of PlexinB2 expression in paw tissue e or L3-L4 DRGs f in naive mice or at 24 h after intraplantar injection of CFA. Data are represented as fold-increase of the ratio of Plexin-B2 over housekeeping gene signal. All data are presented as mean ± s.e.m. n = 8 for paw tissues, n = 9 for DRGs. P < 0.05 indicated by † as compared to basal

Article Snippet: DRGs were stained with anti-β-galactosidase antibodies (rabbit 1:700; MP Biomedicals, 559762 and chicken 1:800; Abcam, ab9361), anti-PlexinB2 (rabbit 1:25, CSB-PA347467, CusAb-Cusabio), anti-CGRP antibody (1:2000; 24112, Immunostar), biotinylated Isolectin B 4 (1:200; B-1205, Vector), and anti-NF200 antibody (1:300, N4142, Sigma).

Techniques: Expressing, Immunofluorescence, Knock-In, Immunolabeling, Marker, Staining, Injection, Western Blot

Journal: Nature Communications

Article Title: Semaphorin 4C Plexin-B2 signaling in peripheral sensory neurons is pronociceptive in a model of inflammatory pain

doi: 10.1038/s41467-017-00341-w

Figure Lengend Snippet: Sema4C is expressed in adult DRG and paw skin and plays a functional role in inflammatory pain. a Expression of Sema4c via β-galactosidase (LacZ) staining in adult DRG and plantar paw skin using LacZ reporter knock-in mice. Typical examples ( upper ) and quantitative summary (panel) of LacZ staining in the basal state or following CFA-induced paw inflammation. Scale bars , 50 µm. b Expression of Sema4C and its co-localization with immune cells markers via double immunofluorescence staining in plantar paw skin of mice at 24 h after vehicle or CFA injection, using anti-Sema4C antibody and antibodies against immune cells (CD3 to mark infiltrating T cells, upper ; or GR-1 to target macrophages, lower ). Higher magnification views of dermis are shown on extreme right to illustrate cells showing co-localization ( arrowheads ). Scale bars , 25 µm. c Quantitative measurement of intensity of Sema4C immunoreactivity paw tissue 24 h after intraplantar injection of vehicle or CFA; n = 3 mice/group. d , e Examples ( left ) and densitometric quantifications ( right ) of western blot analysis of Sema4C signal in lysates of L3-L4 DRGs d or paw tissue e 24 h after intraplantar CFA injection; n = 8 for DRGs, n = 9 for paw tissues. f Analysis of inflammatory mechanical hypersensitivity following hindpaw CFA injection in mice lacking Sema4C (Sema4C −/− ) and their wild-type littermates. Frequency of paw withdrawal in response to application of 0.07 g force via a von Frey filament is shown. g Changes in paw response latency to radiant heat following CFA injection in Sema4C −/− mice and their wild-type controls littermates. n = 5 (Sema4C −/− mice) and n = 7 (wild-type littermates) mice/group. Student’s t -test was performed in a – e and two-way ANOVA for repeated measures followed by Tukey’s test was performed in f and g . In f , P < 0.01 upon comparing the entire curves with each other. In all panels, P < 0.05 indicated by * as compared to the corresponding control groups and by † as compared to basal. Error bars represent s.e.m

Article Snippet: DRGs were stained with anti-β-galactosidase antibodies (rabbit 1:700; MP Biomedicals, 559762 and chicken 1:800; Abcam, ab9361), anti-PlexinB2 (rabbit 1:25, CSB-PA347467, CusAb-Cusabio), anti-CGRP antibody (1:2000; 24112, Immunostar), biotinylated Isolectin B 4 (1:200; B-1205, Vector), and anti-NF200 antibody (1:300, N4142, Sigma).

Techniques: Functional Assay, Expressing, Staining, Knock-In, Double Immunofluorescence Staining, Injection, Western Blot