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: Paw sections were stained with anti-CGRP antibody (1:800; 24112, Immunostar), anti-PGP9.5 antibody (1:500, RA96101, Ultraclone), anti-Gr-1 antibody (1:500, 553125, BD Biosciences), anti-CD3 antibody (1:100, 555273, BD Biosciences) or anti-Sema4C antibody (rabbit 1:25, CSB-PA787154, CusAb-Cusabio).

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

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: Intraplantar paw injection of Sema4C causes rapid nociceptive sensitization. a A schematic representation of unilateral intraplantar Sema4C application in adult wild-type mice. b Analysis of mechanical sensitivity following a single injection of Sema4C at various doses or vehicle. Response frequency to five applications of von Frey filaments exerting graded force measured at 3 h following Sema4C injection is shown. c Time-course analyses of the effect of a single intraplantar dose of 1 ng Sema4C on sensitivity to 0.07 g of von Frey hair force. d Frequency of paw withdrawal in response to application of 0.07 g force via a von Frey filament upon single intraplantar dose of 1 ng Sema4C in mice lacking Plexin-B2 conditionally in adult DRG (AAV-DRG-PB2 −/− ) and their corresponding AAV-DRG-PB2 fl/fl controls ( n = 7 mice/group). In all panels, two-way ANOVA for repeated measures followed by Tukey’s test was performed, and P values comparing entire curves are represented. 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: Paw sections were stained with anti-CGRP antibody (1:800; 24112, Immunostar), anti-PGP9.5 antibody (1:500, RA96101, Ultraclone), anti-Gr-1 antibody (1:500, 553125, BD Biosciences), anti-CD3 antibody (1:100, 555273, BD Biosciences) or anti-Sema4C antibody (rabbit 1:25, CSB-PA787154, CusAb-Cusabio).

Techniques: Injection, Control

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 activates the RhoA-ROCK pathway in peripheral sensory neurons to induce mechanical hypersensitivity. a Schematic representation of the diverse components of Sema4C/Plexin-B2 signaling pathway tested in this study b Mechanical hypersensitivity to von Frey stimuli (0.07 g shown) induced by plantar application of Sema4C in the presence or absence of a ROCK 1/2 inhibitor (Y-27632, 2 ng), an inhibitor of Met signaling (PHA 665752, 3.3 μg) or vehicle ( n = 8 mice/group for Sema4C + Y-27632 treatment, n = 9 for Sema4C + PHA 665752 treatment and n = 10 each for vehicle or Sema4C treatment). c Examples ( left ) and densitometric quantification ( right ) of western blot analysis of Met phosphorylation (P-Met) in paw tissue 15 min after intraplantar injection of recombinant mouse Hepatocyte growth factor (50 ng) in the presence of a Met inhibitor (PHA 665752, 3.3 μg) or vehicle. Data are represented as fold-increase of the ratio of P-Met over GAPDH expression over vehicle-treated group; n = 3 independent blots and mice/group. P < 0.05 indicated by * as compared to vehicle-treated group. d Analysis of active RhoA in cultured DRG neurons upon treatment with Sema4C (150 nM) or vehicle ( n = 6 independent culture experiments). e Analysis of mechanical hypersensitivity induced by plantar application of Sema4C in the presence or absence of a TRPA1 inhibitor (AP-18, 10 nmol) or vehicle ( n = 4 mice/group for Sema4C + AP-18 treatment, n = 9 each for vehicle or Sema4C treatment). Frequency of paw withdrawal in response to application of 0.07 g force via a von Frey filament is shown. f Analysis of Sema4C-induced mechanical hypersensitivity in mice lacking TRPA1 ( TRPA1 −/− ) and wild-type (wt) controls, expressed as % change over baseline sensitivity ( n = 8 mice/group). ANOVA for random ( c ) and repeated ( b , d , e and f ) measures followed by Tukey’s test, and P values comparing entire curves with each other are represented. In all panels, P < 0.05 indicated by * as compared to the corresponding control groups and by † as compared to basal and by # as compared to Sema4C-treated group. Error bars represent s.e.m

Article Snippet: Paw sections were stained with anti-CGRP antibody (1:800; 24112, Immunostar), anti-PGP9.5 antibody (1:500, RA96101, Ultraclone), anti-Gr-1 antibody (1:500, 553125, BD Biosciences), anti-CD3 antibody (1:100, 555273, BD Biosciences) or anti-Sema4C antibody (rabbit 1:25, CSB-PA787154, CusAb-Cusabio).

Techniques: Western Blot, Phospho-proteomics, Injection, Recombinant, Expressing, Cell Culture, Control

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: Modulation of TRPA1 channels by Sema4C/Plexin-B2/RhoA-ROCK signaling. a Modulation of calcium transients evoked in DRG sensory neurons by AITC (50 µM) by pretreatment with either vehicle or Sema4C (150 nM) in the presence or absence of ROCK inhibitor (50 μg/ml); n = 3 independent culture experiments. Note that AITC was applied together with Sema4C or vehicle treatment and again 30 min later alone - the responses elicited by a second application of AITC are shown in a . b Sema4C-induced changes in subcellular localization of myc-tagged TRPA1 in cultured DRG neurons. Shown are typical examples, line profiles on confocal images and quantification of fluorescence intensity at the cell membrane relative to cytoplasmic localization ( n = 40 cells/group for Sema4C treatment and n = 43 for vehicle treatment). Scale bar , 25 µm. c Nocifensive responses to intraplantar application of Allyl isothiocyanate (AITC, 3.5 mM), a TRPA1 agonist, 3–4 h following intraplantar injection of vehicle or Sema4C ( n = 5 mice/group for vehicle treatment, n = 8 for Sema4C treatment). d , e Nocifensive responses to intraplantar application of AITC following intraplantar injection of vehicle or Sema4C in mice lacking Plexin-B2 conditionally in adult DRG (AAV-DRG-PB2 −/− ) and their corresponding AAV-DRG-PB2 fl/fl controls ( n = 8 mice/group), d or in mice lacking Sema4C (Sema4C −/− ) and their wild-type littermates ( n = 6 mice/group), e . ANOVA for random measures followed by Tukey’s test was performed in a , d and e , Student’s t -test was performed in b and c . In all panels, P < 0.05 indicated by * as compared to the corresponding control groups and by † as compared to basal and by # as compared to Sema4C-treated group. Error bars represent s.e.m

Article Snippet: Paw sections were stained with anti-CGRP antibody (1:800; 24112, Immunostar), anti-PGP9.5 antibody (1:500, RA96101, Ultraclone), anti-Gr-1 antibody (1:500, 553125, BD Biosciences), anti-CD3 antibody (1:100, 555273, BD Biosciences) or anti-Sema4C antibody (rabbit 1:25, CSB-PA787154, CusAb-Cusabio).

Techniques: Cell Culture, Fluorescence, Membrane, Injection, Control

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: RhoA-ROCK signaling downstream of Plexin-B2 activation mediates Sema4C-induced mechanical hypersensitivity and contributes to inflammatory hypersensitivity in vivo. a , b Schematic representation of the allelic series of transgenic Plexin-B2 mice carrying specific mutations in the intracellular domain resulting in loss-of-function (LOF) of the RhoA activation pathway. c , d Examples ( left ) and densitometric quantifications ( right ) of western blot analysis of PlexinB2 signal in lysates of L3-L4 DRGs ( c , n = 4 mice/group) or Sciatic nerve tissue ( d , n = 3 mice/group) of transgenic Plexin-B2 mice carrying specific mutations in the intracellular domain resulting in LOF of the RhoA activation pathway (PB2 −/− ; PB2-LOF RhoA ) and the control mice carrying wild-type (wt) Plexin-B2 (PB2 −/− ; PB2 wt ). Data are represented as fold changes of the ratio of PlexinB2 over loading control signal β-TubulinIII, p > 0.05. e Analysis of Sema4C-induced mechanical hypersensitivity to plantar application of 0.07 g von Frey force in PB2 −/− ; PB2-LOF RhoA and control mice. f Analysis of inflammatory hypersensitivity, represented as response frequency to 0.07 g force before and at 24, 48, 72 and 96 h following hindpaw CFA injection. N = 5 mice/group for PB2 −/− ; PB2-LOF RhoA mice and n = 8 for control mice in all experiments e and f . g Nocifensive responses to intraplantar application of AITC following intraplantar injection of vehicle or Sema4C in PB2 −/− ; PB2-LOF RhoA and control PB2 −/− ; PB2 wt mice ( n = 8 mice/group). Student t -test was performed in c and d , ANOVA for repeated e , f or random g measures followed by Tukey’s test was performed. In e and f , P values upon comparing entire curves with each other are represented. 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: Paw sections were stained with anti-CGRP antibody (1:800; 24112, Immunostar), anti-PGP9.5 antibody (1:500, RA96101, Ultraclone), anti-Gr-1 antibody (1:500, 553125, BD Biosciences), anti-CD3 antibody (1:100, 555273, BD Biosciences) or anti-Sema4C antibody (rabbit 1:25, CSB-PA787154, CusAb-Cusabio).

Techniques: Activation Assay, In Vivo, Transgenic Assay, Western Blot, Control, Injection