Journal: bioRxiv

Article Title: PI16 is a non-neuronal regulator of neuropathic pain

doi: 10.1101/696542

Figure Lengend Snippet: Representative sciatic nerve section from naïve WT mice showing double immunostaining of PI16 (red) with markers of different cell types (green) present in and around meningeal sheath. (A) Fibroblast markers α-SMA and P4HB (B) perineurial marker GLUT-1, (C) endothelial markers CD31 and CLDN1. Top panel of (C) shows PI16-positive fibroblasts and endothelial cells are closely positioned within 1 μm distance. Middle panel of (C) shows staining of whole mount DRG. Bright field (BF) image merged with other channels is shown. Scale bar indicates 25 μm.

Article Snippet: The following primary antibodies were used in the study: PI16 (1:750, R&D Systems AF4929), α-SMA (1:1500, Abcam ab5694), β-Actin (1:50,000, Sigma A3854), a-Tubulin (1:2000, Cell Signaling 2144S), pMLC2 (Cell Signaling 3674S, 3675S), Chemerin (R&D Systems AF2325).

Techniques: Double Immunostaining, Marker, Staining

Journal: bioRxiv

Article Title: PI16 is a non-neuronal regulator of neuropathic pain

doi: 10.1101/696542

Figure Lengend Snippet: (A) Representative image showing immunostaining of PI16 (red) and GLUT-1 (green) in lumbar DRG section from side contralateral (contra) and ipsilateral (ipsi) to SNI surgery. BF (bright field) image merged with PI16 and GLUT-1 is shown. The zoom panel show a magnified view of the area outlined by the square. Perineurial GLUT-1 (green) staining marks the border of DRG and the dotted white line marks the edge of the meningeal sheath. Note the PI16 staining in the meningeal sheath. Note increased PI16 staining outside GLUT-1 in the meninges of ipsilateral DRGs compared to contralateral. Scale bar indicates 25 μm. (B) Representative merged images showing immunostaining of PI16 (red), GLUT-1 (green), and DAPI (blue) in sciatic nerve longitudinal section from ipsilateral and contralateral side after SNI. Note increase PI16 staining (arrow) outside GLUT-1 in ipsilateral nerve compared to contralateral. Scale bar indicates 100 μm. Note increased thickness of epi/perineurium on the right panel (double headed arrow) in sciatic nerve longitudinal (top) and cross section (bottom). Scale bar 100 μm (top), 25 μm (bottom). (C) Representative image showing PI16 (red) immunostaining in α-SMA (green) positive fibroblast in sciatic nerve from contralateral and ipsilateral side. Note expansion of α-SMA positive fibroblasts co-expressing PI16 in the ipsilateral nerve. BF (bright field) image merged with PI16, α-SMA, and DAPI (blue) is shown. Scale bar indicates 25 μm.

Article Snippet: The following primary antibodies were used in the study: PI16 (1:750, R&D Systems AF4929), α-SMA (1:1500, Abcam ab5694), β-Actin (1:50,000, Sigma A3854), a-Tubulin (1:2000, Cell Signaling 2144S), pMLC2 (Cell Signaling 3674S, 3675S), Chemerin (R&D Systems AF2325).

Techniques: Immunostaining, Staining, Expressing

Journal: bioRxiv

Article Title: PI16 is a non-neuronal regulator of neuropathic pain

doi: 10.1101/696542

Figure Lengend Snippet: (A) Mouse fibroblasts cultured from postnatal day 8 mouse sciatic nerve were serum starved for 24 hr and treated with TGF-β1 (5ng/ml) for 48 hr or 96 hr followed by Western blot analysis of total cell lysate or TCA precipitated culture supernatant. Representative western blots are shown. α-SMA was used as a myofibroblast marker and α-Tubulin was loading control. (B) Quantification of PI16 and α-SMA for panel B. Bar graph depicts means ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01 (analyzed using t test). (C) Live imaging of mouse fibroblasts (L cells) transfected with mTurquoise2 (endoplasmic reticulum marker, ER-turq) and PI16 tagged with turbo-GFP. Raw black and white images are shown in bottom panel. Note PI16 staining in endoplasmic reticulum vesicles and tubules (arrowhead) in the magnified view in the white box. Scale bar indicates 25 μm.

Article Snippet: The following primary antibodies were used in the study: PI16 (1:750, R&D Systems AF4929), α-SMA (1:1500, Abcam ab5694), β-Actin (1:50,000, Sigma A3854), a-Tubulin (1:2000, Cell Signaling 2144S), pMLC2 (Cell Signaling 3674S, 3675S), Chemerin (R&D Systems AF2325).

Techniques: Cell Culture, Western Blot, Marker, Imaging, Transfection, Staining

Journal: PLoS Biology

Article Title: Targeting miR-27a/VE-cadherin interactions rescues cerebral cavernous malformations in mice

doi: 10.1371/journal.pbio.3000734

Figure Lengend Snippet: (A) (i) CD5-2 increased VE-cadherin expression in CCM lesions from mouse samples given the control drug (Ctrl) or CD5-2. (ii) CD5-2 improves VE-cadherin localization in CCM lesions compared with mice given the control drug (Ctrl). In Ctrl, the white arrows point to disruptive VE-cadherin staining. In CD5-2, the red arrows point to junctional VE-cadherin. (B) In situ detection of CD5-2 in the blood vessels in the hindbrain of P6 Ccm2 ECKO animal, 6 hours after IP injection of CD5-2. The image without CD5-2 probe was used as background control (top). CD5-2 was detected as the color of cyan (bottom) with ECs stained with CD31. Bar, 50 μm (left); 10 μm (right). Values are shown as mean ± SEM, ** P < 0.01, determined by Student t test. For the raw data used for quantification, see Fig 2 in . CCM, cerebral cavernous malformation; EC, endothelial cell; IP, intraperitoneal; miR-27a, microRNA-27a; TSB, target site blocker; VE-cadherin, vascular endothelial cadherin.

Article Snippet: For primary staining, the following antibodies were used for immunostaining: rat CD31 (1:200, 553370, BD Biosciences, San Jose, CA), rabbit ICAM-1 (1:200, ab124759, Abcam,Cambridge, United Kingdom), rabbit pMLC2 (1:200, 3674S, Cell Signaling Technology, Danvers, MA), rat CD144 (1:200, 555289, BD Biosciences, San Jose, CA), goat VE-cadherin (1:1,000, sc-6458, Santa Cruz Biotechnology, Dallas, TX), and rat CD11b (1:200, 53-0112-82, eBiosciences, San Diego, CA).

Techniques: Expressing, Staining, In Situ, Injection

Journal: PLoS Biology

Article Title: Targeting miR-27a/VE-cadherin interactions rescues cerebral cavernous malformations in mice

doi: 10.1371/journal.pbio.3000734

Figure Lengend Snippet: (A) CD5-2 effects on ICAM-1 expression in CCM lesion. Sections were triple-stained for DAPI (blue) or CD31 (green), ICAM-1 (red); 3 mice were utilized for each condition. Representative sections are shown. Bar, 8 μm. (B-C) Western blot analysis of ICAM-1 (B) and phospho-p65 (C) in brain microvascular ECs (hCMEC/D3) treated with 10 nM siRNAs for scramble control (Ctrl), CCM1, or CCM2 for 4 hours, followed by transfection of 15 nM CD5-2 or controls then cultured overnight. Molecular weights in kilodaltons are shown. ICAM-1 antibody (Cell signalling #4915) gave two bands (89 and 92 kDa). This antibody recognizes the ICAM-1 C-terminal portion, which is dense with ubiquitination and phosphorylation sites and, as such, #4915 can detect multiple sized ICAM moieties. Representative blots are shown ( n = 3) with α-tubulin or β-actin rabbit used as loading control. (D) Representative images of dynamic adhesion of neutrophils to control- or CD5-2–treated ECs following the stimulation with TNF-α for 4 hours. Quantification of number of adherent neutrophils with control or CD5-2–treated ECs is given ( n = 3). (E) Representative images of paracellular transmigration of neutrophils (green) through TNF-α–stimulated endothelium (VE-cadherin, red) in the presence of control and CD5-2 in vitro. Transmigrated neutrophils appear GFP dim . (F) (i) Heatmap of all genes between control (Ctrl) and CD5-2 (CD5-2) in VE-cadherin–expressing ECs (VE-cadherin +/+ ). (ii) GSEA analysis of differentially expressed genes (DEGs) between Ctrl- and CD5-2–treated ECs. (iii) Bar plot of selected inflammation-related DEGs inhibited by CD5-2. Values are shown as mean ± SEM. * P < 0.05, ** P < 0.01, N.S, not significant, determined by Student t test or one-way ANOVA with Tukey correction. For the raw data used for quantification, see Fig 6 in ; Fig 6B and 6C in . CCM, cerebral cavernous malformation; CD31, cluster of differentiation 31; EC, endothelial cell; GFP, green fluorescent protein; GSEA, Gene Set Enrichment Analysis; hCMEC/D3, human cerebral microvascular endothelial cell line; ICAM-1, intercellular adhesion molecule 1; NF-κB, nuclear factor kappa B; PDGF, platelet-derived growth factor; siRNA, small interfering RNA; TNF-α, tumor necrosis factor-α; TSB, target site blocker; VE-cadherin, vascular endothelial cadherin.

Article Snippet: For primary staining, the following antibodies were used for immunostaining: rat CD31 (1:200, 553370, BD Biosciences, San Jose, CA), rabbit ICAM-1 (1:200, ab124759, Abcam,Cambridge, United Kingdom), rabbit pMLC2 (1:200, 3674S, Cell Signaling Technology, Danvers, MA), rat CD144 (1:200, 555289, BD Biosciences, San Jose, CA), goat VE-cadherin (1:1,000, sc-6458, Santa Cruz Biotechnology, Dallas, TX), and rat CD11b (1:200, 53-0112-82, eBiosciences, San Diego, CA).

Techniques: Expressing, Staining, Western Blot, Transfection, Cell Culture, Transmigration Assay, In Vitro, Derivative Assay, Small Interfering RNA