Journal: Diabetologia

Article Title: Enhanced VEGF signalling mediates cerebral neovascularisation via downregulation of guidance protein ROBO4 in a rat model of diabetes

doi: 10.1007/s00125-017-4214-6

Figure Lengend Snippet: ROBO4 regulates VEGF-induced angiogenic signal. (a) Silencing Robo4 in BMVECs isolated from control Wistar rats (Wis, light grey bars) resulted in a significant increase in migration and tube formation compared with cells transfected with scrambled (Sc) siRNA (white bars). BMVECs isolated from diabetic GK rats (black bars) showed significant increases in migration and tube formation. Overexpression of ROBO4 in diabetic BMVECs (dark grey bars) significantly decreased endothelial cell migration and tube formation (n=3 in duplicate, *p<0.05 vs Wistar+Sc siRNA, †p<0.05 vs GK+GFP). (b) Transwell permeability assay showed that overexpression of ROBO4 (black bars) significantly decreased BMVEC permeability in diabetes compared with control (n=3 in duplicate, †p<0.05 vs GK+GFP). (c) Representative image of brain and adenovirus stereotactic injection site. ROBO4 was overexpressed by 50% compared with empty vector control. (n=3, *p<0.05 vs empty vector). Scale bar, 10 μm. (d) Representative image and quantification of neovascularisation indices after ROBO4 overexpression. ROBO4 overexpression (Robo4 AD) significantly reduced all neovascularisation indices (n=4, *p<0.05 vs contralateral side). Scale bars, 50 μm

Article Snippet: ROBO4 overexpression In vivo experiments Robo4 adenovirus (3 μl of 1.85 × 10 12 viral particles/ml, Ad- Gfp -h Robo4 , ADV-221472; Vector Biolabs, Philadelphia, PA, USA) was injected over 6 min via a 30-gauge needle adjacent to the MCA at stereotactic coordinates (bregma: anterior–posterior, −0.5 mm; mediolateral −1 mm; dorsoventral, −4 mm).

Techniques: Isolation, Control, Migration, Transfection, Over Expression, Permeability, Injection, Plasmid Preparation

Journal: Diabetologia

Article Title: Enhanced VEGF signalling mediates cerebral neovascularisation via downregulation of guidance protein ROBO4 in a rat model of diabetes

doi: 10.1007/s00125-017-4214-6

Figure Lengend Snippet: VEGFR-2 inhibitor, SKLB1002, increases ROBO4 availability. Diabetic GK rats were treated with vehicle or SLKB1002 (5, 10 or 15 mg/kg per day i.p.) for 2 weeks. (a) Western blot analysis of VEGFR-2 activation. GK rats treated with SLKB1002 showed significant decrease in VEGFR-2 phosphorylation compared with control vehicle-treated rats (n=3, *p<0.05 vs vehicle). (b) Western blot analysis of ROBO4 expression in GK rats treated with SLKB1002 showed increased expression of ROBO4 (n=3 or 4, *p<0.05 vs vehicle). (c) Western blot analysis of VEGF-induced phosphorylation of β3 integrin (β-3). Treatment with SLKB1002 or ROBO4 overexpression (ROBO4 AD) significantly reduced VEGF-induced phosphorylation of β3 integrin in GK rats. (n=3 or 4, *p<0.05 vs GK). (d) Immunoprecipitation (IP) of brain homogenate from GK rats treated with SKLB1002 or overexpressing ROBO4. Both treatments significantly decreased the ROBO4–β3 integrin interaction and binding in GK brain homogenate (n=3 or 4, *p<0.05 vs GK). (e) Representative images and quantification of cell migration and tube formation. Dashed white lines represent migration after 18 h. Silencing the β3 integrin gene significantly decreased cellular tube formation and migration in BMVECs isolated from diabetic GK rats compared with GK BMVECs treated with scrambled siRNA (Sc siRNA) (n=3 in duplicate, *p<0.05 vs GK+Sc siRNA)

Article Snippet: ROBO4 overexpression In vivo experiments Robo4 adenovirus (3 μl of 1.85 × 10 12 viral particles/ml, Ad- Gfp -h Robo4 , ADV-221472; Vector Biolabs, Philadelphia, PA, USA) was injected over 6 min via a 30-gauge needle adjacent to the MCA at stereotactic coordinates (bregma: anterior–posterior, −0.5 mm; mediolateral −1 mm; dorsoventral, −4 mm).

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Control, Expressing, Over Expression, Immunoprecipitation, Binding Assay, Migration, Isolation

Journal: Diabetologia

Article Title: Enhanced VEGF signalling mediates cerebral neovascularisation via downregulation of guidance protein ROBO4 in a rat model of diabetes

doi: 10.1007/s00125-017-4214-6

Figure Lengend Snippet: Diabetes decreases the expression of ROBO4 in cerebral vasculature. Diabetic GK rats were injected with FITC–dextran via the jugular vein. Brain sections were reacted with anti-ROBO4 antibody. (a) Co-localised ROBO4 (red) on FITC-filled cerebral vasculature (green) was compared between diabetic GK rats and control Wistar rats. Diabetic GK rats showed decreased ROBO4 expression (n=4 or 5, *p<0.05 vs Wistar). (b) Western blot of BMVECs isolated from control and diabetic rats, showing a significant reduction in ROBO4 expression in diabetes (n=3 or 4, *p<0.05 vs Wistar). Scale bars, 10 μm

Article Snippet: ROBO4 overexpression In vivo experiments Robo4 adenovirus (3 μl of 1.85 × 10 12 viral particles/ml, Ad- Gfp -h Robo4 , ADV-221472; Vector Biolabs, Philadelphia, PA, USA) was injected over 6 min via a 30-gauge needle adjacent to the MCA at stereotactic coordinates (bregma: anterior–posterior, −0.5 mm; mediolateral −1 mm; dorsoventral, −4 mm).

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

Journal: Diabetologia

Article Title: Enhanced VEGF signalling mediates cerebral neovascularisation via downregulation of guidance protein ROBO4 in a rat model of diabetes

doi: 10.1007/s00125-017-4214-6

Figure Lengend Snippet: ROBO4 overexpression increases vascular stability. (a) Nuclear morphology was used to assess the pericyte-to-endothelial cell ratio. Dotted lines represent the elliptical endothelial cells nuclei and solid circles represent pericytes nuclei. Overexpression of ROBO4 (Robo4 AD) significantly increased this ratio in the cerebral vasculature (n=4, *p<0.05 vs contralateral side). Scale bars, 10 μm. (b) Representative images of brain sections showing FITC-filled vessels (green), nuclear stain (DAPI, blue), pericyte marker, PDGF receptor-β antibody (red) and merged images. Scale bars, 10 μm

Article Snippet: ROBO4 overexpression In vivo experiments Robo4 adenovirus (3 μl of 1.85 × 10 12 viral particles/ml, Ad- Gfp -h Robo4 , ADV-221472; Vector Biolabs, Philadelphia, PA, USA) was injected over 6 min via a 30-gauge needle adjacent to the MCA at stereotactic coordinates (bregma: anterior–posterior, −0.5 mm; mediolateral −1 mm; dorsoventral, −4 mm).

Techniques: Over Expression, Staining, Marker

Journal: Diabetologia

Article Title: Enhanced VEGF signalling mediates cerebral neovascularisation via downregulation of guidance protein ROBO4 in a rat model of diabetes

doi: 10.1007/s00125-017-4214-6

Figure Lengend Snippet: Increased VEGF signalling in cerebral vessels in diabetes decreases the expression and availability of ROBO4 via binding with VEGF-activated β3 integrin. Overexpression of ROBO4 reduces the augmented VEGF-induced angiogenic signal and decreases cerebral neovascularisation in diabetes. Inhibition of the VEGF angiogenic signal by SKLB1002, a selective VEGFR-2 antagonist, decreases ROBO4–β3 integrin interaction, increases the expression and availability of ROBO4 and prevents/repairs cerebral neovascularisation in diabetes

Article Snippet: ROBO4 overexpression In vivo experiments Robo4 adenovirus (3 μl of 1.85 × 10 12 viral particles/ml, Ad- Gfp -h Robo4 , ADV-221472; Vector Biolabs, Philadelphia, PA, USA) was injected over 6 min via a 30-gauge needle adjacent to the MCA at stereotactic coordinates (bregma: anterior–posterior, −0.5 mm; mediolateral −1 mm; dorsoventral, −4 mm).

Techniques: Expressing, Binding Assay, Over Expression, Inhibition

Journal: EBioMedicine

Article Title: Destabilization of Lysophosphatidic Acid Receptor 1 Reduces Cytokine Release and Protects Against Lung Injury

doi: 10.1016/j.ebiom.2016.07.020

Figure Lengend Snippet: USP11 promotes LPA1-CD14 interaction and LPS-induced lung inflammation. a . MLE12 cells were transfected with USP11-HA plasmid for 48 h, and then cells were treated with LPA (5 μM) for 30 min. Cell lysates were subjected to immunoprecipitation with LPA1 antibody, followed by CD14 and LPA1 immunoblotting. Input lysates were analyzed by immunoblotting with CD14, HA, LPA1, and β-actin antibodies. b . MLE12 cells were transfected with usp11 shRNA for 72 h, and then cells were treated with LPS (10 μg/ml) for 30 min. Cell lysates were analyzed by immunoblotting with p-Erk1&2, Erk1&2, and USP11 antibodies. Representative immunoblots were from at least three independent times. c . MLE12 cells were transfected with Usp11 shRNA for 72 h, and then cells were treated with LPS (10 μg/ml) for 3 h. Cell culture supernatants were collected and KC levels in the media were analyzed by Elisa. d – h . C57/BL6 mice (6–8/group) were intratracheally infected with control lentivirus (Lenti cont, 1 × 10 9 cfu/mice) or lentiviral vector carrying usp11 shRNA (Lenti USP11 shRNA Lenti cont, 1 × 10 9 cfu/mice) for 7 days, followed by i.t. administration with LPS (2 mg/kg body weight) for 24 h. Lung tissue lysates were analyzed by immunoblotting with USP11 and β-actin antibodies ( d ). Lung tissue were fixed and stained with H&E ( e ). BAL was collected, and then protein levels were examined by protein assay ( f ), IL-6 ( g ), and KC ( h ) were examined by Elisa.

Article Snippet: Mouse Usp11 shRNA was inserted into a pLVX-IRES vector (Clontech); Lenti-shUSP11 viral and control viral vectors were generated by using a lentivirus packaging system (Clontech).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, shRNA, Cell Culture, Enzyme-linked Immunosorbent Assay, Infection, Staining

Journal: iScience

Article Title: Primary cilia are required for the persistence of memory and stabilization of perineuronal nets

doi: 10.1016/j.isci.2021.102617

Figure Lengend Snippet: The role of the primary cilium in recent and remote memory (A) Experimental outline: AAV vector expressing control (GFP) or shIFT88 (IFT88) RNA was infused into CA1. Six weeks later mice were fear conditioned, and tested 4, 21 and 35 days later. (B) Effect of the primary cilium depletion from CA1 on memory. Mice infused into CA1 with control or shIFT88 AAV were fear conditioned, and tested at recent and remote time points. Data are represented as mean ± SEM (repeated measures two-way ANOVA, n = 10/group, effect of virus: F 2, 36 = 1.351, p = 0.0070; effect of day: F 1.917, 34.51 = 14.13, P<0.0001; post-hoc: ∗p = 0.0110 D35 GFP vs IFT88, ‡ p = 0.0251 IFT88 D35 vs D4, ## p = 0.0025 IFT88 D21 v D35). (C) Representative images demonstrating the depletion of primary cilia from CA1 region of DH. Size bar: 5 μm. (D) Quantification of the shIFT88 RNA effect. Data are represented as mean ± SEM. ∗∗∗∗ P < 0.0001 (unpaired t test, two-tailed; n = 8/group; t 1 4 = 8.466). (E) Effect of the primary cilium depletion from CA1 on remote memory. Mice infused into CA1 with control or shIFT88 AAV were fear conditioned, and tested at remote time point only. Data are represented as mean ± SEM (unpaired t test, two-tailed; n = 9 (GFP), n = 10 (IFT88)/group; t 1 7 = 7.674; ∗∗∗∗ P < 0.0001). (F) Effect of cyclopamine infusion into CA1 on recent and remote memory retrieval. Data are represented as mean ± SEM (two-way ANOVA, n = 9/group, effect of treatment F 1, 14 = 3.182 p = 0.0962; effect of day F 1, 14 = 1.755, p = 0.2065). See also A and S3B.

Article Snippet: The viral vectors carrying a construct coding for an shRNA targeting mouse IFT88 (IFT88: AAV-DJ/8-U6-GFP-m-IFT88-shRNA; Vector Biolabs), or control construct (GFP: pAAV2-CAG-GFP; Addgene) were bilaterally infused into the dorsal hippocampus (1.8 mm posterior, ±1.0 mm lateral and 2.0 mm ventral to bregma).

Techniques: Plasmid Preparation, Expressing, Control, Virus, Two Tailed Test

Journal: iScience

Article Title: Primary cilia are required for the persistence of memory and stabilization of perineuronal nets

doi: 10.1016/j.isci.2021.102617

Figure Lengend Snippet:

Article Snippet: The viral vectors carrying a construct coding for an shRNA targeting mouse IFT88 (IFT88: AAV-DJ/8-U6-GFP-m-IFT88-shRNA; Vector Biolabs), or control construct (GFP: pAAV2-CAG-GFP; Addgene) were bilaterally infused into the dorsal hippocampus (1.8 mm posterior, ±1.0 mm lateral and 2.0 mm ventral to bregma).

Techniques: Virus, Plasmid Preparation, Recombinant, Staining, Blocking Assay, Fluorsave, Isolation, Reverse Transcription, SYBR Green Assay, Sample Prep, Software, Real-time Polymerase Chain Reaction