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: BMVECs were transfected with either scrambled, Robo4 small interfering RNA (siRNA) or β3 integrin ( Itgb3 ) siRNA (Santa Cruz) using Amaxa Nucleofector kit, following the manufacturer’s protocol (Lonza).

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: 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: BMVECs were transfected with either scrambled, Robo4 small interfering RNA (siRNA) or β3 integrin ( Itgb3 ) siRNA (Santa Cruz) using Amaxa Nucleofector kit, following the manufacturer’s protocol (Lonza).

Techniques: Expressing, Binding Assay, Over Expression, Inhibition

Journal: International Journal of Nanomedicine

Article Title: Targeting integrins with RGD-conjugated gold nanoparticles in radiotherapy decreases the invasive activity of breast cancer cells

doi: 10.2147/IJN.S137833

Figure Lengend Snippet: Internalization and colocalization of RGD/P-AuNPs with integrins in breast cancer cells. Notes: ( A ) Protein expression of RGD receptors (α5β1-, αvβ5- and αvβ3-integrins) in breast cancer cell lines. Total cell lysates were subjected to Western blotting using the indicated antibodies. ( B ) Phase-contrast microscopic images of MDA-MB-231, Hs578T and SK-BR-3 cells cultured with or without AuNPs. Bar, 20 μm. ( C ) Confocal immunofluorescence images show localization of FITC-conjugated AuNPs (green) and integrins (red) in cells. MDA-MB-231 cells were cultured with FITC-conjugated AuNPs, and then integrins were labeled with an anti-α5-integrin or αv-integrin antibody coupled to Alexa 568-labeled anti-mouse IgG antibody. White arrows: colocalized FITC/RGD/P-AuNPs or integrins. Bar, 25 μm. Abbreviations: RGD/P-AuNP, polyethylene-glycolylated gold nanoparticle (P-AuNP) conjugated with Arg–Gly–Asp (RGD) peptides; FITC, fluorescein isothiocyanate; IgG, Immunoglobulin G.

Article Snippet: The following primary antibodies were used for immunoblotting: anti-α5-integrin (Merck Millipore), anti-αv-integrin (Abcam, Cambridge, UK), anti-β1-integrin (BD Transduction Laboratories, San Jose, CA, USA), anti-β3-integrin (Merck Millipore), anti-β5-integrin (Abcam), anti-pERK (Cell Signaling Technology, Danvers, MA, USA), anti-ERK (Cell Signaling Technology), anti-LAMP1 (Cell Signaling Technology) and anti-β-actin (Sigma-Aldrich).

Techniques: Expressing, Western Blot, Cell Culture, Immunofluorescence, Labeling

Journal: International Journal of Nanomedicine

Article Title: Targeting integrins with RGD-conjugated gold nanoparticles in radiotherapy decreases the invasive activity of breast cancer cells

doi: 10.2147/IJN.S137833

Figure Lengend Snippet: Colocalization of RGD/P-AuNPs with protein markers of early/late endosomes and lysosomes in MDA-MB-231. Notes: ( A ) Confocal immunofluorescence images show localization of FITC-conjugated RGD/P-AuNPs (FITC/RGD/P-AuNPs) (green) and protein markers of endosome/lysosome (red) in cells. MDA-MB-231 cells were cultured with FITC/RGD/P-AuNPs, and then markers of early endosomes (EEA1/Rab5) and late endosomes (Rab7/Rab9) were labeled with antibodies. Bar, 25 μm. ( B ) Colocalization analysis of FITC/RGD/P-AuNPs and proteins of endosome were quantified by Pearson’s correlation coefficient using ImageJ software. RGD/P-AuNPs were incubated with MDA-MB-231 cells for 1 day or 3 days. Columns, mean (n>10), bars, SD, ** P <0.01. ( C ) Immunofluorescence images of FITC/RGD/P-AuNPs (green), integrins (blue) and LAMP1 (red). Integrins were labeled with an anti-α5-integrin or αv-integrin antibody coupled to an Alexa 647-labeled anti-mouse IgG antibody. LAMP1 was labeled with anti-LAMP1 rabbit monoclonal antibody coupled to an Alexa 567-labeled anti-rabbit IgG antibody, which served as a marker of lysosomes. Bar, 25 μm. Abbreviations: RGD/P-AuNP, polyethylene-glycolylated gold nanoparticle (P-AuNP) conjugated with Arg–Gly–Asp (RGD) peptides; FITC, fluorescein isothiocyanate; EEA1, early endosome antigen 1; SD, standard deviation; LAMP1, lysosomal-associated membrane protein 1; IgG, Immunoglobulin G.

Article Snippet: The following primary antibodies were used for immunoblotting: anti-α5-integrin (Merck Millipore), anti-αv-integrin (Abcam, Cambridge, UK), anti-β1-integrin (BD Transduction Laboratories, San Jose, CA, USA), anti-β3-integrin (Merck Millipore), anti-β5-integrin (Abcam), anti-pERK (Cell Signaling Technology, Danvers, MA, USA), anti-ERK (Cell Signaling Technology), anti-LAMP1 (Cell Signaling Technology) and anti-β-actin (Sigma-Aldrich).

Techniques: Immunofluorescence, Cell Culture, Labeling, Software, Incubation, Marker, Standard Deviation