Journal: International Journal of Nanomedicine

Article Title: Enhanced anticancer efficacy of paclitaxel through multistage tumor-targeting liposomes modified with RGD and KLA peptides

doi: 10.2147/IJN.S122859

Figure Lengend Snippet: Illustration of the multistage tumor-targeting liposomes modified using cRGD and KLA peptides (RGD-KLA/PTX-Lips) for anticancer drug delivery. Notes: Main components of the RGD-KLA/PTX-Lips ( A ). Schematic illustration of RGD-KLA/PTX-Lips delivery in vivo ( B ). Abbreviations: PTX, paclitaxel; PEG, polyethylene glycol; EPR, enhanced permeability and retention.

Article Snippet: cRGD peptide with a terminal cysteine (cRGD-(D-Tyr)-K, MW =722.84, cRGD-SH) and KLA peptide with a terminal cysteine ( D -(KLAKLAK) 2 -Cys, MW =1,627, KLA-SH) were purchased from SciLight Biotechnology, LLC (Beijing, China).

Techniques: Modification, In Vivo, Permeability

Journal: BMC Biology

Article Title: Targeting the Tie2–α v β 3 integrin axis with bi-specific reagents for the inhibition of angiogenesis

doi: 10.1186/s12915-018-0557-9

Figure Lengend Snippet: Binding of Ang2-BD bi-specific variants to recombinant and cell-expressed human Tie2 and α v β 3 integrin. a Representative SPR sensorgram of Ang2-BD bi-specific variants (400 nM) binding to both α v β 3 integrin (immobilized) and soluble Tie2 (400 nM). Ang2-BD variants [ Ang2-BD WT (blue), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red)] were allowed to flow for 400 s followed by an additional 150 s flow of Tie2. Injection of running buffer followed by rhTie2 in IBB served as negative control (black). Injection steps are indicated on the sensorgrams. b Binding of Ang2-BD bi-specific variants to TIME cells. 1 × 10 5 cells were incubated with Ang2-BD WT (blue), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red) for 2 h at 4 °C with gentle agitation. Mean fluorescence values were determined by flow cytometry using fluorescently labeled antibodies against a FLAG epitope tag. “*” indicates a P value < 0.05 by one-way ANOVA upon comparing results between Ang2-BD variants at the same concentration. c Competitive binding of 1 μM Ang2-BD WT (blue), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red), alone or with a combination of 1000 ng/ml of FL-Ang1 (checkered bars), 10 μM cRGD (horizontally lined bars) and both 1000 ng/ml FL-Ang1 and 10 μM cRGD (vertically-lined bars). Mean fluorescence values were determined by flow cytometry using fluorescently labeled antibodies against a FLAG epitope tag. “*” indicates a P value < 0.05 by one-way ANOVA upon comparing results between Ang2-BD variants alone and with FL-Ang1 and cRGD competitors. Data shown represent the average of triplicates from independent experiments, and error bars represent the standard error of the mean

Article Snippet: TIME cells were seeded (7500 cells per well) on a human vitronectin-coated 96-well microplate (R&D Systems) and incubated at 37 °C/5% CO 2 for 24 h. The medium was then replaced with fresh Vascular Cell Basal Medium supplemented with 2% FBS and growth factor supplements, and the cells were incubated with 500 ng/ml of rhAng1 either alone or with 2 μM Ang2 - BD WT , Ang2-BD BC5 , Ang2-BD BC6 , Ang2-BD BC10 , or cRGD peptide (Merck Millipore).

Techniques: Binding Assay, Recombinant, Injection, Negative Control, Incubation, Gentle, Fluorescence, Flow Cytometry, Labeling, FLAG-tag, Concentration Assay

Journal: BMC Biology

Article Title: Targeting the Tie2–α v β 3 integrin axis with bi-specific reagents for the inhibition of angiogenesis

doi: 10.1186/s12915-018-0557-9

Figure Lengend Snippet: Inhibition of TIME cells adhesion and phosphorylation of Tie2, Akt, and FAK. a 5 × 10 4 TIME cells were incubated alone (cells only; black), with 500 ng/ml of FL-Ang1 (gray), or with a combination of 500 ng/ml FL-Ang1 and 1 μM cRGD (purple), Ang2-BD WT (blue), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red) for 2 h on vitronectin-coated 96-well plates. b 5 × 10 4 TIME cells were incubated alone (cells only; black) or with 1 μM cRGD (purple), Ang2-BD WT (blue), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red) for 2 h on vitronectin-coated 96-well plates. c For determining Tie2 phosphorylation, TIME cells were treated with control buffer (basal level; black), 500 ng/ml FL-Ang1 (gray), or a combination of 500 ng/ml FL-Ang1 with 1 μM cRGD (purple), Ang2-BD WT (blue), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red) for 15 min on vitronectin-coated 12-well plates. d Cell lysates were analyzed by Western blot using antibodies against phosphorylated Tie2 (pTie2), Tie2, and β-actin. e Akt phosphorylation was determined as in c except that the cells were treated with a combination of 500 ng/ml FL-Ang1 with 0.5 μM of the above proteins and the incubation time was 30 min. f Cell lysates were analyzed by Western blot using antibodies against phosphorylated Akt (pAkt), Akt, and β-actin. g FAK phosphorylation was determined as in c . h Cell lysates were analyzed by Western blot using antibodies against phosphorylated FAK (pFAK), FAK, and β-actin. “*” indicates a P value < 0.05 upon comparing the results between the cells-only control ( b ) and the tested proteins. “*” indicates a P value < 0.05 upon comparing the results between FL-Ang1 alone ( a , c , e , h ) and a combination of FL-Ang1 with the tested proteins. “&” indicates a P value < 0.05 upon comparing the results between cRGD ( a , b , c , e , h ) and the tested proteins. “#” indicates a P value < 0.05 upon comparing the results between Ang2-BD WT ( a , b , c , e , h ) and the tested proteins

Article Snippet: TIME cells were seeded (7500 cells per well) on a human vitronectin-coated 96-well microplate (R&D Systems) and incubated at 37 °C/5% CO 2 for 24 h. The medium was then replaced with fresh Vascular Cell Basal Medium supplemented with 2% FBS and growth factor supplements, and the cells were incubated with 500 ng/ml of rhAng1 either alone or with 2 μM Ang2 - BD WT , Ang2-BD BC5 , Ang2-BD BC6 , Ang2-BD BC10 , or cRGD peptide (Merck Millipore).

Techniques: Inhibition, Phospho-proteomics, Incubation, Control, Western Blot

Journal: BMC Biology

Article Title: Targeting the Tie2–α v β 3 integrin axis with bi-specific reagents for the inhibition of angiogenesis

doi: 10.1186/s12915-018-0557-9

Figure Lengend Snippet: Endothelial cell tube formation and inhibition of cell viability by Ang2-BD bi-specific variants. a 3.25 × 10 4 TIME cells were treated with the indicated proteins overnight and washed, and pictures were taken using a × 2 objective. Scale bar, 500 μm. b Images of tube structures were analyzed for the number of generated junctions and the number of meshes in control buffer (cells only; black), 500 ng/ml FL-Ang1 (gray), or a combination of 500 ng/ml FL-Ang1 with 1 μM cRGD (purple), Ang2-BD WT (blue), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red). c Cell viability was determined by the XTT assay for control buffer (cells only; black), 500 ng/ml FL-Ang1 (gray), or a combination of 500 ng/ml FL-Ang1 with 2 μM cRGD (purple), Ang2-BD WT (blue), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red). “*” indicates a P value < 0.05 upon comparing the results between FL-Ang1 and the tested proteins. “&” indicates a P value < 0.05 upon comparing the results between cRGD and the tested proteins. “#” indicates a P value < 0.05 upon comparing the results between Ang2-BD WT and the tested proteins ( b , c )

Article Snippet: TIME cells were seeded (7500 cells per well) on a human vitronectin-coated 96-well microplate (R&D Systems) and incubated at 37 °C/5% CO 2 for 24 h. The medium was then replaced with fresh Vascular Cell Basal Medium supplemented with 2% FBS and growth factor supplements, and the cells were incubated with 500 ng/ml of rhAng1 either alone or with 2 μM Ang2 - BD WT , Ang2-BD BC5 , Ang2-BD BC6 , Ang2-BD BC10 , or cRGD peptide (Merck Millipore).

Techniques: Inhibition, Generated, Control, XTT Assay

Journal: BMC Biology

Article Title: Targeting the Tie2–α v β 3 integrin axis with bi-specific reagents for the inhibition of angiogenesis

doi: 10.1186/s12915-018-0557-9

Figure Lengend Snippet: Inhibition of endothelial cell invasiveness by Ang2-BD bi-specific variants. a TIME cells were treated with the indicated proteins in Boyden chambers. Scale bar, 200 μm. b The invading cells accumulating at the bottom of the membrane were counted in 16 frames for each membrane and analyzed for the number of cells for control buffer (cells only; black), 500 ng/ml FL-Ang1 (gray), or a combination of 500 ng/ml FL-Ang1 with 1 μM cRGD (purple), Ang2-BD WT (blue), cRGD together with Ang2-BD WT (orange), Ang2-BD BC5 (green), Ang2-BD BC6 (yellow), or Ang2-BD BC10 (red). “*” indicates a P value < 0.05 upon comparing the results between FL-Ang1 and the tested proteins. “&” indicates a P value < 0.05 upon comparing the results between cRGD and the tested proteins. “#” indicates a P value < 0.05 upon comparing the results between Ang2-BD WT and the tested proteins. “$” indicates a P value < 0.05 upon comparing the results between cRGD + Ang2-BD WT and the tested proteins. The data in Figs. , , and 6 were analyzed by one-way ANOVA. Data shown represent the average of triplicates from independent experiments, and error bars represent the standard error of the mean

Article Snippet: TIME cells were seeded (7500 cells per well) on a human vitronectin-coated 96-well microplate (R&D Systems) and incubated at 37 °C/5% CO 2 for 24 h. The medium was then replaced with fresh Vascular Cell Basal Medium supplemented with 2% FBS and growth factor supplements, and the cells were incubated with 500 ng/ml of rhAng1 either alone or with 2 μM Ang2 - BD WT , Ang2-BD BC5 , Ang2-BD BC6 , Ang2-BD BC10 , or cRGD peptide (Merck Millipore).

Techniques: Inhibition, Membrane, Control

Journal: BMC Biology

Article Title: Targeting the Tie2–α v β 3 integrin axis with bi-specific reagents for the inhibition of angiogenesis

doi: 10.1186/s12915-018-0557-9

Figure Lengend Snippet: Inhibition percentage of capillary tube formation, viability, and invasiveness of endothelial TIME cells growing on Matrigel vs FL-Ang1-treated controls

Article Snippet: TIME cells were seeded (7500 cells per well) on a human vitronectin-coated 96-well microplate (R&D Systems) and incubated at 37 °C/5% CO 2 for 24 h. The medium was then replaced with fresh Vascular Cell Basal Medium supplemented with 2% FBS and growth factor supplements, and the cells were incubated with 500 ng/ml of rhAng1 either alone or with 2 μM Ang2 - BD WT , Ang2-BD BC5 , Ang2-BD BC6 , Ang2-BD BC10 , or cRGD peptide (Merck Millipore).

Techniques: Inhibition