Journal: Molecular Cancer Therapeutics

Article Title: A Human Monoclonal Anti-ANG2 Antibody Leads to Broad Antitumor Activity in Combination with VEGF Inhibitors and Chemotherapy Agents in Preclinical Models

doi: 10.1158/1535-7163.mct-09-0554

Figure Lengend Snippet: Figure 1. Antibody 3.19.3 binds human Ang2 and blocks Tie2-mediated phosphorylation and downstream signaling. A, the ability of antibody 3.19.3 to selectively bind Ang2 in a competitive Tie2-Fc ELISA assay was determined with an IC50 value of 506 pmol/L, whereas Ang1 binding was essentially undetectable. B, inhibition of Ang2 binding to human Tie2 in a cell-based HEK293 assay. Antibody 3.19.3 was tested for the ability to block Ang2 interactions with Tie2 expressed on the cell surface using biotinylated human Ang1 or Ang2. Percentage of inhibition of binding was calculated as a fraction of the maximum signal obtained without antibody and suggested EC50 values of 150 pmol/L affinity to Ang2, whereas the Ang1 binding did not reach saturation up to concentrations of 1 μmol/L antibody. C, inhibition of Ang2-induced Tie2 phosphorylation in HEK293/Tie2 cells. IgG control antibody (lanes 1, 2) was compared alongside 3.19.3 (lanes 3, 4) for the ability to block recombinant Ang2 (1 μg/mL)–mediated Tie2 phosphorlylation at 2:1 molar ratios (lanes 1, 3) or 1:2 molar ratios (lanes 2, 4) corresponding to 1.35 μg/mL or 5.4 μg/mL antibody, respectively. Controls for maximal Ang2 induction are shown (lane 5) or no treatment (lane 6), indicating baseline pTie2 activity in this assay.

Article Snippet: Cells were harvested and lysed, and 0.8 mg total protein was immunoprecipitated using an anti-Tie2 antibody (MAB313, R&D Systems).

Techniques: Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Binding Assay, Inhibition, Blocking Assay, Control, Recombinant, Activity Assay

Journal: Frontiers in cell and developmental biology

Article Title: Inhibiting Heat Shock Protein 90 Protects Nucleus Pulposus-Derived Stem/Progenitor Cells From Compression-Induced Necroptosis and Apoptosis.

doi: 10.3389/fcell.2020.00685

Figure Lengend Snippet: FIGURE 1 | Compression induced necrotic cell death of NPSCs. (A) IHC staining of Tie2 for marking NPSCs in non-degenerated (37 years old, male, grade II) and degenerated (43 years old, male, grade IV) human NP tissues (original magnification: ×400). (B) Cell viability of NPSCs examined by CCK-8 assays. (C) The relative release of LDH at different time points. (D) Representative dot plots of PI staining obtained from flow cytometry analysis of NPSCs. (E) The statistical analysis of PI positive ratio of NPSCs. (F) The morphological ultrastructural appearance of NPSCs observed by TEM. The NPSCs exposed to 48 h of compression displayed necrotic morphological changes, such as severe vacuolation, swelling of organelles and disruption of the plasma membrane. The data were expressed as mean ± SD from at least three independent experiments, and they were analyzed by a two-tailed t-test or ANOVA. (**P < 0.01, ***P < 0.001 vs. 0 h).

Article Snippet: Cells were then permeabilized with 0·5% Triton X-100 (Beyotime) for 15 min at room temperature (for staining of Tie2, the permeabilization was not performed), and blocked with goat serum albumin for 1 h. Next, samples were washed with Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 August 2020 | Volume 8 | Article 685 PBS and incubated with the mixture of mouse anti-HSP90 antibody (1:200, Santa Cruz Biotechnology) and rabbit antiphosphorylated MLKL (P-MLKL) antibody (Ser358, 1:200, Affinity Biosciences, OH, United States), the rabbit anti-HSP70 antibody (1:500, ABclonal, Wuhan, China), or rabbit anti-Tie2 antibody (1:100, Proteintech Group, Wuhan, China) at 4◦C overnight, followed by incubation with fluorophore-conjugated secondary antibody (1:200, Proteintech Group).

Techniques: Immunohistochemistry, CCK-8 Assay, Staining, Cytometry, Disruption, Clinical Proteomics, Membrane, Two Tailed Test

Journal: Frontiers in cell and developmental biology

Article Title: Inhibiting Heat Shock Protein 90 Protects Nucleus Pulposus-Derived Stem/Progenitor Cells From Compression-Induced Necroptosis and Apoptosis.

doi: 10.3389/fcell.2020.00685

Figure Lengend Snippet: FIGURE 9 | Inhibiting HSP90 attenuated the exhaustion of NPSCs in vivo. Hematoxylin and eosin staining (original magnification: ×25) and the IHC staining of Tie2 (original magnification: ×25 and 400) for labeling endogenous NPSCs of IVDs.

Article Snippet: Cells were then permeabilized with 0·5% Triton X-100 (Beyotime) for 15 min at room temperature (for staining of Tie2, the permeabilization was not performed), and blocked with goat serum albumin for 1 h. Next, samples were washed with Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 August 2020 | Volume 8 | Article 685 PBS and incubated with the mixture of mouse anti-HSP90 antibody (1:200, Santa Cruz Biotechnology) and rabbit antiphosphorylated MLKL (P-MLKL) antibody (Ser358, 1:200, Affinity Biosciences, OH, United States), the rabbit anti-HSP70 antibody (1:500, ABclonal, Wuhan, China), or rabbit anti-Tie2 antibody (1:100, Proteintech Group, Wuhan, China) at 4◦C overnight, followed by incubation with fluorophore-conjugated secondary antibody (1:200, Proteintech Group).

Techniques: In Vivo, Staining, Immunohistochemistry, Labeling

Journal: Journal of cell science

Article Title: Rat aorta-derived mural precursor cells express the Tie2 receptor and respond directly to stimulation by angiopoietins.

doi: 10.1242/jcs.00629

Figure Lengend Snippet: Fig. 5. Tie2 expression in MPCs. (Top) Western-blot analysis (WB) of MPC protein extract immunoreacted for Tie2 demonstrated a 140 kDa band that migrated with the Tie2 band of the endothelial cell control (EC). RT-PCR confirmed expression of Tie2 in MPCs at the mRNA level. Endothelial cells (EC) were used as positive control. No PCR bands were detected in the absence of reverse transcriptase (data not shown). Specificity of the PCR products was confirmed by sequence analysis. (Bottom) Analysis of MPC protein extracts immunoprecipitated (IP) with an anti-Tie2 antibody and reacted with an anti-phosphotyrosine antibody (α-pY) showed phosphorylation of the MPC Tie2 receptor upon stimulation with both Ang-1 and Ang-2. Stripping and reprobing of the membrane with an anti-Tie2 antibody (α-Tie2) showed that equal amounts of Tie2 were present in each lane.

Article Snippet: Tie2 protein expression in isolated cells was demonstrated in separate experiments with two rabbit polyclonal anti- Journal of Cell Science 116 (17) 3637Angiopoietins and Tie2+ mural precursor cells Tie2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA; Alpha Diagnostics International, San Antonio, TX).

Techniques: Expressing, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Positive Control, Reverse Transcription, Sequencing, Immunoprecipitation, Phospho-proteomics, Stripping Membranes, Membrane

Journal: Journal of cell science

Article Title: Rat aorta-derived mural precursor cells express the Tie2 receptor and respond directly to stimulation by angiopoietins.

doi: 10.1242/jcs.00629

Figure Lengend Snippet: Fig. 6. Photomicrographs of planar cultures of isolated MPCs (A-E) and endothelial cells (F), and of histologic sections of native rat aorta (G,H) immunostained for Tie2 by immunoperoxidase (A,B,F-H) or immunofluorescence (C-E). Planar cultures: (A) shows a group of confluent MPCs whereas (B) shows a single MPC at higher magnification. Immunoperoxidase staining of cultured cells demonstrated Tie2 in both MPCs (A,B) and control endothelial cells (F). The positive staining reaction for Tie2 was predominantly localized at the cell periphery (arrows). Confocal images of MPCs double stained for Tie2 (C, green fluorescence) and α-SMA (D, red fluorescence) showed coexpression of Tie2 and α-SMA in the same cells (E, green and red fluorescence overlay). Cultured cells reacted with nonimmune IgG were negative (data not shown). Scale bars, 50 µM (A,C-E), 30 µM (B,F). Histological sections of native rat aorta: the intimal and subintimal layers of the aorta contain Tie2+ nonendothelial mesenchymal cells (G,H, arrows). Arrowheads highlight the endothelial lining of the aortic intima, which serves as a positive internal control. Scale bar, 100 µm.

Article Snippet: Tie2 protein expression in isolated cells was demonstrated in separate experiments with two rabbit polyclonal anti- Journal of Cell Science 116 (17) 3637Angiopoietins and Tie2+ mural precursor cells Tie2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA; Alpha Diagnostics International, San Antonio, TX).

Techniques: Isolation, Immunoperoxidase Staining, Cell Culture, Control, Staining