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: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: 5-Aminolaevulinic Acid-Based Photodynamic Therapy Restrains Pathological Hyperplasia of Fibroblasts

doi: 10.12659/MSM.898221

Figure Lengend Snippet: ALA-PDT reduces the growth-promoting effect of fibroblasts on HUVEC cells. ( A ) MTT assay was carried out to test the cell viability of HUVEC cells cultured together with fibroblasts. ( B, C ) The expression levels of vascular endothelial growth factor-A (VEGFA) and basic fibroblast growth factor (bFGF) in culture supernatant were tested by ELISA. ( D, E ) The expression levels of VEGFA and bFGF in fibroblasts were detected by Western blot analysis. The data were analyzed by one-way ANOVA test, with the fibroblasts in scar tissue group as the control. * P<0.05, ** P<0.01, *** P<0.001, ns – no significance.

Article Snippet: After blocking with 5% skim milk (YILI, Hohhot, Inner Mongolia, China) at room temperature for 1 h, the membrane was incubated with the following antibodies at 4°C overnight: rabbit anti-TGF-β1 (1:200) (Santa Cruz, CA), mouse anti-α-SMA (1:400) (Boster, Wuhan, Hubei, China), rabbit anti-Collagen I (1:400) (Boster), mouse anti-Collagen III (1:400) (Boster), rabbit anti-vascular endothelial factor-A (VEGFA) (1:400) (Boster), and rabbit anti-basic fibroblast growth factor (bFGF) (1:200) (Santa Cruz).

Techniques: MTT Assay, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control