Journal: Circulation journal : official journal of the Japanese Circulation Society

Article Title: Angiopoietin-1 mediates adipose tissue-derived stem cell-induced inhibition of neointimal formation in rat femoral artery.

doi: 10.1253/circj.cj-12-0930

Figure Lengend Snippet: Figure 8. Tie2-expressing cells are not recruited to the neointimal layer. AdGFP or AdAng1 was ap- plied to the femoral artery from the adventitial side after wire injury. The femoral arteries were harvest- ed 14 days after the injury for im- munohistochemical analysis of Tie2. Bars=50 μm. Ang1, angiopoietin-1; GFP, green fluorescence protein.

Article Snippet: Anti-Tie2 antibody was purchased from Santa-Cruz Biotechnology (Santa-Cruz, CA, USA).

Techniques: Expressing, Fluorescence

Journal: International Journal of Nanomedicine

Article Title: Delivery of curcumin by directed self-assembled micelles enhances therapeutic treatment of non-small-cell lung cancer

doi: 10.2147/IJN.S128921

Figure Lengend Snippet: CUR micelles inhibited the migration and invasion of A549 cells in vitro. Notes: ( A and C ) CUR micelles inhibited A549 cell migration in wound-healing assay. ( B and D ) CUR micelles inhibited A549 cell invasion in transwell assay. Images in ( A and B ) were taken by microscope under ×100 magnification. ( E ) The VEGF, MMP-2 and MMP-9 were analyzed by Western blotting assay, with GAPDH acted as loading controls. Blank micelles, blank mPEG–PLA micelles; CUR micelles, CUR/mPEG–PLA micelles. Abbreviations: CUR, curcumin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MMP, matrix metalloproteinase; mPEG–PLA, methoxy polyethylene glycol–polylactide; VEGF, vascular endothelial growth factor.

Article Snippet: Anti-Bcl-2, anti-Bax, anti-MMP-2 and anti-MMP-9 were provided by Abcam (Cambridge, UK), while anti-vascular endothelial growth factor (VEGF) was obtained from Boster (Wuhan, China).

Techniques: Migration, In Vitro, Wound Healing Assay, Transwell Assay, Microscopy, Western Blot

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: Blood

Article Title: Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer.

doi: 10.1182/blood-2006-10-053504

Figure Lengend Snippet: Figure 1. A subset of human monocytes express the TIE2 angiopoietin receptor. (A) Flow cytometry analysis of PB granulocytes (G), lymphocytes (L), and monocytes (M) identified on the basis of physical gating (dot plot on the left; gates indicated by dashed line) shows TIE2 expression in a subset of monocytes (open red line in the histogram plots on the right; filled line IgG isotype con- trol). Percentages of marker-positive cells are indicated. (B) The TIE2 PBMCs (red gate in left panels) are mostly CD14 monocytes (dot plots on the right). Representa- tive analysis of at least 16 performed on different donors. (C) The vast majority of TIE2 PBMCs do not express the CEC/EPC markersAC133 or CD146. Rare TIE2AC133

Article Snippet: Neutralizing anti-TIE2 antibodies (R&D Systems) were preincubated with cells for 20 minutes at 37°C.

Techniques: Flow Cytometry, Expressing, Marker

Journal: Blood

Article Title: Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer.

doi: 10.1182/blood-2006-10-053504

Figure Lengend Snippet: Figure 3. Characterization of the TIE2 monocytes. (A) TIE2 cells in unfractioned PBMCs were gated and analyzed for the expression of a panel of hematopoietic markers, as indicated in the histogram plots. The red open line shows expression of the indicated marker in the gated TIE2 population (with the percentage of marker-positive cells in red); the filled line depicts expression of the indicated marker in the TIE2 cell population (with the percentage of marker-positive cells in black). The TIE2 cells were CD45, CD11b, CD11c, CD16, CD33, CD115, and CD13, which are all markers of monocytic cells. In addition, the TIE2 cells were CCR2, CD62L (L-selectin), and CCR5, a surface profile previously associated with resident monocytes. As expected, the TIE2 cells were CD56, CD3, and CD19 and thus distinct from natural killer cells and T and B lymphocytes. Representative analysis of 3 to 6 experiments per- formed on different donors.

Article Snippet: Neutralizing anti-TIE2 antibodies (R&D Systems) were preincubated with cells for 20 minutes at 37°C.

Techniques: Expressing, Marker

Journal: Blood

Article Title: Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer.

doi: 10.1182/blood-2006-10-053504

Figure Lengend Snippet: Figure 2. TIE2 receptor expression by TIE2 monocytes. (A) TaqMan analyses of TIE2 and VEGFR2 transcripts in FACS-sorted monocyte subsets showing Ct values over endogenous control GAPDH. The lower the Ct, the higher the expression of the transcript in the target cell population. Ct values are expressed as mean standard error. Note that TIE2 transcript is clearly expressed in CD14lowCD16 (14low16) resident but nearly undetectable in CD14CD16 (14 16) inflammatory mono- cytes. (B) Relative quantification values of TIE2 transcript in FACS-sorted monocyte subsets. TIE2 transcript is significantly enriched in CD14TIE2 (14TIE2) TEMs compared with the resident monocytes. For each relative value, an interval of confidence was calculated; confidence intervals that do not overlap indicate statisti- cally significant differences (P .05). (C) Western-blot analysis of TIE2 protein expression in the indicated cell populations. Blots were probed with C-terminus specific anti-TIE2 rabbit (top panels) or mouse anti- actin (bottom panels) antibod- ies. The expected migration of each protein relative to molecular weight standards is indicated. Representative experiment of 3 performed. (D) TIE2 immunoprecipitated from CD14lowCD16 resident monocytes is phosphorylated on tyrosine. Representa- tive experiment of 2 performed.

Article Snippet: Neutralizing anti-TIE2 antibodies (R&D Systems) were preincubated with cells for 20 minutes at 37°C.

Techniques: Expressing, Control, Western Blot, Migration, Molecular Weight, Immunoprecipitation

Journal: Blood

Article Title: Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer.

doi: 10.1182/blood-2006-10-053504

Figure Lengend Snippet: Figure 5. TIE2 immunohistochemistry of human cancer cryosections. After TIE2 immunostaining, the sections were counterstained with hematoxylin and eosin and shown at lower (left) or higher (right) magnification. (A) Colon adenocarcinoma. In addition to vascular ECs (arrowheads), TIE2 staining highlights the presence of stromal mononuclear elements morphologically consistent with monocytes (arrows). These cells appear inhomogeneously distributed, with foci of high density (arrows). (B) Gastric undifferentiated adenocarcinoma. Many TIE2 mononuclear cells are found in the tumor stroma (arrows). Blood vessels are indicated by arrowheads. (C) Pancreatic adenocarcinoma. The majority of TIE2 structures in the left panel are blood vessels (arrowheads). An individual TIE2 mononuclear cell is shown (arrow) in the right panel.

Article Snippet: Neutralizing anti-TIE2 antibodies (R&D Systems) were preincubated with cells for 20 minutes at 37°C.

Techniques: Immunohistochemistry, Immunostaining, Staining

Journal: Blood

Article Title: Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer.

doi: 10.1182/blood-2006-10-053504

Figure Lengend Snippet: Figure 4. TIE2 monocytes are found in human tumors. Flow cytometry analysis of the indicated tumor specimens, processed and analyzed as described in the text. FITC-conjugated anti-CD31 or anti-CD14, PE-conjugated anti-TIE2, or APC- conjugated anti-CD45 antibodies were used. (A) Breast carcinoma. Note that the gated CD31CD45 tumor-derived ECs are TIE2 (red open line in the histogram on the right; filled line is the IgG isotype control). (B) Renal carcinoma. In this tumor specimen, approximately 2% of the tumor-derived cells are CD31 (CD45, not depicted) ECs. The wide majority of these CD31 tumor-derived ECs are TIE2. Note that a fraction of the tumor-derived cells are CD31TIE2 non-ECs (top right dot plot). In the same tumor sample, approximately 10% of tumor-derived cells are CD45

Article Snippet: Neutralizing anti-TIE2 antibodies (R&D Systems) were preincubated with cells for 20 minutes at 37°C.

Techniques: Flow Cytometry, Derivative Assay, Control

Journal: Blood

Article Title: Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer.

doi: 10.1182/blood-2006-10-053504

Figure Lengend Snippet: Figure 6. Confocal immunofluorescence analysis of human cancer sections confirms the presence of TIE2CD45CD14 tumor-infiltrating monocytes. (A) Colon adenocarcinoma analyzed for CD31 (green), TIE2 (red), and CD45 (blue). Confocal planes are shown individually and after merging. Several TIE2CD45CD31 hematopoietic cells (merge of red and blue giving purple; arrows) are found within the tumor stroma. Note that TIE2 is expressed by vascular ECs (merge of green and red giving yellow; arrowheads), which are TIE2CD45CD31. (B) Gastric adenocarci- noma analyzed for CD31 (green), TIE2 (red), and CD45 (blue). Some TIE2CD45CD31 hematopoietic cells are found in the tumor stroma (arrows) together with TIE2CD45CD31 tumor blood vessels (arrowhead). Scale bars as indicated. (C) Colon adenocarcinoma analyzed for CD14 (green), TIE2 (red), and CD11b (blue). Several TIE2CD14CD11b monocytes (arrows) are found within the tumor stroma. Note TIE2 expression by TIE2CD14CD11b vascular ECs (arrowheads). (D) Gastric adenocarcinoma analyzed for VWF (green), TIE2 (red), and CD11b (blue). Arrowheads indicate TIE2CD11bVWF monocytes. (E) Pancreatic adeno- carcinoma analyzed for CD16 (green), TIE2 (red), and CD14 (blue). High-magnification photos show the pres- ence of TIE2CD16CD14 monocytes (arrows) in the tumor stroma. (F) Colon adenocarcinoma analyzed for CD14 (green), TIE2 (red), and CD11b (blue). A triple- positive CD14TIE2CD11b TEM with peri-endothelial localization is indicated by the arrow. Note the presence of CD14TIE2CD11b inflammatory cells (arrowheads). Scale bars as indicated. (G) TIE2 expression in nonneo- plastic tissues is restricted to vascular ECs. Nonneoplas- tic colon mucosa adjacent to tumor tissue analyzed by confocal immunofluorescence staining of CD31 or CD14 (green), TIE2 (red), and CD45 (blue). Note that the lamina propria macrophages are CD14TIE2. A single CD14TIE2 monocyte (arrow) is found within a TIE2

Article Snippet: Neutralizing anti-TIE2 antibodies (R&D Systems) were preincubated with cells for 20 minutes at 37°C.

Techniques: Expressing, Staining

Journal: Blood

Article Title: Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer.

doi: 10.1182/blood-2006-10-053504

Figure Lengend Snippet: Figure 7. Circulating TIE2 monocytes migrate toward Ang-2 and have marked proangiogenic activity in vivo. (A) Modified Boyden chamber assays show migration of resident monocytes toward Ang-2. The 2 graphics show parts of 2 independent experiments of 3 performed. Both serum and Ang-2 induced significant migration of resident monocytes (left histograms; P .05 vs control: Medium). Heat inactivation of Ang-2 or treatment of the cells with neutralizing anti-TIE2 antibodies, but not with control immunoglobulins, abrogates cell migration in response to Ang-2 (right histograms). (B) Human glioma U87 cells were injected subcutaneously into nude mice with or without the indicated monocyte populations at a 1:100 or 1:20 ratio. Tumors were grown for 5 to 7 days and analyzed by CD31 immunostaining and confocal analysis to assess angiogenesis. Representative pictures of the tumors are shown. The tumor margin is indicated by a dashed line. (C) The mean vascular area (n 3 tumors/group) was calculated by digital image analysis and expressed as fold increase over the value obtained in tumors from U87 cells only. Error bars indicate SD. Statistical difference between groups was calculated by Student t test.

Article Snippet: Neutralizing anti-TIE2 antibodies (R&D Systems) were preincubated with cells for 20 minutes at 37°C.

Techniques: Activity Assay, In Vivo, Migration, Control, Injection, Immunostaining