Journal: Biochimica et biophysica acta

Article Title: Fibronectin conformation regulates the proangiogenic capability of tumor-associated adipogenic stromal cells.

doi: 10.1016/j.bbagen.2013.03.033

Figure Lengend Snippet: Fig. 2. Decellularized tumor-associated matrices influence adipogenic stromal cell adhesion and proangiogenic factor secretion. 3T3-L1 cells assemble a denser, more fibrillar matrix with increased Fn content when cultured in the presence of TCM [11], which was reflected with decellularized matrices prepared from control (Control) and TCM-treated 3T3-L1s (Tumor) using detergent-based methods. Scale bar = 20 μm (A). Tumor matrices decreased the total number of adherent 3T3-L1 per well as determined by counting trypsinized cells (B) and increased VEGF secretion per cell as analyzed via ELISA followed by normalization to cell number (C). Varied VEGF levels were not due to differential ECM sequestra- tion because ELISA of lysates of decellularized ECMs did not reveal statistically significant differences between conditions (D). * indicates p b 0.05.

Article Snippet: Subsequently, media was removed and analyzed using a human VEGF ELISA (R&D Systems).

Techniques: Cell Culture, Control, Enzyme-linked Immunosorbent Assay

Journal: Biochimica et biophysica acta

Article Title: Fibronectin conformation regulates the proangiogenic capability of tumor-associated adipogenic stromal cells.

doi: 10.1016/j.bbagen.2013.03.033

Figure Lengend Snippet: Fig. 3. Fn influences cell behavior in response to control and tumor ECMs. Addition of the control peptide Del29 during preparation of tumor ECMs does not alter Fn incorporation, whereas addition of the Fn polymerization inhibitor pUR4 markedly decreased Fn incorporation. Scale bar = 20 μm (A). Accordingly, tumor matrices prepared in the presence of pUR4 increased cell adhesion as determined by counting of trypsinized cells (B) and diminished VEGF secretion as analyzed via ELISA followed by normalization to cell number (C) to levels comparable to control matrices. * indicates p b 0.05.

Article Snippet: Subsequently, media was removed and analyzed using a human VEGF ELISA (R&D Systems).

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: Biochimica et biophysica acta

Article Title: Fibronectin conformation regulates the proangiogenic capability of tumor-associated adipogenic stromal cells.

doi: 10.1016/j.bbagen.2013.03.033

Figure Lengend Snippet: Fig. 4. Tumor ECM-induced VEGF secretion by stromal cells stimulates endothelial cell mi- gration. Media collected from 3T3-L1 cells cultured on tumor ECMs increased HUVEC mi- gration in transwell migration assays relative to media collected from control ECMs, as measured via image analysis of DAPI-stained, fixed membranes following migration. Typ- ical micrographs of migrated HUVECs stained with DAPI are shown to the right. Scale bar = 20 μm (A). Blockade of VEGF signaling using a VEGFR2/KDR peptide antagonist confirmed that greater VEGF levels secreted by stromal cells cultured on tumor ECMs con- tributed to changes in HUVEC migration (B). * indicates p b 0.05.

Article Snippet: Subsequently, media was removed and analyzed using a human VEGF ELISA (R&D Systems).

Techniques: Cell Culture, Migration, Control, Staining

Journal: Biochimica et biophysica acta

Article Title: Fibronectin conformation regulates the proangiogenic capability of tumor-associated adipogenic stromal cells.

doi: 10.1016/j.bbagen.2013.03.033

Figure Lengend Snippet: Fig. 5. Conducting polymer films to define the effect of Fn conformation on adhesion and VEGF secretion. Oxidized (+1 V) or reduced (−1 V) PEDOT:PSS surfaces were coated with media containing either fibronectin (Fn) only, or 10% serum (containing Fn), to es- tablish compact and partially unfolded conformations of adsorbed Fn, respectively. The number of 3T3-L1 cells per well adhered to unfolded Fn was decreased relative to compact Fn (A), while an opposite effect was detected for VEGF secretion (B) for both Fn and serum-coated devices. These differences were determined by image analysis of DAPI-stained fixed devices and VEGF ELISA followed by normalization to cell number. * in- dicates p b 0.05.

Article Snippet: Subsequently, media was removed and analyzed using a human VEGF ELISA (R&D Systems).

Techniques: Polymer, Staining, Enzyme-linked Immunosorbent Assay

Journal: Biochimica et biophysica acta

Article Title: Fibronectin conformation regulates the proangiogenic capability of tumor-associated adipogenic stromal cells.

doi: 10.1016/j.bbagen.2013.03.033

Figure Lengend Snippet: Fig. 6. Increased VEGF levels in response to partial Fn unfolding are not due to differential binding to Fn and are relevant to additional cell types. Incubation of cell-free, serum- coated devices with recombinant VEGF followed by ELISA of non-bound VEGF in the media suggested that VEGF preferentially interacts with partially unfolded Fn and, hence, confirms that varied secretions rather than differential binding were responsible for the differences in VEGF levels detected above (A). Similar to 3T3-L1, human bone marrow-derived MSCs enhanced secretion of VEGF when seeded onto substrates with partially unfolded Fn relative to compact Fn as examined via VEGF ELISA followed by nor- malization to cell number (B). * indicates p b 0.05.

Article Snippet: Subsequently, media was removed and analyzed using a human VEGF ELISA (R&D Systems).

Techniques: Binding Assay, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Biochimica et biophysica acta

Article Title: Fibronectin conformation regulates the proangiogenic capability of tumor-associated adipogenic stromal cells.

doi: 10.1016/j.bbagen.2013.03.033

Figure Lengend Snippet: Fig. 7. Varied Fn conformation regulates adhesion and VEGF secretion via altered integrin engagement. Individual and combined blockade of synergy-dependent (α5β1) and -independent (αvβ3) integrins via β1- and αv-function blocking antibodies, respectively, suggested that α5β1 inhibits VEGF secretion of 3T3-L1 in response to compact Fn, which may be compensated by increased engagement of αvβ3 (white bars). Additionally, αvβ3 stimulates VEGF secretion in response to partially unfolded Fn, which is independent of α5β1 usage (black bars). * indicates p b 0.05.

Article Snippet: Subsequently, media was removed and analyzed using a human VEGF ELISA (R&D Systems).

Techniques: Blocking Assay

Journal: PLoS ONE

Article Title: The Effect of Vascular Endothelial Growth Factor on Osteoclastogenesis in Rheumatoid Arthritis

doi: 10.1371/journal.pone.0124909

Figure Lengend Snippet: (A) The synovial fluid samples of 32 RA patients were collected and their VEGF and soluble RANKL concentrations were determined by sandwich ELISA. (B) The serum samples of 32 RA patients were collected and their VEGF and soluble RANKL concentrations were determined by sandwich ELISA. Each dot expresses the results from an individual patient. (C) The synovial tissues of patients with RA and osteoarthritis (OA) were simultaneously labeled with anti-VEGF (green), anti-RANKL (red), and CD55 (white) antibodies and then photographed under appropriate filters. The merged image shows co-localization of the three markers (yellow). Sections were counterstained with DAPI staining. The figures are representative of three independent experiments (original magnification 400×).

Article Snippet: Anti-human VEGF, anti-human VEGFR1, Anti-human VEGFR2 and anti-CD55 antibodies were purchased from R&D Systems.

Techniques: Sandwich ELISA, Labeling, Staining

Journal: PLoS ONE

Article Title: The Effect of Vascular Endothelial Growth Factor on Osteoclastogenesis in Rheumatoid Arthritis

doi: 10.1371/journal.pone.0124909

Figure Lengend Snippet: (A) After RA synovial fibroblasts were cultured with 0–50 ng/ml of VEGF for 72 h, the RANKL mRNA expression determined by RT-PCR. Data were normalized to beta-actin and reported in relative expression units. The figure is representative of three experiments. (B) RA synovial fibroblasts were cultured with VEGF for 72 h, and RANKL concentration in the cultured media was measured by sandwich ELISA. (C) RA synovial fibroblasts were cultured with VEGF for 72 h and then stained with anti-RANKL antibodies (red) (original magnification 400×). The figures are representative of three independent experiments. (D) Triplicate wells of RA synovial fibroblasts were transfected with 1 μg of pGL3-RANKL reporter plasmids and 1 μg of pRLTk control plasmid. Both firefly and renilla luminescence were measured after 24 h incubation with 20ng/ml of VEGF. (E) After RA synovial fibroblasts were cultured with VEGF for 72 h, the concentrations of IL-1β, TNF-α, and IL-6 in the cultured media was determined by sandwich ELISA. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: Anti-human VEGF, anti-human VEGFR1, Anti-human VEGFR2 and anti-CD55 antibodies were purchased from R&D Systems.

Techniques: Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Sandwich ELISA, Staining, Transfection, Control, Plasmid Preparation, Incubation

Journal: PLoS ONE

Article Title: The Effect of Vascular Endothelial Growth Factor on Osteoclastogenesis in Rheumatoid Arthritis

doi: 10.1371/journal.pone.0124909

Figure Lengend Snippet: (A) RA synovial fibroblasts were pretreated with anti-VEGFR1 (20 ng/ml), anti-VEGFR2 (20 ng/ml), SB203580, a p38 MAPK inhibitor (10 nM), Src inhibitor (10 nM), or PKC inhibitor (5 nM) for 1 h, and then cultured with 20 ng/ml VEGF for 72 h. The expression of RANKL mRNA was determined by real time-PCR. Data were normalized to beta-actin and reported in relative expression units. (B) RA synovial fibroblasts were stimulated with 20 ng/ml VEGF, the phosphorylated forms of Src, PKC, and ERK were detected by western blotting. The figures are representative of three independent experiments. (C) Stimulation of RA synovial fibroblasts with VEGF activated the phosphorylation of p-Src, Src, p-PKC, PKC, p-ERK and ERK as detected by Western blotting and shown by the ratio of phosphorylated to total proteins. Data were normalized to beta-actin and reported in relative expression units. The figure represents one of three independent experiments. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: Anti-human VEGF, anti-human VEGFR1, Anti-human VEGFR2 and anti-CD55 antibodies were purchased from R&D Systems.

Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Phospho-proteomics

Journal: PLoS ONE

Article Title: The Effect of Vascular Endothelial Growth Factor on Osteoclastogenesis in Rheumatoid Arthritis

doi: 10.1371/journal.pone.0124909

Figure Lengend Snippet: (A) CD14+ monocytes isolated from peripheral blood of RA patients were cultured with 25 ng/ml M-CSF and 0–50 ng/ml VEGF or 30 ng/ml RANKL. After maximal 21 days of culturing, TRAP-positive multinucleated cells were counted. The figures represent one of three independent experiments. (B) The gene expression of osteoclast markers such as TRAP, RANK, CTR, cathepsin K, and MMP-9 from differentiated osteoclasts measured by real-time PCR. Data were normalized to beta-actin and reported in relative expression units. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: Anti-human VEGF, anti-human VEGFR1, Anti-human VEGFR2 and anti-CD55 antibodies were purchased from R&D Systems.

Techniques: Isolation, Cell Culture, Gene Expression, Real-time Polymerase Chain Reaction, Expressing

Journal: PLoS ONE

Article Title: The Effect of Vascular Endothelial Growth Factor on Osteoclastogenesis in Rheumatoid Arthritis

doi: 10.1371/journal.pone.0124909

Figure Lengend Snippet: (A) CD14+ monocytes were cultured with M-CSF and 20 ng/ml of VEGF in the presence of 20ng/ml of anti-VEGFR1, 20ng/ml of anti-VEGFR2, or 10nM of p38 MAPK inhibitor. After 21 days of culturing, TRAP+ multinucleated cells were counted. The figure represents one of three independent experiments. (B) CD14+ monocytes were cultured with M-CSF and 20 ng/ml of VEGF in the presence of Src inhibitor (10 nM), or PKC inhibitor (5 nM). After 21 days of culturing, TRAP+ multinucleated cells were counted. The figure represents one of three independent experiments. (C) The gene expression of TRAP, RANK, CTR, cathepsin K, and MMP-9 from differentiated osteoclasts was measured by real-time PCR. Data were normalized to beta-actin and reported in relative expression units. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: Anti-human VEGF, anti-human VEGFR1, Anti-human VEGFR2 and anti-CD55 antibodies were purchased from R&D Systems.

Techniques: Cell Culture, Gene Expression, Real-time Polymerase Chain Reaction, Expressing

Journal: PLoS ONE

Article Title: The Effect of Vascular Endothelial Growth Factor on Osteoclastogenesis in Rheumatoid Arthritis

doi: 10.1371/journal.pone.0124909

Figure Lengend Snippet: (A) RA synovial fibroblasts were cultured with 20 ng/ml of VEGF for 72 h and RANKL production was quantified using ELISA in the cultured media. (B) RA synovial fibroblasts were preincubated with 20 ng/ml of VEGF for 72 h and Src inhibitor (10 nM) or PKC inhibitor (5 nM) and then cocultured with CD14+ monocytes from the peripheral blood in the presence of M-CSF. After 21 days of culturing, TRAP-positive multinucleated cells were counted. The figure represents one of three independent experiments. (B) The gene expressions of TRAP, RANK, CTR, cathepsin K, and MMP-9 from differentiated osteoclasts measured by real-time PCR. Data were normalized to beta-actin and reported in relative expression units. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: Anti-human VEGF, anti-human VEGFR1, Anti-human VEGFR2 and anti-CD55 antibodies were purchased from R&D Systems.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: Molecular medicine reports

Article Title: Intraperitoneal injection of thalidomide alleviates early osteoarthritis development by suppressing vascular endothelial growth factor expression in mice.

doi: 10.3892/mmr.2018.8980

Figure Lengend Snippet: Figure 2. mRNA expression levels of VEGF and MMP‑13 in the knee articular cartilage of mice among the Sham, Dmm and Dmm+Th groups (n=4 in each group). (A) Relative mRNA expression levels of VEGF in the medial articular cartilage. (B) Relative mRNA expression levels of MMP‑13 in the medial articular cartilage. The values are presented as the mean ± standard deviation. *P<0.05 compared with the Sham group; #P<0.05 compared with the Dmm group. Dmm, destabilization of the medial meniscus; MMP‑13, matrix metalloproteinase‑13; Th, thalidomide; VEGF, vascular endothelial growth factor.

Article Snippet: An ELISA kit of VEGF (E-EL-M1292c) was purchased from Elabscience Biotechnology Co., Ltd., Wuhan, China.

Techniques: Expressing, Standard Deviation

Journal: Molecular medicine reports

Article Title: Intraperitoneal injection of thalidomide alleviates early osteoarthritis development by suppressing vascular endothelial growth factor expression in mice.

doi: 10.3892/mmr.2018.8980

Figure Lengend Snippet: Figure 3. Immunohistochemical analysis of VEGF expression in the knee articular cartilage of mice among the Sham, Dmm and Dmm+Th groups (n=4 in each group). (A) Immunohistochemistry staining of VEGF in the articular cartilage of the medial tibial plateau (magnification, x400, scale bar=100 µm). (B) Quantification of VEGF positive cells, based on the results of immunohistochemistry staining. The values are presented as the mean ± standard deviation. *P<0.05 compared with the Sham group; #P<0.05 compared with the Dmm group. Dmm, destabilization of the medial meniscus; Th, thalidomide; VEGF, vascular endothelial growth factor.

Article Snippet: An ELISA kit of VEGF (E-EL-M1292c) was purchased from Elabscience Biotechnology Co., Ltd., Wuhan, China.

Techniques: Immunohistochemical staining, Expressing, Immunohistochemistry, Staining, Standard Deviation

Journal: Molecular medicine reports

Article Title: Intraperitoneal injection of thalidomide alleviates early osteoarthritis development by suppressing vascular endothelial growth factor expression in mice.

doi: 10.3892/mmr.2018.8980

Figure Lengend Snippet: Figure 5. ELISA analysis of serum VEGF concentration of mice among the Sham, Dmm and Dmm+Th groups (n=8 in each group). The values are presented as the mean ± standard deviation. *P<0.05 compared with the Sham group; #P<0.05 compared with the Dmm group. Dmm, destabilization of the medial meniscus; Th, thalidomide; VEGF, vascular endothelial growth factor.

Article Snippet: An ELISA kit of VEGF (E-EL-M1292c) was purchased from Elabscience Biotechnology Co., Ltd., Wuhan, China.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Standard Deviation

Journal: iScience

Article Title: Induced retinal pigment epithelial cells with anti-epithelial-to-mesenchymal transition ability delay retinal degeneration

doi: 10.1016/j.isci.2022.105050

Figure Lengend Snippet: iRPE cells have similar phenotype and functions as iPSC-RPE cells (A) Schematic for the transforming process. A cocktail of TF-expressing retroviruses was used to transfect De-iPSC-RPE cells. After seven days, iRPE clone was observed in the culture and picked out for subculturing. (B and C) RPE-specific and EMT-associated markers detected by (B) immunostaining and (C) western blotting after cells were cultured for 8 days. The expression pattern of these markers in iRPE cells is more similar to that in iPSC-RPE cells. Scale bar = 50 μm. (D) Electron micrographs of iPSC-RPE cells, De-iPSC-RPE cells, and iRPE cells demonstrated that quite a few microvilli were on the surface of iRPE and iPSC-RPE cells. Scale bar = 0.5 μm. (E and F) The bound and phagocyted POSs (pointed by arrows) in iPSC-RPE, De-iPSC-RPE, and iRPE cells (E) and quantification of phagocytosis (F) as determined by the number of bound and phagocyted POS per field. Scale bar = 50 μm. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n ≥ 5. (G and H) TER analysis (G) and HRP permeability assay (H) showed that iRPE cells maintained the same epithelial integrity as iPSC-RPE cells. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 6. (I) Expression levels of PEDF and VEGF from upper and lower chambers were determined by ELISA. iRPE cells and iPSC-RPE cells demonstrated similar secretion patterns. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 3.

Article Snippet: PEDF and VEGF were quantified by PEDF ELISA kit (Elabscience, Wuhan, China) and VEGF ELISA kit (Proteintech).

Techniques: Expressing, Subculturing Assay, Immunostaining, Western Blot, Cell Culture, Permeability, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Induced retinal pigment epithelial cells with anti-epithelial-to-mesenchymal transition ability delay retinal degeneration

doi: 10.1016/j.isci.2022.105050

Figure Lengend Snippet: BMP7 and FOXF2 are critical regulators of EMT in iRPE cells (A) The higher level of BMP7 secreted by iRPE cells compared with De-iPSC-RPE cells was determined by ELISA. Data are mean ± SD, unpaired two-sided t-tests, n = 4. (B and C) The reduced expression level of FOXF2 in iRPE compared with that in De-iPSC-RPE cells was determined by (B) immunostaining and (C) western blotting. Scale bar = 50 μm. (D) The efficiency of bmp7 knockdown was determined by qRT-PCR; shBmp7-1 was slightly more efficient at reducing the mRNA level of bmp7 than shBmp7-2; therefore, it was selected for subsequent experiments. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 4. (E) The shBmp7 construct contained the ZsGreen expression element to indicate successful transfection. Scale bar = 50 μm. (F and G) FLAG-FOXF2 overexpression (ov-FOXF2) in (F) iRPE cells (G) shBmp7-iRPE cells. Scale bar = 50 μm. (H and I) The expression levels of RPE-specific markers and EMT markers were determined by (H) western blotting and (I) quantitative analysis. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 3. (J) BMP7 expression levels in shCont-iRPE, shBmp7-iRPE, ov-FOXF2- iRPE, and shBmp7 + ov-FOXF2-iRPE. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 4. (K) The immunostaining of RPE-specific markers and EMT markers. Overexpression of FOXF2 exhibited similar effects to knockdown of bmp7 in iRPE cells by downregulating RPE markers and upregulating EMT marker. Scale bar = 50 μm.

Article Snippet: PEDF and VEGF were quantified by PEDF ELISA kit (Elabscience, Wuhan, China) and VEGF ELISA kit (Proteintech).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Immunostaining, Western Blot, Knockdown, Quantitative RT-PCR, Construct, Transfection, Over Expression, Marker

Journal: iScience

Article Title: Induced retinal pigment epithelial cells with anti-epithelial-to-mesenchymal transition ability delay retinal degeneration

doi: 10.1016/j.isci.2022.105050

Figure Lengend Snippet: Four TFs transcriptionally regulate bmp7 , foxf2 , lin7a , pard6b , and ppm1a in a direct or indirect manner (A) ELISA analysis demonstrated that BMP7 levels was reduced in 4TFs−nr2e1-RPE, 4TFs−mitf-a-RPE, 4TFs−c-myc-RPE, and 4TFs−crx-RPE cells compared with that in iRPE cells. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 4. (B and C) The levels of FOXF2, LIN7A, PARD6B, and PPM1A were determined by (B) western blotting and (C) quantitative analysis. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 3. (D) Generation of FLAG-MITF-A-iRPE, FLAG-CRX-iRPE, FLAG-NR2E1-iRPE, and FLAG-C-MYC-iRPE cells. Scale bar = 50 μm. (E–I) Enriched peaks of CRX binding to the target genes and fold enrichment of CRX immunoprecipitation compared with IgG control, as determined by qRT-PCR. Data are mean ± SD, unpaired two-sided t-tests, n = 3. (J–M) Enriched peaks of MITF-A and NR2E1 binding to the target gene lin7a and fold enrichment of MITF-A and NR2E1 immunoprecipitation compared with IgG control, as determined by qRT-PCR. Data are mean ± SD, unpaired two-sided t-tests, n = 3. (N) Schematic model for the regulation of EMT and MET processes by the four TFs. CRX, MITF-A, NR2E1, and C-MYC directly or indirectly regulated the expression of bmp7 , lin7a , pard6b, ppm1a , and foxf2 to inhibit EMT and promote MET.

Article Snippet: PEDF and VEGF were quantified by PEDF ELISA kit (Elabscience, Wuhan, China) and VEGF ELISA kit (Proteintech).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Binding Assay, Immunoprecipitation, Control, Quantitative RT-PCR, Expressing

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD34 Hybrid Cells Promote Endothelial Colony-Forming Cell Bioactivity and Therapeutic Potential for Ischemic Diseases

doi: 10.1161/atvbaha.112.301052

Figure Lengend Snippet: Figure 1. The role of CD34− accessory cells in CD34+ stem cell–derived endothelial progenitor cell (EPC) commitment. A, A schematic diagram of the human EPC colony-forming assay (CFA) used to evaluate the effect of functional CD34− cells on EPC colony-forming units (CFUs). B, Differentiation of 2 types of EPC colony clusters, small EPC-CFUs and large EPC-CFUs, from 3 kinds of cell populations. Small EPC-CFUs were round-shaped, and large EPC-CFUs were spindle-shaped (magnification, ×40). C, Standard quantification of EPC-CFUs was performed by counting the number of small, large, and total EPC-CFUs. Results are shown as the mean±SEM (*P<0.05 and **P<0.01 vs CD34+ cells). D, Schematic diagram of the insert culture assay used to assess the effect of functional CD34− cells on CD34+ cell- mediated endothelial cell (EC)-lineage commitment. E, Standard quantification of EPC-CFUs was performed by calculating the number of expanded EC progenitor colonies using CD34+ cells (lower chamber) cocultured with CD34− cells, CD11b+/CD34− cells (macrophage), or CD11b−/CD34− cells (upper chamber). The results were shown as the mean±SEM (*P<0.05 and **P<0.01 vs CD34+ cells). F, Expression of angiogenic cytokines in tumor necrosis factor (TNF)α-treated or untreated CD34− cell–derived macrophages. G, Effect of stromal cell– derived factor (SDF)-1α and vascular endothelial growth factor (VEGF) on human EPC-CFUs. In response to stimulation with SDF-1α and VEGF, the frequency of large EPC-CFUs was significantly increased (*P<0.05 and **P<0.01 vs basal control cytokines: stem cell factor, VEGF, interleukin-3, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-1). H, Expression of endothelial lin- eage markers for kinase insert domain receptor, C-X-C chemokine receptor 4, and Tie2 on transwell-cultured cells with or without CD34− cells. HUCB indicates human umbilical cord blood; and MNC, mononuclear cell.

Article Snippet: SDF-1α and VEGF levels were determined by Quantikine ELISA Human SDF-1α Immunoassay and Quantikine ELISA Human VEGF Immunoassay (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Derivative Assay, Functional Assay, Expressing, Control, Cell Culture

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD34 Hybrid Cells Promote Endothelial Colony-Forming Cell Bioactivity and Therapeutic Potential for Ischemic Diseases

doi: 10.1161/atvbaha.112.301052

Figure Lengend Snippet: Figure 2. Characterization and functional capacity of CD34−/CD34+ cell–derived endothelial colony-forming cells (hybrid-dECFCs) and CD34+ cell–derived ECFCs (stem-dECFCs). A, Hybrid-dECFCs and stem-dECFCs are spindle-shaped cells (similar to outgrowth endothelial cells [OECs]; magnification, ×40). B, Human ECFCs were characterized by the endothelial cell (EC) markers CD31, vascular endothelial growth factor (VEGFR)2 (KDR), and von Willebrand factor (vWF), as well as pivotal markers of functional ECFCs, including phosphor-protein kinase B, endothelial nitric oxide synthase (eNOS), and p-eNOS. C, Expression of surface markers for CD34, CD133, kinase insert domain receptor (KDR), C-X-C chemokine receptor 4 (CXCR4), and c-Kit on 2 types of ECFCs measured by flow cytometry. Fluorescence-activated cell sorter (FACS) analysis indicated the ratios of CD34/CD133, KDR, CXCR4, or c-Kit positive cells. D, Hybrid- dEPC (passages 6–20) and stem-dEPC (passages 6–16) lysates containing equal amounts of total protein were analyzed by Western blot- ting using anti-cyclin-dependent kinase (Cdk)-2, anticyclin E, anti-Cdk4, and anticyclin D1 antibodies. E, Both types of ECFCs (passages 6–16) were treated with VEGF (100 ng/mL), and cell proliferation was examined via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetra- zolium bromide assay after 24 hours. F, Senescence-associated β-galactosidase (SA-β-gal) activity of the 2 types of ECFCs (passages 6–16). The green cells were counted as positive for senescence (magnification, ×40). G, Hybrid-dECFC (passages 6–20) and stem-dECFC (passages 6–16) lysates containing equal amounts of total protein were analyzed by Western blotting using senescence markers with anti-senescence marker protein (SMP)-30 and antip21 antibodies. Results are shown as the mean±SEM (*P<0.05 and **P<0.01 vs hybrid- dECFCs). DAPI indicates 4',6-diamidino-2-phenylindole; EBM, endothelial basal medium; and SMP, senescence marker protein.

Article Snippet: SDF-1α and VEGF levels were determined by Quantikine ELISA Human SDF-1α Immunoassay and Quantikine ELISA Human VEGF Immunoassay (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Functional Assay, Derivative Assay, Expressing, Flow Cytometry, Fluorescence, Western Blot, Activity Assay, Marker

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD34 Hybrid Cells Promote Endothelial Colony-Forming Cell Bioactivity and Therapeutic Potential for Ischemic Diseases

doi: 10.1161/atvbaha.112.301052

Figure Lengend Snippet: Figure 5. Proliferation, survival, and secretion of angiogenic growth factors of endothelial colony-forming cells (ECFCs) in hind-limb isch- emia. At day 3 after surgery, samples harvested from hind-limb ischemic tissues were stained to determine the proliferation, survival, and secretion of angiogenic growth factors of the transplanted ECFCs (passage 6). A, Proliferative cells in ischemic injury sites visualized by immunofluorescent staining for proliferating cell nuclear antigen (PCNA; red). B, Standard quantification of proliferating cells represented as the number of PCNA/4',6-diamidino-2-phenylindole (DAPI) double-positive cells per high-power field. Results are shown as the mean±SEM (*P<0.05 and **P<0.01 vs sham, ##P<0.05 vs CD34+ cell–derived ECFCs [stem-dECFCs]). C, Proliferative transplanted cells at the ischemic injury sites indicated by human nuclear antigen (HNA; red), Ki-67 (green), and DAPI (blue) triple-positive cells. White color indicates triple-positive cells in the merged images. D, Apoptotic transplanted cells in ischemic injury shown as HNA (red), caspase-3 (green), and DAPI (blue) triple-positive cells. E, Standard quantification of proliferating transplanted cells represented as the number of HNA/Ki-67/DAPI triple-positive cells (white) per high-power field. F, Standard quantification of apoptotic cells represented as the num- ber of HNA/caspase-3/DAPI triple-positive cells (white) per high-power field. Results are shown as the mean±SEM (**P<0.01 vs CD34−/ CD34+ cell–derived ECFCs [hybrid-dEPC]). G–K, Secretion of angiogenic growth factor from transplanted ECFCs in injury sites visualized by fibroblast growth factor (FGF-2; G), hepatocyte growth factor (HGF; H), stromal cell–derived factor (SDF)-1α (I), vascular endothelial growth factor (VEGF; J), and interleukin (IL)-8 (K) staining (green) and HNA staining (red). L At day 3 after surgery, samples harvested from hind-limb ischemic tissues were analyzed to confirm the secretion of angiogenic growth factor at the injury sites by Western blot- ting. Western blots of ischemic tissue homogenates indicated secretion of FGF-2, HGF, SDF-1α, VEGF, and IL-8. Hvf indicates high visual field.

Article Snippet: SDF-1α and VEGF levels were determined by Quantikine ELISA Human SDF-1α Immunoassay and Quantikine ELISA Human VEGF Immunoassay (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Staining, Derivative Assay, Western Blot

Journal: Parasites & Vectors

Article Title: Effect of sunitinib against Echinococcus multilocularis through inhibition of VEGFA-induced angiogenesis

doi: 10.1186/s13071-023-05999-4

Figure Lengend Snippet: Efficacy of SU11248 against E. multilocularis metacestodes in vivo. Ten weeks after infection of C57BL/6 mice with protoscoleces, the mice were administered SU11248 intragastrically for 6 weeks. Uninfected, ABZ-treated, or PBS-treated mice served as the blank control, positive control, and negative control, respectively. a Gross morphology of metacestodes. b Intrahepatic manifestation for metacestode liver metastases in different treatment groups. c Echinococcus multilocularis metacestodes in different treatment groups were observed by scanning electron microscopy (SEM). The red arrow indicates the cells of the germinal layer. Scale bar = 20 μm. d Statistical analysis of mouse body weight changes. e Statistical analysis of liver weight changes. f Statistical analysis of spleen weight changes. g Statistical analysis of the intraperitoneal metacestode weight changes. h , i Cytokine expression in serum was measured by enzyme-linked immunosorbent assay. Serum levels of interleukin (IL)-4 ( h ) and vascular endothelial growth factor A (VEGFA) ( i ). The means ± SD are shown

Article Snippet: VEGFA and IL-4 levels in the culture supernatants or the serum were determined using mouse VEGFA-specific or IL-4-specific ELISA kits (Cloud-Clone Corp., Wuhan, China), following the manufacturer’s instructions.

Techniques: In Vivo, Infection, Positive Control, Negative Control, Electron Microscopy, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Parasites & Vectors

Article Title: Effect of sunitinib against Echinococcus multilocularis through inhibition of VEGFA-induced angiogenesis

doi: 10.1186/s13071-023-05999-4

Figure Lengend Snippet: SU11248 inhibition of VEGFA-induced phosphorylation of VEGFR2 in liver cells from C57BL/6 mice. The hepatic cells were co-cultured with protoscoleces, and then SU11248 was added directly at final concentrations of 0, 40, 100, and 500 nM, with further incubation for another 48 h. The hepatic cell group co-cultured without protoscoleces was treated with 0.1% DMSO for 48 h. a Cell supernatants were analyzed for VEGFA by ELISA. The means ± SD of triplicate experiments are shown. b Western blotting was used to detect the protein expression of VEGFA, VEGFR2, and p-VEGFR2. c Quantitative analysis of VEGFA, VEGFR2, and p-VEGFR2 protein levels. The means ± SD of triplicate experiments are shown. d Messenger RNA expression levels of VEGFA and VEGFR2 were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The data are presented as the mean ± SD of triplicate experiments. For other abbreviations, see Fig.

Article Snippet: VEGFA and IL-4 levels in the culture supernatants or the serum were determined using mouse VEGFA-specific or IL-4-specific ELISA kits (Cloud-Clone Corp., Wuhan, China), following the manufacturer’s instructions.

Techniques: Inhibition, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, RNA Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Clinical and Experimental Obstetrics & Gynecology

Article Title: Predicting Tumour Progression of ID-8 Syngeneic Mouse Ovarian Cancer with Blood Biomarkers of CA-125, IL-6 and VEGF: A Prospective Laboratory-Based Study

doi: 10.31083/j.ceog5010212

Figure Lengend Snippet: Fig. 5. Levels of biomarkers in blood and ascitic fluids of mice with tumour. Levels of CA-125 (A), IL-6 (B), VEGF (C) and ascitic

Article Snippet: Mouse IL-6 DuoSet and mouse VEGF DuoSet were purchased from R&D systems (Minneapolis, MN, USA).

Techniques:

Journal: bioRxiv

Article Title: ASK1/p38-mediated NLRP3 inflammasome signaling pathway contributed to aberrant retinal angiogenesis in diabetic retinopathy

doi: 10.1101/763102

Figure Lengend Snippet: (A) The inflammatory related cytokine IL-6, TNF-α and IL-1β mRNA expression levels were enhanced in DR group. (B) The expression of vascular marker CD31 was increased in DR group by confocal detection. Meanwhile, (C) the VEGF secretion levels in DR group was increased compared with control group. Values are expressed as the mean ± SD (n = 6, *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control).

Article Snippet: The retinal tissue from control group, HG group and HG treated with inhibitor group were added in PBS and homogenized; the homogenate was centrifuged at 12000 g at 4°C for 10 min. Then, the supernatant was tested for VEGF by mouse VEGF ELISA kit (R&D Systems, MN, USA).

Techniques: Expressing, Marker, Control

Journal: bioRxiv

Article Title: ASK1/p38-mediated NLRP3 inflammasome signaling pathway contributed to aberrant retinal angiogenesis in diabetic retinopathy

doi: 10.1101/763102

Figure Lengend Snippet: (A) The protein expression level of NLRP3 was enhanced in DR groups; moreover, (B) blocking NLRP3, the mRNA expression of IL-6, TNF-α and IL-1β was down-regulated; meanwhile, (C) the vascular marker CD31 expression level and (D) VEGF secretion level were decreased through inhibiting NLRP3. In addition, in HRMEC cell model, (E) high glucose enhanced the protein expression of NLRP3, which was inhibited by NLPR3 inhibitor. (F) The inflammatory related cytokine IL-6, TNF-α and IL-1β was enhanced in HG-induced HRMEC cell model and was inhibited through blocking NLRP3. (G) The VEGF secretion level was enhanced, and was decreased after inhibiting NLRP3. Data are presented as the mean ± standard deviation from triplicate wells. *P < 0.05 and **P < 0.01 compared with the control. #P < 0.05 and ## P < 0.01 compared with the relative DR animal model group or HG-induced HRMEC cell group.

Article Snippet: The retinal tissue from control group, HG group and HG treated with inhibitor group were added in PBS and homogenized; the homogenate was centrifuged at 12000 g at 4°C for 10 min. Then, the supernatant was tested for VEGF by mouse VEGF ELISA kit (R&D Systems, MN, USA).

Techniques: Expressing, Blocking Assay, Marker, Standard Deviation, Control, Animal Model

Journal: bioRxiv

Article Title: ASK1/p38-mediated NLRP3 inflammasome signaling pathway contributed to aberrant retinal angiogenesis in diabetic retinopathy

doi: 10.1101/763102

Figure Lengend Snippet: (A) The protein expression levels of AKS1 and p38 were up-regulated in DR animal group. (B)The result of HG-induced HRMEC group was similar with DR animal group. Moreover, (C) the NLPR3, IL-6, TNF-α, IL-1β and VEGF protein expression levels were inhibited by (C) NLPR3 inhibitor and (D) p38 inhibitor.

Article Snippet: The retinal tissue from control group, HG group and HG treated with inhibitor group were added in PBS and homogenized; the homogenate was centrifuged at 12000 g at 4°C for 10 min. Then, the supernatant was tested for VEGF by mouse VEGF ELISA kit (R&D Systems, MN, USA).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: USP12 promotes breast cancer angiogenesis by maintaining midkine stability

doi: 10.1038/s41419-021-04102-y

Figure Lengend Snippet: A The relative expression of VEGF-A and VEGF-C in the supernatant of MDA-MB-231 cells overexpressing USP12 was detected by ELISA. B The relative expression of VEGF-A and VEGF-C in the supernatant of MDA-MB-231 cells with USP12 knockdown was detected by ELISA. C , D Migration of HUVECs treated with supernatant from MDA-MB-231 cells with USP12 overexpression ( C ) and USP12 knockdown ( D ). Representative images are shown in the left panel. The quantitative results are shown in the right panel. E , F . Tube formation assay of HUVECs treated with supernatant from MDA-MB-231 cells with USP12 overexpression ( E ) and USP12 knockdown ( F ). Representative images are shown in the left panel. The quantitative results are shown in the right panel. The experiments were repeated three times. ** p < 0.01, *** p < 0.001; mean ± SD.

Article Snippet: Matrigel matrix (356234, Corning), the human VEGF-A ELISA kit (EKO588-96, Boster) and the human VEGF-C ELISA KIT (EKO539-96, Boster) were also used.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Migration, Over Expression, Tube Formation Assay