Journal: Scientific Reports

Article Title: An efficient protein production system via gene amplification on a human artificial chromosome and the chromosome transfer to CHO cells

doi: 10.1038/s41598-019-53116-2

Figure Lengend Snippet: Anti-VEGF antibody production via the IR/MAR HAC system. ( A ) The VEGF loxP MAR vector includes the RPS7 promoter to drive anti-VEGF antibody Hc and Lc gene expression, the IR/MAR sequence, and the loxP site. ( B ) The site-specific insertion event was detected in G418-resistant clones with genomic PCR analysis. A primer was designed to span the HAC insertion junction-specific site. VEGF HAC clones and VEGF MAR HAC clones were G418-resistant CHO DG44 clones. HAC indicates the parental CHO DG44 cells including an empty HAC, which was used as a negative control. Cropped gels were used in this figure. Original full-length gels are presented in Supplementary Fig. . ( C ) Relative copy number analysis of anti-VEGF antibody Hc and Lc genes on the HAC vector with qPCR. Data were normalized to NV1 . The copy number of anti-VEGF antibody genes in VEGF HAC clone 1 was arbitrarily at set as 1. Bars correspond to the means ± SD of three independent experiments. ( D,E ) Two-color FISH analysis of the HAC vector in G418-resistant CHO DG44 clones was performed with the digoxigenin-labeled hCot-1 DNA (red) and biotin-labeled anti-VEGF antibody gene (green). The arrow indicates the HAC, and the inset shows enlarged images of the HAC (anti-VEGF antibody probe-specific green signals are indicated by the arrowheads). Panel (D) shows VEGF HAC control clones. The left panel shows clone 1. The right panel shows clone 2. Panel (E) shows VEGF MAR-HAC clones. The left panel shows clone 1. The right panel shows clone 2. ( F ) Viable cell density in CHO DG44 VEGF HAC clones and CHO DG44 VEGF MAR-HAC clones. Bars correspond to the means ± SD of three independent experiments. ( G ) Analysis of anti-VEGF antibody production from CHO DG44 VEGF HAC clones and CHO DG44 VEGF MAR-HAC clones was measured using ELISA from day 2 to day 6. Bars correspond to the means ± SD of three independent experiments.

Article Snippet: For quantification of anti-VEGF antibody production from CHO cells, we used the concentration-defined anti-VEGF antibody, bevacizumab (Medchem Express, Monmouth Junction, NJ, USA) to create a standard curve.

Techniques: Plasmid Preparation, Gene Expression, Sequencing, Clone Assay, Negative Control, Labeling, Control, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: An efficient protein production system via gene amplification on a human artificial chromosome and the chromosome transfer to CHO cells

doi: 10.1038/s41598-019-53116-2

Figure Lengend Snippet: Transfer of the VEGF MAR-HAC vector to CHO K1 cells promotes antibody production. ( A ) Two-color FISH analysis of the VEGF MAR-HAC vector in G418-resistant CHO K1 clones was performed with digoxigenin-labeled hCot-1 DNA (red) and biotin-labeled anti-VEGF antibody gene (green). The arrow indicates the HAC, and the inset shows enlarged images of the HAC (anti-VEGF antibody probe-specific green signals are indicated by arrows). The arrowheads indicate the three large metacentric chromosomes that identify CHO K1 cells. The left panel shows CHO K1R VEGF MAR-HAC cl.1. The right panel shows CHO K1R VEGF MAR-HAC cl.2. ( B ) Analysis of anti-VEGF antibody production with ELISA from CHO K1R VEGF MAR-HAC clones and CHO DG44 VEGF MAR-HAC clones from day 2 to day 8. Bars correspond to the means ± SD of three independent experiments. ( C ) Culture supernatants were analyzed by SDS-PAGE. The protein bands were stained by silver staining. MW, molecular weight markers; line 1, antibody purified from CHO DG44 VEGF MAR-HAC cl.2; line 2, antibody purified from CHO K1R VEGF MAR-HAC cl.2; line 3, antibody purified from CHO K1R VEGF MAR-HAC cl.2; line 4, commercial human anti-VEGF antibody (bevacizumab; 0.1 µg). Hc means heavy chain. Lc means light chain. ( D ) Western blotting of the 19.1 kDa VEGF 165 protein with the purified antibody from the CHO cell clones. Line 1, antibody purified from CHO DG44 VEGF MAR-HAC cl.2; line 2, antibody purified from CHO K1R VEGF MAR-HAC cl.2; line 3, antibody purified from CHO K1R VEGF MAR-HAC cl.2; line 4, commercial human anti-VEGF antibody.

Article Snippet: For quantification of anti-VEGF antibody production from CHO cells, we used the concentration-defined anti-VEGF antibody, bevacizumab (Medchem Express, Monmouth Junction, NJ, USA) to create a standard curve.

Techniques: Plasmid Preparation, Clone Assay, Labeling, Enzyme-linked Immunosorbent Assay, SDS Page, Staining, Silver Staining, Molecular Weight, Purification, Western Blot

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: Journal of extracellular vesicles

Article Title: Anti-angiogenesis triggers exosomes release from endothelial cells to promote tumor vasculogenesis.

doi: 10.1080/20013078.2019.1629865

Figure Lengend Snippet: Figure 4. Inhibition of VEGFR (Flk1) or autophagy releases VEGF-enriched exosomes from HUVECs.

Article Snippet: After determination of protein concentration using a protein determination kit (Cayman Chemical Company, USA), equal amounts (20–30 μg for cells, 10 μg for exosome pellets, and 50 μg for tissues) of protein samples or exosomal proteins from 2.5 mL culture supernatant were size fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, electrotransferred onto a polyvinylidene fluoride membrane (Millipore), blocked with 5% non-fat milk in PBS, and hybridized with antibodies against VEGF (TA500289; Origene, Rockville, MD, USA), Flk1 (#9698; Cell Signaling Technology, Danvers, MA, USA), p-Flk1 (Tyr1175; #19A10; Cell Signaling Technology), LC3BI/II (ab192890; Abcam), TSG101 (14,497–1-AP; Proteinteck; Rosemont, IL, USA), HSP70 (10,995–1-AP; Proteinteck), CD63 (ab134045; Abcam), and GRP94 (ab3674; Abcam) at 4°C overnight.

Techniques: Inhibition

Journal: Journal of extracellular vesicles

Article Title: Anti-angiogenesis triggers exosomes release from endothelial cells to promote tumor vasculogenesis.

doi: 10.1080/20013078.2019.1629865

Figure Lengend Snippet: Figure 7. VEGF levels and Flk1 activation by VEGF-enriched exosomes.

Article Snippet: After determination of protein concentration using a protein determination kit (Cayman Chemical Company, USA), equal amounts (20–30 μg for cells, 10 μg for exosome pellets, and 50 μg for tissues) of protein samples or exosomal proteins from 2.5 mL culture supernatant were size fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, electrotransferred onto a polyvinylidene fluoride membrane (Millipore), blocked with 5% non-fat milk in PBS, and hybridized with antibodies against VEGF (TA500289; Origene, Rockville, MD, USA), Flk1 (#9698; Cell Signaling Technology, Danvers, MA, USA), p-Flk1 (Tyr1175; #19A10; Cell Signaling Technology), LC3BI/II (ab192890; Abcam), TSG101 (14,497–1-AP; Proteinteck; Rosemont, IL, USA), HSP70 (10,995–1-AP; Proteinteck), CD63 (ab134045; Abcam), and GRP94 (ab3674; Abcam) at 4°C overnight.

Techniques: Activation Assay

Journal: Journal of magnetic resonance imaging : JMRI

Article Title: 3D dynamic contrast-enhanced MRI of rectal carcinoma at 3T: correlation with microvascular density and vascular endothelial growth factor markers of tumor angiogenesis.

doi: 10.1002/jmri.21378

Figure Lengend Snippet: Figure 1. a: DCE MRI image of a 76-year-old female shows enhancing rectal adenocarcinoma. ROIs for rectal carcinoma (area 1), normal rectal wall (area 2), gluteal muscle (area 3), correspond to (b) time-intensity curves showing early peak enhancement of the tumor. Rectal carcinoma has (c) higher microvascular density (MVD) (vascular endothelial cells show brown color to identify microvessels, arrow) and (d) strong positive VEGF expression (yellow to brown marks the positive expression of VEGF in cytoplasm of tumor cells, arrow) compared to (e) MVD and (f) negative VEGF expression of normal rectal wall (SP, 100). [Color figure can be viewed in the online issue, which is available at http://www.interscience.wiley.com.]

Article Snippet: MVD and VEGF expression of the tumor were determined using immunohistochemistry (IHC) stain (SP method) with CD34 monoclonal antibody and VEGF monoclonal antibody (Zhongshan Golden Bridge Biotechnology Co. Ltd., Beijing, China) in 24 tumor specimens (one per patient) from 24 patients who had sufficiently large surgical specimens.

Techniques: Expressing

Journal: Journal of magnetic resonance imaging : JMRI

Article Title: 3D dynamic contrast-enhanced MRI of rectal carcinoma at 3T: correlation with microvascular density and vascular endothelial growth factor markers of tumor angiogenesis.

doi: 10.1002/jmri.21378

Figure Lengend Snippet: Figure 2. a: DCE MRI image of an 80-year-old female shows enhancing rectal adenocarcinoma. ROIs for rectal carcinoma (area 1), normal rectal wall (area 2), and gluteal muscle (area 3) correspond to (b) time-intensity curves showing delayed peak enhancement of the tumor. Rectal carcinoma has (c) lower MVD (vascular endothelial cells are stained in brown to identify microvessels, arrow) and (d) weak positive VEGF expression (yellow marks the positive expression of VEGF in cytoplasm of tumor cells, arrow) (SP, 100) compared to Fig. 1c and 1d. [Color figure can be viewed in the online issue, which is available at http://www.interscience.wiley.com.]

Article Snippet: MVD and VEGF expression of the tumor were determined using immunohistochemistry (IHC) stain (SP method) with CD34 monoclonal antibody and VEGF monoclonal antibody (Zhongshan Golden Bridge Biotechnology Co. Ltd., Beijing, China) in 24 tumor specimens (one per patient) from 24 patients who had sufficiently large surgical specimens.

Techniques: Staining, Expressing

Journal: Journal of magnetic resonance imaging : JMRI

Article Title: 3D dynamic contrast-enhanced MRI of rectal carcinoma at 3T: correlation with microvascular density and vascular endothelial growth factor markers of tumor angiogenesis.

doi: 10.1002/jmri.21378

Figure Lengend Snippet: Figure 3. a: DCE MRI image of a 50-year-old female shows enhancing rectal signet cell cancer. ROIs for rectal carcinoma (area 1) and gluteal muscle (area 2) correspond to (b) time-intensity curves showing delayed peak enhancement of signet cell cancer. Rectal signet cell cancer has (c) lower MVD and (d) negative VEGF expression (SP, 100) compared to Fig. 1c and 1d. [Color figure can be viewed in the online issue, which is available at http://www.interscience.wiley.com.]

Article Snippet: MVD and VEGF expression of the tumor were determined using immunohistochemistry (IHC) stain (SP method) with CD34 monoclonal antibody and VEGF monoclonal antibody (Zhongshan Golden Bridge Biotechnology Co. Ltd., Beijing, China) in 24 tumor specimens (one per patient) from 24 patients who had sufficiently large surgical specimens.

Techniques: Expressing