Journal: Chinese Journal of Cancer Research

Article Title: Tim-3 promotes cell aggressiveness and paclitaxel resistance through NF-κB/STAT3 signalling pathway in breast cancer cells

doi: 10.21147/j.issn.1000-9604.2020.05.02

Figure Lengend Snippet: VEGFC and VEGFR2 protein levels in conditioned media of stable cell lines indicated by ELISA. (A) VEGFC; (B) VEGFR2. VEGF, vascular endothelial growth factor; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: ELISA was performed using the human VEGFC (cat. no. E-EL-H1600; Elabscience) and vascular endothelial growth factor receptor 2 (VEGFR2) ELISA kit (cat. no. E-EL-H1603; Elabscience), according to the manufacturer’s protocol.

Techniques: Stable Transfection, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Sestrin2 Coordinates Multiple Protective Pathways to Maintain Angiogenic Function in Diabetes-Associated Endothelial Dysfunction

doi: 10.3390/ijms262311396

Figure Lengend Snippet: SESN2 modulates the NRF2/HO-1 antioxidant axis and the expression of key angiogenic factors at both protein and mRNA levels. ( A ) Representative Western blot analysis showing protein expression of NRF2, SESN2, and HO-1, with GAPDH as loading control. Cells were treated under control conditions (Ctl), SESN2 silencing (Si), or SESN2 overexpression (Oe), with or without 600 μM MGO treatment for 18 h. Blots shown are representative of n = 3 independent experiments. ( B ) Quantitative analysis of NRF2/GAPDH, SESN2/GAPDH, and HO-1/GAPDH protein expression. ( C ) Relative mRNA expression levels of NRF2 target genes, HMOX1 and NQO1 , measured by qRT-PCR. ( D ) Relative mRNA expression levels of key angiogenic factors, VEGFA , VEGFC , and their receptor KDR ( VEGFR2 ), measured by qRT-PCR. For panels C and D, mRNA data were normalized to GAPDH expression and are presented as fold change relative to the control (−MGO) group. ( E ) Quantification of secreted VEGF-C protein in conditioned media measured by ELISA. All quantitative data (panels ( B – E )) are presented as mean ± SD ( n = 3–5 independent experiments). Groups that do not share a common letter are significantly different from one another ( p < 0.05).

Article Snippet: The concentration of secreted VEGF-C in the conditioned cell culture media was quantified using a commercially available Human VEGF-C Enzyme-Linked Immunosorbent Assay (ELISA) kit (R&D Systems ® , Minneapolis, MN, USA; Catalog Number DVE00) according to the manufacturer’s instructions, and concentrations were calculated based on a standard curve.

Techniques: Expressing, Western Blot, Control, Over Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: miRNA-27b Targets Vascular Endothelial Growth Factor C to Inhibit Tumor Progression and Angiogenesis in Colorectal Cancer

doi: 10.1371/journal.pone.0060687

Figure Lengend Snippet: (A) VEGFC is predicted as a novel target of miR-27b. (B) 293T cells were co-transfected with empty pmirGLO Dual-Luciferase reporter plasmids or VEGFC 3′UTR firefly luciferase reporter plasmids and pRL-TK-luciferase plasmids, together with miR-27b mimics or anti-miR-27b. After 48 h, firefly luciferase activity was measured and normalized to that of Renilla luciferase. (C) CRC cells were transfected with NC, miR-27b or anti-miR-27b mimics and expression of VEGFC was detected by western blotting. (D) CRC cells were transfected with NC, miR-27b or anti-miR-27b mimics and VEGFC in culture medium was detected by ELISA. (E) VEGFC protein in xenografts from negative control (NC) and miR-27b mimics was detected by western blotting. Error bars represent the means ± SEM, * P <0.05.

Article Snippet: After blocking with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST) for 60 min, the membrane was incubated with the primary antibodies anti-human-VEGFC (Cell signaling technology, Danvers, MA, USA) (1∶2000) or anti-human GAPDH (KangChen, Shanghai, China) dissolved in 5% bovine serum albumin in TBST overnight at 4°C.

Techniques: Transfection, Luciferase, Activity Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: PLoS ONE

Article Title: miRNA-27b Targets Vascular Endothelial Growth Factor C to Inhibit Tumor Progression and Angiogenesis in Colorectal Cancer

doi: 10.1371/journal.pone.0060687

Figure Lengend Snippet: (A) VEGFC knockdown in anti-miR-27b stable cells was confirmed by western blotting. (B) Cell proliferation rate was determined by measuring the absorbance at 490 nm in a MTS assay. (C and D) Results of a soft-agar colony assay. Colonies were visualized by microscopy after 2 weeks of incubation. Colonies containing >20 cells were counted. Scale bars = 200 µm. (E) Results of a tumorigenesis assay. A representative image of xenograft tumors in nude mice subcutaneously injected with 1×10 6 CRC cells. (F) Comparison of xenograft formation in vivo . Tumor volumes were measured each week. Error bars represent the means ± SEM, * P <0.05.

Article Snippet: After blocking with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST) for 60 min, the membrane was incubated with the primary antibodies anti-human-VEGFC (Cell signaling technology, Danvers, MA, USA) (1∶2000) or anti-human GAPDH (KangChen, Shanghai, China) dissolved in 5% bovine serum albumin in TBST overnight at 4°C.

Techniques: Knockdown, Western Blot, MTS Assay, Colony Assay, Microscopy, Incubation, Injection, Comparison, In Vivo

Journal: Journal of Virology

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Induces Sustained Levels of Vascular Endothelial Growth Factors A and C Early during In Vitro Infection of Human Microvascular Dermal Endothelial Cells: Biological Implications

doi: 10.1128/jvi.00873-07

Figure Lengend Snippet: FIG. 1. Detection of VEGF-A and VEGF-C mRNA and protein in KSHV-infected HMVEC-d cells. HMVEC-d cells grown to 80 to 90% confluence were serum starved for 8 h and infected with KSHV at an MOI of 10. (A and C) Infected and uninfected cells were washed and lysed, and total RNA was prepared. DNase I-treated RNA (250 ng) was subjected to real-time RT-PCR with VEGF-A and VEGF-C gene-specific primers. Known concentrations of DNase I-treated, in vitro-transcribed VEGF-A and VEGF-C transcripts were used in a real-time RT-PCR to construct a standard graph from which the relative copy numbers of transcripts were calculated and normalized, with GAPDH used as the internal control. Each reaction was done in duplicate, and each bar represents the average standard deviation from three independent experiments. The VEGF-A and VEGF-C levels normalized to GAPDH in the uninfected cells were considered as 1 for comparison. (B and D) The levels of VEGF-A and VEGF-C proteins released in the cell-free culture supernatants were measured by enzyme-linked immunosorbent assay. The data were normalized to a 1-mg/ml total protein concentration in the supernatant. Each reaction was done in duplicate, and each point represents the average standard deviation from three independent experiments. VEGF-A and VEGF-C released from uninfected cells were considered as 1 for comparison, and the induction levels in infected cells are indicated.

Article Snippet: The levels of VEGF-A and -C in the culture supernatant of uninfected or KSHV-infected HMVEC-d cells were quantitated using QuantiGlo human VEGF-A and VEGF-C enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN).

Techniques: Infection, Quantitative RT-PCR, In Vitro, Construct, Control, Standard Deviation, Comparison, Enzyme-linked Immunosorbent Assay, Protein Concentration

Journal: Journal of Virology

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Induces Sustained Levels of Vascular Endothelial Growth Factors A and C Early during In Vitro Infection of Human Microvascular Dermal Endothelial Cells: Biological Implications

doi: 10.1128/jvi.00873-07

Figure Lengend Snippet: FIG. 2. Blocking KSHV binding by heparin inhibits VEGF-A expres- sion. (A) KSHV was incubated at 37°C for 1 h with DMEM containing 100 g/ml of soluble heparin. This mixture was then added to a serum- starved (8 h) HMVEC-d cell monolayer and incubated for 4 h at 37°C. The levels of VEGF-A released in the cell-free culture supernatants were measured by enzyme-linked immunosorbent assay. **, statistically signif- icant (P 0.02). (B) HMVEC-d cells grown to 80 to 90% confluence were serum starved for 8 h and either uninfected (control) or infected with KSHV at an MOI of 10. The levels of VEGF-A proteins released in the cell-free culture supernatants collected at the indicated time points were measured by ELISA. The data were normalized to a 1-mg/ml total protein concentration in the supernatant. Each reaction was done in duplicate, and each point represents the average standard deviation from three independent experiments. (C) VEGF-A and VEGF-C enhance KSHV entry. HMVEC-d cells were serum starved for 8 h and either untreated or treated with VEGF-A or VEGF-C (100 and 250 ng/ml) or with human EGF or BSA (250 ng/ml) for 1 h at 37°C in serum-free EBM2 medium and infected (10 DNA copies per cell) for 2 h. Cells were washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) for 5 min, washed, and collected, and total DNA was prepared. The KSHV ORF73 gene in 100 ng of DNA was amplified by real-time DNA PCR, and the copy numbers were calculated from the standard graph generated by the real- time PCR using known concentrations of a cloned ORF73 gene. Each reaction was done in duplicate, and each point represents the average standard deviation of three experiments.

Article Snippet: The levels of VEGF-A and -C in the culture supernatant of uninfected or KSHV-infected HMVEC-d cells were quantitated using QuantiGlo human VEGF-A and VEGF-C enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN).

Techniques: Blocking Assay, Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control, Infection, Protein Concentration, Standard Deviation, Generated, Real-time Polymerase Chain Reaction, Clone Assay

Journal: Journal of Virology

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Induces Sustained Levels of Vascular Endothelial Growth Factors A and C Early during In Vitro Infection of Human Microvascular Dermal Endothelial Cells: Biological Implications

doi: 10.1128/jvi.00873-07

Figure Lengend Snippet: FIG. 4. Induction of VEGF-A by UV-inactivated KSHV and viral glycoproteins gB and gpK8.1A. (A) HMVEC-d cells (80 to 90% confluence) serum starved for 8 h were uninfected or infected with either live KSHV or UV-irradiated KSHV for the indicated times at an MOI of 10 per cell. (B) Serum-starved HMVEC-d cells were uninfected, infected with KSHV (MOI, 10), or induced with KSHV glycoproteins gB and gpK8.1A alone or together for the indicated times. (C) Serum-starved HMVEC-d cells were uninfected, infected with KSHV (MOI, 10), or induced with KSHV glycoprotein gpK8.1A at the indicated concentrations for 0.5 and 1 h. (D) Serum-starved HMVEC-d cells were uninfected, infected with KSHV (MOI, 10), or induced with KSHV glycoproteins gB and gpK8.1A together at the indicated concentrations for 0.5 and 1 h. The levels of VEGF-A released in the cell-free culture supernatants were measured by ELISA. Each reaction was done in duplicate, and each point represents the average standard deviation from three independent experiments.

Article Snippet: The levels of VEGF-A and -C in the culture supernatant of uninfected or KSHV-infected HMVEC-d cells were quantitated using QuantiGlo human VEGF-A and VEGF-C enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN).

Techniques: Infection, Irradiation, Enzyme-linked Immunosorbent Assay, Standard Deviation