Journal: Communications Biology

Article Title: Cell-derived extracellular vesicles can be used as a biomarker reservoir for glioblastoma tumor subtyping

doi: 10.1038/s42003-019-0560-x

Figure Lengend Snippet: Astrocytes (AS), GBM cell lines, and GBM patient-derived stem cells present different in vitro invasion capabilities and specific subtype marker expression. a AS and GBM cells invasiveness and colony formation abilities using a hyaluronic acid (HA)-based hydrogel assay. Cells were incubated within a HA hydrogel for 7 days. Colony counting was then performed. Scale bar = 400 µm. b AS and GBM cell viability in a HA hydrogel-based assay using the CellTiter-Glo ® Luminescent Cell Viability Assay. c Invasion abilities of AS and GBM cells through an extracellular matrix-coated membrane. Cells were seeded in the top chamber and were allowed to invade the matrix for 24 h in presence or absence of FCS in the bottom chamber. Cells that have passed through the matrix were then detached, lysed, and labeled with CyQuant GR Dye. Fluorescence was then read (480/520 nm filter set). Data obtained in presence of FCS was normalized to data obtained without FCS. Representative images are shown. d qRT-PCR analysis of GBM subtype and aggressiveness marker expression in astrocytes, six different GBM cell lines and two different GBM patient-derived stem cells. GAPDH was used as an internal control. Data are shown as normalized to AS data. Heat-map representative of the qRT-PCR data where the data is normalized to the highest level of gene expression. e Western blotting analysis of GBM subtype and aggressiveness marker expression in AS and six different GBM cell lines. β-actin was used as an internal control. f Western blotting analysis of GBM subtype and aggressiveness marker expression in astrocytes and two different GBM patient-derived stem cells. β-actin was used as an internal control. g ELISA analysis of VEGF-A secretion by AS, six different GBM cell lines and two different GBM patient-derived stem cells. Representative images are shown. The mean ± SEM of n = 3 independent experiments is shown. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (ordinary one-way ANOVA)

Article Snippet: Human VEGF DUOSET ELISA (R&D System) was used to measure VEGF-A levels in culture medium according to the manufacturer’s instructions.

Techniques: Derivative Assay, In Vitro, Marker, Expressing, Incubation, Cell Viability Assay, Membrane, Labeling, CyQUANT Assay, Fluorescence, Quantitative RT-PCR, Control, Gene Expression, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Biotechnology letters

Article Title: Ultrasensitive immuno-detection using viral nanoparticles with modular assembly using genetically-directed biotinylation

doi: 10.1007/s10529-014-1555-9

Figure Lengend Snippet: VEGF (26 aM - 2.6 pM) was spiked into 50% BAL fluid (in PBS), and incubated with polyclonal anti-VEGF antibody functionalized magnetic particles. The biotinylated antibody/Neutravidin/phage affinity reagent was added, followed by washing. The sample was then analyzed using real-time PCR with phage-specific primers (n=6, error bars = ±1 SD, Non-template control Ct ≥34). The No-VEGF control (Ct =22.67±0.55) is shown as a solid line with +/− one standard deviation represented by dotted lines.

Article Snippet: The particles were washed, resuspended in coating buffer (1×10 7 particles in 1 ml 0.1 M sodium borate, pH 9.5) and mixed with 240 μl polyclonal anti-human Vascular Endothelial Growth Factor (VEGF) antibody (1 mg/ml, AB-293-NA, R&D Systems) and 240 μl 3 M (NH 4 ) 2 SO 4 .

Techniques: Incubation, Real-time Polymerase Chain Reaction, Control, Standard Deviation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Long non‐coding RNA GAPLINC promotes angiogenesis by regulating miR‐211 under hypoxia in human umbilical vein endothelial cells

doi: 10.1111/jcmm.14678

Figure Lengend Snippet: Effects of the expression level of GAPLINC on VEGFR and DLL4 expression in HUVECs. The treatment condition as indicated. Cells were lysed and proteins were transferred to PVDF membrane and stained with antibodies. A, Representative Western blot of VEGFR and DLL4 was shown. B, The summary of expression of VEGFR and DLL4 in different condition were shown. The results are presented as mean ± SD for three independent experiments. Each experiment performed in triplicate. *: P < .05, **: P < .01, ***: P < .001, ****: P < .0001

Article Snippet: The primary antibodies raised against GAPDH (1:1000, Goodhere), Bcl2 (1:1000, Proteintech), VEGFR (1:2000, Boster) and DLL4 (1:300, Proteintech) were incubated with the membrane overnight.

Techniques: Expressing, Membrane, Staining, Western Blot

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