Journal: Oncogene

Article Title: Oncogenes and tumor angiogenesis: the HPV-16 E6 oncoprotein activates the vascular endothelial growth factor (VEGF) gene promoter in a p53 independent manner.

doi: 10.1038/sj.onc.1203817

Figure Lengend Snippet: Figure 1 Comparison of VEGF mRNA and protein levels in HPV-16- positive and HPV-16- negative cells. (a) Northern blot analysis of VEGF expression in a panel of cell lines including human immortalized HPV-16- negative (HaCaT) and HPV-16- positive (HPK1A) keratinocytes, as well as cervical carcinoma derived HPV-16- negative (C33A) and HPV-16- positive (HeLa, CaSki) cells. VEGF mRNA levels were increased 4 ± 7-fold in HPV-16- positive cells as compared to HPV-16- negative immortalized keratinocytes, and 2 ± 3-fold compared to the HPV-16- negative cervical carcinoma cell line C33A. (b) Northern blot analysis of VEGF expression in HeLa cells as compared to HaCaT keratinocytes. VEGF mRNA levels were increased 7 ± 18-fold in HeLa, with the maximum dierence observed at low serum concentration (0.1%). The 28S and 18S mRNA bands show equal loading and RNA integrity (lower panel). Fold activation was calculated by densitometry with 28S serving as a normalization control. The value obtained for HaCaT keratinocytes in 0.1% FBS was taken as the basal level (1.0) in (b). (c) ELISA assay results showing the increase in VEGF concentration in conditioned medium collected from HeLa cells compared to that from HaCaT keratinocytes. Cells were incubated for 24 h after being seeded at dierent serum concentrations

Article Snippet: Measurement of human VEGF protein levels in conditioned medium (ELISA) A commercially available human VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA) was used to quantitate the level of VEGF obtained from HaCaT, HeLa and A431 cells according to the manufacturer's instructions.

Techniques: Comparison, Northern Blot, Expressing, Derivative Assay, Concentration Assay, Activation Assay, Control, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Oncogene

Article Title: Oncogenes and tumor angiogenesis: the HPV-16 E6 oncoprotein activates the vascular endothelial growth factor (VEGF) gene promoter in a p53 independent manner.

doi: 10.1038/sj.onc.1203817

Figure Lengend Snippet: Figure 2 Eect of blockade of EGFR signaling on the production of VEGF protein by HeLa and A431 cells. (a) Eect of treatment with two anti-EGFR antibodies, C225 and hR3, on the secretion of VEGF by Hela cells. (b) Eect of equal treatments on the production of VEGF protein by A431 cells. No signi®cant dierences among the distinct treatment culture conditions in terms of VEGF secretion were detected in HeLa cells. In contrast, both antibodies down-regulated VEGF protein levels signi®cantly, up to a maximum of 50%, in A431 cells. (c) In agreement with the lack of eect of the EGFR neutralizing antibodies on HeLa, the anti-TGF-a antibody Ab-3 also failed to down-regulate VEGF protein production by HeLa cells. A one-way ANOVA test was used to compare the dierent treatment culture conditions for each experiment. A Dunnet post-test was used in order to compare each result with its respective control

Article Snippet: Measurement of human VEGF protein levels in conditioned medium (ELISA) A commercially available human VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA) was used to quantitate the level of VEGF obtained from HaCaT, HeLa and A431 cells according to the manufacturer's instructions.

Techniques: Control

Journal: Oncogene

Article Title: Oncogenes and tumor angiogenesis: the HPV-16 E6 oncoprotein activates the vascular endothelial growth factor (VEGF) gene promoter in a p53 independent manner.

doi: 10.1038/sj.onc.1203817

Figure Lengend Snippet: Figure 3 Transcriptional activation of VEGF promoter by a plasmid expressing the HPV-16 E6 oncoprotein. (a) Schematic representation of the activator and the reporter construct. The plasmid pJ4O16E6 expresses the E6 gene of HPV-16, driven by MoMu LV LTR promoter (Crook et al., 1991). The reporter 2.6 VEGF is a VEGF promoter (72361 to +298)-Luciferase construct described previously (Mukhopadhyay et al., 1997). (b and c) Eect of HPV-16 E6 gene product on VEGF promoter activity in either HaCaT keratinocytes or NIH3T3 cells, respectively. Signi®cant dierences (P50.01) were detected for both cell lines by applying unpaired t-test. Dose-dependence of VEGF-promoter induction by E6 expression is seen in (b). Plasmid 2.6 VEGF (1.5 mg) was co-transfected into HaCaT cells along with increasing concentrations of either HPV-16 E6 (0 ± 3 mg) expressing plasmid or control plasmid pJ4O (3 mg). Values are presented as averages of three independent experiments in terms of normalized luciferase activity with the standard error (s.e.) of the mean noted. Fold induction is indicated at the top of the bars

Article Snippet: Measurement of human VEGF protein levels in conditioned medium (ELISA) A commercially available human VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA) was used to quantitate the level of VEGF obtained from HaCaT, HeLa and A431 cells according to the manufacturer's instructions.

Techniques: Activation Assay, Plasmid Preparation, Expressing, Construct, Luciferase, Activity Assay, Transfection, Control

Journal: Oncogene

Article Title: Oncogenes and tumor angiogenesis: the HPV-16 E6 oncoprotein activates the vascular endothelial growth factor (VEGF) gene promoter in a p53 independent manner.

doi: 10.1038/sj.onc.1203817

Figure Lengend Snippet: Figure 4 Identi®cation of the E6-responsive region in the human VEGF promoter. Dierent deletion constructs were co- transfected into HaCaT cells with either E6 expression plasmid (&) or control plasmid (&). Left panel presents a schematic representation of the VEGF promoter deletions including consensus binding sites of dierent transcription factors. The E6- responsive region is localized between bp 7194 and 750 of the VEGF gene promoter. Values are presented as averages of three independent experiments in function of normalized luciferase activity with the s.e. of the mean noted. Fold induction in luciferase activity is depicted to the right side of the bars

Article Snippet: Measurement of human VEGF protein levels in conditioned medium (ELISA) A commercially available human VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA) was used to quantitate the level of VEGF obtained from HaCaT, HeLa and A431 cells according to the manufacturer's instructions.

Techniques: Construct, Transfection, Expressing, Plasmid Preparation, Control, Binding Assay, Luciferase, Activity Assay

Journal: Oncogene

Article Title: Oncogenes and tumor angiogenesis: the HPV-16 E6 oncoprotein activates the vascular endothelial growth factor (VEGF) gene promoter in a p53 independent manner.

doi: 10.1038/sj.onc.1203817

Figure Lengend Snippet: Figure 5 Activation of VEGF promotor by E6 in p53 null cells. The plasmid 2.6 VEGF (1.5 mg) was co-transfected into MEF p53+/+ or MEF p537/7 cells together with 3 mg of the HPV- 16 E6 expression plasmid (pJ4O16E6) or 3 mg of the control plasmid (pJ4O). The HPV-16 E6 appears to induce transcription from VEGF promoter even in the absence of a functional p53 gene. Values are presented as averages of three independent experiments in terms of normalized luciferase activity with the s.e. of the mean noted. Fold induction is indicated at the top of the bars

Article Snippet: Measurement of human VEGF protein levels in conditioned medium (ELISA) A commercially available human VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA) was used to quantitate the level of VEGF obtained from HaCaT, HeLa and A431 cells according to the manufacturer's instructions.

Techniques: Activation Assay, Plasmid Preparation, Transfection, Expressing, Control, Functional Assay, Luciferase, Activity Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

doi: 10.1161/01.atv.20.5.1374

Figure Lengend Snippet: Figure 1. A, Effect of hirudin (Hir) and FXa inhibitors on ACSET- induced VEGF secretion. Confluent fibroblasts were incubated with 10 U/mL Hir, 10 mmol/L DX9065a (DX), 50 mg/mL TAP, or Hir in combination with either DX or TAP for 30 minutes at 37°C before a 24-hour incubation at 37°C with 100 nmol/L ACSET. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF in treated fibroblasts com- pared with unstimulated fibroblasts (NS). Each point represents the mean6SD of at least 3 different determinations, each per- formed in triplicate. *P,0.0001 vs unstimulated fibroblasts. lP,0.05 vs ACSET-stimulated fibroblasts. B, Kinetics of thrombin and FXa generation in the culture medium of confluent human fibroblasts incubated with 100 nmol/L ACSET. Thrombin and FXa generation were assessed by hydrolysis of the thrombin-sensitive chromogenic substrate S-2238 and by hydrolysis of the FXa-sensitive chromogenic substrate S-2765, respectively, in culture supernatants. Results of a typical experi- ment are shown.

Article Snippet: Human VEGF Immunoassay Human VEGF concentrations in fibroblast culture supernatants were determined by using the Quantikine human VEGF kit (R&D Systems Europe).11

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

doi: 10.1161/01.atv.20.5.1374

Figure Lengend Snippet: Figure 2. A, Concentration effect of thrombin on VEGF produc- tion. Confluent fibroblasts were incubated for 24 hours with or without 0.5, 1, and 10 U/mL thrombin at 37°C. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF in treated fibroblasts compared with unstimulated fibroblasts (basal VEGF level 124673 pg/mL). Each point represents the mean6SD of 4 different determina- tions, each performed in triplicate. *P,0.05 vs unstimulated fibroblasts. B, Kinetics of thrombin-induced VEGF secretion. Confluent fibroblasts were incubated with 10 U/mL thrombin for 2, 4, 6, 12, and 24 hours at 37°C. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF in thrombin-treated fibroblasts compared with unstimu- lated fibroblasts at the same time. Results of a typical experi- ment are shown. C, Concentration effect of TRAP on VEGF secretion. Confluent fibroblasts were incubated for 24 hours with or without 10, 50, and 100 mmol/L of TRAP at 37°C. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction in VEGF secretion compared with unstimulated fibroblasts. Each point represents the mean6SD of 3 different determinations, each performed in triplicate. *P,0.05 vs unstimulated fibroblasts.

Article Snippet: Human VEGF Immunoassay Human VEGF concentrations in fibroblast culture supernatants were determined by using the Quantikine human VEGF kit (R&D Systems Europe).11

Techniques: Concentration Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

doi: 10.1161/01.atv.20.5.1374

Figure Lengend Snippet: Figure 3. A, Concentration of FXa on VEGF secretion. Conflu- ent fibroblasts were incubated for 24 hours with or without 22.8, 57, 114, and 228 nmol/L of FXa at 37°C. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF secretion compared with unstimulated fibro- blasts. Each point represents the mean6SD of 3 different deter- minations, each performed in triplicate. *P,0.05 vs unstimulated fibroblasts. Insert, Additive effect of thrombin (IIa) and FXa on VEGF secretion. Confluent fibroblasts were incubated at 37°C for 24 hours with 1 U/mL of IIa or 100 nmol/L of FXa or a com- bination of both. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF secre- tion compared with unstimulated fibroblasts (NS). Each point represents the mean6SD of 3 different determinations, each performed in triplicate. lP,0.01 vs NS. B, Effect of FXa inhibi- tors, Hir, and anti-TF antibodies on FXa-induced VEGF produc- tion. Confluent fibroblasts were incubated either with 10 mg/mL anti-TF antibodies (TFab) for 30 minutes at 4°C or with 10 mmol/L DX, 50 mg/mL TAP, or 10 U/mL Hir for 30 minutes at 37°C before 24-hour incubation at 37°C with 114 nmol/L FXa. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF secretion compared with NS. Each point represents the mean6SD of at least 3 different determinations, each performed in triplicate. *P,0.0001 vs NS. lP,0.01 vs FXa-stimulated fibroblasts.

Article Snippet: Human VEGF Immunoassay Human VEGF concentrations in fibroblast culture supernatants were determined by using the Quantikine human VEGF kit (R&D Systems Europe).11

Techniques: Concentration Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

doi: 10.1161/01.atv.20.5.1374

Figure Lengend Snippet: Figure 5. Effect of FXa inhibitors and TFab on FVIIa1FX- induced VEGF secretion. Confluent fibroblasts were incubated with FXa inhibitors (TAP, NAP5, and NAPc2 at 50 mg/mL and DX at 10 mmol/L) and with TFab for 30 minutes at either 37°C or 4°C, respectively, before 24-hour incubation with 100 nmol/L FVIIa and 90 nmol/L FX. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF secretion compared with NS. Each point represents the mean6SD of at least 3 different determinations, each performed in triplicate. *P,0.0001 vs NS. lP,0.0001 vs FXa-stimulated fibroblasts.

Article Snippet: Human VEGF Immunoassay Human VEGF concentrations in fibroblast culture supernatants were determined by using the Quantikine human VEGF kit (R&D Systems Europe).11

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

doi: 10.1161/01.atv.20.5.1374

Figure Lengend Snippet: Figure 6. Effect of activated clotting factors on VEGF mRNA induction. Confluent fibroblasts were incubated for 24 hours at 37°C with 100 nmol/L FVIIa in combination with 90 nmol/L FX, 114 nmol/L FXa, or 1 U/mL thrombin. Cells were preincubated for 30 minutes at 37°C either with 50 mg/mL TAP before incuba- tion with FVIIa-FX and FXa or with 10 U/mL Hir before incuba- tion with thrombin. Five micrograms of total RNA was analyzed by RT-PCR. A, RNA was quantified by scanning the Polaroid negative by laser densitometry. The density of the 180-bp band was normalized to the density of the mimic, and the fold induc- tion of VEGF mRNA, induced in the different conditions of stim- ulation compared with NS, was plotted. Each point represents the mean6SD of 4 experiments. *P,0.05 vs NS. lP,0.05 vs FVIIa-FX, FXa, or thrombin-stimulated fibroblasts. B, Photograph from a representative experiment is shown. Three VEGF mRNA transcripts are seen (180, 312, and 384 bp). The internal stan- dard, mimic (M), is also visible. L indicates 100-bp DNA ladder.

Article Snippet: Human VEGF Immunoassay Human VEGF concentrations in fibroblast culture supernatants were determined by using the Quantikine human VEGF kit (R&D Systems Europe).11

Techniques: Coagulation, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

doi: 10.1161/01.atv.20.5.1374

Figure Lengend Snippet: Figure 7. A, Effect of FVIIa and FVIIai on p44/42 MAP kinase activation. Fibroblasts were incubated with either 100 nmol/L FVIIa or 100 nmol/L FVIIai for 5, 10, and 15 minutes. Phosphor- ylated p44/42 MAP kinases (p44ERK1 and p42ERK2) and total p44/42 MAP kinases (ERK1 and ERK2) were studied by Western blotting. B, Western blot analysis of thrombin and FXa-induced phosphorylation of p44/42 MAP kinases. Fibroblasts were incu- bated with 1 U/mL of thrombin for 2, 5, 7, and 10 minutes or with 100 nmol/L of FXa for 2, 5, 10, and 15 minutes. p44ERK1, p42ERK2, ERK1, and ERK2 are shown. C, Effect of MAP kinase inhibitors on FXa- and thrombin-induced VEGF secretion. Con- fluent fibroblasts were incubated with MAP kinase inhibitors (50 mmol/L PD 98059 [PD] and 10 mmol/L SB 203580 [SB]) for 30 minutes at 37°C before 24-hour incubation with either FXa (114 nmol/L) or thrombin (1 U/mL). Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF secretion compared with unstimulated fibro- blasts. Each point represents the mean6SD of 4 different deter- minations, each performed in triplicate. *P,0.0005 vs NS. lP,0.05 vs FXa- or thrombin- stimulated fibroblasts.

Article Snippet: Human VEGF Immunoassay Human VEGF concentrations in fibroblast culture supernatants were determined by using the Quantikine human VEGF kit (R&D Systems Europe).11

Techniques: Activation Assay, Incubation, Western Blot, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Effects of E7050 on VEGFR2-mediated signaling pathways in VEGF-stimulated HUVECs. Cells were serum-starved for 6 h and then pretreated with E7050 (5 and 10 μM) or vehicle for 1 h, followed by stimulation with VEGF (100 ng/mL) for another 10 min (VEGFR2, PLCγ1, FAK, and Src) or 30 min (Akt, JNK, and p38 MAPK) before proteins were collected. The expression and phosphorylation status of VEGFR2 and its downstream effectors, including PLCγ1, FAK, Src, Akt, JNK, and p38 MAPK, were detected by Western blotting using respective antibodies. ( A ) E7050 inhibited the phosphorylation (Tyr1175) of VEGFR2 induced by VEGF in HUVECs. ( B ) The quantified results show that the ratio of p-VEGFR2 protein normalized to the total amount of VEGFR2 protein, which was measured by densitometry. ( C ) E7050 inhibited the phosphorylation of PLCγ1, FAK, and Src in VEGF-stimulated HUVECs. Calculated ratios of ( D ) p-PLCγ1, ( E ) p-FAK, and ( F ) p-Src normalized to the relative total protein levels are shown. ( G ) E7050 inhibited the phosphorylation of Akt, JNK, and p38 MAPK in VEGF-stimulated HUVECs. The compiled results of the ratios of ( H ) p-Akt, ( I ) p-JNK, and ( J ) p-p38 MAPK normalized to the relative total protein levels are shown. The data are presented as mean ± SEM of three independent experiments. # p < 0.05 compared with the untreated cells. * p < 0.05 compared with the vehicle-treated cells.

Article Snippet: The concentrations of VEGF in the culture supernatants of E7050-treated MES-SA/Dx5 cells were measured using the commercial human VEGF ELISA Kit PicoKine TM according to the instructions of the manufacturer (Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Protein-Protein interactions, Expressing, Phospho-proteomics, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Effect of E7050 on the expression level of HGF in MES-SA/Dx5 cells. ( A ) Cells were treated with various concentrations (5–25 μM) of E7050 for 24 h. Whole cell extracts were prepared and subjected to Western blotting using antibodies against HGF and β-actin. β-actin was used as an internal loading control. ( B ) Densitometric analysis of blots relative to HGF protein after normalization with β-actin. ( C ) Secreted HGF in cell culture media was determined by ELISA. Data are presented as the mean ± SEM of three independent experiments. * p < 0.05 versus vehicle-treated control cells.

Article Snippet: The concentrations of VEGF in the culture supernatants of E7050-treated MES-SA/Dx5 cells were measured using the commercial human VEGF ELISA Kit PicoKine TM according to the instructions of the manufacturer (Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Expressing, Western Blot, Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Effects of E7050 on VEGFR2-mediated signaling pathways in cultured MES-SA/Dx5 cells-derived conditioned medium (CM)-treated HUVECs. Cells were serum-starved for 6 h and pretreated with E7050 (5 and 10 μM) or vehicle for 1 h, followed by the addition of VEGF (100 ng/mL) or CM (from cultured MES-SA/Dx5 cells) for another 10 min (VEGFR2, FAK, and Src) or 30 min (Akt, JNK, and p38 MAPK) before protein extraction. The expression and phosphorylation status of VEGFR2 and its downstream effectors, including FAK, Src, Akt, JNK, and p38 MAPK, were detected by Western blotting using specific antibodies. ( A ) E7050 inhibited the phosphorylation of VEGFR2 and Src in MES-SA/Dx5 CM-induced HUVECs. ( B ) The relative band density of p-VEGFR2 protein was normalized to total VEGFR2 protein, which was measured by densitometry. Calculated ratios of ( C ) p-FAK and ( D ) p-Src normalized to the relative total protein levels are shown. ( E ) E7050 inhibited the phosphorylation of Akt, JNK, and p38 MAPK in MES-SA/Dx5 CM-induced HUVECs. The compiled results of the ratios of ( F ) p-Akt, ( G ) p-JNK, and ( H ) p-p38 MAPK normalized to relative total protein levels are shown. The data are presented as mean ± SEM of three independent experiments. # p < 0.05 compared with the untreated cells. * p < 0.05 compared with the vehicle-treated cells.

Article Snippet: The concentrations of VEGF in the culture supernatants of E7050-treated MES-SA/Dx5 cells were measured using the commercial human VEGF ELISA Kit PicoKine TM according to the instructions of the manufacturer (Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Protein-Protein interactions, Cell Culture, Derivative Assay, Protein Extraction, Expressing, Phospho-proteomics, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Effects of E7050 on tumor growth and angiogenesis in the MES-SA/Dx5 cell line-derived xenograft mouse model. Histological characteristics in the tumor tissue sections of MES-SA/Dx5 xenografts obtained from vehicle- and E7050-treated nude mice on day 28 were measured by H&E staining. The expression levels of CD31, VEGF, and p-VEGFR2 (Tyr1175) in tumor tissue sections were also examined by immunohistochemical analyses. Representative photomicrographs of H&E and immunohistochemical staining in tumor tissue sections from vehicle control and E7050-treated groups of mice are shown. Scale bar: 100 μm.

Article Snippet: The concentrations of VEGF in the culture supernatants of E7050-treated MES-SA/Dx5 cells were measured using the commercial human VEGF ELISA Kit PicoKine TM according to the instructions of the manufacturer (Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Derivative Assay, Staining, Expressing, Immunohistochemical staining, Control

Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Schematic diagram of a proposed mechanism of E7050-induced anti-angiogenic activity. E7050 exerts anti-angiogenic effects in VEGF-stimulated endothelial cells by downregulating the phosphorylation of VEGFR2 and its downstream mediators, including PLCγ1, FAK, Src, Akt, JNK, and p38 MAPK. The blockage of VEGFR2-mediated signaling cascade pathways by E7050 contributes to the inhibition of proliferation, migration, and tube formation in endothelial cells.

Article Snippet: The concentrations of VEGF in the culture supernatants of E7050-treated MES-SA/Dx5 cells were measured using the commercial human VEGF ELISA Kit PicoKine TM according to the instructions of the manufacturer (Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Activity Assay, Phospho-proteomics, Inhibition, Migration

Journal: Regenerative Therapy

Article Title: Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

doi: 10.1016/j.reth.2023.12.014

Figure Lengend Snippet: The bioactivity and targeting ability of KIT-PR1P. (A–D) The angiogenic ability of KIT-PR1P/VEGF and natural VEGF. N = 3. The Scale Bar = 200 μm ∗∗P < 0.01. (E) The targeting ability of KIT-PR1P/VEGF and PR1P/VEGF in hypoxic HK-2 cells was verified by immunofluorescence of the renal tubular injury marker Kim-1 and VEGF co-staining assay. VEGF (green), the renal tubular injury marker Kim-1 (red), DAPI (blue). Yellow staining indicates co-localization. The Scale Bar = 100 μm. (F) Fluorescence Integrated density of VEGF (35.34 ± 18.22, 17.48 ± 11.65, 4.89 ± 5.18) and Kim-1 (47.15 ± 23.47, 30.54 ± 15.99, 32.28 ± 17.85) in the KIT-PR1P/VEGF group, PR1P/VEGF group and PBS group. ∗∗∗∗P < 0.0001, ∗∗P < 0.01.All data are expressed as mean ± SD. (G) Fluorescence co-localisation analysis of Kim-1and VEGF in the KIT-PR1P/VEGF group. (H) Fluorescence co-localisation analysis of Kim-1 and VEGF in the PR1P/VEGF group. (I) Fluorescence co-localisation analysis of Kim-1 and VEGF in the PBS group.

Article Snippet: And then, extracted proteins and serum were analyzed using a human VEGF ELISA kit (Boster, Wuhan, China) according to the protocol.

Techniques: Immunofluorescence, Marker, Staining, Fluorescence

Journal: Regenerative Therapy

Article Title: Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

doi: 10.1016/j.reth.2023.12.014

Figure Lengend Snippet: The targeting ability of KIT-PR1P/VEGF in vivo . (A–B) Immunofluorescence co-staining assay. for VEGF (green) and the renal tubular injury marker Kim-1 (red) at 6 h and 24 h post-administration. DAPI (blue). The Scale Bar = 50 μm. (C) Fluorescence Integrated density of VEGF (24.60 ± 12.33, 10.79 ± 7.95, 4.47 ± 1.91, 3.17 ± 3.54) and Kim-1 (18.63 ± 9.67, 13.61 ± 6.10,13.23 ± 6.75, 13.85 ± 6.62) in the KIT-PR1P/VEGF group, PR1P/VEGF group, VEGF group and PBS group at I/R 6 h. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001.All data are expressed as mean ± SD. (D) Fluorescence Integrated density of VEGF (42.82 ± 25.27, 7.13 ± 6.16, 8.94 ± 5.11, 3.61 ± 3.33) and Kim-1 (14.60 ± 7.62, 19.28 ± 10.51, 13.49 ± 6.20, 8.70 ± 6.06) in the KIT-PR1P/VEGF group, PR1P/VEGF group, VEGF group and PBS group at I/R 24 h. ∗∗∗∗P < 0.0001. All data are expressed as mean ± SD. (E) Fluorescence co-localisation analysis of Kim-1 and VEGF in the KIT-PR1P/VEGF group, PR1P/VEGF group, VEGF group and PBS group at I/R 6 h. (F) Fluorescence co-localisation analysis of Kim-1 and VEGF in the KIT-PR1P/VEGF group, PR1P/VEGF group, VEGF group and PBS group at I/R 24 h. (G)Quantitative ELISA assay for VEGF in kidney at 6 h and 24 h post-injection. At 6 h post-injection, KIT-PR1P/VEGF = 0.616 ± 0.077 μg/g, KIT-PR1P = 0.429 ± 0.057 μg/g, PR1P/VEGF = 0.406 ± 0.033 μg/g, VEGF = 0.449 ± 0.027 μg/g, PBS = 0.266 ± 0.019 μg/g. At 24 h post-injection, KIT-PR1P/VEGF = 0.378 ± 0.041 μg/g, KIT-PR1P = 0.278 ± 0.031 μg/g, PR1P/VEGF = 0.254 ± 0.041 μg/g, VEGF = 0.185 ± 0.025 μg/g, PBS = 0.162 ± 0.060 μg/g. Data are presented as mean ± SD. N = 4, ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05. (H) Quantitative ELISA assay for VEGF in serum. At 6 h post-injection, KIT-PR1P/VEGF = 3.100 ± 2.453 pg/ml, KIT-PR1P = 5.978 ± 0.813 pg/ml, PR1P/VEGF = 12.783 ± 2.710 pg/ml,VEGF = 7.455 ± 2.315 pg/ml, PBS = 8.765 ± 0.885 pg/ml. At 24 h post-injection, KIT-PR1P/VEGF = 8.042 ± 2.113 pg/ml, KIT-PR1P = 16.243 ± 5.550 pg/ml, PR1P/VEGF = 10.776 ± 4.668 pg/ml,VEGF = 13.899 ± 4.964 pg/ml, PBS = 16.059 ± 4.776 pg/ml. Data are presented as mean ± SD. N = 4, ∗P < 0.05. (I) Western blot of VEGF in KIT-PR1P/VEGF group, KIT-PR1P group, PR1P/VEGF group, VEGF group and PBS group at I/R 6 h and 24 h after injection, and Statistical analysis of Western blot of VEGF in KIT-PR1P/VEGF, KIT-PR1P, PR1P/VEGF, VEGF and PBS at I/R 6 h and 24 h after injection. (Mean ± SD, N = 4, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001).

Article Snippet: And then, extracted proteins and serum were analyzed using a human VEGF ELISA kit (Boster, Wuhan, China) according to the protocol.

Techniques: In Vivo, Immunofluorescence, Staining, Marker, Fluorescence, Enzyme-linked Immunosorbent Assay, Injection, Western Blot

Journal: Regenerative Therapy

Article Title: Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

doi: 10.1016/j.reth.2023.12.014

Figure Lengend Snippet: The evaluation of renal function by serum Scr level after KIT-PR1P/VEGF intravenous injection. (A) Summary of Scr data for renal function after administration. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001. ∗∗P < 0.01. (B–D) Histogram of Scr assay for renal function after I/R injury and at 24 h, 72 h and 2w post-administration. All data are expressed as mean ± SD. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05. N = 6.

Article Snippet: And then, extracted proteins and serum were analyzed using a human VEGF ELISA kit (Boster, Wuhan, China) according to the protocol.

Techniques: Injection

Journal: Regenerative Therapy

Article Title: Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

doi: 10.1016/j.reth.2023.12.014

Figure Lengend Snippet: Morphology evaluation of ischemic kidney after KIT-PR1P/VEGF intravenous injection. (A–C) Histopathological staining (H&E) for kidney pathological injury observation. Scale Bar = 20 μm. Red arrow indicated renal tubular injury; green asterisk indicated glomerulus. (D–F) Quantitative analysis of renal tubule injury based on H&E staining. (G–H) Masson staining of ischemic kidney after KIT-PR1P/VEGF intravenous injection. (I–K) Quantitative analysis of the area of renal interstitial fibrosis in obstructed kidneys. All data are expressed as mean ± SD. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01. N = 5.

Article Snippet: And then, extracted proteins and serum were analyzed using a human VEGF ELISA kit (Boster, Wuhan, China) according to the protocol.

Techniques: Injection, Staining

Journal: Regenerative Therapy

Article Title: Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

doi: 10.1016/j.reth.2023.12.014

Figure Lengend Snippet: Immunofluorescence staining of CD31 in ischemic kidney after KIT-PR1P/VEGF intravenous injection. Green fluorescence in this figure indicated areas stained positive for CD31, and blue fluorescence indicated DAPI. (A–C) Immunofluorescence staining for endothelial marker CD31 after I/R injury. Scale Bar = 20 μm. (D–F) Quantitative of Immunofluorescence staining for CD31. All data was expressed as mean ± SD.∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. N = 5.

Article Snippet: And then, extracted proteins and serum were analyzed using a human VEGF ELISA kit (Boster, Wuhan, China) according to the protocol.

Techniques: Immunofluorescence, Staining, Injection, Fluorescence, Marker

Journal: Regenerative Therapy

Article Title: Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

doi: 10.1016/j.reth.2023.12.014

Figure Lengend Snippet: TUNEL staining and Immunofluorescence staining of cleaved-Caspase3 in ischemic kidney after KIT-PR1P/VEGF intravenous injection. (A–C) TUNEL staining for apoptosis cells assessment (green). Scale Bar = 100 μm. (D–F) Quantitative of kidney TUNEL-positive cells. All data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. N = 5. (H–J) Immunofluorescence staining of cleaved-Caspase3 in ischemic kidney after KIT-PR1P/VEGF intravenous injection. Red fluorescence indicated areas stained positive for cleaved-Caspase3, and blue fluorescence indicated DAPI. Immunofluorescence staining for cleaved-Caspase3 after I/R injury. Scale Bar = 50 μm. (K–M) Quantitative of Immunofluorescence staining for cleaved-Caspase3. All data was expressed as mean ± SD.∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. N = 5.

Article Snippet: And then, extracted proteins and serum were analyzed using a human VEGF ELISA kit (Boster, Wuhan, China) according to the protocol.

Techniques: TUNEL Assay, Staining, Immunofluorescence, Injection, Fluorescence

Journal: Regenerative Therapy

Article Title: Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

doi: 10.1016/j.reth.2023.12.014

Figure Lengend Snippet: Downstream pathway of KIT-PR1P/VEGF. (A) Immunofluorescence staining of p-VEGFR in ischemic kidney at 24 h intravenous injection of KIT-PR1P/VEGF after I/R injury and administration. Scale Bar = 20 μm. (B) Quantitative of IHF staining for p-VEGFR. All data are expressed as mean ± SD.∗P < 0.05, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. N = 5. (C) Western blotting assay of ischemia-reperfused kidneys for p-VEGFR, AKT, phospho-AKT, ERK, phospho-ERK at 24 h post administration. GAPDH was used as a control; (D) Statistical analysis of relative value for Western blot of p-VEGFR; (E) Statistical analysis of relative value for Western blot of AKT; (F) Statistical analysis of relative value for Western blot of p-AKT. (G) Statistical analysis of relative value for Western blot of ERK. (H) Statistical analysis of relative value for Western blot of p-ERK. N = 5. ∗∗∗∗p < 0 0.0001, ∗∗∗p < 0 0.001, ∗∗p < 0 0.01, ∗p < 0.05.

Article Snippet: And then, extracted proteins and serum were analyzed using a human VEGF ELISA kit (Boster, Wuhan, China) according to the protocol.

Techniques: Immunofluorescence, Staining, Injection, Western Blot, Control