Journal: Cell & Bioscience

Article Title: Vascular NRP2 triggers PNET angiogenesis by activating the SSH1-cofilin axis

doi: 10.1186/s13578-020-00472-6

Figure Lengend Snippet: NRP2 modulates angiogenesis by promoting HUVEC migration via a VEGF/VEGFR2-independent pathway. a HUVECs were cultured in the presence or absence of conditioned medium from BON cells (treatment and control, respectively) and then transfected with a vector control or NRP2 overexpression plasmid before they were seeded for the capillary tube formation assay. Representative images at 4, 12 and 24 h after plating are shown. b Quantification of the number of complete and broken tubes at 6 h from a representative experiment. Data are shown as the mean ± SD of three independent experiments. * P ≤ 0.05 by Student’s t test. c HUVECs were treated with conditioned medium from BON cells for 24 h and then transfected with a vector or NRP2 overexpression plasmid before they were subjected to a CCK8 assay. d After HUVECs were cultured in the presence or absence of conditioned medium from BON cells (treatment and control, respectively) and transduced with the NRP2-overexpressing plasmid, flow cytometry was performed to assess apoptosis. e Representative images for the wound-healing assay at 0, 24 and 48 h after scratching for the 4 different cell groups (HUVECs with or without NRP2 overexpression cultured in the presence or absence of conditioned medium from BON cells). f Quantification of the healing rate at 48 h after wound-healing assays in HUVECs cultured in the presence or absence of conditioned medium from BON cells followed by transfection with empty vector or NRP2 plasmid. The data are shown as the means ± SD of three independent experiments. *** P ≤ 0.001 by Student’s t test. g HUVECs were transfected with empty vector or an NRP2 overexpression plasmid and then treated with the VEGFR2-specific inhibitor KI8751. Western blotting assays were performed to determine the levels of VEGFR2 phosphorylation at Tyr951 as well as the total protein levels of VEGFR2, CD31, CD34 and GAPDH. h Control and NRP2-overexpressing HUVECs were treated with KI8751 and PBS and evaluated for tube formation. i After HUVECs were cultured in the presence or absence of conditioned medium from BON cells for 24 h, they were transfected with a vector or NRP2 overexpression plasmid. These cells were subsequently treated with PBS (control) or KI8751, and a wound-healing assay was performed. Representative image of three independent experiments is shown. j Qualification of the wound-healing rate at 48 h in HUVEC-vector or HUVEC-NRP2 cells treated with PBS or KI8751. The data are shown as the mean ± SD of three independent experiments. ** P ≤ 0.01 by Student’s t test. k After HUVECs were transfected with empty vector or an NRP2 overexpression plasmid, they were treated with PBS or KI8751 and subjected to the CCK8 assay at 24, 48 and 72 h after treatment. l Flow cytometry assay was performed in the 4 cell groups described in Fig. 2k

Article Snippet: Western blotting was performed with antibodies targeting the following proteins: VEGF receptor 2 (2479S, Cell Signaling), NRP2 (ab185710, Abcam), phospho-VEGF receptor 2 (Tyr951) (4991S, Cell Signaling), CD31 (ab28364, Abcam), CD34 (ab81289, Abcam), GAPDH (5174S, Cell Signaling), cofilin (5175S, Cell Signaling), phosphorylated cofilin (Ser3) (3313S, Cell Signaling), and SSH1 (ab76943, Abcam).

Techniques: Migration, Cell Culture, Control, Transfection, Plasmid Preparation, Over Expression, Capillary Tube Formation Assay, CCK-8 Assay, Transduction, Flow Cytometry, Wound Healing Assay, Western Blot, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: Poly(I-C) induces endothelial cell apoptosis. A, poly(I-C) induced the detachment of primary HUVECs. Cells, pretreated with 1 μg/ml poly(I-C) for 24 h, were re-stimulated with or without 10 μg/ml poly(I-C) for 24 h, and the cells in representative fields were photographed. Cells, treated with 2 μm staurosporine, were photographed as a positive control. B, poly(I-C) induced apoptosis in primary HUVECs. Cells, pretreated with 1 μg/ml poly(I-C) for 24 h, were re-stimulated with the indicated concentrations of poly(I-C) for 24 h. Unfixed cells were stained with FITC-annexin V/PI. Cell apoptosis was measured by flow cytometry analysis. C, poly(I-C) induced the dose-dependent cell apoptosis in immortalized HUVECs. Cells were treated with the indicated concentrations of poly(I-C) for 24 h. Cell apoptosis was measured as in B. Data are shown as percentages and represented as mean ± S.D. of triplicates. Cells treated with 1 μm staurosporine were used as a positive control. *, p < 0.05 compared with the control group. D, poly(I-C) (10 μg/ml) induced time-dependent cell apoptosis in immortalized HUVECs. *, p < 0.05 compared with the control group.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Positive Control, Staining, Flow Cytometry, Control

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: Poly(I-C) induces TLR3-dependent apoptosis. A, poly(I-C) up-regulated gene expression of TLR3. Immortalized HUVECs, treated with the indicated concentrations of poly(I-C) for 24 h, were harvested, and the gene expression of TLR3 was measured by RT-PCR. GAPDH transcript was measured as a loading control. B, immortalized HUVECs expressed TLR3 protein. Cells, cultured in the presence or absence of 10 μg/ml poly(I-C) for 24 h, were harvested. The expression of TLR3 protein was measured by flow cytometry. Mouse isotype Ig was used as a control. C, poly(I-C) up-regulated the expression of TLR3 protein in primary HUVECs. Cells, treated with the indicated concentrations of poly(I-C) for 24 h, were harvested and lysed with sample buffer. Equal amounts of total proteins were electrophoresed and blotted for the detection of TLR3 protein expression. β-Actin protein was detected as a loading control. D, poly(I-C) significantly up-regulated the gene expression of TLR3 in primary HUVECs. Cells were treated as in Fig. 1B. The gene transcript of TLR3 was measured by qRT-PCR. GAPDH gene expression was detected as an endogenous control. *, p < 0.05 compared with the control group. E, poly(I-C) induced the phosphorylation of the NF-κB p65 subunit. Immortalized HUVECs, starved overnight with serum-free medium, were cultured in the presence of 2 μg/ml poly(I-C) at 37 °C and lysed at the indicated time points. Phospho-p65 (P-p65) and total p65 (T-p65) were detected by Western blot. F, RT-PCR results show that poly(I-C) up-regulated the gene expression of cytokines in immortalized HUVECs. G, TLR3 neutralization abrogated poly(I-C)-induced cell apoptosis. Immortalized HUVECs (iHUVEC) and 1 μg/ml poly(I-C) pretreated (37 °C for 24 h) primary HUVECs (pHUVEC) were cultured in the absence or presence of mouse anti-human TLR3 antibody at 37 °C for 1 h and then treated with or without 2 μg/ml poly(I-C) for 24 h. Cell apoptosis was tested as in Fig. 1B. Mouse isotype Ig was used as a control. Data are represented as mean ± S.D. of triplicates. *, p < 0.05 compared with the poly(I-C) treatment group. H, TLR3 down-regulation by RNA interference abrogated poly(I-C)-induced cell apoptosis in immortalized HUVECs. Wild-type (Wt), nonspecific shRNA (NSsi), and TLR3 shRNA (TLR3si)-transfected cells were treated with 2 μg/ml poly(I-C) for 24 h. Cell apoptosis was detected as in Fig. 1B. *, p < 0.05 compared with the control group.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Control, Cell Culture, Expressing, Flow Cytometry, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Neutralization, shRNA, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: Poly(I-C) activates both the intrinsic and extrinsic pathways. A, poly(I-C) activated both caspases 8 and 9. Immortalized HUVECs, treated with the indicated concentrations of poly(I-C) for 24 h, were lysed with sample buffer. The cleaved fragments from caspases 8 and 9 were detected by Western blot. 2 μm staurosporine (SP) was used as a control. B, caspase inhibitors down-regulated the cell apoptosis induced by poly(I-C). Immortalized HUVECs were cultured in the absence or presence of caspase 8 inhibitor (Z-IETD-FMK), caspase 9 inhibitor (Z-LEHD-FMK), or pan-caspase inhibitor (Z-VAD-FMK) at 37 °C for 1 h, followed by the treatment with or without 2 μg/ml poly(I-C) for 24 h. Cell apoptosis was tested as in Fig. 1B. Data are represented as mean ± S.D. of triplicates. *, p < 0.05 compared with the poly(I-C) treatment group. C, Western blot results show that poly(I-C) activated the caspase downstream molecule PARP. D, PARP inhibitors down-regulated the cell apoptosis induced by poly(I-C). *, p < 0.05 compared with the poly(I-C) treatment group.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Western Blot, Control, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: TNFα and IFNβ do not trigger cell apoptosis. A, poly(I-C) up-regulated the expression of TNFα in immortalized HUVECs. Cells were treated with the indicated concentrations of poly(I-C) for 24 h. The supernatant was harvested to detect the TNFα secretion by ELISA. Data are represented as mean ± S.D. of triplicates. *, p < 0.05 compared with the medium control. B, TNFα (37 °C for 24 h) did not induce cell apoptosis in immortalized HUVECs. C, TNFα (50 ng/ml) induced NF-κB signaling in immortalized HUVECs. D, TNFα neutralization did not inhibit poly(I-C)-induced cell apoptosis in immortalized HUVECs. E, IFNβ (37 °C for 24 h) did not induce cell apoptosis in immortalized HUVECs. F, IFNβ neutralization did not inhibit poly(I-C)-induced cell apoptosis in immortalized HUVECs.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control, Neutralization

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: TRAIL-DR4/5 and Noxa trigger the extrinsic and intrinsic pathways, respectively. A and B, RT-PCR results show that poly(I-C) (37 °C for 24 h) up-regulated the gene expression of TRAIL, DR4, and DR5 in immortalized (A) and 1 μg/ml poly(I-C) pretreated primary (B) HUVECs. C, poly(I-C) up-regulated the protein expression of TRAIL in primary HUVECs. Cells, pretreated with 1 μg/ml poly(I-C), were re-treated with the indicated concentrations of poly(I-C) for 24 h. TRAIL in the cell lysates was assayed by ELISA. *, p < 0.05 compared with the control. D, TRAIL neutralization repressed the cell apoptosis induced by poly(I-C) in immortalized HUVECs. *, p < 0.05 compared with the poly(I-C) treatment group. E and F, RT-PCR results show the effect of poly(I-C) (37 °C for 24 h) on the gene expression of Bcl-2 and Noxa in immortalized (E) and primary (F) HUVECs. G and H, Western blot results show the effect of poly(I-C) on the protein expression of Bcl-2 and Noxa in immortalized (G) and primary (H) HUVECs. I, inhibition of TLR3 repressed the poly(I-C)-induced down-regulation of Bcl-2 and up-regulation of Noxa in immortalized HUVECs. Cells were transiently transfected with human TLR3 shRNA plasmid and then treated with 2 μg/ml poly(I-C) for 24 h. The protein expression of TLR3, Bcl-2 and Noxa was detected by Western blot.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Control, Neutralization, Western Blot, Inhibition, Transfection, shRNA, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: Roles of p53 and p63 in poly(I-C)-induced cell apoptosis. A, RT-PCR results show that poly(I-C) up-regulated p21 but neither MDM2 nor p53 gene expression. B, quantitative expression of p21, MDM2, and p53 in A by qRT-PCR. *, p < 0.05 compared with the control group. C, effect of p53 inhibitor pifithrin-α (PFT) on poly(I-C)-induced cell apoptosis in immortalized HUVECs. D, p53 expression was down-regulated by RNA interference in immortalized HUVECs. Cells were transiently transfected with p53 siRNA. p53 gene expression was detected 48 h after the transfection. E, effect of p53 down-regulation on poly(I-C)-induced cell apoptosis in immortalized HUVECs. Cells were transiently transfected with p53 siRNA and cultured for 24 h and then were treated with poly(I-C) for 24 h for the detection of cell apoptosis. F and G, poly(I-C) up-regulated the gene expression of TAp63α. Immortalized (F) and 1 μg/ml poly(I-C) pretreated primary (G) HUVECs were treated with the indicated concentrations of poly(I-C) for 24 h. The gene expression of p63 was measured by RT-PCR using a primer pair in the N terminus to detect the transactivation domain and a primer pair in the C terminus to detect p63α, -β, and -γ splices. H and I, Western blot results show that poly(I-C) up-regulated the protein expression of TAp63α in immortalized (H) and primary (I) HUVECs. J, Western blot results show that the TLR3 down-regulation by TLR3 shRNA transient transfection inhibited the expression of TAp63α in primary HUVECs.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Expressing, Quantitative RT-PCR, Control, Transfection, Cell Culture, Western Blot, shRNA

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: Modulation of p63 expression represses poly(I-C)-induced cell apoptosis. A, p63 was down-regulated by RNA interference in immortalized HUVECs. Wild-type (Wt), nonspecific shRNA (NSsi), and p63 shRNA (p63si) stably transfected cells were treated with 2 μg/ml poly(I-C) for 24 h. TAp63α protein was measured by Western blot. B, p63 down-regulation repressed poly(I-C)-induced cell apoptosis in immortalized HUVECs. *, p < 0.05 compared with the Wt group. C, Western blot results show that p63 RNA interference inhibited the down-regulation of Bcl-2 and the up-regulation of Noxa in immortalized HUVECs. D, Western blot results show that p63 RNA interference inhibited the activation of caspases 8 and 9 and PARP in immortalized HUVECs. E, RT-PCR results show that p63 RNA interference inhibited the expression of TRAIL, DR4, and DR5 in immortalized HUVECs. F, ΔNp63α was up-regulated by gene transfection in immortalized HUVECs. Cells were transiently transfected with pCDNA3+ (Mock) or pCDNA3+/ΔNp63α and harvested at 48 h post-transfection. The gene expression of ΔNp63α was detected by RT-PCR. G, ΔNp63α overexpression inhibited poly(I-C)-induced cell apoptosis in immortalized HUVECs. Cells were transfected as in F and cultured for 24 h, treated with 2 μg/ml poly(I-C) for another 24 h, and then assayed for cell apoptosis. *, p < 0.05 compared with control groups.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, shRNA, Stable Transfection, Transfection, Western Blot, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Over Expression, Cell Culture, Control

Journal: Advanced Science

Article Title: Chaperonin–Dendrimer Conjugates for siRNA Delivery

doi: 10.1002/advs.201600046

Figure Lengend Snippet: Cell uptake of THS. a) Flow cytometry of different cell lines incubated with THS–Atto647: MCF‐7 (red), PC‐3 (brown), HUVEC (light green), CHO‐K1 (dark green), U87 (orange) and as a control U87 in the absence of THS–Atto647 (blue). b) Confocal microscopy images of U87 cells incubated with THS–Atto647 for 30 min and 2 h: THS–Atto647 (green), nucleus (blue), left: cell membrane (red), right: acidic compartments (red); scale bars: 20 μm.

Article Snippet: Following cell lines were purchased from ATCC (USA): breast adenocarcinoma cells MCF‐7 (ATCC HTB‐22), Chinese hamster ovary cells CHO‐K1 (ATCC CCL‐61), vascular endothelium cells HUVEC (ATCC CRL‐1730), glyoblastoma cells U‐87 MG (ATCC HTB‐14) and cervical cancer cells HeLa (ATCC CCL‐2).

Techniques: Flow Cytometry, Incubation, Control, Confocal Microscopy, Membrane

Journal: PLoS ONE

Article Title: Multiple Delivery of siRNA against Endoglin into Murine Mammary Adenocarcinoma Prevents Angiogenesis and Delays Tumor Growth

doi: 10.1371/journal.pone.0058723

Figure Lengend Snippet: Expression of endoglin in endothelial cells, tumor cells and tumors, expressed as threshold cycle values (Ct) obtained by qRT-PCR in comparison to reference gene.

Article Snippet: Murine endothelial cell line 2H11 (American Type Culture Collection, Manassas, VA) was cultured in Advanced DMEM medium (Gibco) supplemented with 5% fetal bovine serum, 10 mM L-glutamine, 50 μg/ml gentamicin and 100 U/ml crystacillin in a 5% CO2 humidified incubator at 37°C.

Techniques: Expressing, Comparison

Journal: PLoS ONE

Article Title: Multiple Delivery of siRNA against Endoglin into Murine Mammary Adenocarcinoma Prevents Angiogenesis and Delays Tumor Growth

doi: 10.1371/journal.pone.0058723

Figure Lengend Snippet: (A) qRT-PCR analysis of endoglin mRNA level after the transfection of endothelial cells HMEC-1 with 3 different siRNA molecules (h_siRNA 529, h_siRNA 240, h_siRNA 241). (B) qRT-PCR analysis of endoglin mRNA level after the transfection of endothelial cells 2H11 with 3 different siRNA molecules (m_siRNA 868, m_siRNA 869, m_siRNA 150). mRNA level of endoglin in cells treated with siRNA molecules was normalized to the mRNA level of endoglin in cells treated with Lipofectamine RNAiMAX only (% of LF); *P<0.05 vs. untreated control cells (HMEC-1 or 2H11), LF and negative control siRNA (siRNA Ctrl). Data represent arithmetic mean±SEM. (C) Immunofluorescence staining of endoglin after the transfection of endothelial cells HMEC-1 with 3 different siRNA molecules (h_siRNA 529, h_siRNA 240, h_siRNA 241). (D) Immunofluorescence staining of endoglin after the transfection of endothelial cells 2H11 with 3 different siRNA molecules (m_siRNA 868, m_siRNA 869, m_siRNA 150). (E) Mean fluorescence intensity of HMEC cells stained for endoglin determined with flow cytometry analysis after the transfection of cells with 3 different siRNA molecules (h_siRNA 529, h_siRNA 240, h_siRNA 241). (F) Mean fluorescence intensity of 2H11 cells stained for endoglin determined with flow cytometry analysis after the transfection of cells with 3 different siRNA molecules (m_siRNA 868, m_siRNA 869, m_siRNA 150). Values are normalized to Lipofectamine RNAiMAX group (% of LF). *P<0.05 vs. untreated control cells (HMEC-1, 2H11), LF and siRNA Ctrl.

Article Snippet: Murine endothelial cell line 2H11 (American Type Culture Collection, Manassas, VA) was cultured in Advanced DMEM medium (Gibco) supplemented with 5% fetal bovine serum, 10 mM L-glutamine, 50 μg/ml gentamicin and 100 U/ml crystacillin in a 5% CO2 humidified incubator at 37°C.

Techniques: Quantitative RT-PCR, Transfection, Control, Negative Control, Immunofluorescence, Staining, Fluorescence, Flow Cytometry

Journal: PLoS ONE

Article Title: Multiple Delivery of siRNA against Endoglin into Murine Mammary Adenocarcinoma Prevents Angiogenesis and Delays Tumor Growth

doi: 10.1371/journal.pone.0058723

Figure Lengend Snippet: (A) Proliferation of HMEC-1 cells after the transfection with Lipofectamine RNAiMAX only (LF), h_siRNA 529, h_siRNA 240, h_siRNA 241 and negative control siRNA (siRNA Ctrl). (B) Proliferation of 2H11 cells after the transfection with Lipofectamine RNAiMAX only (LF), m_siRNA 868, m_siRNA 869, m_siRNA 150 and negative control siRNA (siRNA Ctrl). (C) Proliferation of TS/A cells after the transfection with Lipofectamine RNAiMAX only (LF), m_siRNA 868, m_siRNA 869, m_siRNA 150 and negative control siRNA (siRNA Ctrl). Proliferation of cells in each experimental group was normalized to day 0. *P<0.05 vs. untreated control cells (HMEC-1, 2H11 or TS/A), LF and siRNA Ctrl. Note that due to the slower growth of HMEC-1 cells, the y-axis has different range.

Article Snippet: Murine endothelial cell line 2H11 (American Type Culture Collection, Manassas, VA) was cultured in Advanced DMEM medium (Gibco) supplemented with 5% fetal bovine serum, 10 mM L-glutamine, 50 μg/ml gentamicin and 100 U/ml crystacillin in a 5% CO2 humidified incubator at 37°C.

Techniques: Transfection, Negative Control, Control

Journal: PLoS ONE

Article Title: Multiple Delivery of siRNA against Endoglin into Murine Mammary Adenocarcinoma Prevents Angiogenesis and Delays Tumor Growth

doi: 10.1371/journal.pone.0058723

Figure Lengend Snippet: Original images of tubular complexes of HMEC-1 cells (A) and corresponding binary masks (B) 2 days after the transfection with different siRNA molecules: untreated control cells (HMEC-1), cells transfected with Lipofectamine RNAiMAX only (LF), h_siRNA 529, h_siRNA 240, h_siRNA 241 and negative control siRNA (siRNA Ctrl). Original images of tubular complexes of 2H11 cells (C) and corresponding binary masks (D) 2 days after the transfection with different siRNA molecules: untreated control cells (2H11), cells transfected with Lipofectamine RNAiMAX only (LF), m_siRNA 868, m_siRNA 869, m_siRNA 150 and negative control siRNA (siRNA Ctrl). Scale bar: 100 µm.

Article Snippet: Murine endothelial cell line 2H11 (American Type Culture Collection, Manassas, VA) was cultured in Advanced DMEM medium (Gibco) supplemented with 5% fetal bovine serum, 10 mM L-glutamine, 50 μg/ml gentamicin and 100 U/ml crystacillin in a 5% CO2 humidified incubator at 37°C.

Techniques: Transfection, Control, Negative Control

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Fucosyltransferase 1 mediates angiogenesis in rheumatoid arthritis.

doi: 10.1002/art.38648

Figure Lengend Snippet: Figure 4. A, Purity of wild-type mouse lung endothelial cells (MLECs), as determined by flow cytometry. The purity of C57BL/6 wild-type (WT) mouse lung ECs was 76.3%, as determined by CD146 staining via flow cytometric analysis. Blocking fucosyltransferase 1 (FUT1) in ECs reduced EC chemotaxis. Green represents CD146-positive cells; black represents control IgG. Results are representative of 2 independent assays. B, Reduction in cell migration in ECs completely lacking FUT1 expression. FUT1 gene–deficient mouse lung ECs had significantly less migration compared to wild-type mouse lung ECs in response to mouse vascular endothelial cell growth factor (VEGF; 10 nM and 100 nM). hpfs high-power fields. The n value is the number of experiments. C, Marked increase in Flt1/VEGF receptor 1 (VEGFR1) induced by tumor necrosis factor (TNF; 25 ng/ml) in wild-type mouse lung ECs compared to FUT1 gene–deficient mouse lung ECs at 8, 16, and 24 hours. Results are representative of 2 independent assays. KDR kinase insert domain receptor. D, FUT1 antisense oligonucleotide (ODN)–transfected HMVEC–conditioned medium reduces HMVEC migration compared to FUT1 sense ODN–transfected HMVEC–conditioned medium. Phosphate buffered saline (PBS) and VEGF were used as negative and positive controls, respectively. The mean SEM number of HMVECs migrating toward PBS was 5 1 and the number migrating toward VEGF was 40 6. Bars show the mean SEM. The n value is the number of experiments. Color figure can be viewed in the online issue which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.38648/abstract.

Article Snippet: The controls used were vascular endothelial cell growth factor (VEGF) (10 nM; R&D Systems) as a positive control and PBS as a negative control.

Techniques: Flow Cytometry, Staining, Blocking Assay, Chemotaxis Assay, Control, Migration, Expressing, Transfection, Saline

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: Endothelial cells characterization regarding morphological, immunophenotyping, and vessel-like structures assay. ( A ) Phase contrast micrography demonstrating the polygonal morphology of aortic artery endothelial cells (PAEC), coronary artery endothelial cells (CAEC), human umbilical vein endothelial cells (HUVEC), and pulmonary artery endothelial cells (HPAEC) cells (100× magnification). ( B ) Immunophenotyping of ECs by flow cytometry. ( C ) All endothelial cells (PAEC, CAEC, HUVEC, and HPAEC) were able to form vessel-like structures when grown in matrigel, evidencing characteristics typical of CEs (40× and 100× magnification).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques: Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: Characterization of EndMT induction by TGF-β2 (10 ng/mL) in cell lines ( A ) PAEC, ( B ) CAEC, ( C ) HPAEC, and ( D ) HUVECs (non-treated or treated with TGF-β2). Immunofluorescence microscopy of cell lines induced to EndMT shows a decrease in the fluorescent intensity of CD31 (green) in PAECs, CAECs, and HUVECs cells. The nuclei were stained with DAPI (blue) and F-actin were stained with Phalloidin (red) (scale bar 50 µM; representative image of one replicate of each sample).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques: Immunofluorescence, Microscopy, Staining

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: TGF-β2 decrease formation of vessel-like structures in the cell lines (CAEC, PAEC, HPAEC, and HUVEC). The cells were treated with TGF-β2 and evaluated the capacity formation of vessel-like structures. This inhibitory effect was observed mainly in PAECs (representative image of one replicate; n = 3).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: Effect of EndMT on the activation of the Erk pathway. The cells (CAEC, PAEC, HUVEC and HPAEC) were cultured for five days in presence TGF-β2 (10 ng/mL). Aliquots were withdrawn after the treatment and evaluated by ( A ) Multiplex technique analysis Array Kit ( n = 3, * p ≤ 0.05) and ( B ) western blotting using phospho-Erk1/2 (Thr202/Tyr204) and ERK1/2. β-actin were used as endogenous controls (representative image of one replicate of each sample). ( C ) Chemical inhibitor against MEK1/2 (U0126; 1 μM) inhibits the increase of ERK1/2 phosphorylation in the PAECs treated with TGF-β2. 1) U0126; 2) U0126-15′ TGF-β2; 3) U0126-30′ TGF-β2; 4) TGF-β2-15′; 5) TGF-β2-30′. GAPDH were used as endogenous controls (representative image of one replicate of each sample).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques: Activation Assay, Cell Culture, Multiplex Assay, Western Blot, Phospho-proteomics

Journal: Journal of Inflammation (London, England)

Article Title: ERK2 and Akt are negative regulators of insulin and Tumor Necrosis Factor-α stimulated VCAM-1 expression in rat aorta endothelial cells

doi: 10.1186/s12950-016-0115-6

Figure Lengend Snippet: Insulin and TNFα increase expression of total VCAM-1 in rat aorta endothelial cells over time. RAEC were cultured in Complete Growth Medium (CGM) until 90 % confluent. Subsequently, cells were stimulated for indicated times with insulin (10 nM) or TNFα (10 ng/mL) alone or in combination. Total VCAM-1 protein was determined by Western blot analysis. Analysis was performed by the Licor Odyssey detection system. Total VCAM-1 is expressed as the percent of controls and represents the mean ± SEM of six independent experiments. *, P < 0.05 vs negative controls. **, P < 0.05 vs TNFα alone

Article Snippet: Rat aorta vascular endothelial cells (RAEC) (CRL-1444) were from ATCC (Manassas, VA) and culture medium was from Life Technologies (Grand Island, NY).

Techniques: Expressing, Cell Culture, Western Blot

Journal: Journal of Inflammation (London, England)

Article Title: ERK2 and Akt are negative regulators of insulin and Tumor Necrosis Factor-α stimulated VCAM-1 expression in rat aorta endothelial cells

doi: 10.1186/s12950-016-0115-6

Figure Lengend Snippet: Different clones of shRNA for Akt and ERK2 differentially downregulate their respective proteins. Akt and ERK2 shRNA clones provided by manufacturer were amplified in E. coli and isolated by plasmid preparation. Individual clones of each shRNA were transfected into different groups of RAEC as described in Methods. (Panel a ) Akt and ERK2 proteins were quantified in unstimulated cells by Western blot analysis in untreated cells. “0” is negative control clone, and numbers 1–4 are different clones provide by the manufacturer. Intensity of bands are proportional to expression of designated protein in the presence of each shRNA clone. (Panel b ) Relative protein expression is compared to controls (negative control clone)

Article Snippet: Rat aorta vascular endothelial cells (RAEC) (CRL-1444) were from ATCC (Manassas, VA) and culture medium was from Life Technologies (Grand Island, NY).

Techniques: Clone Assay, shRNA, Amplification, Isolation, Plasmid Preparation, Transfection, Western Blot, Negative Control, Expressing

Journal: Journal of Inflammation (London, England)

Article Title: ERK2 and Akt are negative regulators of insulin and Tumor Necrosis Factor-α stimulated VCAM-1 expression in rat aorta endothelial cells

doi: 10.1186/s12950-016-0115-6

Figure Lengend Snippet: RAEC transfected with shERK2 (clone #2) and then stimulated with insulin or TNFα alone or in combination demonstrates the negative regulation of ERK2 in VCAM-1 expression. RAEC were stably transfected with shERK2 (ERK2 KD) and then stimulated with insulin (10 nM, 1 h) or TNFα (10 ng/mL, 6 h) alone or in combination. Total VCAM-1 protein was analyzed by SDS-PAGE and determined by Western blot analysis. (Upper Panel) “NST”, cells not stimulated with insulin or TNFα. “Ins” cells stimulated with insulin. “TNFα” cells stimulated with TNFα. “C” control cells not transfected with shRNA, while “sh” cells were transfected with shERK2. (Lower Panel) “C” control RAEC not transfected (open bars). ERK2 KD are RAEC stably transfected with shERK2 (black bars). “NST” cells not stimulated with insulin or TNFα. Total VCAM-1 is expressed as the percent of controls and represents the mean ± SEM of six independent experiments. Alpha-tubulin was used to normalize VCAM-1 profiles. *, P < 0.05 vs non-stimulated cells

Article Snippet: Rat aorta vascular endothelial cells (RAEC) (CRL-1444) were from ATCC (Manassas, VA) and culture medium was from Life Technologies (Grand Island, NY).

Techniques: Transfection, Expressing, Stable Transfection, SDS Page, Western Blot, Control, shRNA

Journal: Journal of Inflammation (London, England)

Article Title: ERK2 and Akt are negative regulators of insulin and Tumor Necrosis Factor-α stimulated VCAM-1 expression in rat aorta endothelial cells

doi: 10.1186/s12950-016-0115-6

Figure Lengend Snippet: RAEC transfected with shAkt (clone #2) and then stimulated with insulin or TNFα alone or in combination demonstrates the negative regulation of Akt in VCAM-1 expression. REAC were stably transfected with shAkt (Akt KD) and then stimulated with insulin (10 nM, 1 h) or TNFα (10 ng/mL, 6 h) alone or in combination. Total VCAM-1 protein was analyzed by SDS-PAGE and determined by Western blot analysis. (Upper Panel) “NST”, cells not stimulated with insulin or TNFα. “Ins” cells stimulated with insulin. “TNFα” cells stimulated with TNFα. “C” control RAEC not transfected with shRNA, while “sh” cells were transfected with shAkt. (Lower Panel) “C” control RAEC not transfected (open bars). Akt KD are RAEC stably transfected with shAkt (black bars). “NST” cells not stimulated with insulin or TNFα. Total VCAM-1 is expressed as the percent of controls and represents the mean ± SEM of six independent experiments. Alpha-tubulin was used to normalize VCAM-1 profiles. *, P < 0.05 vs non-stimulated cells

Article Snippet: Rat aorta vascular endothelial cells (RAEC) (CRL-1444) were from ATCC (Manassas, VA) and culture medium was from Life Technologies (Grand Island, NY).

Techniques: Transfection, Expressing, Stable Transfection, SDS Page, Western Blot, Control, shRNA

Journal: Journal of Inflammation (London, England)

Article Title: ERK2 and Akt are negative regulators of insulin and Tumor Necrosis Factor-α stimulated VCAM-1 expression in rat aorta endothelial cells

doi: 10.1186/s12950-016-0115-6

Figure Lengend Snippet: RAEC transfected with shAkt and shERK2 and then stimulated with insulin or TNFα alone or in combination exhibit increased VCAM-1 protein expression greater than stimulated, mock transfected cells, but not greater than cells transfected with shAkt alone. RAEC were stably transfected with shAKT and then transiently transfected with shERK2. Subsequently, cells were stimulated with insulin (10 nM, 1 h) or TNFα (10 ng/mL, 6 h) or in combination. (Upper Panel) C, control RAEC, no shRNA. A, cells transfected with shAkt alone. AE, cells stably transfected with shAkt and then transiently transfected with shERK2. (Lower Panel) Open bars are control cells not transfected. Stippled bars (Akt KD) RAEC stably transfected with shAkt. Black bars (Akt KD + shERK2) RAEC stably transfected with shAkt and transiently transfected with shERK2. “NST”, cells not stimulated with insulin or TNFα. Total VCAM-1 is expressed as fold increase above negative controls and represents the mean ± SEM of five independent experiments. Alpha-tubulin was used to normalize VCAM-1 profiles. #, P < 0.05 verses non-stimulated controls. *, P < 0.05 versus stimulated controls of similar treatment group without shRNA

Article Snippet: Rat aorta vascular endothelial cells (RAEC) (CRL-1444) were from ATCC (Manassas, VA) and culture medium was from Life Technologies (Grand Island, NY).

Techniques: Transfection, Expressing, Stable Transfection, Control, shRNA

Journal: Journal of Inflammation (London, England)

Article Title: ERK2 and Akt are negative regulators of insulin and Tumor Necrosis Factor-α stimulated VCAM-1 expression in rat aorta endothelial cells

doi: 10.1186/s12950-016-0115-6

Figure Lengend Snippet: RAEC transfected with shERK2 and shAkt and then stimulated with insulin or TNFα alone or in combination exhibit increased VCAM-1 protein expression greater than stimulated mock transfected cells, but not greater than cells transfected with one shERK2 alone. RAEC were stably transfected with shERK2 and then transiently transfected with shAkt. Subsequently, cells were stimulated with insulin (10 nM, 1 h) or TNFα (10 ng/mL, 6 h) or in combination. (Upper Panel) C, control RAEC, no shRNA. E, cells transfected with shERK2 alone. EA, cells stably transfected with shERK2 and then transiently transfected with shAkt. (Lower Panel) Open bars are control cells not transfected. Stippled bars (ERK2 KD) RAEC stably transfected with shERK2. Black bars (ERK2 KD + shAkt) RAEC stably transfected with shERK2 and transiently transfected with shAkt. “NST”, cells not stimulated with insulin or TNFα. Total VCAM-1 is expressed as fold increase above negative controls and represents the mean ± SEM of five independent experiments. Alpha-tubulin was used to normalize VCAM-1 profiles. #, P < 0.05 verses non-stimulated controls. *, P < 0.05 versus stimulated controls of similar treatment group without shRNA

Article Snippet: Rat aorta vascular endothelial cells (RAEC) (CRL-1444) were from ATCC (Manassas, VA) and culture medium was from Life Technologies (Grand Island, NY).

Techniques: Transfection, Expressing, Stable Transfection, Control, shRNA

Journal: Journal of Inflammation (London, England)

Article Title: ERK2 and Akt are negative regulators of insulin and Tumor Necrosis Factor-α stimulated VCAM-1 expression in rat aorta endothelial cells

doi: 10.1186/s12950-016-0115-6

Figure Lengend Snippet: RAEC transfected with shAkt and shERK2 exhibit increased insulin and TNFα-stimulated VCAM-1 at the cell surface as determined by flow cytometery. (Panel a ) RAEC were stably transfected with shAkt (Akt KD) or (Panel b ) shERK2 (ERK2 KD) and then transiently transfected with shERK2 or shAkt, respectively. Subsequently, cells were stimulated without or with insulin (10 nM) or TNFα (10 ng/mL) or in combination and surface VCAM-1 was determined by flow cytometry. C, non-transfected RAEC (no shRNA). NST, no stimulation. Akt KD, stable cell lines with shAkt. ERK2 KD, stable cell lines with shERK2. Akt KD + shERK2, stable shAkt cells lines transiently transfected with shERK2. ERK2 KD + shAkt, stable cell lines with shERK2 transiently transfected with shAkt. Surface VCAM-1 is expressed as percent of respective controls (not stimulated, No Stim) and represents the mean ± SEM of five independent experiments. *, P < 0.05 compared to their respective unstimulated controls. #, P < 0.05 compared to stimulated non-transfected controls

Article Snippet: Rat aorta vascular endothelial cells (RAEC) (CRL-1444) were from ATCC (Manassas, VA) and culture medium was from Life Technologies (Grand Island, NY).

Techniques: Transfection, Stable Transfection, Flow Cytometry, shRNA

Journal: Journal of Inflammation (London, England)

Article Title: ERK2 and Akt are negative regulators of insulin and Tumor Necrosis Factor-α stimulated VCAM-1 expression in rat aorta endothelial cells

doi: 10.1186/s12950-016-0115-6

Figure Lengend Snippet: RAEC transfected with shAkt and shERK2 exhibit increased insulin and TNFα-stimulated cell surface VCAM-1 as determined by confocal microscopy. RAEC were plated and stimulated as described in Methods. Surface VCAM-1 was detected by immunocytochemistry as described in Methods and visualized by confocal microscopy. CON, RAEC controls, no shRNA; No Stim, no stimulation by insulin or TNFα; Akt KD, stable cell lines transfected with shAkt. ERK2 KD, stable cell lines transfected with shERK2. shAkt, cells transiently transfected with shAkt. shERK2, cells transiently transfected with shERK2. Scale bar is 50 μm

Article Snippet: Rat aorta vascular endothelial cells (RAEC) (CRL-1444) were from ATCC (Manassas, VA) and culture medium was from Life Technologies (Grand Island, NY).

Techniques: Transfection, Confocal Microscopy, Immunocytochemistry, shRNA, Stable Transfection

Journal: bioRxiv

Article Title: Immunoregulatory subtype of dermal lymphatic endothelial cells at capillary terminals drives lymphatic malformations

doi: 10.1101/2022.05.22.492950

Figure Lengend Snippet: ( A-E ) Quantification of the CD45 + area ( A , B ) and F4/80 + area ( C - E ) in the ear skin showing increase in Pik3ca H1047R ;Vegfr3-CreER T2 ( A, C, D ) but not in Pik3ca H1047R ;Vegfr1-CreER T2 ( B, C, E ) mice. Data represent mean (CD45: n =4-6 images from n =3-5 mice per genotype; F4/80: n =3-7 images from n =2-3 mice per genotype) ± s.e.m. Representative binary images are shown below the graphs. ( F, G ) Flow cytometry analysis of innate ( F ) and adaptive ( G ) immune cells in the ear skin of 4-OHT-treated 5-week-old and 10-week-old Pik3ca H1047R ; Vegfr3-CreER T2 mice and littermate controls. Mac, macrophage; Mono, monocyte; DC, dendritic cell; Neu, neutrophil; NK, natural killer cell. Data represent relative cell frequency (of live cells) relative to the control ( n =3-9 mice) ± s.e.m. ( H ) Multiplex ELISA analysis of pro-inflammatory cytokines and chemokines associated with recruitment and/or activation of myeloid cells in whole ear skin lysates from Pik3ca H1047R ;Vegfr3-CreER T2 mice and littermate controls. Data represent mean protein levels relative to control ( n =6-11 mice) ± s.e.m. ( I ) qRT-PCR analysis (left) and ELISA analysis (right) of Vegfc /VEGF-C levels in the ear skin lysates of 4-OHT-treated 5-week-old Pik3ca H1047R ;Vegfr3-CreER T2 and littermate control mice. qRT-PCR data represent mean relative expression (normalized to Hprt ; n =7-10 mice) ± s.e.m. Transcript and protein levels are presented relative to controls. ( J ) qRT-PCR analysis of Vegfc in CD45 + Cd11b + F4/80 + macrophages and CD45 + Cd11b + F4/80 + Ly6C + monocytes, FACS-sorted from the ear skin of 4-OHT-treated 5-week-old Pik3ca H1047R ;Vegfr3-CreER T2 and littermate control mice. qRT-PCR data represent mean relative expression (normalized to Hprt ; n =3-5 mice) ± s.e.m. Transcript and protein levels are presented relative to macrophages/monocytes in control mice. p -value in (A, B, D-I) obtained using Two-tailed unpaired Student’s t-test. Scale bar: 100 μm (A-E).

Article Snippet: Mouse Vascular Endothelial Cell Growth Factor C (VEGF-C) ELISA Kit from CUSABIO (CSB-E07361m) was used for detection of VEGF-C protein concentration.

Techniques: Flow Cytometry, Control, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Immunoregulatory subtype of dermal lymphatic endothelial cells at capillary terminals drives lymphatic malformations

doi: 10.1101/2022.05.22.492950

Figure Lengend Snippet: ( A ) Flow cytometry analysis of the number of CD45 + CD11b + F4/80 + macrophages in the ear skin of 4-OHT-treated 5-week-old Pik3ca H1047R ;Vegfr3-CreER T2 ( n =5) and control ( n =3) mice. Data represent mean cell number per gram tissue ± s.e.m. p -value, Mann-Whitney U Test. ( B ) Flow cytometry analysis of the frequency of CD45 + CD11b + F4/80 + antigen-presenting myeloid cells in the ear skin of 4-OHT-treated 5-week-old Pik3ca H1047R ;Vegfr1-CreER T2 ( n =6) and control ( n =5) mice. Data represent relative cell frequency (of live cells) relative to the control ± s.e.m. p -value obtained using Two-tailed unpaired Student’s t-test. ( C ) Flow cytometry analysis of innate (left) and adaptive (right) immune cells in the ear skin of 4-OHT-treated 10-week-old Pik3ca H1047R ; Vegfr1-CreER T2 mice and littermate controls. Mac, macrophage; Mono, monocyte; DC, dendritic cell; Neu, neutrophil; NK, natural killer cell. Data represent relative cell frequency (of live cells) relative to the control ( n =5-8 mice for innate panel, n=7-9 mice for adaptive panel) ± s.e.m. p -value obtained using Two-tailed unpaired Student’s t-test. ( D, E ) Multiplex ELISA analysis of pro-inflammatory cytokines and chemokines associated with recruitment and/or activation of myeloid cells or T-cells and B-cells in whole ear skin lysates from Pik3ca H1047R ;Vegfr1-CreER T2 ( D ) and Pik3ca H1047R ;Vegfr3-CreER T2 ( E ) mice, and respective littermate controls. ( F ) Similar analysis of TNFα and INFγ in blood serum of Pik3ca H1047R ;Vegfr3-CreER T2 mice. Data in ( D-E ) represent mean protein levels ( n =3-9 mice) ± s.e.m.

Article Snippet: Mouse Vascular Endothelial Cell Growth Factor C (VEGF-C) ELISA Kit from CUSABIO (CSB-E07361m) was used for detection of VEGF-C protein concentration.

Techniques: Flow Cytometry, Control, MANN-WHITNEY, Two Tailed Test, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Activation Assay