Journal: Nature Communications

Article Title: Directed self-assembly of a xenogeneic vascularized endocrine pancreas for type 1 diabetes

doi: 10.1038/s41467-023-36582-1

Figure Lengend Snippet: A Immunofluorescence image of BOEC endothelial phenotyping CD31 (green) and Von Willebrand factor (vWF-red), VE-cadherin (green), vascular endothelial growth factor receptor 2 (VEGF-R2-red) and DAPI (blue). Scale bar in µm. Two independent experiments; the representative images were shown; B Flow cytometry BOECs phenotyping for CD31, VE-cadherin, CD34 and KDR (blue and red: histogram for isotype control and positive cells respectively); C WST-1 assay for BOEC proliferation in the presence of standard BOEC culture medium (BCM) or VEP media combination, ( n = 8 biological replicates) for 24 or 48 h. Values presented as mean ± SD. BCM vs . VEP media combination 24 h *** p = 0.0002; BCM vs . VEP media combination 48 h *** p = 0.0002; Mann-Whitney U-test D , E BOECs tube formation assay: D Morphology of BOECs tube formation assay in the presence of VEP media, MSM Modified Stabilization Medium, AM Angiogenic Medium, and BCM magnification 4×. Two independent experiments; the representative images are shown. E Analysis of the formed vascular mesh for vascular density analysis in the presence of BCM, AM, MSM and VEP media combination, ( n = 10 biological replicates), values presented as mean ± SD (BCM vs . Angiogenic medium * p = 0.0166; BCM vs . VEP media combination * p = 0.0235), one way ANOVA with Dunn’s correction. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies included antibodies against human CD31 (1:40, DAKO, M0823, clone: JC70A), human vWF (1:200, DAKO, A0082, polyclonal), human VE-Cadherin (1:40, R&D system,AF938, polyclonal), human VEGF-R2 (1:200, Cell Signaling, 2479 S, polyclonal), pig Insulin (1:200; DAKO, A0564, polyclonal), pig Chromogranin A (1:200, Abcam, AB15160, polyclonal), mouse CD31(1:300, Biolegend, 102501, clone: MEC13.3), mouse CD11b-PE (1:100, Biolegend, 101208, clone: M1/70), mouse LY6G-AF647(1:100, Biolegend, 127610, clone: 1A8), mouse anti-tubulin β3-AF647 (1:100 Biolegend, 801210, clone: TUJ1), anti-pig somatostatin (H-11) (1:100 Santa Cruz Biotechnology, Sc-74556, clone H-11), anti-pig glucagon (1:200 Invitrogen, PA5-83353, polyclonal).

Techniques: Immunofluorescence, Flow Cytometry, WST-1 Assay, MANN-WHITNEY, Tube Formation Assay, Modification

Journal: bioRxiv

Article Title: Apelin improves angiogenesis and blood flow reperfusion following lower limb ischemia in diabetic mice

doi: 10.1101/2023.02.08.527688

Figure Lengend Snippet: BAECs were incubated with normal glucose (NG; 5.6 mmol/L; white bars) or high glucose (HG; 25 mmol/L; black bars) for up to 48h and then stimulated with either VEGF-A or Pyr-apelin-13 for 16h (A-C) , 4h (D-E) , 5 min (VEGF) or 1h (Pyr-apelin-13) (F) under hypoxia (1% O 2 ) for the last 16h of treatment in all experiments. (A) Representative images of the cell migration assay using the Ibidi’s insert. (B) The percentage of the surface area occupied by the BAECs was quantified. (C) BAECs were fixed and stained with DAPI (4’,6-diamidino-2-phenylindole) and then cells were counted using the NIS-Elements software of Nikon eclipse Ti microscope. (D) Representative images of the tubule formation abilities of endothelial cells using Ibidi’s μ -slide rack. (E) Tubule formation was quantified by measuring the total number of closed circles in the entire well, normalized on the NG condition. (F) Protein expression of Akt phosphorylation was detected by immunoblot analysis and the densitometry quantification was measured. Results are presented as the mean ± SD of 8 independent cell experiments.

Article Snippet: BAECs were stimulated with either VEGF-A (10 ng/mL) for 5 minutes or Pyr-apelin-13 (200 nM) for 1h (immunoblotting and cell signaling experiments) or 24h prior to RNA extraction and quantitative PCR analysis.

Techniques: Incubation, Cell Migration Assay, Staining, Software, Microscopy, Expressing, Western Blot

Journal: bioRxiv

Article Title: Apelin improves angiogenesis and blood flow reperfusion following lower limb ischemia in diabetic mice

doi: 10.1101/2023.02.08.527688

Figure Lengend Snippet: Expression levels of (A) APJ, (B) Apelin, (C) Flk-1, (D) VEGF-A, ( E) PDGFR- β and (F) PDGF-B mRNA in the ischemic muscle of nondiabetic (NDM; white bars), diabetic (DM; black bars) and diabetic mice receiving Pyr-apelin-13 (DM+Pyr-ape-13; grey bars). GAPDH gene was used for mRNA normalization. Results are presented as the mean ± SD of 9 mice per group.

Article Snippet: BAECs were stimulated with either VEGF-A (10 ng/mL) for 5 minutes or Pyr-apelin-13 (200 nM) for 1h (immunoblotting and cell signaling experiments) or 24h prior to RNA extraction and quantitative PCR analysis.

Techniques: Expressing

Journal: Nature Communications

Article Title: Angiotensin-converting enzyme inhibitor promotes angiogenesis through Sp1/Sp3-mediated inhibition of notch signaling in male mice

doi: 10.1038/s41467-023-36409-z

Figure Lengend Snippet: A Western blot analysis of Notch1, Notch intracellular domain (NICD), DLL4, Sp1, and Sp3 protein levels in isolated retinal endothelial cells from CTR and dKO mice. B qPCR analysis of NOTCH1, HES1, HEY1, and DLL4 mRNA levels in isolated retinal endothelial cells from CTR and dKO mice. n = 6. C Representative microscopy images and quantification of immunofluorescence staining for Notch1 in isolectin-B4 + vessels of retinas from CTR and dKO mice. Scale bar: 30 μm. n = 5. D Representative microscopy images and quantification of immunofluorescence staining for phospho-VEGFR2 (Y1175) in isolectin-B4 + vessels of retinas from CTR and dKO mice. Scale bar: 30 μm. n = 5. E Retinal whole-mount staining of isolectin-B4 in P5 CTR and dKO mice, with or without the DAPT (γ-secretase inhibitor). Red arrowheads show tip cell sprouting and filopodia. Bottom, quantification of vascular/total retinal area, vasculature length, branch points per field, tip cell number per field, tip sprout number per field, and filopodia number per sprout. Scale bars: 500 (row 1), 100 (row 2), 50 (row 3), and 10 μm (row 4). n = 6. F Representative laser Doppler images of legs on days –1 (before surgery), 0 (immediately after surgery), 7, 14, 21, and 28. Bottom, quantification of blood flow recovery after hindlimb ischemia as determined by the ratio of foot perfusion between ischemic (left) and non-ischemic (right) legs in CTR and dKO mice with or without DAPT. n = 6. G IHC analysis of CD31 + staining (capillary density) in the ischemic gastrocnemius muscle. Right, quantification of CD31 + vessels per mm 2 in CTR and dKO mice, with or without DAPT. Scale bar: 50 μm. n = 6. H Western blot analysis of phosphorylated VEGFR2 (Y1175, Y1059), VEGFR2, PLCγ (Y783), PLCγ, ERK1/2 (Thr202/Tyr204) and ERK1/2 in mithramycin or Ad-NICD treated HUVECs. Two-tailed Student’s unpaired t-test was used for B – D . One-way ANOVA followed by Bonferroni multiple-comparison analysis was employed for E and G . Two-way ANOVA followed by Bonferroni multiple-comparison analysis was performed for F . Data are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Antibodies against GAPDH (#5173, dilution: 1:1000), Notch1 (#3608, dilution: 1:1000), DLL4 (#96406, dilution: 1:1000), cleaved Notch1 (NICD, #4147, dilution: 1:1000), phospho-VEGF Receptor 2 (Tyr1175) (#2478, dilution: 1:1000), phospho-VEGF Receptor 2 (Tyr1059) (#3817, dilution: 1:1000), VEGF receptor 2 (#2479, dilution: 1:1000), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4370, dilution: 1:1000), p44/42 MAPK (Erk1/2) (#4695, dilution: 1:1000), phospho-PLCγ1 (Tyr783) (#14008, dilution: 1:1000), PLCγ1 (#5690, dilution: 1:1000), rabbit IgG (#7074), ubiquitin (#3936, dilution: 1:1000), FLAG (#14793, dilution: 1:1000), HA (#3724, dilution: 1:1000), Myc (#2276, dilution: 1:1000), HIF-1α (#14179, dilution: 1:1000), were from Cell Signaling Technology (Boston, MA, USA).

Techniques: Western Blot, Isolation, Microscopy, Immunofluorescence, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Angiotensin-converting enzyme inhibitor promotes angiogenesis through Sp1/Sp3-mediated inhibition of notch signaling in male mice

doi: 10.1038/s41467-023-36409-z

Figure Lengend Snippet: A Western blot analysis of Notch1, NICD, DLL4, Sp1, and Sp3 protein levels in HUVECs transfected with CTR, Sp1, Sp3, or Sp1 + Sp3 siRNA. n = 6. B qPCR analysis of NOTCH1, HES1, HEY1, and DLL4 mRNA levels in HUVECs transfected with siRNAs for CTR, Sp1, Sp3, or Sp1 + Sp3. n = 6. C Chromatin immunoprecipitation (ChIP) assay showing the binding of Sp1 or Sp3 to the NOTCH1 promoter in HUVECs. D Relative luciferase activity was shown by the indicated serial promoter deletions of NOTCH1 in HEK293T cells infected with Ad-GFP, Ad-Sp1, or Ad-Sp3. n = 6. E Relative luciferase activity of wild-type (NOTCH1-WT) and mutant constructs (NOTCH1-mutant) of the NOTCH1 promoter in HEK293T cells infected with Ad-GFP, Ad-Sp1, Ad-Sp3, or Ad-Sp1+Ad-Sp3. n = 6. F Relative luciferase activity of VEGFR2 promoter in HUVECs with different treatments. n = 5. G Retinal whole-mount staining of isolectin-B4 in P5 CTR, Sp1 ECKO , Sp3 ECKO , and dKO mice. n = 6. Red arrowheads show tip cell sprouting and filopodia. Right, quantification of vascular/total retinal area, vasculature length, branch points per field, tip cell number per field, tip sprout number per field, and filopodia number per sprout. Scale bars: 500 (row 1), 100 (row 2), 50 (row 3), and 10 μm (row 4). One-way ANOVA followed by Bonferroni multiple-comparison analysis was used for A – G . Data are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Antibodies against GAPDH (#5173, dilution: 1:1000), Notch1 (#3608, dilution: 1:1000), DLL4 (#96406, dilution: 1:1000), cleaved Notch1 (NICD, #4147, dilution: 1:1000), phospho-VEGF Receptor 2 (Tyr1175) (#2478, dilution: 1:1000), phospho-VEGF Receptor 2 (Tyr1059) (#3817, dilution: 1:1000), VEGF receptor 2 (#2479, dilution: 1:1000), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4370, dilution: 1:1000), p44/42 MAPK (Erk1/2) (#4695, dilution: 1:1000), phospho-PLCγ1 (Tyr783) (#14008, dilution: 1:1000), PLCγ1 (#5690, dilution: 1:1000), rabbit IgG (#7074), ubiquitin (#3936, dilution: 1:1000), FLAG (#14793, dilution: 1:1000), HA (#3724, dilution: 1:1000), Myc (#2276, dilution: 1:1000), HIF-1α (#14179, dilution: 1:1000), were from Cell Signaling Technology (Boston, MA, USA).

Techniques: Western Blot, Transfection, Chromatin Immunoprecipitation, Binding Assay, Luciferase, Activity Assay, Infection, Mutagenesis, Construct, Staining

Journal: Nature Communications

Article Title: Angiotensin-converting enzyme inhibitor promotes angiogenesis through Sp1/Sp3-mediated inhibition of notch signaling in male mice

doi: 10.1038/s41467-023-36409-z

Figure Lengend Snippet: A Western blot analysis of Notch1, Notch intracellular domain (NICD), DLL4, Sp1, and Sp3 protein levels in isolated retinal endothelial cells from CTR and dKO mice. B qPCR analysis of NOTCH1, HES1, HEY1, and DLL4 mRNA levels in isolated retinal endothelial cells from CTR and dKO mice. n = 6. C Representative microscopy images and quantification of immunofluorescence staining for Notch1 in isolectin-B4 + vessels of retinas from CTR and dKO mice. Scale bar: 30 μm. n = 5. D Representative microscopy images and quantification of immunofluorescence staining for phospho-VEGFR2 (Y1175) in isolectin-B4 + vessels of retinas from CTR and dKO mice. Scale bar: 30 μm. n = 5. E Retinal whole-mount staining of isolectin-B4 in P5 CTR and dKO mice, with or without the DAPT (γ-secretase inhibitor). Red arrowheads show tip cell sprouting and filopodia. Bottom, quantification of vascular/total retinal area, vasculature length, branch points per field, tip cell number per field, tip sprout number per field, and filopodia number per sprout. Scale bars: 500 (row 1), 100 (row 2), 50 (row 3), and 10 μm (row 4). n = 6. F Representative laser Doppler images of legs on days –1 (before surgery), 0 (immediately after surgery), 7, 14, 21, and 28. Bottom, quantification of blood flow recovery after hindlimb ischemia as determined by the ratio of foot perfusion between ischemic (left) and non-ischemic (right) legs in CTR and dKO mice with or without DAPT. n = 6. G IHC analysis of CD31 + staining (capillary density) in the ischemic gastrocnemius muscle. Right, quantification of CD31 + vessels per mm 2 in CTR and dKO mice, with or without DAPT. Scale bar: 50 μm. n = 6. H Western blot analysis of phosphorylated VEGFR2 (Y1175, Y1059), VEGFR2, PLCγ (Y783), PLCγ, ERK1/2 (Thr202/Tyr204) and ERK1/2 in mithramycin or Ad-NICD treated HUVECs. Two-tailed Student’s unpaired t-test was used for B – D . One-way ANOVA followed by Bonferroni multiple-comparison analysis was employed for E and G . Two-way ANOVA followed by Bonferroni multiple-comparison analysis was performed for F . Data are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Antibodies against GAPDH (#5173, dilution: 1:1000), Notch1 (#3608, dilution: 1:1000), DLL4 (#96406, dilution: 1:1000), cleaved Notch1 (NICD, #4147, dilution: 1:1000), phospho-VEGF Receptor 2 (Tyr1175) (#2478, dilution: 1:1000), phospho-VEGF Receptor 2 (Tyr1059) (#3817, dilution: 1:1000), VEGF receptor 2 (#2479, dilution: 1:1000), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4370, dilution: 1:1000), p44/42 MAPK (Erk1/2) (#4695, dilution: 1:1000), phospho-PLCγ1 (Tyr783) (#14008, dilution: 1:1000), PLCγ1 (#5690, dilution: 1:1000), rabbit IgG (#7074), ubiquitin (#3936, dilution: 1:1000), FLAG (#14793, dilution: 1:1000), HA (#3724, dilution: 1:1000), Myc (#2276, dilution: 1:1000), HIF-1α (#14179, dilution: 1:1000), were from Cell Signaling Technology (Boston, MA, USA).

Techniques: Western Blot, Isolation, Microscopy, Immunofluorescence, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Angiotensin-converting enzyme inhibitor promotes angiogenesis through Sp1/Sp3-mediated inhibition of notch signaling in male mice

doi: 10.1038/s41467-023-36409-z

Figure Lengend Snippet: A Western blot analysis of Notch1, NICD, DLL4, Sp1, and Sp3 protein levels in HUVECs transfected with CTR, Sp1, Sp3, or Sp1 + Sp3 siRNA. n = 6. B qPCR analysis of NOTCH1, HES1, HEY1, and DLL4 mRNA levels in HUVECs transfected with siRNAs for CTR, Sp1, Sp3, or Sp1 + Sp3. n = 6. C Chromatin immunoprecipitation (ChIP) assay showing the binding of Sp1 or Sp3 to the NOTCH1 promoter in HUVECs. D Relative luciferase activity was shown by the indicated serial promoter deletions of NOTCH1 in HEK293T cells infected with Ad-GFP, Ad-Sp1, or Ad-Sp3. n = 6. E Relative luciferase activity of wild-type (NOTCH1-WT) and mutant constructs (NOTCH1-mutant) of the NOTCH1 promoter in HEK293T cells infected with Ad-GFP, Ad-Sp1, Ad-Sp3, or Ad-Sp1+Ad-Sp3. n = 6. F Relative luciferase activity of VEGFR2 promoter in HUVECs with different treatments. n = 5. G Retinal whole-mount staining of isolectin-B4 in P5 CTR, Sp1 ECKO , Sp3 ECKO , and dKO mice. n = 6. Red arrowheads show tip cell sprouting and filopodia. Right, quantification of vascular/total retinal area, vasculature length, branch points per field, tip cell number per field, tip sprout number per field, and filopodia number per sprout. Scale bars: 500 (row 1), 100 (row 2), 50 (row 3), and 10 μm (row 4). One-way ANOVA followed by Bonferroni multiple-comparison analysis was used for A – G . Data are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Antibodies against GAPDH (#5173, dilution: 1:1000), Notch1 (#3608, dilution: 1:1000), DLL4 (#96406, dilution: 1:1000), cleaved Notch1 (NICD, #4147, dilution: 1:1000), phospho-VEGF Receptor 2 (Tyr1175) (#2478, dilution: 1:1000), phospho-VEGF Receptor 2 (Tyr1059) (#3817, dilution: 1:1000), VEGF receptor 2 (#2479, dilution: 1:1000), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4370, dilution: 1:1000), p44/42 MAPK (Erk1/2) (#4695, dilution: 1:1000), phospho-PLCγ1 (Tyr783) (#14008, dilution: 1:1000), PLCγ1 (#5690, dilution: 1:1000), rabbit IgG (#7074), ubiquitin (#3936, dilution: 1:1000), FLAG (#14793, dilution: 1:1000), HA (#3724, dilution: 1:1000), Myc (#2276, dilution: 1:1000), HIF-1α (#14179, dilution: 1:1000), were from Cell Signaling Technology (Boston, MA, USA).

Techniques: Western Blot, Transfection, Chromatin Immunoprecipitation, Binding Assay, Luciferase, Activity Assay, Infection, Mutagenesis, Construct, Staining

Journal: Nature Communications

Article Title: Angiotensin-converting enzyme inhibitor promotes angiogenesis through Sp1/Sp3-mediated inhibition of notch signaling in male mice

doi: 10.1038/s41467-023-36409-z

Figure Lengend Snippet: A Western blot analysis of Notch1, Notch intracellular domain (NICD), DLL4, Sp1, and Sp3 protein levels in isolated retinal endothelial cells from CTR and dKO mice. B qPCR analysis of NOTCH1, HES1, HEY1, and DLL4 mRNA levels in isolated retinal endothelial cells from CTR and dKO mice. n = 6. C Representative microscopy images and quantification of immunofluorescence staining for Notch1 in isolectin-B4 + vessels of retinas from CTR and dKO mice. Scale bar: 30 μm. n = 5. D Representative microscopy images and quantification of immunofluorescence staining for phospho-VEGFR2 (Y1175) in isolectin-B4 + vessels of retinas from CTR and dKO mice. Scale bar: 30 μm. n = 5. E Retinal whole-mount staining of isolectin-B4 in P5 CTR and dKO mice, with or without the DAPT (γ-secretase inhibitor). Red arrowheads show tip cell sprouting and filopodia. Bottom, quantification of vascular/total retinal area, vasculature length, branch points per field, tip cell number per field, tip sprout number per field, and filopodia number per sprout. Scale bars: 500 (row 1), 100 (row 2), 50 (row 3), and 10 μm (row 4). n = 6. F Representative laser Doppler images of legs on days –1 (before surgery), 0 (immediately after surgery), 7, 14, 21, and 28. Bottom, quantification of blood flow recovery after hindlimb ischemia as determined by the ratio of foot perfusion between ischemic (left) and non-ischemic (right) legs in CTR and dKO mice with or without DAPT. n = 6. G IHC analysis of CD31 + staining (capillary density) in the ischemic gastrocnemius muscle. Right, quantification of CD31 + vessels per mm 2 in CTR and dKO mice, with or without DAPT. Scale bar: 50 μm. n = 6. H Western blot analysis of phosphorylated VEGFR2 (Y1175, Y1059), VEGFR2, PLCγ (Y783), PLCγ, ERK1/2 (Thr202/Tyr204) and ERK1/2 in mithramycin or Ad-NICD treated HUVECs. Two-tailed Student’s unpaired t-test was used for B – D . One-way ANOVA followed by Bonferroni multiple-comparison analysis was employed for E and G . Two-way ANOVA followed by Bonferroni multiple-comparison analysis was performed for F . Data are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Antibodies against GAPDH (#5173, dilution: 1:1000), Notch1 (#3608, dilution: 1:1000), DLL4 (#96406, dilution: 1:1000), cleaved Notch1 (NICD, #4147, dilution: 1:1000), phospho-VEGF Receptor 2 (Tyr1175) (#2478, dilution: 1:1000), phospho-VEGF Receptor 2 (Tyr1059) (#3817, dilution: 1:1000), VEGF receptor 2 (#2479, dilution: 1:1000), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4370, dilution: 1:1000), p44/42 MAPK (Erk1/2) (#4695, dilution: 1:1000), phospho-PLCγ1 (Tyr783) (#14008, dilution: 1:1000), PLCγ1 (#5690, dilution: 1:1000), rabbit IgG (#7074), ubiquitin (#3936, dilution: 1:1000), FLAG (#14793, dilution: 1:1000), HA (#3724, dilution: 1:1000), Myc (#2276, dilution: 1:1000), HIF-1α (#14179, dilution: 1:1000), were from Cell Signaling Technology (Boston, MA, USA).

Techniques: Western Blot, Isolation, Microscopy, Immunofluorescence, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Angiotensin-converting enzyme inhibitor promotes angiogenesis through Sp1/Sp3-mediated inhibition of notch signaling in male mice

doi: 10.1038/s41467-023-36409-z

Figure Lengend Snippet: A Western blot analysis of Notch1, NICD, DLL4, Sp1, and Sp3 protein levels in HUVECs transfected with CTR, Sp1, Sp3, or Sp1 + Sp3 siRNA. n = 6. B qPCR analysis of NOTCH1, HES1, HEY1, and DLL4 mRNA levels in HUVECs transfected with siRNAs for CTR, Sp1, Sp3, or Sp1 + Sp3. n = 6. C Chromatin immunoprecipitation (ChIP) assay showing the binding of Sp1 or Sp3 to the NOTCH1 promoter in HUVECs. D Relative luciferase activity was shown by the indicated serial promoter deletions of NOTCH1 in HEK293T cells infected with Ad-GFP, Ad-Sp1, or Ad-Sp3. n = 6. E Relative luciferase activity of wild-type (NOTCH1-WT) and mutant constructs (NOTCH1-mutant) of the NOTCH1 promoter in HEK293T cells infected with Ad-GFP, Ad-Sp1, Ad-Sp3, or Ad-Sp1+Ad-Sp3. n = 6. F Relative luciferase activity of VEGFR2 promoter in HUVECs with different treatments. n = 5. G Retinal whole-mount staining of isolectin-B4 in P5 CTR, Sp1 ECKO , Sp3 ECKO , and dKO mice. n = 6. Red arrowheads show tip cell sprouting and filopodia. Right, quantification of vascular/total retinal area, vasculature length, branch points per field, tip cell number per field, tip sprout number per field, and filopodia number per sprout. Scale bars: 500 (row 1), 100 (row 2), 50 (row 3), and 10 μm (row 4). One-way ANOVA followed by Bonferroni multiple-comparison analysis was used for A – G . Data are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Antibodies against GAPDH (#5173, dilution: 1:1000), Notch1 (#3608, dilution: 1:1000), DLL4 (#96406, dilution: 1:1000), cleaved Notch1 (NICD, #4147, dilution: 1:1000), phospho-VEGF Receptor 2 (Tyr1175) (#2478, dilution: 1:1000), phospho-VEGF Receptor 2 (Tyr1059) (#3817, dilution: 1:1000), VEGF receptor 2 (#2479, dilution: 1:1000), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4370, dilution: 1:1000), p44/42 MAPK (Erk1/2) (#4695, dilution: 1:1000), phospho-PLCγ1 (Tyr783) (#14008, dilution: 1:1000), PLCγ1 (#5690, dilution: 1:1000), rabbit IgG (#7074), ubiquitin (#3936, dilution: 1:1000), FLAG (#14793, dilution: 1:1000), HA (#3724, dilution: 1:1000), Myc (#2276, dilution: 1:1000), HIF-1α (#14179, dilution: 1:1000), were from Cell Signaling Technology (Boston, MA, USA).

Techniques: Western Blot, Transfection, Chromatin Immunoprecipitation, Binding Assay, Luciferase, Activity Assay, Infection, Mutagenesis, Construct, Staining