Journal: Circulation Research

Article Title: Inhibition of Delta-Like-4–Mediated Signaling Impairs Reparative Angiogenesis After Ischemia

doi: 10.1161/circresaha.110.221663

Figure Lengend Snippet: Figure 1. Upregulation of Dll4 in ische- mic tissues. A, Time course of Dll4 tran- scription in skeletal muscles measured by quantitative PCR. *P0.05, ***P0.001 vs control (n6 each time point). B, Confocal images of adductor muscles showing the induction of Dll4 expression in newly forming capillaries, 5 days postischemia. Dll4 (green), IsolectinB4 (marker of ECs, red), DAPI (nucleus marker, blue). Scale bar: 100 m. Bot- tom images, Higher magnification images showing the high expression of Dll4 in new capillary sprouts. Scale bar: 50 m. C, Confocal images showing costaining of Dll4 (green) and PECAM-1 (EC marker, red). Scale bar: 100 m. Bottom images, High magnification of upper panels. Scale bar: 50 m. D, Dll4 expression in the myocardium 5 days after myocardial infarction. Scale bar: 50 m. The dotted line delimits the infarct border zone. Arrows point ECs double positive for Dll4 and PECAM-1.

Article Snippet: Briefly, following incubation with blocking buffer, the samples were incubated with primary antibody: Goat polyclonal anti-mouse Dll4 (1:25, R&D systems), Rat monoclonal anti-mouse CD31 (1:25, BD Bioscience), Goat anti-mouse Podocalyxin (1:100, R&D systems), Rabbit anti-NG2 (1:20, Chemicon), Rat monoclonal anti-mouse CD45 (1:50, BD Bioscience) or Rat monoclonal anti-mouse CD11b (1:50, R&D system) overnight at 4°C.

Techniques: Muscles, Real-time Polymerase Chain Reaction, Control, Expressing, Marker

Journal: Circulation Research

Article Title: Inhibition of Delta-Like-4–Mediated Signaling Impairs Reparative Angiogenesis After Ischemia

doi: 10.1161/circresaha.110.221663

Figure Lengend Snippet: Figure 2. Inhibition of Dll4-mediated signaling by Ad-sDll4. A, Western blot- ting showing the soluble protein of Dll4 in supernatant of Ad-sDll4–infected HUVECs. B, Real-time PCR results showing the induction of Hes-1 mRNA levels in HUVECs seeded on immobilized Dll4 and the inhibition of Hes-1 upregu- lation by coculture with HUVECs trans- fected with Ad-sDll4. Values are fold changes relative to Hes-1/-actin ratio in control (BSA, noninfected); n3 repli- cates. *P0.05, **P0.01. C, Represen- tative photograph of exposed hindlimb muscles showing distribution of -galactosidase (-gal) 5 days after Ad--gal injection (arrow shows site of femoral artery ligation; *adductor mus- cle). Right, Longitudinal section of adductor muscle showing -gal staining counterstained with eosin. Scale bar: 200 m. D, Time course of transgenic Dll4 mRNA levels in whole homogenized adductor muscles following Ad-sDll4 injection. E, Immunoblot represents the presence of extracellular domain of Dll4 (sDll4) (57 KDa only in Ad-sDll4–injected muscles). F, Induction of Notch target genes in adductor muscles at 3 days postischemia and inhibition of Hey-1 and Nrarp-1 upregulation by Ad-sDll4; §P0.001 vs contralateral muscles; *P0.05 vs Ad-Null; n6 each group.

Article Snippet: Briefly, following incubation with blocking buffer, the samples were incubated with primary antibody: Goat polyclonal anti-mouse Dll4 (1:25, R&D systems), Rat monoclonal anti-mouse CD31 (1:25, BD Bioscience), Goat anti-mouse Podocalyxin (1:100, R&D systems), Rabbit anti-NG2 (1:20, Chemicon), Rat monoclonal anti-mouse CD45 (1:50, BD Bioscience) or Rat monoclonal anti-mouse CD11b (1:50, R&D system) overnight at 4°C.

Techniques: Inhibition, Western Blot, Infection, Real-time Polymerase Chain Reaction, Control, Muscles, Injection, Ligation, Staining, Transgenic Assay

Journal: Circulation Research

Article Title: Inhibition of Delta-Like-4–Mediated Signaling Impairs Reparative Angiogenesis After Ischemia

doi: 10.1161/circresaha.110.221663

Figure Lengend Snippet: Figure 4. Dll4 Inhibition impairs muscle regeneration. A, Cross-section showing the whole perimeter of adductor muscles harvested at 14 days postischemia and stained with hematoxy- lin/eosin. Lower panel: higher magnification of upper panel. B) Considerable loss of muscle fibers in Ad-sDll4–injected ischemic muscles. Myocytes (phalloidin, green), microvessels (Isolect- inB4, red). Scale bar: 100 m. Bottom images, Lipid deposit is increased in Ad-sDll4–injected ischemic muscles as revealed by oil red O staining (red). Scale bar: 200 m. C, Quantification of muscle fibers, lipid degeneration, and necrosis area. *P0.05; n6 per group.

Article Snippet: Briefly, following incubation with blocking buffer, the samples were incubated with primary antibody: Goat polyclonal anti-mouse Dll4 (1:25, R&D systems), Rat monoclonal anti-mouse CD31 (1:25, BD Bioscience), Goat anti-mouse Podocalyxin (1:100, R&D systems), Rabbit anti-NG2 (1:20, Chemicon), Rat monoclonal anti-mouse CD45 (1:50, BD Bioscience) or Rat monoclonal anti-mouse CD11b (1:50, R&D system) overnight at 4°C.

Techniques: Inhibition, Muscles, Staining, Injection

Journal: Circulation Research

Article Title: Inhibition of Delta-Like-4–Mediated Signaling Impairs Reparative Angiogenesis After Ischemia

doi: 10.1161/circresaha.110.221663

Figure Lengend Snippet: Figure 6. Dll4 inhibition enhances leukocyte infiltration. A, Confocal microscopy images and bar graph show increased number of infiltrating leukocytes in Ad-sDll4–injected muscles (CD45, green; IsolectinB4, red). Contralateral normoperfused muscles are shown for reference (control). Scale bar: 100 m. ***P0.0001; n10 per group. B, High magnifications showing the linear alignment of leuko- cytes along the vessel long-axis in Ad-null–injected ischemic muscles and the loss of structured patterning in Ad-sDll4–injected ische- mic muscles. Scale bar: 50 m. C, Identification of CD11b leukocytes in ischemic muscles (CD11b, green; IsolectinB4, red). D, Flow cytometric analysis of single cell suspensions from skeletal muscle digests shows the increased abundance of CD45 leukocytes in Ad-sDll4–injected muscles. *P0.05; n6 per group. E, Confocal images and bar graph showing induction of leukocyte infiltration in normoperfused Ad-sDll4–injected muscles (CD45, green; IsolectinB4, red). *P0.001 vs Ad.gal; n6 per group.

Article Snippet: Briefly, following incubation with blocking buffer, the samples were incubated with primary antibody: Goat polyclonal anti-mouse Dll4 (1:25, R&D systems), Rat monoclonal anti-mouse CD31 (1:25, BD Bioscience), Goat anti-mouse Podocalyxin (1:100, R&D systems), Rabbit anti-NG2 (1:20, Chemicon), Rat monoclonal anti-mouse CD45 (1:50, BD Bioscience) or Rat monoclonal anti-mouse CD11b (1:50, R&D system) overnight at 4°C.

Techniques: Inhibition, Confocal Microscopy, Injection, Muscles, Control

Journal: Circulation Research

Article Title: Inhibition of Delta-Like-4–Mediated Signaling Impairs Reparative Angiogenesis After Ischemia

doi: 10.1161/circresaha.110.221663

Figure Lengend Snippet: Figure 7. Dll4 inhibition increases the release of CXCL1/IL-8. A, Ad-sDll4 increases the circulating levels of CXCL1 in mice with limb ischemia. *P0.05, **P0.001 vs Ad-Null; #P0.05, **P0.01 vs nonischemic mice (control); n6 each group. B, Left, IL-8 concentrations in condi- tioned media of THP1 monocytes cultured on immobilized Dll4 in the presence of TNF- or vehicle. To block Notch, DAPT or vehicle (DMSO) was added. Right, Hes1 expression levels in the same experiment. *P0.001 vs BSA; #P0.001 vs vehicle; n4. n.d. indicates not detectable. C, Left, IL-8 concentrations in conditioned media of THP1 monocytes cocultured with trans- fected HUVECs. Right, Hes1 expression levels in the same experiment. *P0.001 vs BSA; #P0.01 vs Ad-Null; n4. D, Time- lapse video microscopy images illustrating the endothelial network formation by HUVECs on Matrigel in the presence of DiI- labeled THP1 cells (red). HUVECs were transfected with Ad-Null or Ad-sDll4 (100 plaque-forming units/cell).

Article Snippet: Briefly, following incubation with blocking buffer, the samples were incubated with primary antibody: Goat polyclonal anti-mouse Dll4 (1:25, R&D systems), Rat monoclonal anti-mouse CD31 (1:25, BD Bioscience), Goat anti-mouse Podocalyxin (1:100, R&D systems), Rabbit anti-NG2 (1:20, Chemicon), Rat monoclonal anti-mouse CD45 (1:50, BD Bioscience) or Rat monoclonal anti-mouse CD11b (1:50, R&D system) overnight at 4°C.

Techniques: Inhibition, Control, Cell Culture, Blocking Assay, Expressing, Microscopy, Labeling, Transfection

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 1. Haploinsufficiency of Dll4 decreased brain weights at P10 compared to Dll4+/+ littermate controls Data are presented as the mean ± SD. A, brain weights at P10 (n = 8–13 mice/sex/group). B, body weights at P10 (n = 8–13 mice/sex/group). C, brain weights at P21 (n = 7–13 mice/sex/group). D, body weights at P21 (n = 5–20 mice/sex/group). E, brain weights at P100 (n = 3–4 mice/sex/group). F, body weights at P100 (n = 8–15 mice/sex/group). ∗P < 0.05, ∗∗P < 0.01.

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques:

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 2. DLL4 expression pattern in brain A–F, representative images of X-gal and CD31 double staining in the brains of Dll4+/+ (A, C and E) and Dll4+/LacZ

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques: Expressing, Double Staining

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 3. DLL4 was differentially expressed in different cell types in mouse brain A–R, immunofluorescence double staining with antibodies against DLL4 and different cell type markers in P21 Dll4+/+ mouse brain. Colocalization of DLL4 (green) and cell type marker (red) makes yellow. A–C, DLL4 double staining with CD31 (endothelial cell marker). D–F, DLL4 double staining with GFAP (astrocyte marker). G–I, DLL4 double staining with NeuN (neuronal marker). J–L, DLL4 double staining with CD11b (microglia marker). M–O, DLL4 double staining with Nestin (stem cell marker). P–R, DLL4 double staining with SOX10 (oligodendrocyte marker). S, X-gal (blue) double staining with venous marker NR2F2 (brown). T, X-gal (blue) double staining with venous marker EPHB$ (brown). Images were taken using a Keyence microscope at 20× magnification. Scale bar = 100 μm. U, Dll4 mRNA levels in different cell types of P21 Dll4+/+ female (F) and male (M) mouse brain. Data are presented as the mean ± SD. N = 3 mice/sex/group.

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques: Immunofluorescence, Double Staining, Marker, Microscopy

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 4. Haploinsufficiency of Dll4 decreased capillary diameter and increased vessel densities in P7, P21, and P100 mouse brains compared to Dll4+/+ littermate controls A–J, representative images of CD31 staining in female cortex (CTX, A and B), hippocampus (HP, C and D), mid brain (MB, E and F), cerebellum (CB, G and H) and brain stem (BS, I and J) regions of Dll4+/+ (A, C, E, G and I)

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques: Staining

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 5. Haploinsufficiency of Dll4 altered gene expression in the EC of Dll4+/Lacz mouse brain at P10 and impaired BBB integrity at P21 compared to Dll4+/+ littermate controls Data are presented as the mean ± SD. A, mRNA levels of Dll4, Hey1, Jag1, Hes1, Vegfa, Vegfr1-3, Nrp1-2 and Ephb4 in the EC of Dll4+/+ vs. Dll4+/Lacz female (F) and male (M) mouse brains. N = 5 mice/sex/group, ∗P < 0.05. B, protein levels of DLL4, NICD, VEGFA, VEGFR2, JAG1 and HES1 in the EC of Dll4+/+ vs. Dll4+/Lacz mouse brains. N = 4 mice/sex/group. C, Albumin levels in whole brain lysates of Dll4+/+ vs. Dll4+/Lacz mice at P7. N = 4–5 mice/sex/group, female P = 0.413, male P = 0.73. D, Albumin levels in whole brain lysates of Dll4+/+ vs. Dll4+/Lacz

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques: Gene Expression

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 6. DLL4 insufficiency led to a hypersprouting angiogenic phenotype in HBMEC and increased HBMEC permeability in vitro Data are presented as the mean ± SD. A–F, EC phenotype confirmation in HBMEC-Im cells by LDL uptake (J and K), CDH5 (L and M), and ERG (N and O) staining. Images were taken using a Keyence microscope at a 60× magnification. Scale bar = 100 μm. G and H, DLL4 insufficiency caused hyperbranching network in 3-D cell culture (H) compared to the controls, red arrow heads point to branches (G). Images were taken using a Keyence microscope at a 10× magnification. Scale bar = 100 μm. I, quantitative analysis of cells per bead in a 3-D angiogenesis assay. N = 17–19 beads/group across three different experiments. ∗∗∗∗P < 0.0001. J, quantitative analysis of length of tube formation. N = 13–14 beads/group across three different experiments. ∗∗∗∗P < 0.0001. K, mRNA levels of DLL4 signalling genes in DLL4 sh-RNA transduced cells relative to scRNA transduced cells. Data were collected from four different experiments. L, protein levels of DLL4 signalling genes in DLL4 sh-RNA trans- duced cells relative to scRNA transduced cells. Data were collected from four different experiments. ∗P < 0.05, ∗∗P < 0.01. M, normalized TEER values in scRNA transduced HBMEC (circles, top) vs. DLL4-shRNA transduced cells (triangles, bottom). Changes in the TEER were normalized to the initial values. Each plotted data point is presented as the mean ± SD. Data were collected from three different experiments. ∗P < 0.05. ∗∗P < 0.01, ∗∗∗P < 0.001. N, HMBEC-Im cells transduced with either scRNA or DLL4-shRNA were seeded and cultured in growth medium until a monolayer was formed and confirmed by cell membrane marker wheat germ agglutinin (WGA, green) and nuclei marker Hoechst (blue) double staining. O, non-monolayer controls. Images were taken using a Keyence microscope at 20× magnification. Scale bar = 100 μm. P, quantitative analysis of fluorescence value (485/535 nm excitation/emission). Data were collected from three different experiments, ∗P = 0.0256.

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques: Permeability, In Vitro, Staining, Microscopy, Cell Culture, Angiogenesis Assay, shRNA, Transduction, Membrane, Marker, Double Staining, Fluorescence

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 7. DLL4 insufficiency decreased tight junction protein CLDN5 expression in HBMEC through NOTCH-NICD-RBPJ-CLDN5 signalling Data are presented as the mean ± SD. A, mRNA levels of CLDN5 and TJP1 in HBMEC-im treated with scRNA vs. DLL4-shRNA. Data were collected from four different experiments, CLDN5, ∗P = 0.0104; TJP, P = 0.697. B, protein levels of CLDN5 and TJP1 in HBMEC-im treated with scRNA vs. DLL4-shRNA. Data were collected from four different experiments, CLDN5, ∗P = 0.0255; TJP, P = 0.113. C, mRNA levels of DLL4 and CLDN5 in primary HBMEC. Data were collected from four different experiments. DLL4, ∗∗P = 0.00202; CLDN5, ∗P = 0.0312. D, schematic representation of DLL4-NOTCH-NICD-CLDN5 pathway. E, schematic representation of human CLDN5 promoter with RBPJ site at –2168 bp upstream of transcription start site set as +1. F, sequences around RBPJ binding site of CLDN5 promoter among different species with the sequence for RBPJ bonding site capitalized and bold. G, NOTCH density at RBPJ site of CLDN5 promoter in HBMEC-im. Data were collected from three different experiments. ∗P = 0.0141. H, CLDN5 promoter reporter activity assay in HEK293T cells. Data were collected from three different experiments. ∗∗P = 0.0058 or 0.0059 compared to either empty vector pGL4.10-pcDNA 3.1 or vector + NICD only or vector + CLDN5 only. I, CLDN5 and HEY1 mRNA fold change in HBMEC-im treated with y-secretase inhibitors DAPT and DBZ. Data were collected from three different experiments. ∗∗∗∗P < 0.0001.

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques: Expressing, shRNA, Binding Assay, Sequencing, Activity Assay, Plasmid Preparation

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 8. Neonatal hyperoxia exposure decreased cerebral vessel density and DLL4 expression in the EC of mouse brain Data are presented as the mean ± SD. A–J, representative images of CD31 staining in male cortex (CTX, A and B), hippocampus (HP, C and D), mid brain (MB, E and F), cerebellum (CB, G and H) and brain stem (BS, I and J) regions of RA (A, C, E, G and I) and HOX (B, D, F, H and J). Images were taken using a Keyence light microscope at 20× magnification. Scale bar = 200 μm. K, quantitative analysis of vessel densities in different brain regions, female (F) and male (M). N = 4 mice/sex/group. ∗P < 0.05. L, Dll4 mRNA fold change in brain EC of room air (RA) vs. hyperoxia exposure (HOX) relative to Tubb5. N = 6 mice/sex/group; female, ∗∗P = 0.0079; male, ∗∗P = 0.0098. M, DLL4 protein levels in brain EC of RA vs. HOX relative vinculin. N = 4–5 mice/sex/group, female P = 0.382, male ∗P = 0.0317. N, representative immunofluorescence double staining of CD31 (green) and DLL4 (red) images in the CTX of female mice exposed to RA (top) and HOX (bottom) in females. Images were taken using a Keyence microscope at 20× magnification. Scale bar = 100 μm. O, quantitative analysis of DLL4 densities in cortex. N = 4 mice/sex/group; female, ∗P = 0.0286; male, ∗P = 0.0286.

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques: Expressing, Staining, Light Microscopy, Immunofluorescence, Double Staining, Microscopy

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 9. Neonatal hyperoxia exposure decreased Cldn5 mRNA levels in the EC and protein abundance in CTX region of mouse brain Data are presented as the mean ± SD. A, Cldn5 mRNA fold change in brain EC of room air (RA) vs. hyperoxia exposure (HOX) relative to Tubb5, female (F) and male (M). N = 5 mice/sex/group; female, ∗P < 0.05; male, ∗∗P < 0.01. B, representative immunofluorescence double staining images of CLDN5 (green) in the CTX of female mice exposed to RA (top) and HOX (bottom) in females. Images were taken using a Keyence microscope at 20× magnification. Scale bar = 100 μm. C, quantitative analysis of CLDN5 densities in cortex. N = 4 mice/sex/group. ∗P < 0.05 for both sexes. D, representative immunofluorescence triple staining images of DLL4 (green), CLDN5 (red) and CD31 (white) in the CTX of male mice exposed to RA (top) and HOX (bottom). Red arrows pointed to DLL4 positive vessels. Images were taken using a Keyence microscope at 60× magnification. Image exposure times for both the RA and HOX groups were configured as: 5 s for green, 1.2 s for red, 8 s for white and 1/3 s for blue, respectively. Image contrast was adjusted less than 10% for all images. E, DLL4 mRNA time course change in HBMEC exposed to normoxia vs. hyperoxia. Data were collected from three different experiments. ∗P < 0.05.

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques: Quantitative Proteomics, Immunofluorescence, Double Staining, Microscopy, Staining