Journal: Chinese Herbal Medicines

Article Title: Qishen Granule protects against myocardial ischemia by promoting angiogenesis through BMP2-Dll4-Notch1 pathway

doi: 10.1016/j.chmed.2023.12.007

Figure Lengend Snippet: Effects of QSG on angiogenesis of BMP2-Dll4-Notch1 pathway in myocardial ischemic rats. (A−B) Representative immunoblot images and quantitative analysis of BMP2, Dll4 and Notch1 in different groups. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control. (C) Representative immunofluorescent images were shown. CD31 was used to identify endothelial cells, DAPI to visualize cell nuclei. Scale bar, 100 μm (× 20). Data were presented as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs model group.

Article Snippet: Marker (5 μL) and protein sample (10 μL) were added into the sample hole of the prepared concentrated glue, which was separated at 110 V for 90 min, transferred at 300 mA for 90 min, and blocked with 5% milk for 2 h. The film was incubated in anti-BMP2 (YT5651; Immunoway), anti-Dll4 (bs-5909R; Bioss) and anti-Notch1 (10062–2-AP; ProteinTech) at 4 °C overnight, and anti-rabbit IgG H&L (ab16284; Abcam) was incubated at room temperature for 1 h. The film was placed in a gel imager, the luminescent liquid was uniformly dropped on the film, and the image was obtained and saved.

Techniques: Western Blot, Control

Journal: Chinese Herbal Medicines

Article Title: Qishen Granule protects against myocardial ischemia by promoting angiogenesis through BMP2-Dll4-Notch1 pathway

doi: 10.1016/j.chmed.2023.12.007

Figure Lengend Snippet: Effects of QSG on protein expressions in BMP2-Dll4-Notch1 pathway in OGD-induced HUVECs model. (A) Expression of BMP2 in HUVECs detected by Western blots. (B) Release of BMP2 in supernatant of HUVECs detected by ELISA. (C−D) Representative immunoblots and corresponding quantification of Dll4 and Notch1 protein levels in HUVECs. Data were presented as mean ± SEM ( n = 3). ** P < 0.01, *** P < 0.001 vs model group.

Article Snippet: Marker (5 μL) and protein sample (10 μL) were added into the sample hole of the prepared concentrated glue, which was separated at 110 V for 90 min, transferred at 300 mA for 90 min, and blocked with 5% milk for 2 h. The film was incubated in anti-BMP2 (YT5651; Immunoway), anti-Dll4 (bs-5909R; Bioss) and anti-Notch1 (10062–2-AP; ProteinTech) at 4 °C overnight, and anti-rabbit IgG H&L (ab16284; Abcam) was incubated at room temperature for 1 h. The film was placed in a gel imager, the luminescent liquid was uniformly dropped on the film, and the image was obtained and saved.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: DLL4 and PDGF-BB regulate migration of human iPSC-derived skeletal myogenic progenitors

doi: 10.1101/2021.02.28.431778

Figure Lengend Snippet: (A) Principal Component Analysis (PCA) showing mMuSC-derived myoblasts (left), human myoblasts (centre) and hiMPs (right). 4 cell lines were analysed with RNAseq in treated and untreated conditions for each cell population. Each point on the PCA represents a cell population. Additional information in Table S1,2. (B) Volcano plots visualising differentially expressed genes between untreated and DLL4 & PDGFBB-treated mMuSCs, human myoblasts and hiMPs. Red dots represent genes which display a positive fold-change in expression upon treatment with DLL4 & PDGF-BB whilst violet dots represent genes which are significantly downregulated. Differentially expressed genes required a P value of ≤ 0.05. (C) Heatmaps showing changes in expression of key myogenic ( MYOD, MYOGENIN ), perivascular ( PDGFRB, NG2, CD146, ALPL ) and NOTCH target ( HEY1, HES1 ) genes upon treatment with DLL4 & PDGF-BB in mMuSC-derived myoblasts (left), human myoblasts (middle) and hiMPs (right). Clustering was performed by genes/probes with Pearson correlation. Colour scale based on z-scores: red regions indicate high expression whilst blue regions indicate low expression. Dendrograms indicate the similarity of clusters as well as the orders in which clusters were assembled. (D) Validation of RNAseq data of panel (C) by real-time PCR analysis of the same myogenic, perivascular and NOTCH target transcripts in treated and untreated hiMPs (N=3; error bars; S.E.M.). Statistical analysis (paired t test) performed on ΔCt values whilst graphs were produced as fold change relative to untreated controls. (E) Curated dot plot Gene Ontology (GO; left), Kyoto Encyclopaedia of Genes and Genomes (KEGG; centre) and Reactome (right) enrichment analyses showing shared gene functions amongst the cell groups; numbers in brackets: genes analysed with a p value threshold set at 0.05; full lists in a dedicated spreadsheet available in Supplemental Information.

Article Snippet: Recombinant human DLL4 (DLL4 fused with the Fc domain of human IgG; R&D Systems; 1506-D4) was resuspended to a final concentration of 10 μg/ml in sterile PBS containing 1% wt/vol bovine serum albumin (BSA; Sigma-Aldrich; A9418-10G) as a carrier protein.

Techniques: Derivative Assay, Expressing, Biomarker Discovery, Real-time Polymerase Chain Reaction, Produced

Journal: bioRxiv

Article Title: DLL4 and PDGF-BB regulate migration of human iPSC-derived skeletal myogenic progenitors

doi: 10.1101/2021.02.28.431778

Figure Lengend Snippet: (A) Top cellular and molecular functions associated with DLL4 & PDGFBB modulation generated via ingenuity pathway analysis (IPA). Genes upregulated in the DLL4 & PDGFBB-treated hiMPs relative to the untreated control were subjected to IPA to reveal the predicted most significant associated functions. (B) Fluorescence microscopy images depicting Hoechst-positive nuclei of each cell at sequential time points. Coloured tails represent the locations of the nuclei at previous time points. (C) Unsupervised hierarchical clustering (Ward’s method) visualised with a t-SNE plot showing two distinct clusters (Silhouette Si = 0.19) (n = 408) (perplexity = 35). Cells pooled from 3 independent experimental replicates for each condition were used (untreated and DLL4 & PDGF-BB-treated). (D) Bar chart demonstrating normalised values for comparison of motility phenotypes between cells within the two clusters (mean ±SEM). Statistical significance based on Bonferroni-corrected t -test: all parameters except “hurst_RS” and “autocorr” are statistically significant between the two groups p<0.01 (data points: single cells pooled together from 3 independent experiments). (E) Bar graph displaying the proportions of control and DLL4 & PDGF-BB-treated cells within each cluster. (F) Functional protein association network analysis ( https://string-db.org ). The network view summarises predicted associations for proteins positively regulating cell migration common to all three datasets. The nodes are proteins and the edges represent the predicted functional associations. Red line: fusion evidence; Green line: neighbourhood evidence; Blue line: co-occurrence evidence; Purple line: experimental evidence; Yellow line: text mining evidence; Light blue line: database evidence; Black line: co-expression evidence. Blue nodes: GO:0030335 positive regulation of cell migration, Count in gene set: 8 of 452, false discovery rate: 0.0156. (G) P value-adjusted hierarchical clustering heatmap displaying hierarchical clustering of genes associated with leukocyte trans-endothelial migration (KEGG pathway: hsa04670; P set at 0.05). (H) Assessment of DLL4 & PDGF-BB-treated WT and genetically corrected DMD hiMP migration through a layer of endothelial cells. Representative images showing the lower side of the trans-well membrane on which treated and untreated hiMPs (stained with the transient dye CFDA, in green) are simultaneously seeded on HUVECs for 8 hours. Bar graphs quantifying the average number of CFDA-positive cells/ mm 2 , that have migrated through the endothelial layer in each considered condition. (N = 3). A minimum of 10 (1.5 mm 2 ) fields per condition was quantified (mean ±SEM). Scale bar: 250 μm. (I) Bar graph showing fold-change in trans-endothelial migration (mean ±SEM). Statistical significance based on one-way ANOVA with Bonferroni’s multiple comparison.

Article Snippet: Recombinant human DLL4 (DLL4 fused with the Fc domain of human IgG; R&D Systems; 1506-D4) was resuspended to a final concentration of 10 μg/ml in sterile PBS containing 1% wt/vol bovine serum albumin (BSA; Sigma-Aldrich; A9418-10G) as a carrier protein.

Techniques: Generated, Control, Fluorescence, Microscopy, Comparison, Functional Assay, Migration, Expressing, Membrane, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Nrf2 acts cell-autonomously in endothelium to regulate tip cell formation and vascular branching

doi: 10.1073/pnas.1309276110

Figure Lengend Snippet: Deletion of endothelial Nrf2 leads to increased Dll4/Notch signaling. (A–C) VEGF protein levels at P5 (n = 5). (D) Quantitative RT-PCR analysis of Dll4 and Notch target genes in the retinas of Nrf2−/− and WT mice at P5 (n = 4). (E) Quantitative RT-PCR analysis of Dll4 and Notch target genes in the retinas of Nrf2fl/fl;Six3-Cre and control mice at P5 (n = 6). (F, Upper) Laser-capture microdissection of blood vessels. (Scale bar, 100 µm.) (F, Lower) Quantitative RT-PCR analysis of Dll4 and Notch target genes in laser-capture microdissected blood vessels from Nrf2fl/fl;Cdh5-Cre and control retinas at P5 (n = 5). (G) Increased Dll4 expression was observed in the angiogenic front (arrowheads) in Nrf2fl/fl;Cdh5-Cre retinas compared with control at P5. (Scale bar, 25 µm.) Data are presented as mean ± SEM (*P < 0.05 and **P < 0.01; NS, not significant).

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-Nrf2 (5 ng/μL; R&D Systems), monoclonal rat anti-mouse PECAM-1(1:50; BD Pharmingen), and polyclonal goat anti-mouse Dll4 (15 ng/μL; R&D Systems).

Techniques: Quantitative RT-PCR, Control, Laser Capture Microdissection, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Nrf2 acts cell-autonomously in endothelium to regulate tip cell formation and vascular branching

doi: 10.1073/pnas.1309276110

Figure Lengend Snippet: Inhibition of Dll4/Notch signaling abrogates the restrained sprouting angiogenesis in Nrf2-deficient mice. (A and C) PECAM-1–stained P5 retina from Nrf2−/− and WT mice after 24 h treatment (P4–P5) with Dll4 antibody (intravitreous injection) (A) or DAPT (i.p. injection) (C). Dll4 antibody or DAPT administration results in comparable vascular hyperplasia in the affected region (between dashed lines) in Nrf2−/− and WT retinas. (B and D) Quantification of vascular density in P5 retinas after 24 h treatment (P4–P5) with Dll4 antibody (intravitreous injection; B) or DAPT (i.p. injection; D); n = 6 per group for Dll4 antibody injection; n = 5 per group for DAPT injection. (E) Aortic ring explants from Nrf2fl/fl;Cdh5-Cre and control mice treated with Dll4 antibody or DAPT, respectively. (F) Quantification of outgrowth area of aortic explants shown in E (n = 7). (Scale bar, 200 µm.) Data are presented as mean ± SEM (*P < 0.05 and **P < 0.01; NS, not significant).

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-Nrf2 (5 ng/μL; R&D Systems), monoclonal rat anti-mouse PECAM-1(1:50; BD Pharmingen), and polyclonal goat anti-mouse Dll4 (15 ng/μL; R&D Systems).

Techniques: Inhibition, Staining, Injection, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Nrf2 acts cell-autonomously in endothelium to regulate tip cell formation and vascular branching

doi: 10.1073/pnas.1309276110

Figure Lengend Snippet: Deletion of the Nrf2 repressor Keap1 in ECs promotes sprouting angiogenesis via suppression of Dll4/Notch signaling. (A) Visualization of blood vessels by PECAM-1 staining of control (Keap1fl/fl) and Keap1fl/fl;Cdh5-Cre retinas at P5. Enhanced vascular density (B), branch points (C), and hypersprouting characterized by increased tip cell numbers (D) and filopodia (E) were observed in Keap1fl/fl;Cdh5-Cre retina (n = 6). (F and G) Isolectin B4 (green) and BrdU labeling (red) of control and Keap1fl/fl;Cdh5-Cre retinas at P5 (n = 4). (Scale bar, 50 µm.) (H and I) Increased area of the deep vascular plexus was observed in Keap1fl/fl;Cdh5-Cre retina at P9 (n = 6). (Scale bar, 500 µm.) (J and K) Keap1 knockdown enhanced HREC tube formation. (Scale bar, 100 µm.) (L) Increased Dll4 expression was observed at the angiogenic front (arrowheads) in Nrf2fl/fl;Cdh5-Cre retinas at P5. (Scale bar, 25 µm.) (M) Quantitative RT-PCR analysis of Dll4 and Notch target genes in HRECs cultured in a collagen-sandwich gel. (N) Immunoblot analysis of NICD and quantitative RT-PCR analysis of Dll4 in HRECs stimulated with rDll4. GAPDH was detected as a loading control. (O and P) Keap1 knockdown enhanced HREC spheroid sprouting with lower or higher level of VEGF. DAPT corrected the differential sprouting between control siRNA and Keap1 siRNA-transfected HRECs. (Scale bar, 50 µm.) (Q) Immunoblot analysis of Nrf2, HIF-1α, HIF-2α, Dll4, and NICD in HRECs under normoxia or hypoxia. (R and S) Quantitative RT-PCR analysis of HIF-2α in HRECs under hypoxia (R) or in blood vessels (S) from Keap1fl/fl;Cdh5-Cre mice at P5 (n = 4). Data are presented as mean ± SEM (*P < 0.05 and **P < 0.01; NS, not significant).

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-Nrf2 (5 ng/μL; R&D Systems), monoclonal rat anti-mouse PECAM-1(1:50; BD Pharmingen), and polyclonal goat anti-mouse Dll4 (15 ng/μL; R&D Systems).

Techniques: Staining, Control, Labeling, Knockdown, Expressing, Quantitative RT-PCR, Cell Culture, Western Blot, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Nrf2 acts cell-autonomously in endothelium to regulate tip cell formation and vascular branching

doi: 10.1073/pnas.1309276110

Figure Lengend Snippet: Nrf2 activation by Keap1 knockdown inhibits PI3K/Akt-dependent Notch signaling in ECs. (A) PI3K activity in HRECs. Knockdown of Keap1 blocked VEGF-induction of PI3K activity. (B) Immunoblot analysis of VEGFR2, Akt, and Erk in HRECs transfected with control siRNA or Keap1 siRNA in the presence or absence of VEGF. Knockdown of Keap1 inhibited VEGF-induced phosphorylation of VEGFR2 and Akt, but not Erk. (C) Immunoblot analysis of Dll4, NICD, Akt in lung or retina homogenates from Keap1fl/fl;Cdh5-Cre mice at P5. (D and E) Quantitative RT-PCR analysis of Dll4 (D) and Hey1 (E) in HRECs treated with PI3K inhibitor LY294002. (F) Immunoblot analysis of Dll4 and NICD in HRECs expressing constitutively active Akt. Suppression of Dll4 and NICD by Keap1 knockdown was reversed by adenovirus-mediated enhancement of Akt activity. GAPDH was detected as a loading control. (G) Proposed schematic of Nrf2 in developmental angiogenesis. Nrf2 activation by release from Keap1 suppresses Dll4/Notch signaling via inhibition of VEGF-induced VEGFR2-PI3K/Akt and down-regulation of HIF-2α, leading to the enhanced sprouting angiogenesis. Data are presented as mean ± SEM (*P < 0.05 and **P < 0.01; NS, not significant).

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-Nrf2 (5 ng/μL; R&D Systems), monoclonal rat anti-mouse PECAM-1(1:50; BD Pharmingen), and polyclonal goat anti-mouse Dll4 (15 ng/μL; R&D Systems).

Techniques: Activation Assay, Knockdown, Activity Assay, Western Blot, Transfection, Control, Phospho-proteomics, Quantitative RT-PCR, Expressing, Inhibition