Journal: iScience

Article Title: Chemical inhibition of oxygen-sensing prolyl hydroxylases impairs angiogenic competence of human vascular endothelium through metabolic reprogramming

doi: 10.1016/j.isci.2022.105086

Figure Lengend Snippet: DMOG suppresses endothelial cell proliferation, migration, and tube formation (A) Representative bright field images of formazan crystal formed after 3 h incubation of MTT with vehicle or DMOG-treated (1 mM) HPAEC. Right graph shows relative HPAEC proliferation assessed by MTT assay. Scale bar, 100μm. (B) Representative images of BrdU immunostaining. Right graph shows semi-quantitative analysis of BrdU positive cells/hpf. Scale bar, 50μm. (C) Representative histograms of cell cycle analysis for control and DMOG-treated cells. Right side graph demonstrates relative percentages of cell populations in G0/G1, S, and G2/M cell cycle phases. (D) Representative images of 2D scratch wound assay of control and DMOG-treated cells and semi-quantitative analysis of healed area after 24 h. Scale bar, 200μm. (E) Representative images of tubes formed at indicated time points in control and DMOG-treated cells and semi-quantitative analysis of different parameters at 20 h time point. Scale bar, 200μm. Data are pooled from 3 independent experiments and represented as mean ± SEM. Statistics were determined by two-tailed t-test. ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001; ns, not statistically significant. See also Figure S1 .

Article Snippet: Primary Pulmonary Artery Endothelial Cells; Normal, Human (HPAEC) , ATCC , Cat# PCS-100-022.

Techniques: Migration, Incubation, MTT Assay, Immunostaining, Cell Cycle Assay, Control, Scratch Wound Assay Assay, Two Tailed Test

Journal: iScience

Article Title: Chemical inhibition of oxygen-sensing prolyl hydroxylases impairs angiogenic competence of human vascular endothelium through metabolic reprogramming

doi: 10.1016/j.isci.2022.105086

Figure Lengend Snippet: DMOG alters the endothelial cell metabolome (A) Shown are the top 25 downregulated (upper graph) and upregulated (lower graph) metabolic pathways detected by metabolites set enrichment analysis in DMOG-treated cells compared to control. Scaled intensity values indicating relative levels of metabolites related to glycolysis (B) and TCA cycle (C). (D) NAD + /NADH ratio in cells treated with vehicle or DMOG. Scaled intensity values indicating relative levels of lipid metabolites (E), nucleotides (F), and amino acids (G). n = 5 independent samples per condition. All statistical data are represented as mean ± SEM and statistics were determined by a Welch’s two sample t-test. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; ns, not significant. G6P, glucose-6-phosphate; FBP, fructose 1,6 bisphosphate; DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate; AKG, alpha-ketoglutarate; DPA, docosapentaenoate; DHLA, dihomolinolenate; ALC, acetylcarnitine; CHOP, choline phosphate; GPC, glycerophosphorylcholine; PEA, phosphoethanolamine; GPEA, glycerylphosphorylethanolamine; G3P, glycerol 3-phosphate; 5′-AMP, adenosine-5′-monophosphate; 5′-ADP, adenosine-5′-diphopshate; 5′-CMP, cytidine 5′-monophosphate; CDP, cytidine diphosphate; 2′,3′-cCMP, cytidine 2′,3′-cyclic monophosphate; 5′-UDP, uridine-5-diphosphate; UTP, uridine 5′-triphosphate. See also Figure S4 and Table S1 .

Article Snippet: Primary Pulmonary Artery Endothelial Cells; Normal, Human (HPAEC) , ATCC , Cat# PCS-100-022.

Techniques: Control

Journal: iScience

Article Title: Chemical inhibition of oxygen-sensing prolyl hydroxylases impairs angiogenic competence of human vascular endothelium through metabolic reprogramming

doi: 10.1016/j.isci.2022.105086

Figure Lengend Snippet: Citrate supplementation partially rescues the DMOG-induced defects in endothelial migration and tube formation capacity (A) Representative bright field images of formazan crystal formed after MTT incubation with control, DMOG (1mM), DMOG + citrate and citrate (0.5mM)-treated HPAEC. Right graph shows relative HPAEC proliferation calculated by MTT assay. Scale bar, 100μm. (B) Representative images of BrdU immunostaining under the conditions indicated in A. Right graph shows semi-quantitative analysis of BrdU positive cells per hpf. Scale bar, 50μm. (C) Quantitative analysis of cell cycle showing relative percentage of cell population in G0/G1, S, and G2/M cell cycle phase. (D) Representative images of 2D scratch wound assay of control or DMOG-treated cells and semi-quantitative analysis of healed area after 24 h. Scale bar, 200μm. (E) Representative images of tubes formed at the indicated time points in control, DMOG, DMOG + citrate, and citrate-treated cells and semi-quantitative analysis of different parameters at 20 h time point. Scale bar, 200μm. Data are pooled from 3 independent experiments and represented as mean ± SEM. Statistics were determined by one-way ANOVA with Sidak correction for multiple comparisons. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; ns, not significant. Asterisks above bars indicate significant difference between control and treated group, whereas asterisks above lines indicate significant difference between DMOG and DMOG + citrate treated groups. See also Figures S5–S7 .

Article Snippet: Primary Pulmonary Artery Endothelial Cells; Normal, Human (HPAEC) , ATCC , Cat# PCS-100-022.

Techniques: Migration, Incubation, Control, MTT Assay, Immunostaining, Scratch Wound Assay Assay

Journal: iScience

Article Title: Chemical inhibition of oxygen-sensing prolyl hydroxylases impairs angiogenic competence of human vascular endothelium through metabolic reprogramming

doi: 10.1016/j.isci.2022.105086

Figure Lengend Snippet: Nicotinamide Riboside supplementation partially rescues the DMOG-induced defects in endothelial migration and tube formation capacity (A) Representative bright field images of formazan crystal formed after incubation with MTT in control, DMOG (1mM), DMOG + NR and NR (200μM)-treated HPAEC. Right graph shows relative HPAEC proliferation assessed by MTT assay. Scale bar, 100μm. (B) Representative images of BrdU immunostaining. Right graph shows semi-quantitative analysis of BrdU positive cells/hpf. Scale bar, 50μm. (C) Representative images of 2D scratch wound assay and semi-quantitative analysis of healed area after 24 h. Scale bar, 200μm. (D) Representative images of tubes formed at different time points in control, DMOG, DMOG + NR and NR-treated ECs and semi-quantitative analysis of different parameters at 20 h time point. Scale bar, 200μm. Data are pooled from 3 independent experiments and represented as mean ± SEM. Statistics were determined by one-way ANOVA with Sidak correction for multiple comparisons. ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; ns, not significant. Asterisks above bars indicate significant difference between control and treated group, whereas asterisks above lines indicate significant difference between DMOG and DMOG + NR-treated groups. NR, nicotinamide riboside.

Article Snippet: Primary Pulmonary Artery Endothelial Cells; Normal, Human (HPAEC) , ATCC , Cat# PCS-100-022.

Techniques: Migration, Incubation, Control, MTT Assay, Immunostaining, Scratch Wound Assay Assay

Journal: iScience

Article Title: Chemical inhibition of oxygen-sensing prolyl hydroxylases impairs angiogenic competence of human vascular endothelium through metabolic reprogramming

doi: 10.1016/j.isci.2022.105086

Figure Lengend Snippet:

Article Snippet: Primary Pulmonary Artery Endothelial Cells; Normal, Human (HPAEC) , ATCC , Cat# PCS-100-022.

Techniques: Purification, Recombinant, Lysis, Extraction, Protease Inhibitor, Angiogenesis Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Activity Assay, Bicinchoninic Acid Protein Assay, Software, Western Blot, Membrane