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

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: Bone morphogenetic protein (BMP)-4, transforming growth factor (TGF)-β1, serotonin (or 5-hydroxytryptamine; 5-HT), endothelin (ET)-1, and glycogen synthase kinase (GSK)-3β inhibitors increase pulmonary smooth muscle cell size and protein synthesis. A: change in forward scatter in human pulmonary artery smooth muscle cells treated with PBS, BMP-4, TGF-β1, 5-HT, ET-1, LiCl, SB-216763, and EGF. B: overall protein synthesis of cells treated with PBS, BMP-4, TGF-β1, 5-HT, ET-1, LiCl, or SB-216763, as assessed by [3H]leucine incorporation (cpm/well). C: Overall DNA synthesis of cells treated with PBS, BMP-4, TGF-β1, 5-HT, ET-1, LiCl, or SB-216763, as assessed by [3H]thymidine incorporation (cpm/well); n = 3, means ± SE; *P < 0.05, ANOVA.

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: DNA Synthesis

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: Phosphorylation of GSK-3β is required for BMP-4-, TGF-β1-, 5-HT-, and ET-1-induced hypertrophy. A: representative immunoblots for phospho-GSK-3β and total GSK-3β in human pulmonary artery smooth muscle cells treated with BMP-4, TGF-β1, 5-HT, ET-1, LiCl, and SB-216763. B: GSK-3β-A9 was expressed in A7R5 cells via retroviral gene transfer. Expression of GSK-3β-A9 acts as a “dominant-negative,” decreasing the binding of upstream kinases and scaffolding proteins to native GSK-3β. This leads to a relative reduction of phosphorylated, inactive GSK-3β, and an increase in GSK-3β activity. C: effect of GSK-3β-A9 overexpression on the size of cells treated with BMP-4, TGF-β1, 5-HT, ET-1, LiCl, or SB-216763 (*different from MSCV-transduced cells, P < 0.05, ANOVA).

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: Western Blot, Expressing, Dominant Negative Mutation, Binding Assay, Scaffolding, Activity Assay, Over Expression

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: Mechanism of GSK-3β-mediated cell hypertrophy. A: representative immunoblots for phospho- and total eIF2B in pulmonary artery smooth muscle cells treated with BMP-4, TGF-β1, 5-HT, ET-1, and GSK-3β inhibitors. B: effect of BMP-4, TGF-β1, 5-HT, ET-1, LiCl, and SB-216763 on serum response factor (SRF) reporter activity. A7R5 cells were transiently transfected with SV40 Renilla luciferase vector and SRF-luc. Forty-eight hours after treatment, cells were lysed and luciferase activity determined. Each stimulus increased SRF activity (n = 8, means ± SE; *different from control cells, P < 0.05, ANOVA). C: effect of BMP-4, TGF-β1, 5-HT, ET-1, LiCl, and SB-216763 on α-actin mRNA in human pulmonary artery cells. Cells were treated for 4 days and processed for qPCR analysis of α-actin mRNA levels relative to GAPDH mRNA. Each stimulus increased α-actin mRNA (n = 3, means ± SE, *different from control cells, P < 0.05, ANOVA).

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: Western Blot, Activity Assay, Transfection, Luciferase, Plasmid Preparation

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: BMP-4, TGF-β1, 5-HT, and ET-1 activate the p70S6K signaling pathway. A: representative immunoblots for phospho-p70S6K, total p70S6K (top), phospho-S6, and total S6 (bottom) in pulmonary artery smooth muscle cells treated with BMP-4, TGF-β1, 5-HT, and ET-1. B: group mean data (n = 3, ± SE, *different from unstimulated cells, P < 0.05, ANOVA). C: specific siRNAs against p70S6K (top) and S6 (bottom) block phosphorylation of these proteins. D: group mean data (n = 3, ± SE, *different from nontargeting siRNA, P < 0.05, ANOVA).

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: Western Blot, Blocking Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: Activation of the p70S6K pathway is required for cell hypertrophy. Pulmonary artery smooth muscle cells were transfected with either nontargeting siRNA, specific siRNA against p70S6K (A), or siRNA against S6 (B), and treated with BMP-4, TGF-β1, 5-HT, or ET-1. Cell size was measured by flow cytometry. C: representative immunoblots for α-actin and β-actin from cells transfected with either nontargeting siRNA, p70S6K siRNA, or S6 siRNA. D: group mean data for p70S6K siRNA experiments (n = 3, ± SE, *different from nontargeting siRNA, P < 0.05, ANOVA). E: group mean data for S6 siRNA experiments (n = 3, ± SE, *different from nontargeting siRNA, P < 0.05, ANOVA).

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: Activation Assay, Transfection, Flow Cytometry, Western Blot

Journal: Cancers

Article Title: The Potential of Human Pulmonary Mesenchymal Stem Cells as Vectors for Radiosensitizing Metallic Nanoparticles: An In Vitro Study †

doi: 10.3390/cancers16183239

Figure Lengend Snippet: Dose- and time-dependent effects of Fe 3 O 4 @Au nanoparticles on the viability of HPMSCs. Cells were incubated with increasing concentrations of Fe 3 O 4 @Au NPs (10–500 µg/mL). ( A ) The mitochondrial metabolic activity of HPMSCs was quantified via an MTT assay after 24 h, ( B ) 48 h, and ( C ) 72 h of incubation with Fe 3 O 4 @Au NPs, and ( D ) cell counting was performed via the trypan blue exclusion method on cells after 24 h, ( E ) 48 h, or ( F ) 72 h of incubation with Fe 3 O 4 @Au NPs. Data are expressed as the mean ± SEM of three independent experiments. *: p < 0.05, **: p < 0.01, ***: p < 0.001, and ****: p < 0.0001 as compared to control conditions.

Article Snippet: Primary human pulmonary mesenchymal stem cells (HPMSCs) isolated from human lung tissue were purchased from the ScienCell research laboratory (Carlsbad, CA, USA) and were cultured in Modified Eagle’s Medium (MEM; PAN Biotech, Aidenbach, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS; PAN Biotech), 1 mM of sodium pyruvate (PAN Biotech), 0.5 μg/mL of amphotericin B (PAN Biotech), 2 mM of L-glutamine (Biochrom AG, Berlin, Germany), and 0.1 mg/mL penicillin–streptomycin (PAN Biotech).

Techniques: Incubation, Activity Assay, MTT Assay, Cell Counting, Control

Journal: Cancers

Article Title: The Potential of Human Pulmonary Mesenchymal Stem Cells as Vectors for Radiosensitizing Metallic Nanoparticles: An In Vitro Study †

doi: 10.3390/cancers16183239

Figure Lengend Snippet: Evaluation of the cellular uptake of Fe 3 O 4 @Au nanoparticles by HPMSCs. Cellular internalization was assessed using Prussian blue staining. The internalization of Fe 3 O 4 @Au NPs was determined on ( A ) control cells and ( B ) cells exposed to 100 µg/mL Fe 3 O 4 @Au NPs for 24 h, as well as ( C ) control cells and ( D ) cells exposed to 100 µg/mL Fe 3 O 4 @Au for 72 h. Blue staining is consistent with the intracellular uptake of Fe 3 O 4 @Au NPs by HPMSCs.

Article Snippet: Primary human pulmonary mesenchymal stem cells (HPMSCs) isolated from human lung tissue were purchased from the ScienCell research laboratory (Carlsbad, CA, USA) and were cultured in Modified Eagle’s Medium (MEM; PAN Biotech, Aidenbach, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS; PAN Biotech), 1 mM of sodium pyruvate (PAN Biotech), 0.5 μg/mL of amphotericin B (PAN Biotech), 2 mM of L-glutamine (Biochrom AG, Berlin, Germany), and 0.1 mg/mL penicillin–streptomycin (PAN Biotech).

Techniques: Staining, Control

Journal: Cancers

Article Title: The Potential of Human Pulmonary Mesenchymal Stem Cells as Vectors for Radiosensitizing Metallic Nanoparticles: An In Vitro Study †

doi: 10.3390/cancers16183239

Figure Lengend Snippet: Effect of Fe 3 O 4 @Au nanoparticles on the necrosis and apoptosis markers of HPMSCs. ( A ) The cytotoxic effect of 100 µg/mL Fe 3 O 4 @Au NPs on cells was assessed using an LDH assay after 24 h and ( B ) 72 h of exposure to nanoparticles. The mean expression of genes encoding ( C ) caspase 3, ( D ) caspase 9, and ( E ) Bcl2 was obtained via RT-qPCR on HPMSCs incubated or not with Fe 3 O 4 @Au NPs for 24 h and 72 h. Data are indicated as the mean ± SEM of three independent experiments. *: p < 0.05, **: p < 0.01, and ****: p < 0.0001 as compared to the corresponding control conditions.

Article Snippet: Primary human pulmonary mesenchymal stem cells (HPMSCs) isolated from human lung tissue were purchased from the ScienCell research laboratory (Carlsbad, CA, USA) and were cultured in Modified Eagle’s Medium (MEM; PAN Biotech, Aidenbach, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS; PAN Biotech), 1 mM of sodium pyruvate (PAN Biotech), 0.5 μg/mL of amphotericin B (PAN Biotech), 2 mM of L-glutamine (Biochrom AG, Berlin, Germany), and 0.1 mg/mL penicillin–streptomycin (PAN Biotech).

Techniques: Lactate Dehydrogenase Assay, Expressing, Quantitative RT-PCR, Incubation, Control

Journal: Cancers

Article Title: The Potential of Human Pulmonary Mesenchymal Stem Cells as Vectors for Radiosensitizing Metallic Nanoparticles: An In Vitro Study †

doi: 10.3390/cancers16183239

Figure Lengend Snippet: Effect of Fe 3 O 4 @Au nanoparticles on the redox status of HPMSCs. The mean normalized expression of genes encoding redox markers was determined by RT-qPCR. The expressions of genes coding for ( A ) Nox4, ( B ) HMOX-1, and ( C ) Nrf2 in HPMSCs were measured after 24 h and 72 h of exposure to 100 µg/mL Fe 3 O 4 @Au NPs. ( D ) ROS production was determined in HPMSCs exposed to 100 µg/mL Fe 3 O 4 @Au NPs for 2 h by using the DCFH-DA probe. Data are expressed as the mean ± SEM of three independent experiments. **: p < 0.01 and ****: p < 0.0001 as compared to the corresponding control conditions.

Article Snippet: Primary human pulmonary mesenchymal stem cells (HPMSCs) isolated from human lung tissue were purchased from the ScienCell research laboratory (Carlsbad, CA, USA) and were cultured in Modified Eagle’s Medium (MEM; PAN Biotech, Aidenbach, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS; PAN Biotech), 1 mM of sodium pyruvate (PAN Biotech), 0.5 μg/mL of amphotericin B (PAN Biotech), 2 mM of L-glutamine (Biochrom AG, Berlin, Germany), and 0.1 mg/mL penicillin–streptomycin (PAN Biotech).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Cancers

Article Title: The Potential of Human Pulmonary Mesenchymal Stem Cells as Vectors for Radiosensitizing Metallic Nanoparticles: An In Vitro Study †

doi: 10.3390/cancers16183239

Figure Lengend Snippet: Effect of Fe 3 O 4 @Au nanoparticles on proinflammatory cytokines and chemokines secreted by HPMSCs. The proinflammatory response of HPMSCs was evaluated through ELISA. The levels of ( A ) TNF-α, ( B ) IL-1β, ( C ) IL-6, ( D ) CXCL8, and ( E ) CCL5 secreted by HPMSCs were quantified after 24 or 72 h of exposure to 100 µg/mL of Fe 3 O 4 @Au NPs. Data are presented as the mean ± SEM of three independent experiments. *: p < 0.05, **: p < 0.01, ***: p < 0.001, and ****: p < 0.0001 as compared to the corresponding control condition.

Article Snippet: Primary human pulmonary mesenchymal stem cells (HPMSCs) isolated from human lung tissue were purchased from the ScienCell research laboratory (Carlsbad, CA, USA) and were cultured in Modified Eagle’s Medium (MEM; PAN Biotech, Aidenbach, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS; PAN Biotech), 1 mM of sodium pyruvate (PAN Biotech), 0.5 μg/mL of amphotericin B (PAN Biotech), 2 mM of L-glutamine (Biochrom AG, Berlin, Germany), and 0.1 mg/mL penicillin–streptomycin (PAN Biotech).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Cancers

Article Title: The Potential of Human Pulmonary Mesenchymal Stem Cells as Vectors for Radiosensitizing Metallic Nanoparticles: An In Vitro Study †

doi: 10.3390/cancers16183239

Figure Lengend Snippet: Effect of Fe 3 O 4 @Au nanoparticles on the expression and production of protumorigenic factors by the HPMSCs. The gene expression and secreted levels of protumorigenic markers were measured on cells exposed to 100 µg/mL Fe 3 O 4 @Au nanoparticles for 24 and 72 h. First, the mean expression of genes encoding ( A ) PI3K, ( B ) AMPK, ( C ) PDGF, and ( D ) VEGF were determined via the use of RT-qPCR. Then, ( E ) the level of VEGF secreted by HPMSCs was quantified through an ELISA. Data are expressed as the mean ± SEM of three independent experiments. *: p < 0.05 and ****: p < 0.0001 as compared to the corresponding control conditions.

Article Snippet: Primary human pulmonary mesenchymal stem cells (HPMSCs) isolated from human lung tissue were purchased from the ScienCell research laboratory (Carlsbad, CA, USA) and were cultured in Modified Eagle’s Medium (MEM; PAN Biotech, Aidenbach, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS; PAN Biotech), 1 mM of sodium pyruvate (PAN Biotech), 0.5 μg/mL of amphotericin B (PAN Biotech), 2 mM of L-glutamine (Biochrom AG, Berlin, Germany), and 0.1 mg/mL penicillin–streptomycin (PAN Biotech).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control