Journal: Antioxidants

Article Title: Endothelial Nitric Oxide Synthase-Dependent Mechanism of Hydroxyurea-Induced S-Phase Arrest in Erythroid Cells

doi: 10.3390/antiox15040435

Figure Lengend Snippet: Hydroxyurea induces NOS3 expression and activity in HEL92.1.7 cells. ( A ) Immunocytochemistry for endothelial nitric oxide synthase (NOS3) protein in HEL92.1.7 cells treated with 10, 50, and 100 µM hydroxyurea (HU) or vehicle (Ctrl) for 48 h and quantification of NOS3-positive cells. Scale bar = 80 µm. ( B ) Western blot for total and phosphorylated (S1177) NOS3 protein in HEL92.1.7 cells treated with the indicated concentrations of HU or vehicle (Ctrl) for 48 h. Quantification of band intensity normalized to Ctrl, where β-actin was used as a loading control. ( C ) Quantification of phospho-to-total protein ratio normalized to Ctrl. Concentrations of ( D ) nitrite and ( E ) citrulline in HEL92.1.7 cells treated for 48 h with 5 μM of the NOS3 inhibitor Caveolin-1 scaffolding domain peptide (CSD), as well as NOS3 kd cells treated for 48 h with 100 µM HU or vehicle. ( F ) In silico model of HU and NOS3 interaction showing binding at amino acids ASN366 and ARG372 and with the substrate L-arginine (ARG700). In the hydroxyurea molecule, white spheres represent hydrogen, blue spheres nitrogen, red spheres oxygen, and black sphere carbon. ( G ) Concentrations of nitrite or citrulline measured after in vitro NOS3 enzymatic assay with the indicated concentrations of HU and incubation times. ( H ) Western blot for phospho-AKT1 (Ser473) and total AKT1 protein in HEL92.1.7 cells treated with 100 μM HU for 5, 15, or 30 min. Quantification of phospho-to-total protein ratio normalized to Ctrl. ( I ) Western blot for NOS3 protein in HEL92.1.7 cells treated with the indicated concentrations of HU and/or 30 μM of the AKT inhibitor uprosertib (UPS). Quantification of band intensity with β-actin used as a loading control and normalized to Ctrl. n = 3; mean + SEM; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Ctrl or as indicated. ns—non-significant.

Article Snippet: Human recombinant NOS3 protein (OriGene, TP309228M, Rockville, MD, USA) was added to reach a final concentration of 0.5 μg/μL in the presence of 0, 10, 50, or 100 μM HU.

Techniques: Expressing, Activity Assay, Immunocytochemistry, Western Blot, Control, Scaffolding, In Silico, Binding Assay, In Vitro, Enzymatic Assay, Incubation

Journal: Antioxidants

Article Title: Endothelial Nitric Oxide Synthase-Dependent Mechanism of Hydroxyurea-Induced S-Phase Arrest in Erythroid Cells

doi: 10.3390/antiox15040435

Figure Lengend Snippet: NOS3 deletion or inhibition shifts cells from S to G 0 /G 1 phase and regulates apoptosis under hydroxyurea treatment. ( A ) Sorting of GFP-positive endothelial nitric oxide synthase knock-down (NOS3 kd ) HEL92.1.7 cells after transduction with lentiviral particles containing shRNA against NOS3 and GFP. ( B ) NOS3 kd was confirmed by quantifying NOS3-positive cells upon immunocytochemistry staining. ( C ) Quantification of NOS1- and NOS2-positive cells in NOS3 kd and control HEL92.1.7 cells after immunocytochemistry staining. HEL92.1.7 and NOS3 kd HEL92.1.7 cells were treated with 100 μM hydroxyurea (HU), 1 μM of the NOS3 inhibitor Caveolin-1 scaffolding domain peptide (CSD), or vehicle. ( D ) Immunocytochemistry for Ki67 protein and ( E ) quantification of Ki67-positive cells. ( F ) Percentage of cells in the G 0 /G 1 , S, and G 2 /M phases of the cell cycle were analyzed by flow cytometry after PI staining. ( G ) Immunocytochemistry for ssDNA and ( H ) quantification of ssDNA-positive cells; percentage of ( I ) early and ( J ) late apoptotic cells were analyzed by flow cytometry after Annexin V/PI staining. ( B – E , G , H ) n = 5; ( E , I , J ) n = 3; mean + SEM; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control (Ctrl) or as indicated (red line refers to S phase). ns—non-significant. Scale bar = 80 µm.

Article Snippet: Human recombinant NOS3 protein (OriGene, TP309228M, Rockville, MD, USA) was added to reach a final concentration of 0.5 μg/μL in the presence of 0, 10, 50, or 100 μM HU.

Techniques: Inhibition, Knockdown, Transduction, shRNA, Immunocytochemistry, Staining, Control, Scaffolding, Flow Cytometry

Journal: Antioxidants

Article Title: Endothelial Nitric Oxide Synthase-Dependent Mechanism of Hydroxyurea-Induced S-Phase Arrest in Erythroid Cells

doi: 10.3390/antiox15040435

Figure Lengend Snippet: Nos3 deficiency impairs hydroxyurea-induced protein nitrosylation and alters hematopoietic lineage commitment in vivo. ( A ) Schematic representation of experimental setup: endothelial nitric oxide synthase null mice (Nos3) -/- or wild-type (WT) mice were treated orally with 1 mg/mL hydroxyurea (HU) or vehicle in drinking water for 2 weeks. Bone marrow cells were used for NO and citrulline measurements, biotin switch assay for the detection of nitrosylated proteins, and colony formation assay. Concentrations of ( B ) nitrite and ( C ) citrulline in the bone marrow of WT and Nos3 -/- mice treated with HU or vehicle. ( D ) Quantification of nitrosylated proteins in bone marrow of WT, Nos2 -/- , and Nos3 -/- mice treated with HU or vehicle. Total protein was used as a loading control, and protein levels were normalized to the levels of WT mice. ( E ) Nitrosylation of proteins was visualized using anti-streptavidin-HRP antibody after the biotin switch assay, while Coomassie blue staining was used to detect total proteins. ( F ) Colony formation assay showing the number of late erythroid (CFU-E), early erythroid (BFU-E), and ( G ) granulocyte/macrophage progenitors (CFU-GM) in the bone marrow of WT and Nos3 -/- mice treated with vehicle and HU, respectively, for 2 weeks. n = 3; mean + SEM; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. untreated WT or as indicated. ns—non-significant.

Article Snippet: Human recombinant NOS3 protein (OriGene, TP309228M, Rockville, MD, USA) was added to reach a final concentration of 0.5 μg/μL in the presence of 0, 10, 50, or 100 μM HU.

Techniques: In Vivo, Biotin Switch Assay, Colony Assay, Control, Staining

Journal: Antioxidants

Article Title: Endothelial Nitric Oxide Synthase-Dependent Mechanism of Hydroxyurea-Induced S-Phase Arrest in Erythroid Cells

doi: 10.3390/antiox15040435

Figure Lengend Snippet: In vivo NOS3 depletion or inhibition impairs hydroxyurea-mediated S-phase blockage and alters apoptosis. ( A ) Schematic representation of experimental setup: endothelial nitric oxide synthase (Nos3) -/- and wild-type (WT) mice were treated orally with 1 mg/mL hydroxyurea (HU) and vehicle, respectively, in drinking water for 2 weeks. WT mice were treated with 0.5 mg/kg CSD intraperitoneally on days 12–14 of HU treatment. Mouse erythroid progenitors (mERPs) isolated from WT and Nos3 -/- mice based on CD71 and Ter119 expression and used for immunostaining, and cell cycle and apoptosis analysis. ( B ) Immunocytochemistry for endothelial nitric oxide synthase (NOS3) protein and quantification of NOS3-positive cells. ( C ) Immunocytochemistry for Ki67 and ( D ) quantification of Ki67-positive cells. ( E ) Cell cycle distribution by flow cytometry showing percentages of cells in the G 0 /G 1 , S, and G 2 /M phases. ( F ) Immunocytochemistry for caspase 3 (Cas3) and ( G ) quantification of Cas3-positive cells. Annexin V/PI assay showing percentages of: ( H ) early and ( I ) late apoptotic cells. n = 3; mean + SEM; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. untreated WT or as indicated (red line refers to S phase). ns—non-significant. Scale bar = 40 µm.

Article Snippet: Human recombinant NOS3 protein (OriGene, TP309228M, Rockville, MD, USA) was added to reach a final concentration of 0.5 μg/μL in the presence of 0, 10, 50, or 100 μM HU.

Techniques: In Vivo, Inhibition, Isolation, Expressing, Immunostaining, Immunocytochemistry, Flow Cytometry

Journal: Antioxidants

Article Title: Endothelial Nitric Oxide Synthase-Dependent Mechanism of Hydroxyurea-Induced S-Phase Arrest in Erythroid Cells

doi: 10.3390/antiox15040435

Figure Lengend Snippet: Dual NOS2/NOS3 inhibition impairs hydroxyurea-induced proliferation block in erythroid cells. HEL92.1.7 cells were treated for 48 h with the indicated concentrations of diphenyleneiodonium chloride (DPI), an NADPH oxidase (NOX)/inducible nitric oxide synthase (NOS2)/endothelial nitric oxide synthase (NOS3) inhibitor alone or in combination with 100 µM of hydroxyurea (HU). ( A ) Immunocytochemistry for Ki67 and ( B ) quantification of Ki67-positive cells were performed. Scale bar = 80 µm. ( C ) Percentages of HEL92.1.7 cells in G 0 /G 1 , S, and G 2 /M cell cycle phases were analyzed by flow cytometry after PI staining. ( D ) Schematic representation of experimental setup: wild-type (WT) mice were treated orally with 1 mg/mL HU or vehicle in drinking water for 14 days. On days 12, 13, and 14, the mice were injected with 1 mg/kg DPI twice daily. Mouse erythroid progenitors (mERPs) were isolated from WT and Nos3 -/- mice based on CD71 and Ter119 expression and used for nitrite and citrulline measurements, immunostaining, and apoptosis analysis. Concentrations of ( E ) nitrite and ( F ) citrulline in the bone marrow of WT mice treated with HU, DPI, or a combination of both. mERPs were isolated from WT mice treated with DPI or vehicle and ( G ) immunocytochemistry for Ki67 and quantification of Ki67-positive cells were performed. Scale bar = 40 µm. ( H ) Percentages of mERPs in G 0 /G 1 , S, and G 2 /M phases of cell cycle were analyzed by flow cytometry after staining with PI. n = 3; mean + SEM; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. WT or as indicated (red line refers to S phase). ns—non-significant.

Article Snippet: Human recombinant NOS3 protein (OriGene, TP309228M, Rockville, MD, USA) was added to reach a final concentration of 0.5 μg/μL in the presence of 0, 10, 50, or 100 μM HU.

Techniques: Inhibition, Blocking Assay, Immunocytochemistry, Flow Cytometry, Staining, Injection, Isolation, Expressing, Immunostaining

Journal: Antioxidants

Article Title: Endothelial Nitric Oxide Synthase-Dependent Mechanism of Hydroxyurea-Induced S-Phase Arrest in Erythroid Cells

doi: 10.3390/antiox15040435

Figure Lengend Snippet: Dual NOS2/NOS3 inhibition impairs hydroxyurea-induced apoptosis of erythroid cells in a context-dependent manner. HEL92.1.7 cells were treated for 48 h with the indicated concentrations of diphenyleneiodonium chloride (DPI), an NADPH oxidase (NOX)/inducible nitric oxide synthase (NOS2)/endothelial nitric oxide synthase (NOS3) inhibitor alone or in combination with 100 µM hydroxyurea (HU). ( A ) Immunocytochemistry for ssDNA and ( B ) quantification of ssDNA-positive cells were performed. Scale bar = 80 µm. Annexin V/PI assay showing percentages of ( C ) early and ( D ) late apoptotic cells. Mouse erythroid progenitors (mERPs) isolated from WT mice treated with 1 mg/mL HU, 1 mg/kg DPI, or a combination of both were used for: ( E ) immunocytochemistry for caspase-3 (Cas3) and quantification of Cas3-positive cells. Scale bar = 40 µm. Annexin V/PI assay showing percentages of ( F ) early and ( G ) late apoptotic mERPs. n = 3; mean + SEM; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. WT or as indicated. ns—non-significant.

Article Snippet: Human recombinant NOS3 protein (OriGene, TP309228M, Rockville, MD, USA) was added to reach a final concentration of 0.5 μg/μL in the presence of 0, 10, 50, or 100 μM HU.

Techniques: Inhibition, Immunocytochemistry, Isolation

Journal: Antioxidants

Article Title: Endothelial Nitric Oxide Synthase-Dependent Mechanism of Hydroxyurea-Induced S-Phase Arrest in Erythroid Cells

doi: 10.3390/antiox15040435

Figure Lengend Snippet: Nitric oxide synthases (NOSs) mediate hydroxyurea (HU)-induced reduction in cell proliferation and enhancement of apoptosis in erythroid cells. Compared to individual HU treatment, NOS2 and NOS3 mediate the HU effects on ( A ) cell proliferation (confirmed by the level of the Ki67 marker during active cell cycle: G 1 , S, G 2 , and mitosis) and S-phase arrest (flow cytometry); ( B ) early (flow cytometry Annexin V + /propidium iodide (PI) - population), late (flow cytometry—Annexin V + /PI + population), and total (ssDNA for HEL92.1.7 cells and caspase 3 for mice) apoptosis. The ssDNA accumulation may indicate DNA damage or replication stress, whereas apoptosis is more reliably supported by markers such as Caspase-3 activation and Annexin V positivity. Red line—studies on mouse erythroid progenitors (wild-type mice treated with NOS inhibitors and Nos knockout (NOS ko ) mice); blue line—studies on HEL92.1.7 erythroleukemic cells (NOS inhibitors and NOS knockdown (NOS kd )); black line—studies on both models. Lines with an arrow (stimulation) and inhibition arc (reduction) represent the effects of NOS inhibitors and genetic modifications. The full lines below and above the NOS boxes correspond to the NOS2 and/or NOS3 isoforms.

Article Snippet: Human recombinant NOS3 protein (OriGene, TP309228M, Rockville, MD, USA) was added to reach a final concentration of 0.5 μg/μL in the presence of 0, 10, 50, or 100 μM HU.

Techniques: Marker, Flow Cytometry, Activation Assay, Knock-Out, Knockdown, Inhibition

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Low albumin levels increase endothelial NO production and decrease vascular NO sensitivity.

doi: 10.1093/ndt/gfl443

Figure Lengend Snippet: Fig. 5. Representative western blots and group data (average of western blots; six rats/strain) depicting thoracic aorta endothelial NO synthase (eNOS) protein abundance in the Sprague–Dawley rat (SDR) and the Nagase analbuminemic rat (NAR).

Article Snippet: Representative western blots and group data (average of western blots; six rats/strain) depicting thoracic aorta endothelial NO synthase (eNOS) protein abundance in the Sprague–Dawley rat (SDR) and the Nagase analbuminemic rat (NAR).

Techniques: Western Blot, Quantitative Proteomics