Journal: American journal of physiology. Renal physiology

Article Title: Role of γ-adducin in actin cytoskeleton rearrangements in podocyte pathophysiology.

doi: 10.1152/ajprenal.00423.2020

Figure Lengend Snippet: Figure 3. Validation of primary podocytes isolated from fawn-hooded hypertensive (FHH) and FHH.Add3 transgenic rats. A: representative images of freshly isolated glomeruli on day 0 (D0), cobblestone-like undifferentiated podocytes outgrowth from the decapsulated glomerulus on D5, and fully dif- ferentiated arborized podocytes on D10. Images were captured using a Lionheart FX Automated Microscope with 10 objective (total magnification of 350). B: representative images of nephrin-, synaptopodin (SYNPO)-, and podocin-positive primary podocytes by immunocytochemistry. C, top: repre- sentative images of c-adducin (ADD3) expression and distribution in primary podocytes. Podocytes were isolated from 6–9 male rats in each strain. Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope with 60 oil immersion objective and a 3 digi- tal zoom (total magnification of 3,960). C, bottom: representative images and quantitation of ADD3 expression in podocytes by Western blot. BP, block- ing peptide; M, molecular mass. Mean values ± SE are presented. P < 0.05 from the corresponding values in age-matched FHH rats.

Article Snippet: Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope or a Nikon Eclipse 55i fluorescence microscope equipped with a DS-Fil1 color camera (Nikon).

Techniques: Biomarker Discovery, Isolation, Transgenic Assay, Microscopy, Immunocytochemistry, Expressing, Inverted Microscopy, Quantitation Assay, Western Blot, Blocking Assay

Journal: American journal of physiology. Renal physiology

Article Title: Role of γ-adducin in actin cytoskeleton rearrangements in podocyte pathophysiology.

doi: 10.1152/ajprenal.00423.2020

Figure Lengend Snippet: Figure 4. Validation of conditionally immortalized mouse podocytes and the efficiency of knockdown of the expression of c-adducin (ADD3) with Add3 Dicer-substrate short interfering RNA (DsiRNA). A: representative images of nephrin-, synaptopodin (SYNPO)-, and podocin-positive conditionally immor- talized mouse podocytes by immunocytochemistry. B and C: representative images and quantitation of ADD3 immunostaining (B) and Western blots (C) of the efficiency of knockdown of the expression of ADD3 in conditionally immortalized mouse podocytes 36 h posttransfection with Add3 DsiRNA. Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope with 20 objective (total magnification of 440). Experiments were repeated 4–5 times in triplicate in each experiment. Mean values ± SE are presented. P < 0.05 from the corresponding val- ues in NC1 negative controls (Ctrl).

Article Snippet: Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope or a Nikon Eclipse 55i fluorescence microscope equipped with a DS-Fil1 color camera (Nikon).

Techniques: Biomarker Discovery, Knockdown, Expressing, Small Interfering RNA, Immunocytochemistry, Quantitation Assay, Immunostaining, Western Blot, Inverted Microscopy

Journal: American journal of physiology. Renal physiology

Article Title: Role of γ-adducin in actin cytoskeleton rearrangements in podocyte pathophysiology.

doi: 10.1152/ajprenal.00423.2020

Figure Lengend Snippet: Figure 5. Downregulation of c-adducin (ADD3) enhances F-actin net formation in podocytes. A: representative images (left) and quantitation (right) of im- munostaining for comparison of the expression and distribution of F/G-actin and the F/G-actin ratio in Add3 Dicer-substrate short interfering RNA (DsiRNA) and NC1 scrambled siRNA transfected immortalized mouse podocytes B: representative images (left) and quantitation (right) of immunostaining for comparison of the expression and distribution of F/G-actin and F/G-actin ratio in primary podocytes isolated from FHH and FHH.Add3 transgenic rats. C: representative images of immunostaining for comparison of the expression and distribution of ARPC1B (left) and quantitation with Western blots (right) in primary podocytes isolated from male FHH and FHH.Add3 transgenic rats. Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope with 60 oil immersion objective and a 3 digital zoom (total magnification of 3,960). Experiments using condition- ally immortalized mouse podocytes were repeated 3 times in triplicate in each experiment. Podocytes were isolated from 6–9 male rats in each strain. ARPC1B, actin-related protein 2/3 complex subunit 1B. Mean values ± SE are presented. P < 0.05 from the corresponding values in age-matched FHH rats or NC1 negative controls (Ctrl).

Article Snippet: Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope or a Nikon Eclipse 55i fluorescence microscope equipped with a DS-Fil1 color camera (Nikon).

Techniques: Quantitation Assay, Comparison, Expressing, Small Interfering RNA, Transfection, Immunostaining, Isolation, Transgenic Assay, Western Blot, Inverted Microscopy

Journal: American journal of physiology. Renal physiology

Article Title: Role of γ-adducin in actin cytoskeleton rearrangements in podocyte pathophysiology.

doi: 10.1152/ajprenal.00423.2020

Figure Lengend Snippet: Figure 6. Reduced c-adducin (ADD3) expression alters the Rho family of small GTPases levels in podocytes in fawn-hooded hypertensive (FHH) rats. A: representative images (left) and quantitation (right) of synaptopodin (SYNPO) immunostaining in the glo- meruli of male FHH and FHH.Add3 rats. Six sections per rat and 30 glomeruli per section were examined; n = 6 rats per group. Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope with 20 objective and 1 digi- tal zoom (left, total magnification of 440) and 4.5 digital zoom (right, total magnification of 1,980). B: representative images (left) and quantitation (right) of SYNPO and RhoA expression in the renal cortex of male FHH and FHH.Add3 rats. C: representative images (left) and quantitation (right) of SYNPO and RhoA expression in the primary podocytes isolated from male FHH and FHH.Add3 rats. D: representa- tive images (left) and quantitation (right) of Rac1 and CDC42 expression in the renal cortex of male FHH and FHH.Add3 rats. E: representative images (left) and quantitation (right) of Rac1 and CDC42 expres- sion in primary podocytes isolated from male FHH and FHH.Add3 rats. Mean values ± SE are presented; n = 6–9 rats in each strain. P < 0.05 from the corre- sponding values in age-matched FHH rats.

Article Snippet: Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope or a Nikon Eclipse 55i fluorescence microscope equipped with a DS-Fil1 color camera (Nikon).

Techniques: Expressing, Quantitation Assay, Immunostaining, Inverted Microscopy, Isolation

Journal: American journal of physiology. Renal physiology

Article Title: Role of γ-adducin in actin cytoskeleton rearrangements in podocyte pathophysiology.

doi: 10.1152/ajprenal.00423.2020

Figure Lengend Snippet: Figure 7. Reduced c-adducin (ADD3) expression attenu- ates slit diaphragm protein expression in fawn-hooded hypertensive (FHH) rats. Validation of the nephrin anti- body by Western blot in the renal cortex of male FHH and FHH.Add3 rats is shown. A and B: immunostaining using conditionally immortalized mouse podocytes (A) with and without the blocking peptide (BP; B). M, molec- ular mass. C: representative images (left) and quantita- tion (right) of nephrin immunostaining in the glomeruli of male FHH compared with FHH.Add3 rats. Six sections per rat and 30 glomeruli per section were examined; n = 6 rats per group. Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope with 20 objective and 1 digital zoom (left, total magnification of 440) and 4.5 digital zoom (right, total magnification of 1,980). D: represen- tative images (left) and quantitation (right) of nephrin im- munostaining in primary podocytes of FHH compared with FHH.Add3 rats. Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope with 60 oil immersion objective and a 3 digital zoom (total magnification of 3,960). E: representative images (left) and quantitation (right) of nephrin and podocin expression by Western blot in the renal cortex of male FHH and FHH.Add3 rats. Mean values ± SE are presented. P < 0.05 from the corresponding values in age-matched FHH rats.

Article Snippet: Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope or a Nikon Eclipse 55i fluorescence microscope equipped with a DS-Fil1 color camera (Nikon).

Techniques: Expressing, Biomarker Discovery, Western Blot, Immunostaining, Blocking Assay, Inverted Microscopy, Quantitation Assay

Journal: American journal of physiology. Renal physiology

Article Title: Role of γ-adducin in actin cytoskeleton rearrangements in podocyte pathophysiology.

doi: 10.1152/ajprenal.00423.2020

Figure Lengend Snippet: Figure 8. Reduced c-adducin (ADD3) expres- sion affects focal adhesion integrins in fawn- hooded hypertensive (FHH) rats. A: representa- tive images (left) and quantitation (right) of integrin-a3 (ITGA30) immunostaining in the glo- meruli obtained from male FHH compared with FHH.Add3 rats. Six sections per rat and 30 glo- meruli per section were examined; n = 6 rats per group. B: representative images (left) and quantitation (right) of integrin-a3 (ITGB1) expres- sion in the renal cortex of male FHH and FHH. Add3 rats. Images in A and B were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope with 20 objective and 1 digital zoom (left, total magni- fication of 440) and 4.5 digital zoom (right, total magnification of 1,980). C: representative images (left) and quantitation (right) of ITGA3 and ITGB1 expression in the glomeruli of male FHH and FHH.Add3 rats. Mean values ± SE are presented. P < 0.05 from the corresponding values in age-matched FHH rats.

Article Snippet: Images were captured using a Nikon C2 laser scanning confocal on an Eclipse Ti2 inverted microscope or a Nikon Eclipse 55i fluorescence microscope equipped with a DS-Fil1 color camera (Nikon).

Techniques: Quantitation Assay, Immunostaining, Inverted Microscopy, Expressing

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