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: Brain and Behavior

Article Title: Propofol Suppresses Ferroptosis via Modulating eNOS/NO Signaling Pathway to Improve Traumatic Brain Injury

doi: 10.1002/brb3.70187

Figure Lengend Snippet: The neuroprotective effect of propofol may be associated with promoting eNOS to produce nitric oxide (NO). (A) Representative immunoblots of eNOS and α‐tubulin proteins from four groups of mice. (B) Comparative analysis of eNOS protein expression among the four groups of mice ( n = 6). (C) Comparison of NO concentrations in brain tissue among the four groups of mice ( n = 6). Data are presented as mean ± standard deviation and were statistically analyzed using ANOVA, with p values indicated on the bar charts.

Article Snippet: In subsequent experiments, before the application of propofol, eNOS protein inhibitor L‐NAME (S2877, Selleck, USA) was administered intraperitoneally at a dose of 30 mg/kg, or an equivalent volume of solvent was injected as a control.

Techniques: Western Blot, Expressing, Comparison, Standard Deviation

Journal: Brain and Behavior

Article Title: Propofol Suppresses Ferroptosis via Modulating eNOS/NO Signaling Pathway to Improve Traumatic Brain Injury

doi: 10.1002/brb3.70187

Figure Lengend Snippet: The eNOS protein inhibitor L‐NAME can reverse the early neuroprotective effects of propofol on TBI. (A) Representative images of Perls staining from two groups of mice are displayed. (B) A comparison of the number of positively stained cells in Perls staining between the two groups of mice ( n = 6 per group) is presented. (C) Representative images of Nissl staining from two groups of mice are shown, with scale bars at 50 or 10 µm. (D, E) Bar graphs illustrate the statistical analysis of Nissl body count and positive area per field of view for four groups of mice ( n = 6 per group). (F) Representative TUNEL fluorescence images from the damaged cortical area, hippocampal CA1 region, CA3 region, and DG region of two groups of mice are displayed. Red labeling indicates neurons, green labeling indicates apoptotic cells, and blue labeling indicates cell nuclei, with a scale bar at 50 µm. (G) A comparison of the number of apoptotic neurons in the damaged cortical area, hippocampal CA1 region, CA3 region, and DG region among four groups of mice ( n = 6 per group) is presented. (H) Representative immunoblots of Gpx4, 4‐HNE, Bak, Bcl‐2, and α‐tubulin proteins from two groups of mice are shown. (I–K) Comparisons of Gpx4 protein expression, 4‐HNE protein expression, and the Bak/Bcl‐2 ratio between the two groups of mice ( n = 6 per group) are provided. (L) Comparison of the expression levels of FTH1 mRNA between the two groups of mice ( n = 6 per group). Data are presented as mean ± standard deviation and statistically analyzed using Student's t ‐test, with p values on the bar charts.

Article Snippet: In subsequent experiments, before the application of propofol, eNOS protein inhibitor L‐NAME (S2877, Selleck, USA) was administered intraperitoneally at a dose of 30 mg/kg, or an equivalent volume of solvent was injected as a control.

Techniques: Staining, Comparison, TUNEL Assay, Fluorescence, Labeling, Western Blot, Expressing, Standard Deviation

Journal: Brain and Behavior

Article Title: Propofol Suppresses Ferroptosis via Modulating eNOS/NO Signaling Pathway to Improve Traumatic Brain Injury

doi: 10.1002/brb3.70187

Figure Lengend Snippet: The eNOS protein inhibitor L‐NAME can reverse the effect of propofol in improving long‐term outcomes after TBI. (A, B) Representative GFAP and Iba1 immunofluorescence images from the damaged cortical area, hippocampal CA1 region, CA3 region, and DG region of two groups of mice are shown. (C, D) Comparisons of GFAP‐positive area and Iba1‐positive cell counts in the damaged cortical area, hippocampal CA1 region, CA3 region, and DG region between two groups of mice are presented ( n = 6 per group). (E) The escape latencies during the MWM training period for two groups of mice were compared, and two‐way repeated measures ANOVA was used for statistical analysis, with **** p < 0.0001. (F) Representative traces of the mice in the MWM are shown. (G, H) Comparisons of platform crossing times and time spent in the target quadrant between two groups of mice are presented ( n = 6 per group). (I) Representative traces of the mice in the NOR test are shown, with N representing the novel object and F representing the familiar object. (J) A comparison of the discrimination index between two groups of mice is presented ( n = 6 per group). Data are expressed as mean ± standard deviation and were statistically analyzed using Student's t ‐test, with p values indicated on the bar charts.

Article Snippet: In subsequent experiments, before the application of propofol, eNOS protein inhibitor L‐NAME (S2877, Selleck, USA) was administered intraperitoneally at a dose of 30 mg/kg, or an equivalent volume of solvent was injected as a control.

Techniques: Immunofluorescence, Comparison, Standard Deviation

Journal: Brain and Behavior

Article Title: Propofol Suppresses Ferroptosis via Modulating eNOS/NO Signaling Pathway to Improve Traumatic Brain Injury

doi: 10.1002/brb3.70187

Figure Lengend Snippet: The neuroprotective effect of propofol may be associated with promoting eNOS to produce nitric oxide (NO). (A) Representative immunoblots of eNOS and α‐tubulin proteins from four groups of mice. (B) Comparative analysis of eNOS protein expression among the four groups of mice ( n = 6). (C) Comparison of NO concentrations in brain tissue among the four groups of mice ( n = 6). Data are presented as mean ± standard deviation and were statistically analyzed using ANOVA, with p values indicated on the bar charts.

Article Snippet: In subsequent experiments, before the application of propofol, eNOS protein inhibitor L‐NAME (S2877, Selleck, USA) was administered intraperitoneally at a dose of 30 mg/kg, or an equivalent volume of solvent was injected as a control.

Techniques: Western Blot, Expressing, Comparison, Standard Deviation

Journal: Brain and Behavior

Article Title: Propofol Suppresses Ferroptosis via Modulating eNOS/NO Signaling Pathway to Improve Traumatic Brain Injury

doi: 10.1002/brb3.70187

Figure Lengend Snippet: The eNOS protein inhibitor L‐NAME can reverse the early neuroprotective effects of propofol on TBI. (A) Representative images of Perls staining from two groups of mice are displayed. (B) A comparison of the number of positively stained cells in Perls staining between the two groups of mice ( n = 6 per group) is presented. (C) Representative images of Nissl staining from two groups of mice are shown, with scale bars at 50 or 10 µm. (D, E) Bar graphs illustrate the statistical analysis of Nissl body count and positive area per field of view for four groups of mice ( n = 6 per group). (F) Representative TUNEL fluorescence images from the damaged cortical area, hippocampal CA1 region, CA3 region, and DG region of two groups of mice are displayed. Red labeling indicates neurons, green labeling indicates apoptotic cells, and blue labeling indicates cell nuclei, with a scale bar at 50 µm. (G) A comparison of the number of apoptotic neurons in the damaged cortical area, hippocampal CA1 region, CA3 region, and DG region among four groups of mice ( n = 6 per group) is presented. (H) Representative immunoblots of Gpx4, 4‐HNE, Bak, Bcl‐2, and α‐tubulin proteins from two groups of mice are shown. (I–K) Comparisons of Gpx4 protein expression, 4‐HNE protein expression, and the Bak/Bcl‐2 ratio between the two groups of mice ( n = 6 per group) are provided. (L) Comparison of the expression levels of FTH1 mRNA between the two groups of mice ( n = 6 per group). Data are presented as mean ± standard deviation and statistically analyzed using Student's t ‐test, with p values on the bar charts.

Article Snippet: In subsequent experiments, before the application of propofol, eNOS protein inhibitor L‐NAME (S2877, Selleck, USA) was administered intraperitoneally at a dose of 30 mg/kg, or an equivalent volume of solvent was injected as a control.

Techniques: Staining, Comparison, TUNEL Assay, Fluorescence, Labeling, Western Blot, Expressing, Standard Deviation

Journal: Brain and Behavior

Article Title: Propofol Suppresses Ferroptosis via Modulating eNOS/NO Signaling Pathway to Improve Traumatic Brain Injury

doi: 10.1002/brb3.70187

Figure Lengend Snippet: The eNOS protein inhibitor L‐NAME can reverse the effect of propofol in improving long‐term outcomes after TBI. (A, B) Representative GFAP and Iba1 immunofluorescence images from the damaged cortical area, hippocampal CA1 region, CA3 region, and DG region of two groups of mice are shown. (C, D) Comparisons of GFAP‐positive area and Iba1‐positive cell counts in the damaged cortical area, hippocampal CA1 region, CA3 region, and DG region between two groups of mice are presented ( n = 6 per group). (E) The escape latencies during the MWM training period for two groups of mice were compared, and two‐way repeated measures ANOVA was used for statistical analysis, with **** p < 0.0001. (F) Representative traces of the mice in the MWM are shown. (G, H) Comparisons of platform crossing times and time spent in the target quadrant between two groups of mice are presented ( n = 6 per group). (I) Representative traces of the mice in the NOR test are shown, with N representing the novel object and F representing the familiar object. (J) A comparison of the discrimination index between two groups of mice is presented ( n = 6 per group). Data are expressed as mean ± standard deviation and were statistically analyzed using Student's t ‐test, with p values indicated on the bar charts.

Article Snippet: In subsequent experiments, before the application of propofol, eNOS protein inhibitor L‐NAME (S2877, Selleck, USA) was administered intraperitoneally at a dose of 30 mg/kg, or an equivalent volume of solvent was injected as a control.

Techniques: Immunofluorescence, Comparison, Standard Deviation