Journal: Molecular Biology of the Cell

Article Title: Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

doi: 10.1091/mbc.e11-09-0811

Figure Lengend Snippet: FIGURE 1: Interaction of Cav-1 and eNOS determined by FRET and coIP. (A) Typical FRET image of Cav-1–YFP and eNOS–CFP expressed in CHO cells. Note that the fluorescence intensity of YFP increases, whereas CFP decreases in the ROI (white box) after addition of 5 μM Ca2+ ionophore A23187. Bar, 10 μm. (B) Normalized FRET measurements in CHO cells expressing Cav-1–YFP and eNOS–CFP after treatment with 5 μM A23187 (mean ± SEM; n = 12) or 4 U/ml thrombin (mean ± SEM; n = 5). YFP/CFP was normalized as 1 by the value at time zero (*p < 0.01 for all times beyond 2 min after thrombin addition; **p < 0.005 for all times beyond 1.5 min after A23187). (C) CoIP between Cav-1–YFP and eNOS–CFP in CHO cells (mean ± SEM; n = 3). (D) Normalized data of coIP (ratio of eNOS-CFP/Cav-1-YFP after IP with anti–Cav-1 antibody; *p < 0.05). (E) Phosphorylation of Cav-1–YFP and eNOS–CFP in CHO cells after treatment with 5 μM A23187 or 4 U/ml thrombin (mean ± SEM; n = 3). Ratio of phosphorylated/ total protein at 0 min was set as 1 (*p < 0.05).

Article Snippet: To create the vector eNOS with CFP tagged at the C-terminus, full-length H. sapiens eNOS cDNA (with GenBank Accession number NM_000603 from OriGene Technologies, Rockville, MD) was used as DNA template in a PCR to generate eNOS PCR fragment that contained AgeI at both 5′ and 3′ ends.

Techniques: Fluorescence, Expressing, Phospho-proteomics

Journal: Molecular Biology of the Cell

Article Title: Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

doi: 10.1091/mbc.e11-09-0811

Figure Lengend Snippet: FIGURE 2: CoIP between eNOS and Cav-1 in mouse lung. (A) Mouse lungs were perfused with medium containing 5 μM A23187 for 0, 3, and 10 min. Lungs were then homogenized and prepared for IP with polyclonal anti–Cav-1. (B) Normalized coIP data (mean ± SEM; n = 4); ratio of eNOS/Cav-1 at 0 min was set as 1 (*p < 0.05). (C) Phosphorylation of eNOS–Ser-1177, Src–Tyr-418, and Cav-1–Tyr-14 (indicated by arrows) in mouse lung homogenates following A23187 treatment (D). Normalized ratio of phosphorylated/total protein, with time 0 set as 1 (*p < 0.05; mean ± SEM; n = 4).

Article Snippet: To create the vector eNOS with CFP tagged at the C-terminus, full-length H. sapiens eNOS cDNA (with GenBank Accession number NM_000603 from OriGene Technologies, Rockville, MD) was used as DNA template in a PCR to generate eNOS PCR fragment that contained AgeI at both 5′ and 3′ ends.

Techniques: Phospho-proteomics

Journal: Molecular Biology of the Cell

Article Title: Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

doi: 10.1091/mbc.e11-09-0811

Figure Lengend Snippet: FIGURE 3: Phosphorylation-dependent interaction of eNOS and Cav-1 in HUVEC. (A) Time course of phosphorylation of eNOS–Ser-1177 and Cav-1–Tyr-14 in HUVECs after addition of 5 μM A23187, 4 U/ml thrombin, or 20 ng/ml VEGF (mean ± SEM; n = 6). Normalized data are shown in the bottom two rows (*p < 0.05). (B) CoIP of eNOS and Cav-1 in HUVEC after stimulation with 5 μM A23187 at indicated times (mean ± SEM; n = 4. *p < 0.05; **p < 0.005). (C, D) CoIP of phosphorylated eNOS and Cav-1 in HUVECs treated with A23187 for 5 min at 37°C (mean ± SEM; n = 3). p-Ser-1177-eNOS antibody was used for IP. Normalized data (p-Tyr-14-Cav-1 vs. t-Cav-1 bound to p-eNOS) is shown in D (*p < 0.05). (E) Effects of l-NAME and PP2 on phosphorylation of eNOS–Ser-1177, Src–Tyr-418, and Cav-1–Tyr-14 in HUVECs (mean ± SEM; n = 9). Confluent HUVECs were pretreated with 1 mM l-NAME or 15 μM PP2 for 30 min at 37°C prior to addition of 5 μM A23187 (§p < 0.001; *p < 0.05; and **p < 0.001). (F) Effect of l-NAME and PP2 on coIP between eNOS and Cav-1 (mean ± SEM; n = 6) in HUVEC (*p < 0.05; **p < 0.005).

Article Snippet: To create the vector eNOS with CFP tagged at the C-terminus, full-length H. sapiens eNOS cDNA (with GenBank Accession number NM_000603 from OriGene Technologies, Rockville, MD) was used as DNA template in a PCR to generate eNOS PCR fragment that contained AgeI at both 5′ and 3′ ends.

Techniques: Phospho-proteomics

Journal: Molecular Biology of the Cell

Article Title: Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

doi: 10.1091/mbc.e11-09-0811

Figure Lengend Snippet: FIGURE 4: Binding of Y14D-Cav-1 with eNOS inhibits eNOS activity. (A) CoIP of Cav-1 and eNOS mutants in HEK cells. WT-eNOS-CFP, eNOS-S1177D-CFP, and eNOS-S1177A-CFP were transiently transfected in HEK cells stably expressing WT-Cav-1-YFP, Cav-1-Y14D-YFP, or Cav-1-Y14F-YFP. Cell lysates were immunoprecipitated with anti–Cav-1 pAb 48 h after transfection of eNOS mutants in the absence of stimulation. Ratio of WT-eNOS-CFP/wt-Cav-1- YFP was set at 1 (*p < 0.05; **p < 0.005). Bar graph shows mean ± SEM (n = 3). (B) Protein levels of Cav-1 and eNOS mutants in transfected HEK cells before IP. (C) Nitrite accumulation in HEK/ eNOS cells transiently transfected with Cav-1 mutant cDNAs (mean ± SEM; n = 11). Cells were treated for 30 min with 5 μM A23187, and supernatants were collected for nitrite assay (*p < 0.001). (D) eNOS activity (representative of four independent experiments) of stable HEK/ eNOS cells transiently transfected with Cav-1 mutants.

Article Snippet: To create the vector eNOS with CFP tagged at the C-terminus, full-length H. sapiens eNOS cDNA (with GenBank Accession number NM_000603 from OriGene Technologies, Rockville, MD) was used as DNA template in a PCR to generate eNOS PCR fragment that contained AgeI at both 5′ and 3′ ends.

Techniques: Binding Assay, Activity Assay, Transfection, Stable Transfection, Expressing, Immunoprecipitation, Mutagenesis, Nitration

Journal: Molecular Biology of the Cell

Article Title: Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

doi: 10.1091/mbc.e11-09-0811

Figure Lengend Snippet: FIGURE 5: NO-dependent Src activation mediates Cav-1–Tyr-14 phosphorylation. (A) Decreased phosphorylation of Src–Tyr-418 and Cav-1–Tyr-14 in HUVECs after eNOS siRNA treatment (mean ± SEM; n = 4). Ratio of phosphorylated to total protein at 0 min was set at 1 (*p < 0.05). (B) The NO donor DEA NONOate induces Src activation in Cav-1–null MEF cells (mean ± SEM; n = 3). After stimulation with 0.2 mM DEA NONOate at indicated times, cells were prepared for Western blotting (*p < 0.05). (C) eNOS/NO-dependent Src activation in HEK cells (mean ± SEM; n = 7). The ratio of p-Tyr-418-Src at 5 min of A23187 treatment vs. untreated in WT-HEK cells was set as 1 (*p < 0.001 vs. WT-HEK). (D) A typical live-cell FRET image of HEK/eNOS cells expressing Src FRET biosensor. Fluorescence intensity of CFP and YFP was measured in the ROI (white box) before and after treatment with 5 μM A23187. Bar, 10 μm. (E) Activation of Src measured by FRET in HEK/eNOS cells expressing Src-FRET biosensor (mean ± SEM; n = 13). After cells were pretreated with medium alone, 1 mM l-NAME, or 15 μM PP2 for 30 min at 37°C, 5 μM A23187 was added and FRET was measured (*p < 0.05 vs. WT-HEK cells; **p < 0.01 all times beyond 2 min after A23187 addition vs. 0 min).

Article Snippet: To create the vector eNOS with CFP tagged at the C-terminus, full-length H. sapiens eNOS cDNA (with GenBank Accession number NM_000603 from OriGene Technologies, Rockville, MD) was used as DNA template in a PCR to generate eNOS PCR fragment that contained AgeI at both 5′ and 3′ ends.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Expressing, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

doi: 10.1091/mbc.e11-09-0811

Figure Lengend Snippet: FIGURE 6: Proposed mechanism of negative feedback regulation of eNOS by phospho–Cav-1. Ca2+-dependent stimulation of endothelial cells induces eNOS activation by phosphorylation on Ser-1177, and NO is released within 1 min. NO induces Src activation and phosphorylation of Cav-1 on Tyr-14, which we propose leads to unmasking of the caveolin scaffolding domain. Phosphorylated Cav-1 then binds to activated eNOS, inhibiting eNOS activity, and release of NO, thus returning eNOS to its basal state. eNOS/Cav-1 interaction can be blocked by eNOS inhibitor l-NAME and Src inhibitor PP2, suggesting that an NO/Src-dependent feedback mechanism mediates eNOS inactivation.

Article Snippet: To create the vector eNOS with CFP tagged at the C-terminus, full-length H. sapiens eNOS cDNA (with GenBank Accession number NM_000603 from OriGene Technologies, Rockville, MD) was used as DNA template in a PCR to generate eNOS PCR fragment that contained AgeI at both 5′ and 3′ ends.

Techniques: Activation Assay, Phospho-proteomics, Scaffolding, Activity Assay

Journal: Human Cell

Article Title: Single-cell dynamic RNA and glycosylation sequencing reveals the mechanism underlying the differentiation of pluripotent stem cells into hematopoietic stem cells

doi: 10.1007/s13577-025-01234-7

Figure Lengend Snippet: The analysis for the heterogeneity of endothelial clusters. a . The gene expression of endothelial, angiogenesis, and hematopoiesis in new RNA. b . The hotspot analyzed the similarity of cells and obtained 12 modules. c–d . The vascular endothelial cell migration pathway was selected from modules 3 and 8 and mapped to UMAP plots. e . The GO analysis of differential genes in high-glucose and low-glucose cells. f . The endothelial and hematopoietic genes in high-glucose and low-glucose cells in clusters 1 and 15

Article Snippet: From day 2 to day 6, 50 ng/mL human vascular endothelial growth factor (VEGF, MCE Catalog #HY- P70458 ), 50 ng/mL human basic fibroblast growth factor (bFGF, MCE Catalog #HY-P7331), 5 ng/mL BMP4, and 10 μM SB-431542 (MCE Catalog #HY-10431) were added to the BDM.

Techniques: Gene Expression, Migration

Journal: Human Cell

Article Title: Single-cell dynamic RNA and glycosylation sequencing reveals the mechanism underlying the differentiation of pluripotent stem cells into hematopoietic stem cells

doi: 10.1007/s13577-025-01234-7

Figure Lengend Snippet: Potential linkages of endothelial subsets in their differentiation fate. a . Clusters 1 and 15 were reclustered to seven new clusters, which were identified by differentially expressed genes, including NEFH, CLDN5, TOP2 A, RUNX1, MCM3, ANGPT2, and IGFBP3. b . The expression of endothelial and hematopoietic-related genes in endothelial subsets. c–d . The NTR and the levels of glycation in endothelial subsets. e . The expression of transcription factors in the endothelial subsets. f . The correlation between clusters 1, 15, and the endothelial subsets using the Spearman correlation analysis

Article Snippet: From day 2 to day 6, 50 ng/mL human vascular endothelial growth factor (VEGF, MCE Catalog #HY- P70458 ), 50 ng/mL human basic fibroblast growth factor (bFGF, MCE Catalog #HY-P7331), 5 ng/mL BMP4, and 10 μM SB-431542 (MCE Catalog #HY-10431) were added to the BDM.

Techniques: Expressing

Journal: International journal of molecular medicine

Article Title: Naringenin modulates the NO‑cGMP‑PKG signaling pathway by binding to AKT to enhance osteogenic differentiation in hPDLSCs.

doi: 10.3892/ijmm.2024.5391

Figure Lengend Snippet: Figure 3. Ability of NAR to promote osteogenesis in human periodontal ligament stem cells is mitigated after L‑NAME (eNOS inhibitor) treatment. (A) Alkaline phosphatase staining in different treatment groups. (B) Alizarin red staining in different treatment groups. (C) NO levels in different treatment groups. (D) Gene expression levels of RUNX2, OPN and sGC in different treatment groups. (E‑G) Protein expression of RUNX2, OPN, sGC and p‑eNOS in various treatment groups. *P<0.05, **P<0.01, ***P<0.001. NAR, naringenin; eNOS, endothelial nitric oxide synthase; RUNX2, Runt‑related transcription factor; OPN, osteopontin; sGC, soluble guanylate cyclase; NO, nitric oxide; p‑, phosphorylated; t‑, total; L‑NAME, NG‑nitro‑L‑arginine methyl ester.

Article Snippet: Cells treated with NAR (10 μmol/l) were also treated with NG‐nitro‐L‐arginine methyl ester (L‐NAME; 70 μM) to inhibit endothelial nitric oxide synthase (eNOS), 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 20 μM) to inhibit soluble guanylate cyclase (sGC) and AKT inhibitor VIII (58 nM) to inhibit AKT, as recommended by the manufacturer (MedChemExpress for all).

Techniques: Staining, Gene Expression, Expressing

Journal: International journal of molecular medicine

Article Title: Naringenin modulates the NO‑cGMP‑PKG signaling pathway by binding to AKT to enhance osteogenic differentiation in hPDLSCs.

doi: 10.3892/ijmm.2024.5391

Figure Lengend Snippet: Figure 6. Impact of AKT inhibitor VIII on NAR‑mediated osteogenic promotion in human periodontal ligament stem cells. (A) Alkaline phosphatase staining in different treatment groups. (B) Alizarin red staining in different treatment groups. (C) Gene expression of RUNX2 and OPN in different treatment groups. (D‑F) Protein levels of RUNX2, OPN, p‑AKT and p‑eNOS in different treatment groups. *P<0.05, **P<0.01, ***P<0.001. NAR, naringenin; RUNX2, Runt‑related transcription factor; OPN, osteopontin; eNOS, endothelial nitric oxide synthase; p‑, phosphorylated; t‑, total.

Article Snippet: Cells treated with NAR (10 μmol/l) were also treated with NG‐nitro‐L‐arginine methyl ester (L‐NAME; 70 μM) to inhibit endothelial nitric oxide synthase (eNOS), 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 20 μM) to inhibit soluble guanylate cyclase (sGC) and AKT inhibitor VIII (58 nM) to inhibit AKT, as recommended by the manufacturer (MedChemExpress for all).

Techniques: Staining, Gene Expression

Journal: International journal of molecular medicine

Article Title: Naringenin modulates the NO‑cGMP‑PKG signaling pathway by binding to AKT to enhance osteogenic differentiation in hPDLSCs.

doi: 10.3892/ijmm.2024.5391

Figure Lengend Snippet: Figure 8. Schematic diagram of the mechanism by which naringenin promotes the osteogenic differentiation of human periodontal ligament stem cells. eNOS, endothelial nitric oxide synthase; p, phosphorylated; NO, nitric oxide; sGC, soluble guanylate cyclase; cGMP, cyclic guanosine monophosphate; TRPC6, transient receptor potential cation channel, subfamily C, member 6; PKG, protein kinase G.

Article Snippet: Cells treated with NAR (10 μmol/l) were also treated with NG‐nitro‐L‐arginine methyl ester (L‐NAME; 70 μM) to inhibit endothelial nitric oxide synthase (eNOS), 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 20 μM) to inhibit soluble guanylate cyclase (sGC) and AKT inhibitor VIII (58 nM) to inhibit AKT, as recommended by the manufacturer (MedChemExpress for all).

Techniques:

Journal: Current Research in Physiology

Article Title: The comparison of endothelial function of moderate intensity interval exercise with continuous exercise in healthy men

doi: 10.1016/j.crphys.2022.07.003

Figure Lengend Snippet: Comparison of vascular markers between intermittent and continuous exercise.

Article Snippet: Then, the enzyme-linked immunosorbent assay (ELISA) method was used for determination of serum concentrations of N-terminal proANP (NTproANP), N-terminal proBNP (NTproBNP), N-terminal proCNP (NTproCNP), endothelial nitric oxide synthase activity, endothelin 1 (ET-1), adiponectin, and leptin (Elabscience Cat. No: E-EL-H1848, Cat. No: E-EL-H0902, Cat. No: E-EL-H2538, Cat. No: E-EL-H0755, Cat. No: E-EL-H0064, Cat. No: E-EL-H0004, Cat. No: E-EL-H0113 respectively).

Techniques: Comparison, Activity Assay

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: Antioxidants

Article Title: 2-(4-Methylthiazol-5-yl) Ethyl Nitrate Hydrochloride Ameliorates Cognitive Impairment via Modulation of Oxidative Stress and Nuclear Factor Kappa B (NF-κB) Signaling Pathway in Chronic Cerebral Hypoperfusion-Associated Spontaneously Hypertensive Rats

doi: 10.3390/antiox13050585

Figure Lengend Snippet: The effects of W1302 on NO production and activities of eNOS or iNOS in SHR-2VO rats. SHR-2VO rats: permanent occlusion of bilateral common carotid arteries in spontaneously hypertensive rats; WKY rats: age-matched Wistar Kyoto rats as non-hypertensive sham-operated control. ( A , B ) The nitric oxide assay kit for detecting the production of NO ( n = 8). ( C ) EC 50 value of cGMP production in SKN cells after 4 h of incubation with W1302 ( n = 4). ( D – G ) eNOS and iNOS were analyzed by Elisa kits ( n = 8). Data were expressed as mean ± S.E.M. # p < 0.05 vs. WKY rats and * p < 0.05, ** p < 0.01, vs. SHR-2VO rats.

Article Snippet: The commercial ELISA kits were purchased from Cozmo-lab Corp. (Shanghai, China) and used for the determination of cyclic guanosine monophosphate (cGMP, Abcam, ab234585), tumor necrosis factor (TNF-α, RayBio ® , Peachtree Corners, GA, USA, Lot#0104190709), interleukin-1 beta (IL-1β, RayBio ® , Lot#0118190721), endothelial nitric oxide synthase (eNOS, CUSABIO ® , Wuhan, China, CSB-E08323r), and inducible nitric oxide synthase (iNOS, CUSABIO ® , Wuhan, China, CSB-E08325r).

Techniques: Control, Nitric Oxide Assay, Incubation, Enzyme-linked Immunosorbent Assay