rabbit polyclonal antibody against inos  (Bioss)

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Hydroxyurea inhibits proliferation and stimulates apoptosis through inducible nitric oxide synthase in erythroid cells.

doi: 10.1016/j.biopha.2024.117723

Figure Lengend Snippet: Fig. 1. Hydroxyurea induces NOS2 expression in erythroid cells. a) Immunocytochemistry for NOS2 protein in HEL92.1.7 cells treated with the indicated con centrations of hydroxyurea (HU) or vehicle (Ctrl) and quantification of NOS2-positive cells. b) Western blot analysis for NOS2 protein in HEL92.1.7 cells treated for 48 h with the indicated concentrations of HU or vehicle (Ctrl). Quantification of band intensity is shown, with β-actin used as a loading control and normalized to Ctrl. c) Western blot for phospho-NFκB p65 (Ser536) and total NFκB p65. d) Western blot for phospho-p38 and total p38. e) Western blot for phospho-ERK1/2 and total ERK1/2, with β-actin, on HEL92.1.7 cells treated with 100 μM HU for 5, 15, or 30 min, or vehicle (Ctrl). Quantification of band intensity is expressed as the phospho-to-total protein ratio and normalized to vehicle-treated cells. f) Western blot for NOS2 protein in HEL92.1.7 cells treated with the indicated concentrations of HU and/or 10 μM of JSH23 for 48 h. Quantification of band intensity is shown with β-actin used as a loading control and normalized to Ctrl. n = 3; mean + SEM, *p < 0.05, **p < 0.01, ***p < 0.001 vs. Ctrl or as indicated.

Article Snippet: Membranes were incubated overnight at 4◦C with antibodies against phospho-NOS2 (Tyr151) (TA325739, OriGene, Rockville, Maryland, USA), NOS2 (E-AB-64331, Elabscience, Houston, Texas, USA), phosphorylated NFκB p65 (p-NFκB p65) (Ser536) (3031, Cell Signaling, Danvers, Massachusetts, USA), total NFκB p65 (E-AB-22066, Elabscience), phospho-p38 (Tyr180/Tyr182) (E-AB-21027, Elabscience), total p38 (E-AB-66279, Elabscience), phospho-p44/42 (extracellular signal-regulated kinase 1⁄2 [Erk1/2]) (Thr202/Tyr204) (9101, Cell Signaling), total p44/42 (Erk1/2) (9102, Cell Signaling), or β-actin (MAB8928, R&D Systems, Minneapolis, Minnesota, USA).

Techniques: Expressing, Immunocytochemistry, Western Blot, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Hydroxyurea inhibits proliferation and stimulates apoptosis through inducible nitric oxide synthase in erythroid cells.

doi: 10.1016/j.biopha.2024.117723

Figure Lengend Snippet: Fig. 2. Hydroxyurea directly activates NOS2 in vitro. (a) Nitrite concentration in HEL92.1.7 cells treated with vehicle (Ctrl), HU, L-NAME, 1400W, or a combination of HU and L-NAME or 1400W. (b) Citrulline concentration in HEL92.1.7 cells treated with vehicle (Ctrl), HU, L-NAME, or a combination of HU and L-NAME. c) Western blot for phospho-NOS2 (Tyr151) and total NOS2 protein in HEL92.1.7 cells treated with the indicated concentrations of HU or vehicle (Ctrl). Quantification of band intensity is expressed as the phospho-NOS2 to total NOS2 ratio and normalized to vehicle-treated cells. d) Nitrite concentration in in vitro NOS enzymatic assays with the indicated concentrations of HU and incubation times. e) Citrulline concentration in in vitro NOS enzymatic assays with the indicated concentrations of HU and incubation times. f) In silico model of HU and NOS2 interaction showing binding at amino acids ASP382, ASP385, and ARG388, and with the substrate L- arginine (ARG700). g) Molecular dynamics analysis showing root mean square deviation (RMSD) of the C–Cα–N backbone versus simulation time for NOS2 in complex with and without HU during 20 ns. h) Root mean square fluctuation (RMSF) values of the NOS2-HU complex plotted against residue numbers. j) Radius of gyration (Rg) plots of NOS2 receptor with and without HU in active sites during 20 ns. a-e) n = 3; mean + SEM, *p < 0.05, **p < 0.01, ***p < 0.001 vs. Ctrl or 0.

Article Snippet: Membranes were incubated overnight at 4◦C with antibodies against phospho-NOS2 (Tyr151) (TA325739, OriGene, Rockville, Maryland, USA), NOS2 (E-AB-64331, Elabscience, Houston, Texas, USA), phosphorylated NFκB p65 (p-NFκB p65) (Ser536) (3031, Cell Signaling, Danvers, Massachusetts, USA), total NFκB p65 (E-AB-22066, Elabscience), phospho-p38 (Tyr180/Tyr182) (E-AB-21027, Elabscience), total p38 (E-AB-66279, Elabscience), phospho-p44/42 (extracellular signal-regulated kinase 1⁄2 [Erk1/2]) (Thr202/Tyr204) (9101, Cell Signaling), total p44/42 (Erk1/2) (9102, Cell Signaling), or β-actin (MAB8928, R&D Systems, Minneapolis, Minnesota, USA).

Techniques: In Vitro, Concentration Assay, Western Blot, Incubation, In Silico, Binding Assay, Residue

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Hydroxyurea inhibits proliferation and stimulates apoptosis through inducible nitric oxide synthase in erythroid cells.

doi: 10.1016/j.biopha.2024.117723

Figure Lengend Snippet: Fig. 3. NOS2 inhibition or knockdown prevents HU-induced inhibition of HEL92.1.7 cell proliferation. HEL92.1.7 cells were treated with the indicated concen trations of NOS2-specific inhibitor 1400W alone or in combination with HU. a) Immunocytochemistry for Ki67 protein and quantification of Ki67-positive cells as a proliferation marker. b) Cell cycle analysis by flow cytometry. Debris and doublets were excluded, and the distribution in the phases of the cell cycle was determined based on the incorporation of PI. c) Percentage of cells in G0/G1, S, or G2/M phases of the cell cycle was quantified. d) Gating of NOS2kd HEL92.1.7 cells based on GFP expression. e) Quantification of NOS1-, NOS2-, or NOS3-positive cells in NOS2kd and control HEL92.1.7 cells after immunocytochemistry. f) Immunocyto chemistry for Ki67 protein in NOS2kd or control HEL92.1.7 cells treated or not with HU, and quantification of Ki67-positive cells. g) Example of gating for cell cycle distribution using PI by flow cytometry. Exclusion of debris, exclusion of doublets, distribution in G0/G1, S, or G2/M phases based on PI incorporation. h) Percentage of cells in G0/G1, S, or G2/M phases of the cell cycle in NOS2kd cells and controls treated or not with HU. n = 5; mean + SEM, *p < 0.05, **p < 0.01, ***p < 0.001 vs. Ctrl or as indicated.

Article Snippet: Membranes were incubated overnight at 4◦C with antibodies against phospho-NOS2 (Tyr151) (TA325739, OriGene, Rockville, Maryland, USA), NOS2 (E-AB-64331, Elabscience, Houston, Texas, USA), phosphorylated NFκB p65 (p-NFκB p65) (Ser536) (3031, Cell Signaling, Danvers, Massachusetts, USA), total NFκB p65 (E-AB-22066, Elabscience), phospho-p38 (Tyr180/Tyr182) (E-AB-21027, Elabscience), total p38 (E-AB-66279, Elabscience), phospho-p44/42 (extracellular signal-regulated kinase 1⁄2 [Erk1/2]) (Thr202/Tyr204) (9101, Cell Signaling), total p44/42 (Erk1/2) (9102, Cell Signaling), or β-actin (MAB8928, R&D Systems, Minneapolis, Minnesota, USA).

Techniques: Inhibition, Knockdown, Immunocytochemistry, Marker, Cell Cycle Assay, Flow Cytometry, Expressing, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Hydroxyurea inhibits proliferation and stimulates apoptosis through inducible nitric oxide synthase in erythroid cells.

doi: 10.1016/j.biopha.2024.117723

Figure Lengend Snippet: Fig. 4. NOS2 inhibition or knockdown prevents HU-induced apoptosis in HEL92.1.7 cells. HEL92.1.7 cells were treated with the indicated concentrations of NOS2- specific inhibitor 1400W alone or in combination with HU. a) Immunocytochemistry for ssDNA and quantification of ssDNA-positive cells. b) Example of gating for the apoptotic assay by flow cytometry. The bottom left quadrant represents early apoptotic cells, and the upper left quadrant represents late apoptotic cells. c) Percentage of early apoptotic cells. d) Percentage of late apoptotic cells. e) Immunocytochemistry for ssDNA on NOS2kd or control HEL92.1.7 cells treated or not with HU and quantification of ssDNA-positive cells. f) Example of gating for the apoptotic assay by flow cytometry with numbers of early and late apoptotic cells. g) Percentage of early and late apoptotic cells. n = 5; mean + SEM, *p < 0.05, **p < 0.01, ***p < 0.001 vs. Ctrl or as indicated.

Article Snippet: Membranes were incubated overnight at 4◦C with antibodies against phospho-NOS2 (Tyr151) (TA325739, OriGene, Rockville, Maryland, USA), NOS2 (E-AB-64331, Elabscience, Houston, Texas, USA), phosphorylated NFκB p65 (p-NFκB p65) (Ser536) (3031, Cell Signaling, Danvers, Massachusetts, USA), total NFκB p65 (E-AB-22066, Elabscience), phospho-p38 (Tyr180/Tyr182) (E-AB-21027, Elabscience), total p38 (E-AB-66279, Elabscience), phospho-p44/42 (extracellular signal-regulated kinase 1⁄2 [Erk1/2]) (Thr202/Tyr204) (9101, Cell Signaling), total p44/42 (Erk1/2) (9102, Cell Signaling), or β-actin (MAB8928, R&D Systems, Minneapolis, Minnesota, USA).

Techniques: Inhibition, Knockdown, Immunocytochemistry, Flow Cytometry, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Hydroxyurea inhibits proliferation and stimulates apoptosis through inducible nitric oxide synthase in erythroid cells.

doi: 10.1016/j.biopha.2024.117723

Figure Lengend Snippet: Fig. 5. In vivo HU treatment of Nos2–/– mice impairs HU inhibition of proliferation in erythroid progenitors. a) Schematic representation of experimental setup: Nos2–/– or wild-type (WT) mice were treated orally with 200 mg/kg HU or drinking water for 2 weeks. WT mice were injected with 20 mg/kg of 1400W twice daily for 3 consecutive days. Mouse erythroid progenitors (mERP) were isolated from bone marrow by immunomagnetic cell separation using anti-CD71-PE and anti- Ter119-FITC antibodies. b) Immunocytochemistry for Nos2 protein in mERP isolated from WT mice treated or not with HU. Quantification of Nos2-positive cells. c) Citrulline concentration in the bone marrow of WT and Nos2–/– mice treated or not with HU. d) Colony formation assay showing the number of late erythroid (CFU-E), early erythroid (BFU-E), or granulocyte/macrophage progenitors (CFU-GM) in the bone marrow of WT or Nos2–/– mice treated or not with HU. e) Immunocytochemistry for Ki67 in mERP cells isolated from WT and Nos2–/– mice treated or not with HU. f) Quantification of Ki67-positive cells. g) Cell cycle distribution by flow cytometry showing the percentage of cells in G0/G1, S, or G2/M phases of the cell cycle. c) n = 3, f) n = 5; mean + SEM, *p < 0.05, **p < 0.01, ***p < 0.001 vs. WT.

Article Snippet: Membranes were incubated overnight at 4◦C with antibodies against phospho-NOS2 (Tyr151) (TA325739, OriGene, Rockville, Maryland, USA), NOS2 (E-AB-64331, Elabscience, Houston, Texas, USA), phosphorylated NFκB p65 (p-NFκB p65) (Ser536) (3031, Cell Signaling, Danvers, Massachusetts, USA), total NFκB p65 (E-AB-22066, Elabscience), phospho-p38 (Tyr180/Tyr182) (E-AB-21027, Elabscience), total p38 (E-AB-66279, Elabscience), phospho-p44/42 (extracellular signal-regulated kinase 1⁄2 [Erk1/2]) (Thr202/Tyr204) (9101, Cell Signaling), total p44/42 (Erk1/2) (9102, Cell Signaling), or β-actin (MAB8928, R&D Systems, Minneapolis, Minnesota, USA).

Techniques: In Vivo, Inhibition, Injection, Isolation, Immunocytochemistry, Concentration Assay, Colony Assay, Flow Cytometry

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Hydroxyurea inhibits proliferation and stimulates apoptosis through inducible nitric oxide synthase in erythroid cells.

doi: 10.1016/j.biopha.2024.117723

Figure Lengend Snippet: Fig. 6. In vivo HU treatment of Nos2–/– mice impairs HU stimulation of apoptosis in erythroid progenitors. a) Immunocytochemistry for Cas3 in mouse erythroid progenitors (mERP) after treatment of WT or Nos2–/– mice with 1400W and/or HU. b) Quantification of Cas3-positive mERP. c) Example of gating for Annexin V/PI apoptotic assay by flow cytometry with numbers of early and late apoptotic mERP cells. d) Percentage of early apoptotic mERP cells. e) Percentage of late apoptotic mERP cells. f) Percentage of total apoptotic mERP cells. n = 5; mean + SEM, *p < 0.05, **p < 0.01, ***p < 0.001 vs. WT.

Article Snippet: Membranes were incubated overnight at 4◦C with antibodies against phospho-NOS2 (Tyr151) (TA325739, OriGene, Rockville, Maryland, USA), NOS2 (E-AB-64331, Elabscience, Houston, Texas, USA), phosphorylated NFκB p65 (p-NFκB p65) (Ser536) (3031, Cell Signaling, Danvers, Massachusetts, USA), total NFκB p65 (E-AB-22066, Elabscience), phospho-p38 (Tyr180/Tyr182) (E-AB-21027, Elabscience), total p38 (E-AB-66279, Elabscience), phospho-p44/42 (extracellular signal-regulated kinase 1⁄2 [Erk1/2]) (Thr202/Tyr204) (9101, Cell Signaling), total p44/42 (Erk1/2) (9102, Cell Signaling), or β-actin (MAB8928, R&D Systems, Minneapolis, Minnesota, USA).

Techniques: In Vivo, Immunocytochemistry, Flow Cytometry