nos2 inhibitor 1400w  (TargetMol)

Journal: bioRxiv

Article Title: Reprogramming of breast tumor-associated macrophages with modulation of arginine metabolism

doi: 10.1101/2023.08.22.554238

Figure Lengend Snippet: A ) Levels of NO (left), PAs (middle) and NO/PA ratios for THP–1 derived M0, M1 and M2- TAMs after treated with DMSO (vehicle) and SEP (100 μM) for 3 days (n = 5). B) One way ANOVA with post hoc Tukey test was used for statistical analysis. Error bars: ±SEM. GraphPad Prism Version 9.5.1. was used to perform all statistical analyses. B ) Levels of NO (left), PAs (middle) and NO/PA ratios for THP–1 derived Mn, M0, M1 and M2-TAMs after treated with DMSO (vehicle), NOS2 inhibitor, 1400W (50 μM), or Arginase 1 (Ang1) inhibitor, nor NOHA (50 μM) for 3 days (n = 5). Note the significant decrease of NO level in 1400W-treated M1-TAMs and significant decrease of PA level in nor-NOHA-treated M2-TAMs. C ) Immune fluorescence imaging of THP–1 derived M0, M1 and M2-TAMs stained for M1 marker (green, TNFα) vs. M2 marker (red, CD206) and counterstained with DAPI (blue). M1-TAMs were treated with DMSO (Control: Ctrl) or NOS2 inhibitor (100μM 1400W), whereas M2-TAMs were treated with DMSO (Ctrl) or ARG1 inhibitor (50μM nor-NOHA) for 3 days (n=3). D ) Levels of Type 1 cytokine IL12 (left) and Type 2 cytokine IL10 (middle) as well as IL12/IL10 ratios for THP-1 derived TAM subsets measured with ELISA. M1-TAMs were treated with DMSO or NOS inhibitors, 1400W (50 μM) and LNAME (2.5 mM). M2-TAMs were treated with DMSO, SEP (100 μM) or positive control LPS (5ng/ml) plus IFNγ (20ng/ml) for 3 days (n=6). The cytokine levels were measured using ELISA and normalized against the total protein levels. Error bars: ±SEM. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001 and ns, p >0.05. E ) Working scheme for the induction of M1 vs. M2 polarization by activation of NOS2 vs. ARG1/OCD1 pathways and M2-to-M1 reprogramming by SEP.

Article Snippet: For NO inhibition, we used Nω-Nitro-L-arginine methyl ester hydrochloride (L–NAME, 2.5 mM, Sigma, Cat. No. N5751-10G); the NOS2 inhibitor: 1400W hydrochloride (100μM, Cayman Chemical, Ann Arbor, MI, Cat. No. 81520); or the NO scavenger: 4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, 50 or 100 μM, Enzo, Cat. No. ALX-430-001-M050).

Techniques: Derivative Assay, Fluorescence, Imaging, Staining, Marker, Enzyme-linked Immunosorbent Assay, Positive Control, Activation Assay

Journal: Cancer Immunology Research

Article Title: Infiltration of Tumors Is Regulated by T cell–Intrinsic Nitric Oxide Synthesis

doi: 10.1158/2326-6066.CIR-22-0387

Figure Lengend Snippet: NO production and NOS expression in CD8 + T cells. A, CD8 + T cells were activated with anti-CD3/CD28 Dynabeads (or SIINFEKL peptide, when using OT-I CD8 + T cells) for 1 to 4 days in 21%, 5%, and 1% O 2 . After activation, NO production and NOS expression were analyzed. B, NO production determined by extracellular quantification of nitrites (NO 2 − , a NO byproduct) in mouse CD8 + T cells activated for 3 days ( N = 11–13). C, Western blot analysis of NOS using a panNOS antibody (all isoforms detected) in lysates of mouse CD8 + T cell activated for 3 days. Results normalized to total protein stain (TPS, top) and representative blot (bottom; N = 3). D, Time course qRT-PCR analysis of Nos2 and Nos3 mRNA expression in activated mouse CD8 + T cells ( N = 3–9). E, Western blot analysis of NOS2 protein levels in mouse OT-I CD8 + T cells treated or untreated with 50 μmol/L FG-4592 and activated for 3 days in 1% O 2 with increasing amounts of SIINFEKL peptide (0.001, 0.1, 1, and 1000 ng/mL). Quantification normalized to TPS (top) and representative blot (bottom). BMDMs polarized to M1 with 100 U/mL LPS were used as positive control for NOS2 expression ( N = 3). F, NO production as determined by extracellular quantification of nitrites in human T cells cultured for 1 day in 21% and 1% O 2 with or without anti-CD3/CD28 beads ( N = 4). G, Time course qRT-PCR analysis of NOS2 and NOS3 mRNA levels in activated human CD8 + T cells. NOS1 mRNA levels were under the detection limit ( N = 5–8). H, Western blot analysis of panNOS (antibody detecting all NOS isoforms) and NOS3 in human CD8+ T cells. HUVEC cells were used as positive control and PPIB was used as loading control (representative of N = 3). Apart from panel D and G , each data point represents an independent animal and results are shown as median ± interquartile range (IQR). *, P < 0.05; Wilcoxon matched-pairs signed-rank test.

Article Snippet: The NOS2 inhibitor 1400W dihydrochloride (Cayman Chemicals) was used at 100 μmol/L in mouse CD8 + T cells transduced with VC or NOS2 OE vectors following enrichment of Thy-1.1 + cells using MACS as described below.

Techniques: Expressing, Activation Assay, Western Blot, Staining, Quantitative RT-PCR, Positive Control, Cell Culture, Control

Journal: Cancer Immunology Research

Article Title: Infiltration of Tumors Is Regulated by T cell–Intrinsic Nitric Oxide Synthesis

doi: 10.1158/2326-6066.CIR-22-0387

Figure Lengend Snippet: In vitro characterization of Nos2 KO CD8 + T cells. A, After activation in 1% O 2 with anti-CD3/CD28 beads, NOS2 expression was determined by Western blot in Nos2 fl/fl (WT, gray) and Nos2 fl/fl dLck Cre (NOS2 KO , orange) cells. Representative blot (top) and quantification normalized to total protein stain (bottom; N = 2). B, WT and NOS2 KO CD8 + T cells were activated for 3 days in 21% or 1% O 2 , and NO production was determined by the extracellular nitrite concentration ( N = 5–8). C, WT and NOS2 KO mouse CD8 + T cells were activated for 72 hours in 21%, 5%, or 1% O 2 . Viable CD8 + T-cell number was determined by flow cytometry using count beads (left); cell proliferation assessed with CTV staining and expressed as division index (right; N = 8–18). D, Proportion of CD62L − CD44 + in cells activated as in C (left) and representative FACS plots for 1% O 2 activated cells (right; N = 11–18). E, Heat map illustrating expression of markers of differentiation determined by flow cytometry in CD8 + T cells activated for 72 hours in 21%, 5%, or 1% O 2 . Increased and reduced expression of proteins are shown in gray and orange, respectively. Rows represent averaged z-scores ( N = 11–18). F, Seahorse metabolic analysis of mouse T cells activated for 3 days in 1% O 2 , as determined by OCR and ECAR after injection of anti-CD3/CD28 beads or antibodies, oligomycin (O), FCCP (F), or rotenone+antimycin A (R+A; left). Effect of T-cell activation on T-cell OCR and ECAR was determined by % change from baseline following injection of anti-CD3/CD28 beads or antibodies. Seahorse analysis was conducted in a hypoxia chamber set to 3%O 2 ( N = 8). G, OT-I CD8 + T cells activated for 3 days in 1% O 2 were cocultured with 10,000 OVA-expressing B16-F10 tumor cells at different effector:target (E:T) ratios. Cytotoxicity was assessed with Alamar blue assay after 14 to 18 hours of coculture at 21% O 2 . A nonlinear regression [(agonist) vs. normalized response] was used to determine dose–response curves (shaded areas: 95% confidence intervals; N = 4–6). H, CD8 + T cells were activated for 6 days and incubated for 48 hours in 1% O 2 before being loaded with calcein-AM and cocultured with mouse endothelial cells in a transwell system. mCCL19 and mCCL21 were added to the lower chamber as chemoattractant. Calcein signal corresponding to T cells migrating through the endothelial barrier was assessed after 3 hours of coculture in a plate reader ( N = 7–9). All results [median ± interquartile range (IQR)] are pooled from a minimum of two independent experiments, and each data point from panels B , F (right), and G and H represent an independent animal. ns, P > 0.05; *, P < 0.05; **, P < 0.01; Mann–Whitney test relative to respective WT control.

Article Snippet: The NOS2 inhibitor 1400W dihydrochloride (Cayman Chemicals) was used at 100 μmol/L in mouse CD8 + T cells transduced with VC or NOS2 OE vectors following enrichment of Thy-1.1 + cells using MACS as described below.

Techniques: In Vitro, Activation Assay, Expressing, Western Blot, Staining, Concentration Assay, Flow Cytometry, Injection, Alamar Blue Assay, Incubation, MANN-WHITNEY, Control

Journal: Cancer Immunology Research

Article Title: Infiltration of Tumors Is Regulated by T cell–Intrinsic Nitric Oxide Synthesis

doi: 10.1158/2326-6066.CIR-22-0387

Figure Lengend Snippet: Tumor growth in animals lacking Nos2 expression in T cells. A, Tumor growth model. 5×10 5 MC38 or B16-F10-OVA were subcutaneously injected in Nos2 fl/fl (WT) Nos2 fl/fl dlck Cre (NOS2 KO ) animals. On day 10 after tumor inoculation, peripheral blood and tumors were processed to single-cell suspensions and analyzed by flow cytometry. Tumor growth was monitored until day 30. B, MC38 (top) and B16-F10-OVA (bottom) tumor growth data. Tumor growth curves in WT and NOS2 KO animals; thin lines represent individual animals and thick line represents an exponential (Malthusian) growth curve (left). Survival curves using 500 mm 3 as threshold (right). Results pooled from two independent experiments ( N = 18–26 animals per group). C, Immune composition was analyzed by flow cytometry on peripheral blood of animals bearing MC38 (top; N = 6–8) and B16-F10-OVA (bottom; N = 13–18) tumors for 10 days. Results expressed as cells per milliliter of blood; median ± interquartile range (IQR). D, Representative flow cytometry plots from CD4 + and CD8 + T-cell infiltration in B16-F10-OVA tumors collected on day 10 following inoculation in WT and NOS2 KO animals (top). Immune cell infiltration in B16-F10-OVA analyzed by flow cytometry and expressed as counts per million CD45 + cells (bottom). Cells pregated on live, singlet, CD45 + events ( N = 9, median ± IQR). Each data point represents an individual animal. ns, P > 0.05; *, P < 0.05; **, P < 0.05; log-rank (Mantel–Cox) test relative to WT animals ( B ) and Mann–Whitney test relative to WT control ( C and D ).

Article Snippet: The NOS2 inhibitor 1400W dihydrochloride (Cayman Chemicals) was used at 100 μmol/L in mouse CD8 + T cells transduced with VC or NOS2 OE vectors following enrichment of Thy-1.1 + cells using MACS as described below.

Techniques: Expressing, Injection, Flow Cytometry, MANN-WHITNEY, Control

Journal: Cancer Immunology Research

Article Title: Infiltration of Tumors Is Regulated by T cell–Intrinsic Nitric Oxide Synthesis

doi: 10.1158/2326-6066.CIR-22-0387

Figure Lengend Snippet: Antitumor function and tissue infiltration capacity of NOS2 KO OT-I T cells. A, ACT model. C57BL/6j mice were injected subcutaneously with 1×10 6 OVA-expressing B16-F10 tumor cells, and 4 days later were lymphodepleted with 300 mg/kg CPA. Mice bearing tumors for 7 days were then intraperitoneally injected with 1×10 6 of 4 days activated WT or NOS2 KO OT-I cells. Tumor growth was monitored every 2 to 3 days until day 60. B, B16-F10-OVA tumor growth after ACT. Tumor growth curves after No ACT or ACT with VC or NOS2 KO OT-I cells; vertical dotted lines represent day of ACT, thin lines represent individual animals, and thick lines represent an exponential (Malthusian) growth curve (left). Survival curves using 500 mm 3 as threshold (right; N = 9–20 animals per group). C, Tumor infiltration model. C57BL/6j mice were injected subcutaneously with 1×10 6 OVA-expressing B16-F10 tumor cells, and 11 days later were lymphodepleted with CPA. Mice bearing tumors for 14 days were then intraperitoneally injected with Nos2 fl/fl (WT) and Nos2 fl/fl dlck Cre (NOS2 KO ) OT-I CD8 + T cells (1×10 6 each in 1:1 NOS2 KO :WT ratio). Spleen, peripheral blood, liver, and tumor were harvested on day 19 and processed to single-cell suspensions for flow cytometric analysis. Endogenous and adoptive populations were distinguished by the allelic variants of CD45. D, Total OT-I T-cell expansion in all analyzed tissues expressed as a ratio between NOS2 KO and WT cell counts (gray horizontal line represents the NOS2 KO /WT ratio at the time of injection; N = 22, median ± interquartile range [IQR]). E , Percentage of cells expressing granzyme B (GZMB) within CD8 + T cells in all tissues analyzed by flow cytometry on day 19 (bottom; N = 19–22, median ± IQR). F, Representative FACS plots (left) and flow cytometry analysis (right) of percentage of CD44 + CD8 + T cells in peripheral blood on day 19 ( N = 19–22, median ± IQR). Results are pooled from at least two independent experiments, and each data point represents an independent animal. *, P < 0.05; **, P < 0.01; ***, P < 0.001; log-rank (Mantel–Cox) test relative to WT animals ( B ), one sample t test relative to 1 ( D ) and Wilcoxon matched-pairs signed-rank test relative to WT control ( E and F ).

Article Snippet: The NOS2 inhibitor 1400W dihydrochloride (Cayman Chemicals) was used at 100 μmol/L in mouse CD8 + T cells transduced with VC or NOS2 OE vectors following enrichment of Thy-1.1 + cells using MACS as described below.

Techniques: Injection, Expressing, Flow Cytometry, Control

Journal: Cancer Immunology Research

Article Title: Infiltration of Tumors Is Regulated by T cell–Intrinsic Nitric Oxide Synthesis

doi: 10.1158/2326-6066.CIR-22-0387

Figure Lengend Snippet: In vivo activation and recall response of NOS2 KO OT-I CD8 + T cells. A, Scheme of in vivo T-cell activation and recall response model. C57BL/6j mice were injected intraperitoneally with 1×10 6 naïve Nos2 fl/fl (WT) and Nos2 fl/fl dlck Cre (NOS2 KO ) OT-I CD8 + T (1:1 NOS2 KO :WT ratio). The next day, mouse BMDMs differentiated for 7 days and polarized with LPS for 24 hours were pulsed with SIINFEKL peptide for 4 hours prior to intraperitoneal injection. Peripheral blood was sampled at days 7 and 10 after BMDM transfer and analyzed by flow cytometry. SIINFEKL-pulsed BMDMs (or PBS controls) were administered again on day 30. At day 37, the spleen, inguinal lymph nodes, and a liver portion were harvested and analyzed by flow cytometry. Endogenous and adoptive populations were distinguished by the allelic variants of CD45. B, OT-I T-cell expansion in peripheral blood expressed as the relative ratio between KO and WT cell counts on days 7 and 10 after BMDM injections (horizontal gray line represents the range of the initial NOS2 KO /WT ratio; N = 32–36). C, Percentage of CD62L-CD44+ cells (left) and CD8 and CD127 MFI (right) of WT, KO, and endogenous CD8 + T cells harvested from peripheral blood on day 7 ( N = 31). D, Recall response as determined by the ratio between WT and KO OT-I CD8 + T-cell infiltration in the spleen, lymph node, and liver 7 days after recall with SIINFEKL-pulsed BMDMs (+) or with PBS control (−). Horizontal gray line represents the range of the initial NOS2 KO /WT ratio ( N = 17–21). E, Recall response as determined by amount of CD44 + WT and NOS2 KO OT-I T cells per million CD45 + cells infiltrated in the spleen, lymph node, and liver 7 days after recall with BMDMs or PBS control ( N = 9–18). F, Flow cytometry analysis of CD44 in CD8 + T cells infiltrating the spleen, lymph node, and liver on day 37. All results [median ± interquartile range (IQR)] are pooled from three independent experiments and each data point represents an independent animal. *, P < 0.05; **, P < 0.01; ***, P < 0.001. One sample t test relative to 1 ( B and D ), Tukey multiple comparisons paired test ( C – F ). #, P < 0.05; ##, P < 0.01; ###, P < 0.001; Unpaired t test.

Article Snippet: The NOS2 inhibitor 1400W dihydrochloride (Cayman Chemicals) was used at 100 μmol/L in mouse CD8 + T cells transduced with VC or NOS2 OE vectors following enrichment of Thy-1.1 + cells using MACS as described below.

Techniques: In Vivo, Activation Assay, Injection, Flow Cytometry, Control

Journal: Journal of Clinical Investigation

Article Title: 3D model of harlequin ichthyosis reveals inflammatory therapeutic targets

doi: 10.1172/jci132987

Figure Lengend Snippet: Figure 4. Decrease in antiinflammatory response and activation of NOS2 pathway in the in vitro ABCA12 KO 3D model. (A) qPCR analysis of IL37 in ABCA12 WT and KO 3D models. n = 3. Data are represented as mean ± SD. NS, P = 0.068, unpaired t test. qPCR analysis of (B) SOCS1 and (C) SOCS3 in ABCA12 WT and KO 2D model cell lysates. Each dot represents the mean of 3 technical replicates. n = 3. Data are represented as mean ± SD. **P ≤ 0.01, unpaired t test. (D) Representative immuno- blot of p-STAT1 (Y701), total STAT1, and GAPDH proteins in untreated (–) or stimulated (+) with IFN-γ ABCA12 WT and KO cell lysates and (E) associated p-STAT1 quantita- tive analysis. n = 4. Data are represented as mean ± SD. *P ≤ 0.05, unpaired t test. The p-STAT1 blot was run in parallel, contemporaneously, with total STAT1 and GAPDH blots. (F) Representative NOS2 (green channel) and DAPI (blue channel) staining images of in vitro WT and HI 3D models, and (G) associated quantitative NOS2 analysis. Each dot represents the mean of relative NOS2 intensity from 3 independent images. Scale bars: 100 μm. n = 3. Data are represented as mean ± SD. *P ≤ 0.05, unpaired t test.

Article Snippet: Daily treatment used 1 μM of the selective and potent NOS2 inhibitor 1400W (47) (1415, Tocris), 100 nM of the JAK1/3 inhibitor tofacitinib (35) (sc-364726, Insight Biotechnology), or 50 μM of the NO-releasing agent S-Nitroso-N-acetyl-DL-penicillamine (SNAP) (51) (sc-200319, Santa Cruz Biotechnology), which was added to the 3D models for 8 days.

Techniques: Activation Assay, In Vitro, Staining

Journal: Journal of Clinical Investigation

Article Title: 3D model of harlequin ichthyosis reveals inflammatory therapeutic targets

doi: 10.1172/jci132987

Figure Lengend Snippet: Figure 5. Inflammation and activation of the STAT1/NOS2 pathway in HI skin. Representative (A) IL-37, (C) IL-36α, (E) IL-36γ, (G) STAT1, (I) p-STAT1, (J) and NOS2 (green channel) and DAPI (blue chan- nel) staining images of control skin and HI patient skin. Arrows indicate granular layer. Associated quantitative analysis of (B) IL-37, (D) IL-36α, (F) IL-36γ, (H) granular layer STAT1, and (K) NOS2 protein expression in control skin and HI patient skin. Each dot represents the mean of relative protein intensity from 3 independent images. n = 3 or 4. Data are represented as mean ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001, unpaired t test. Scale bars: 100 μm.

Article Snippet: Daily treatment used 1 μM of the selective and potent NOS2 inhibitor 1400W (47) (1415, Tocris), 100 nM of the JAK1/3 inhibitor tofacitinib (35) (sc-364726, Insight Biotechnology), or 50 μM of the NO-releasing agent S-Nitroso-N-acetyl-DL-penicillamine (SNAP) (51) (sc-200319, Santa Cruz Biotechnology), which was added to the 3D models for 8 days.

Techniques: Activation Assay, Staining, Control, Expressing

Journal: Journal of Clinical Investigation

Article Title: 3D model of harlequin ichthyosis reveals inflammatory therapeutic targets

doi: 10.1172/jci132987

Figure Lengend Snippet: Figure 6. NO release caused epidermal acanthosis and inhibition of NOS2 resulted in normalization of the skin barrier in the HI 3D model. (A) Repre- sentative H&E (bright-field channel) images of in vitro WT and HI 3D models untreated (UT) or treated with SNAP compound. (B) Quantitative analysis of intracellular NO in in vitro WT and HI 3D models, with or without 1400 W inhibitor. Each dot represents the mean of 3 technical replicates. n = 3. Data are represented as mean ± SD. **P ≤ 0.01; ***P ≤ 0.001, 2-way ANOVA with Tukey’s multiple comparisons test. Representative (C) H&E (bright-field channel), (D) Lucifer yellow (LY) (green channel), (E) polar/neutral (red/green channel), (F) GluCer (green channel), and DAPI (blue channel) staining images of in vitro WT and HI 3D models from 3 independent biological replicates. Scale bars: 100 μm.

Article Snippet: Daily treatment used 1 μM of the selective and potent NOS2 inhibitor 1400W (47) (1415, Tocris), 100 nM of the JAK1/3 inhibitor tofacitinib (35) (sc-364726, Insight Biotechnology), or 50 μM of the NO-releasing agent S-Nitroso-N-acetyl-DL-penicillamine (SNAP) (51) (sc-200319, Santa Cruz Biotechnology), which was added to the 3D models for 8 days.

Techniques: Inhibition, In Vitro, Staining