cgp3466b  (MedChemExpress)

Journal: Frontiers in Neurology

Article Title: Therapeutic potential of blocking GAPDH nitrosylation with CGP3466b in experimental autoimmune encephalomyelitis

doi: 10.3389/fneur.2022.979659

Figure Lengend Snippet: CGP3466b prevents GAPDH nitrosylation within the CNS in the MOG 35 − 55 EAE mouse model of MS. (A) Nitrosylated GAPDH (SNO-GAPDH) was assayed by biotin switch from spinal cord lysates in control (CFA alone) mice at day 15 and at onset (post-immunization day 10), peak (post-immunization day 15), and chronic (post-immunization day 28) stages of EAE. Total GAPDH in tissue lysate was included as loading control. (Left) Representative immunoblot. ( Right ) Quantification of SNO-GAPDH at peak EAE. The ratio of SNO-GAPDH to total GAPDH band intensity was used for quantification, and results were normalized to CFA alone control. Data represent mean ± SEM of three mice per group. (B) CGP3466b was administered at indicated doses by daily i.p. injection beginning on post-immunization day 0, and nitrosylated GAPDH and β-tubulin were assayed by biotin switch on post-immunization daiy 15. ( Left ) Representative immunoblot. “No asc” = no ascorbate control. ( Right ) Quantification of SNO-GAPDH levels following treatment with vehicle or CGP3466b 4 mg/kg. SNO-GAPDH levels were quantified as described in (A) . Data represent mean ± SEM of three mice per group. ** p < 0.01 by student's t -test (A) or one-way ANOVA with Tukey's multiple comparisons test (B) . Full blots are shown in .

Article Snippet: CGP3466b maleate (Tocris, cat # 2966) stock solution (50 mM in DMSO) was diluted to 4% DMSO in PBS as a working solution (final CGP3466b concentration 20–800 μg/ml depending on dosage to be delivered).

Techniques: Control, Western Blot, Injection

Journal: Frontiers in Neurology

Article Title: Therapeutic potential of blocking GAPDH nitrosylation with CGP3466b in experimental autoimmune encephalomyelitis

doi: 10.3389/fneur.2022.979659

Figure Lengend Snippet: Blocking SNO-GAPDH with CGP3466b is neuroprotective in MOG 35 − 55 EAE. (A, B) CGP3466b 4 mg/kg was administered by daily i.p. injection beginning on post-immunization day 0 (prophylactic paradigm). Clinical scoring was performed by a blinded observer. Prophylactic treatment with CGP3466b attenuated neurologic deficits (A) without impacting weight loss (B) . Data represent n = 17 (vehicle) and n = 10 (CGP3466b) mice per group. (C) CGP3466b 4 mg/kg was administered by daily i.p. injection beginning on post-immunization day 10 (therapeutic paradigm), and clinical scoring was performed by a blinded observer. Data represent n = 20 (vehicle) and n = 26 (CGP3466b) mice per group. (D) To quantify axonal injury, mice were treated with vehicle or CGP3466b 4 mg/kg by daily i.p. injection beginning on post-immunization day 0, and SMI-32+ axonal spheroids were examined via immunofluorescence staining of proximal optic nerve at post-immunization day 28. A representative image is shown (left), along with quantification performed from n = 3 mice per group (right). (E) Mice were treated with vehicle or CGP3466b 4 mg/kg by daily i.p. injection beginning on post-immunization day 10, and SMI-32+ axonal spheroids were examined via immunofluorescence staining of proximal optic nerve at post-immunization day 28. A representative image is shown ( left ), along with quantification performed from n = 3 mice per group ( right ). * p < 0.05, ** p < 0.01 by Mann-Whitney U-test (A, C) or student's t -test (D, E) . Data shown as mean ± SEM.

Article Snippet: CGP3466b maleate (Tocris, cat # 2966) stock solution (50 mM in DMSO) was diluted to 4% DMSO in PBS as a working solution (final CGP3466b concentration 20–800 μg/ml depending on dosage to be delivered).

Techniques: Blocking Assay, Injection, Immunofluorescence, Staining, MANN-WHITNEY

Journal: Frontiers in Neurology

Article Title: Therapeutic potential of blocking GAPDH nitrosylation with CGP3466b in experimental autoimmune encephalomyelitis

doi: 10.3389/fneur.2022.979659

Figure Lengend Snippet: CGP3466b has minimal direct effects on myeloid and lymphoid cells in vitro . (A) Bone marrow derived macrophages (BMMs) were treated with LPS (100 ng/mL) plus vehicle or the indicated concentrations of CGP3466b for 24 h. Cytokine concentrations in media were determined by ELISA. Data represent mean ± SEM of n = 3 or n = 4 independent experiments performed in triplicate. (B) BMMs and (C) Bone marrow derived dendritic cells (BMDCs) were treated with LPS (100 ng/mL) plus vehicle or the indicated concentrations of CGP3466b, and expression of activation markers was measured by flow cytometry. Data are a representative example of n = 3 independent experiments. (D) CD4+ lymphocytes were stimulated with anti-CD3/CD28 antibodies (3 ug/mL) in the presence of vehicle or the indicated concentrations of CGP3466b. Percentage of CD4+ lymphocytes expressing the given activation markers was measured by flow cytometry. Data represent mean ± SEM of three biological replicates.

Article Snippet: CGP3466b maleate (Tocris, cat # 2966) stock solution (50 mM in DMSO) was diluted to 4% DMSO in PBS as a working solution (final CGP3466b concentration 20–800 μg/ml depending on dosage to be delivered).

Techniques: In Vitro, Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Activation Assay, Flow Cytometry

Journal: Frontiers in Neurology

Article Title: Therapeutic potential of blocking GAPDH nitrosylation with CGP3466b in experimental autoimmune encephalomyelitis

doi: 10.3389/fneur.2022.979659

Figure Lengend Snippet: CGP3466b does not impact CNS immune infiltration in MOG 35 − 55 EAE. (A) CD45+ infiltrates in lumbar spinal cord at post-immunization day 18. Mice were treated with vehicle or 4 mg/kg CGP3466b daily starting on post-immunization day 0. A representative image is shown ( left ), along with quantification performed from n = 3 mice per group ( right ). (B) Iba1+ infiltrates in optic nerve at post-immunization day 28. Mice (the same cohort depicted in Figure 2D) were treated with vehicle or 4 mg/kg CGP3466b daily starting on post-immunization day 0. A representative image is shown ( left ), along with quantification performed from n = 3 mice per group ( right ). (C) Infiltrating macrophages and (D) resident microglia in the brain and spinal cord were examined via flow cytometry on post-immunization day 18 for expression of arginase-1 as a percentage of total macrophages or microglia, respectively. Data represent mean ± SEM of 3 mice per group. (E) Infiltrating macrophages and (F) resident microglia were examined via flow cytometry on post-immunization day 18 for expression of MHC II, shown as mean fluorescence intensity. Data shown from 3 mice per group. (G) Th1 cells and (H) Th17 cells were quantified via flow cytometry on post-immunization day 18 in the brain and spinal cord as a percentage of total CD4+ cells. Data represent mean ± SEM of 3 mice per group. All statistical analyses performed with student's t -test.

Article Snippet: CGP3466b maleate (Tocris, cat # 2966) stock solution (50 mM in DMSO) was diluted to 4% DMSO in PBS as a working solution (final CGP3466b concentration 20–800 μg/ml depending on dosage to be delivered).

Techniques: Flow Cytometry, Expressing, Fluorescence