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cb2r-selective antagonist sr144528  (Tocris)


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    Tocris cb2r-selective antagonist sr144528
    Cb2r Selective Antagonist Sr144528, supplied by Tocris, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cb2r-selective antagonist sr144528  (Tocris)
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    Tocris cb2r-selective antagonist sr144528
    Cb2r Selective Antagonist Sr144528, supplied by Tocris, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    selective cb2r antagonists sr144528  (Tocris)
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    <t>CB2R-dependent</t> inhibition of FSK-stimulated cAMP and CB2R-dependent recruitment of βarrestin2. CHO cells stably expressing h CB2R were treated with 0.10 nM–10 μM compounds for 90 min, and cAMP inhibition (a,b) or βarrestin2 recruitment (c,d) was measured. cAMP and βarrestin2 recruitment data are expressed as the % CP55,940 response. Data were fitted to a nonlinear regression (three-parameter model, GraphPad v. 9.0). Data are mean ± S.E.M. of 6 independent experiments performed in triplicate. Data from these graphs is presented in Table . Statistical data for these graphs are presented in Table S1 .
    Selective Cb2r Antagonists Sr144528, supplied by Tocris, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    selective cb2r antagonist sr144528  (Santa Cruz Biotechnology)
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    Santa Cruz Biotechnology selective cb2r antagonist sr144528
    <t>CB2R-dependent</t> inhibition of FSK-stimulated cAMP and CB2R-dependent recruitment of βarrestin2. CHO cells stably expressing h CB2R were treated with 0.10 nM–10 μM compounds for 90 min, and cAMP inhibition (a,b) or βarrestin2 recruitment (c,d) was measured. cAMP and βarrestin2 recruitment data are expressed as the % CP55,940 response. Data were fitted to a nonlinear regression (three-parameter model, GraphPad v. 9.0). Data are mean ± S.E.M. of 6 independent experiments performed in triplicate. Data from these graphs is presented in Table . Statistical data for these graphs are presented in Table S1 .
    Selective Cb2r Antagonist Sr144528, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    selective cb2r antagonist sr144528  (Tocris)
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    Tocris selective cb2r antagonist sr144528
    Radioligand binding data of CB91, LV58, LV62, and LV50 a .
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    the selective cb2r antagonist sr144528  (Tocris)
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    Tocris the selective cb2r antagonist sr144528
    Radioligand binding data of CB91, LV58, LV62, and LV50 a .
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    selective cb2r antagonist sr144528  (Cayman Chemical)
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    Radioligand binding data of CB91, LV58, LV62, and LV50 a .
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    CB2R-dependent inhibition of FSK-stimulated cAMP and CB2R-dependent recruitment of βarrestin2. CHO cells stably expressing h CB2R were treated with 0.10 nM–10 μM compounds for 90 min, and cAMP inhibition (a,b) or βarrestin2 recruitment (c,d) was measured. cAMP and βarrestin2 recruitment data are expressed as the % CP55,940 response. Data were fitted to a nonlinear regression (three-parameter model, GraphPad v. 9.0). Data are mean ± S.E.M. of 6 independent experiments performed in triplicate. Data from these graphs is presented in Table . Statistical data for these graphs are presented in Table S1 .

    Journal: Journal of Medicinal Chemistry

    Article Title: Design, Synthesis, and Biological Activity of New CB2 Receptor Ligands: from Orthosteric and Allosteric Modulators to Dualsteric/Bitopic Ligands

    doi: 10.1021/acs.jmedchem.2c00582

    Figure Lengend Snippet: CB2R-dependent inhibition of FSK-stimulated cAMP and CB2R-dependent recruitment of βarrestin2. CHO cells stably expressing h CB2R were treated with 0.10 nM–10 μM compounds for 90 min, and cAMP inhibition (a,b) or βarrestin2 recruitment (c,d) was measured. cAMP and βarrestin2 recruitment data are expressed as the % CP55,940 response. Data were fitted to a nonlinear regression (three-parameter model, GraphPad v. 9.0). Data are mean ± S.E.M. of 6 independent experiments performed in triplicate. Data from these graphs is presented in Table . Statistical data for these graphs are presented in Table S1 .

    Article Snippet: The selective CB2R antagonists SR144528 (Tocris Bioscience, UK) and MC21 were dissolved in saline solution with 5% DMSO and 5% Tween 20.

    Techniques: Inhibition, Stable Transfection, Expressing

    CB2R-dependent inhibition of FSK-stimulated cAMP CHO cells stably expressing h CB2R. cAMP inhibition data are expressed as the % CP55,940 response. Cells were treated with ligands simultaneously as indicated. 10 nM FM-6b (a), 50 nM FD-22a (b), and 5 nM FD-24a (c) were chosen after the completion of preliminary experiments with compounds alone for ease of calculations to approximate the EC 50 for each compound alone. Addition of 100 nM SR144528 to 0.1 nM–10 μM of FD-22a (d) or of FD-24a (e). Data were fitted to a nonlinear regression (three-parameter model, GraphPad v. 9.0). Data are mean ± S.E.M. of 3–6 independent experiments performed in triplicate. Data from these graphs is presented in Table . Statistical data for these graphs are presented in Table S2 .

    Journal: Journal of Medicinal Chemistry

    Article Title: Design, Synthesis, and Biological Activity of New CB2 Receptor Ligands: from Orthosteric and Allosteric Modulators to Dualsteric/Bitopic Ligands

    doi: 10.1021/acs.jmedchem.2c00582

    Figure Lengend Snippet: CB2R-dependent inhibition of FSK-stimulated cAMP CHO cells stably expressing h CB2R. cAMP inhibition data are expressed as the % CP55,940 response. Cells were treated with ligands simultaneously as indicated. 10 nM FM-6b (a), 50 nM FD-22a (b), and 5 nM FD-24a (c) were chosen after the completion of preliminary experiments with compounds alone for ease of calculations to approximate the EC 50 for each compound alone. Addition of 100 nM SR144528 to 0.1 nM–10 μM of FD-22a (d) or of FD-24a (e). Data were fitted to a nonlinear regression (three-parameter model, GraphPad v. 9.0). Data are mean ± S.E.M. of 3–6 independent experiments performed in triplicate. Data from these graphs is presented in Table . Statistical data for these graphs are presented in Table S2 .

    Article Snippet: The selective CB2R antagonists SR144528 (Tocris Bioscience, UK) and MC21 were dissolved in saline solution with 5% DMSO and 5% Tween 20.

    Techniques: Inhibition, Stable Transfection, Expressing

    [ H]CP55, 940 binding to CB1R (a) and CB2R (b). Membranes from CHO cells stably expressing h CB1R or h CB2R were treated with 1 nM [ H]CP55,940 and 0.10 nM–10 μM compounds for 2 h. Data are expressed as %[ H]CP55,940 bound. Data were fitted to a nonlinear regression (three-parameter model, GraphPad v. 9.0). Data are mean ± S.E.M. of 3 independent experiments performed in duplicate. Data from these graphs is presented in Table . Statistical data for these graphs are presented in Table S3 .

    Journal: Journal of Medicinal Chemistry

    Article Title: Design, Synthesis, and Biological Activity of New CB2 Receptor Ligands: from Orthosteric and Allosteric Modulators to Dualsteric/Bitopic Ligands

    doi: 10.1021/acs.jmedchem.2c00582

    Figure Lengend Snippet: [ H]CP55, 940 binding to CB1R (a) and CB2R (b). Membranes from CHO cells stably expressing h CB1R or h CB2R were treated with 1 nM [ H]CP55,940 and 0.10 nM–10 μM compounds for 2 h. Data are expressed as %[ H]CP55,940 bound. Data were fitted to a nonlinear regression (three-parameter model, GraphPad v. 9.0). Data are mean ± S.E.M. of 3 independent experiments performed in duplicate. Data from these graphs is presented in Table . Statistical data for these graphs are presented in Table S3 .

    Article Snippet: The selective CB2R antagonists SR144528 (Tocris Bioscience, UK) and MC21 were dissolved in saline solution with 5% DMSO and 5% Tween 20.

    Techniques: Binding Assay, Stable Transfection, Expressing

    Ability of FD-22a (A,C) and FD-24a (B,D) to decrease the inflammatory phenotype of LPS-stimulated BV2 microglial cells by the modulation of CB2R. Bars represent the release (pg/mL) of ILs in the presence of the drugs. Data represent mean ± (bars) from n = 3 independent experiments performed in duplicate. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01 and *** p < 0.001.

    Journal: Journal of Medicinal Chemistry

    Article Title: Design, Synthesis, and Biological Activity of New CB2 Receptor Ligands: from Orthosteric and Allosteric Modulators to Dualsteric/Bitopic Ligands

    doi: 10.1021/acs.jmedchem.2c00582

    Figure Lengend Snippet: Ability of FD-22a (A,C) and FD-24a (B,D) to decrease the inflammatory phenotype of LPS-stimulated BV2 microglial cells by the modulation of CB2R. Bars represent the release (pg/mL) of ILs in the presence of the drugs. Data represent mean ± (bars) from n = 3 independent experiments performed in duplicate. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01 and *** p < 0.001.

    Article Snippet: The selective CB2R antagonists SR144528 (Tocris Bioscience, UK) and MC21 were dissolved in saline solution with 5% DMSO and 5% Tween 20.

    Techniques: Comparison

    Release of inflammatory (IL-6) (A) and anti-inflammatory (IL-10) (B) interleukins induced by different concentrations of FD-22a . Data represent means ± S.E.M. from n = 3 independent experiments performed in duplicate. The selective CB2R agonist JWH133 was used as positive control. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s multiple comparison test. ** p < 0.01 and *** p < 0.001 compared to cells treated with LPS and TNFα.

    Journal: Journal of Medicinal Chemistry

    Article Title: Design, Synthesis, and Biological Activity of New CB2 Receptor Ligands: from Orthosteric and Allosteric Modulators to Dualsteric/Bitopic Ligands

    doi: 10.1021/acs.jmedchem.2c00582

    Figure Lengend Snippet: Release of inflammatory (IL-6) (A) and anti-inflammatory (IL-10) (B) interleukins induced by different concentrations of FD-22a . Data represent means ± S.E.M. from n = 3 independent experiments performed in duplicate. The selective CB2R agonist JWH133 was used as positive control. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s multiple comparison test. ** p < 0.01 and *** p < 0.001 compared to cells treated with LPS and TNFα.

    Article Snippet: The selective CB2R antagonists SR144528 (Tocris Bioscience, UK) and MC21 were dissolved in saline solution with 5% DMSO and 5% Tween 20.

    Techniques: Positive Control, Comparison

    Ability of FD-22a to decrease the inflammatory phenotype of LPS + TNFα–stimulated HMC3 by the modulation of CB2R. Bars represent the release (pg/mL) of IL-6 (A) and IL-10 (B) in the presence of the drugs at the indicated concentrations. Data represent means ± S.E.M. from n = 3 independent experiments performed in duplicate. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s multiple comparison test.* p < 0.05, ** p < 0.01 and *** p < 0.001.

    Journal: Journal of Medicinal Chemistry

    Article Title: Design, Synthesis, and Biological Activity of New CB2 Receptor Ligands: from Orthosteric and Allosteric Modulators to Dualsteric/Bitopic Ligands

    doi: 10.1021/acs.jmedchem.2c00582

    Figure Lengend Snippet: Ability of FD-22a to decrease the inflammatory phenotype of LPS + TNFα–stimulated HMC3 by the modulation of CB2R. Bars represent the release (pg/mL) of IL-6 (A) and IL-10 (B) in the presence of the drugs at the indicated concentrations. Data represent means ± S.E.M. from n = 3 independent experiments performed in duplicate. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s multiple comparison test.* p < 0.05, ** p < 0.01 and *** p < 0.001.

    Article Snippet: The selective CB2R antagonists SR144528 (Tocris Bioscience, UK) and MC21 were dissolved in saline solution with 5% DMSO and 5% Tween 20.

    Techniques: Comparison

    Effects of CB2 antagonism on FD-22a pain relieving efficacy. The response to a thermal stimulus was evaluated by the cold plate test measuring the latency (s) to pain-related behaviors (lifting or licking of the paw). Mice were daily treated i.p . with oxaliplatin 2.4 mg kg –1 . Tests were performed on day 15. The selective CB2R antagonists MC21 and SR144528 (10 mg kg –1 ) were administered i.p. 15 min before FD-22a (20 mg kg –1 p.o. ). Measurements were performed 15, 30, 45, 60, and 75 min after the injection of FD-22a . Control mice were treated with a vehicle. Each value represents the mean of 16 mice per group performed in 2 different experimental sets. **p < 0.01 vs vehicle + vehicle treated mice ^p < 0.05 and ^^p < 0.01 vs oxaliplatin + vehicle treated mice.

    Journal: Journal of Medicinal Chemistry

    Article Title: Design, Synthesis, and Biological Activity of New CB2 Receptor Ligands: from Orthosteric and Allosteric Modulators to Dualsteric/Bitopic Ligands

    doi: 10.1021/acs.jmedchem.2c00582

    Figure Lengend Snippet: Effects of CB2 antagonism on FD-22a pain relieving efficacy. The response to a thermal stimulus was evaluated by the cold plate test measuring the latency (s) to pain-related behaviors (lifting or licking of the paw). Mice were daily treated i.p . with oxaliplatin 2.4 mg kg –1 . Tests were performed on day 15. The selective CB2R antagonists MC21 and SR144528 (10 mg kg –1 ) were administered i.p. 15 min before FD-22a (20 mg kg –1 p.o. ). Measurements were performed 15, 30, 45, 60, and 75 min after the injection of FD-22a . Control mice were treated with a vehicle. Each value represents the mean of 16 mice per group performed in 2 different experimental sets. **p < 0.01 vs vehicle + vehicle treated mice ^p < 0.05 and ^^p < 0.01 vs oxaliplatin + vehicle treated mice.

    Article Snippet: The selective CB2R antagonists SR144528 (Tocris Bioscience, UK) and MC21 were dissolved in saline solution with 5% DMSO and 5% Tween 20.

    Techniques: Injection, Control

    Inhibition of Forskolin-Stimulated cAMP <xref ref-type= a " width="100%" height="100%">

    Journal: Journal of Medicinal Chemistry

    Article Title: Design, Synthesis, and Biological Activity of New CB2 Receptor Ligands: from Orthosteric and Allosteric Modulators to Dualsteric/Bitopic Ligands

    doi: 10.1021/acs.jmedchem.2c00582

    Figure Lengend Snippet: Inhibition of Forskolin-Stimulated cAMP a

    Article Snippet: The selective CB2R antagonists SR144528 (Tocris Bioscience, UK) and MC21 were dissolved in saline solution with 5% DMSO and 5% Tween 20.

    Techniques: Inhibition

    [ 3 H] CP55,940 Binding <xref ref-type= a " width="100%" height="100%">

    Journal: Journal of Medicinal Chemistry

    Article Title: Design, Synthesis, and Biological Activity of New CB2 Receptor Ligands: from Orthosteric and Allosteric Modulators to Dualsteric/Bitopic Ligands

    doi: 10.1021/acs.jmedchem.2c00582

    Figure Lengend Snippet: [ 3 H] CP55,940 Binding a

    Article Snippet: The selective CB2R antagonists SR144528 (Tocris Bioscience, UK) and MC21 were dissolved in saline solution with 5% DMSO and 5% Tween 20.

    Techniques: Binding Assay

    Radioligand binding data of CB91, LV58, LV62, and LV50 a .

    Journal: International Journal of Molecular Sciences

    Article Title: Anti-Proliferative Properties and Proapoptotic Function of New CB2 Selective Cannabinoid Receptor Agonist in Jurkat Leukemia Cells

    doi: 10.3390/ijms19071958

    Figure Lengend Snippet: Radioligand binding data of CB91, LV58, LV62, and LV50 a .

    Article Snippet: The incubation times used were in the range of 4–72 h. To investigate CB2R involvement, in some experiments, we pretreated Jurkat cells with a selective CB2R antagonist SR144528 (Tocris Bioscience, Bristol, UK), 1 μM was added 2 h before compound.

    Techniques: Binding Assay

    CB2R expression. Whole cell extracts of Jurkat, lymphoblastoid T cell line (CEM), and peripheral blood lymphocytes (PBL) were analyzed by Western blot to detect cannabinoid receptor 2 (CB2R) levels using antibodies specific for CB2R protein. The loading control was evaluated using anti-tubulin mAb. Densitometric CB2R/β-tubulin ratio is shown. The results are represented as the mean ± SD from three independent experiments. Statistical analysis indicated: *** p < 0.001 versus PBL cells.

    Journal: International Journal of Molecular Sciences

    Article Title: Anti-Proliferative Properties and Proapoptotic Function of New CB2 Selective Cannabinoid Receptor Agonist in Jurkat Leukemia Cells

    doi: 10.3390/ijms19071958

    Figure Lengend Snippet: CB2R expression. Whole cell extracts of Jurkat, lymphoblastoid T cell line (CEM), and peripheral blood lymphocytes (PBL) were analyzed by Western blot to detect cannabinoid receptor 2 (CB2R) levels using antibodies specific for CB2R protein. The loading control was evaluated using anti-tubulin mAb. Densitometric CB2R/β-tubulin ratio is shown. The results are represented as the mean ± SD from three independent experiments. Statistical analysis indicated: *** p < 0.001 versus PBL cells.

    Article Snippet: The incubation times used were in the range of 4–72 h. To investigate CB2R involvement, in some experiments, we pretreated Jurkat cells with a selective CB2R antagonist SR144528 (Tocris Bioscience, Bristol, UK), 1 μM was added 2 h before compound.

    Techniques: Expressing, Western Blot, Control

    Preliminary analysis of CB91, LV58, LV62, and LV50 after pretreatment with CB2R antagonist SR144528 a .

    Journal: International Journal of Molecular Sciences

    Article Title: Anti-Proliferative Properties and Proapoptotic Function of New CB2 Selective Cannabinoid Receptor Agonist in Jurkat Leukemia Cells

    doi: 10.3390/ijms19071958

    Figure Lengend Snippet: Preliminary analysis of CB91, LV58, LV62, and LV50 after pretreatment with CB2R antagonist SR144528 a .

    Article Snippet: The incubation times used were in the range of 4–72 h. To investigate CB2R involvement, in some experiments, we pretreated Jurkat cells with a selective CB2R antagonist SR144528 (Tocris Bioscience, Bristol, UK), 1 μM was added 2 h before compound.

    Techniques:

    LV50 decreases cell viability and inhibits cell proliferation. Jurkat, CEM, and PBL cells were cultured with different concentrations of LV50 for 24, 48, and 72 h. The number of viable cells was determined by Trypan Blue exclusion test. ( A , left panel ) Jurkat cells, data are reported as the mean ± SD among ten independent experiments. Statistical analysis indicated: **** p < 0.0001 versus vehicle. ( A , right panel ) Jurkat cells were pretreated with selective antagonist for CB2R (SR144528, 1 μM), exposed to LV50 for 72 h and then analyzed for cell viability. Statistical analysis indicated: **** p < 0.0001 versus vehicle; **** p < 0.0001 versus pretreated with SR144528. ( B , left panel ) CEM cells, data are reported as the mean ± SD among ten independent experiments. Statistical analysis indicated: **** p < 0.0001 versus vehicle. ( B , right panel ) CEM cells were pretreated with selective antagonist for CB2R (SR144528, 1 μM), exposed to LV50 for 72 h and then analyzed for cell viability. Statistical analysis indicated: **** p < 0.0001 versus vehicle; **** p < 0.0001 versus pretreated with SR144528. ( C ) PBL cells, data are reported as the mean ± SD among ten independent experiments. Statistical analysis indicated: LV50 10 μM versus vehicle NS (not significant). ( D , left panel ) Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in Jurkat cells. The results represent the mean ± SD of five independent experiments performed in triplicate and represent cell viability as a percentage of untreated control cells. Statistical analysis indicated: ** p < 0.01 versus vehicle; *** p < 0.001 versus vehicle. ( D , right panel ) Jurkat cells were pretreated with selective antagonist for CB2R (SR144528, 1 μM), exposed to LV50 for 72 h and then analyzed for proliferation. Statistical analysis indicated: **** p < 0.0001 versus vehicle; **** p < 0.0001 versus pretreated with SR144528.

    Journal: International Journal of Molecular Sciences

    Article Title: Anti-Proliferative Properties and Proapoptotic Function of New CB2 Selective Cannabinoid Receptor Agonist in Jurkat Leukemia Cells

    doi: 10.3390/ijms19071958

    Figure Lengend Snippet: LV50 decreases cell viability and inhibits cell proliferation. Jurkat, CEM, and PBL cells were cultured with different concentrations of LV50 for 24, 48, and 72 h. The number of viable cells was determined by Trypan Blue exclusion test. ( A , left panel ) Jurkat cells, data are reported as the mean ± SD among ten independent experiments. Statistical analysis indicated: **** p < 0.0001 versus vehicle. ( A , right panel ) Jurkat cells were pretreated with selective antagonist for CB2R (SR144528, 1 μM), exposed to LV50 for 72 h and then analyzed for cell viability. Statistical analysis indicated: **** p < 0.0001 versus vehicle; **** p < 0.0001 versus pretreated with SR144528. ( B , left panel ) CEM cells, data are reported as the mean ± SD among ten independent experiments. Statistical analysis indicated: **** p < 0.0001 versus vehicle. ( B , right panel ) CEM cells were pretreated with selective antagonist for CB2R (SR144528, 1 μM), exposed to LV50 for 72 h and then analyzed for cell viability. Statistical analysis indicated: **** p < 0.0001 versus vehicle; **** p < 0.0001 versus pretreated with SR144528. ( C ) PBL cells, data are reported as the mean ± SD among ten independent experiments. Statistical analysis indicated: LV50 10 μM versus vehicle NS (not significant). ( D , left panel ) Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in Jurkat cells. The results represent the mean ± SD of five independent experiments performed in triplicate and represent cell viability as a percentage of untreated control cells. Statistical analysis indicated: ** p < 0.01 versus vehicle; *** p < 0.001 versus vehicle. ( D , right panel ) Jurkat cells were pretreated with selective antagonist for CB2R (SR144528, 1 μM), exposed to LV50 for 72 h and then analyzed for proliferation. Statistical analysis indicated: **** p < 0.0001 versus vehicle; **** p < 0.0001 versus pretreated with SR144528.

    Article Snippet: The incubation times used were in the range of 4–72 h. To investigate CB2R involvement, in some experiments, we pretreated Jurkat cells with a selective CB2R antagonist SR144528 (Tocris Bioscience, Bristol, UK), 1 μM was added 2 h before compound.

    Techniques: Cell Culture, MTT Assay, Control

    Effect of LV50 on nuclear fragmentation. Jurkat, CEM, and PBL cells incubated with 5 μM or 10 μM LV50 for 72 h were analyzed by flow cytometric analysis after staining with propidium iodide. Histograms show cell cycle profiles and hypodiploid sub-G1 peak indicates typical DNA fragmentation that defines apoptosis. The percentage of cells in each phase of cell cycle is summarized below the histograms. Vehicle-treated or cells incubated with 5 μM or 10 μM LV50 were analyzed at three incubation times (24, 48, and 72 h). ( A , left panel ) Jurkat cells, results represent mean ± SD of three independent experiments. Statistical analysis indicated: * p < 0.05 versus vehicle; *** p < 0.001 versus vehicle. ( A , right panel ) In Jurkat cells, Propidium Iodide staining was performed in the presence or absence of the selective CB2R antagonist (1 μM). The results represent mean ± SD of three independent experiments. Statistical analysis indicated: ** p < 0.01 versus vehicle; § p < 0.05 versus pretreated with SR144528. ( B , left panel ) CEM cell, results represent mean ± SD of three independent experiments. Statistical analysis indicated: * p < 0.05 versus vehicle; *** p < 0.001 versus vehicle. ( B , right panel ) In CEM cells, propidium iodide staining was performed in the presence or absence of the selective CB2R antagonist (1 μM). The results represent mean ± SD of three independent experiments. Statistical analysis indicated: ** p < 0.01 versus vehicle; §§ p < 0.01 versus pretreated with SR144528. ( C ) PBL cells, results represent mean ± SD of three independent experiments. Statistical analysis indicated: LV50 10 μM versus vehicle NS (not significant).

    Journal: International Journal of Molecular Sciences

    Article Title: Anti-Proliferative Properties and Proapoptotic Function of New CB2 Selective Cannabinoid Receptor Agonist in Jurkat Leukemia Cells

    doi: 10.3390/ijms19071958

    Figure Lengend Snippet: Effect of LV50 on nuclear fragmentation. Jurkat, CEM, and PBL cells incubated with 5 μM or 10 μM LV50 for 72 h were analyzed by flow cytometric analysis after staining with propidium iodide. Histograms show cell cycle profiles and hypodiploid sub-G1 peak indicates typical DNA fragmentation that defines apoptosis. The percentage of cells in each phase of cell cycle is summarized below the histograms. Vehicle-treated or cells incubated with 5 μM or 10 μM LV50 were analyzed at three incubation times (24, 48, and 72 h). ( A , left panel ) Jurkat cells, results represent mean ± SD of three independent experiments. Statistical analysis indicated: * p < 0.05 versus vehicle; *** p < 0.001 versus vehicle. ( A , right panel ) In Jurkat cells, Propidium Iodide staining was performed in the presence or absence of the selective CB2R antagonist (1 μM). The results represent mean ± SD of three independent experiments. Statistical analysis indicated: ** p < 0.01 versus vehicle; § p < 0.05 versus pretreated with SR144528. ( B , left panel ) CEM cell, results represent mean ± SD of three independent experiments. Statistical analysis indicated: * p < 0.05 versus vehicle; *** p < 0.001 versus vehicle. ( B , right panel ) In CEM cells, propidium iodide staining was performed in the presence or absence of the selective CB2R antagonist (1 μM). The results represent mean ± SD of three independent experiments. Statistical analysis indicated: ** p < 0.01 versus vehicle; §§ p < 0.01 versus pretreated with SR144528. ( C ) PBL cells, results represent mean ± SD of three independent experiments. Statistical analysis indicated: LV50 10 μM versus vehicle NS (not significant).

    Article Snippet: The incubation times used were in the range of 4–72 h. To investigate CB2R involvement, in some experiments, we pretreated Jurkat cells with a selective CB2R antagonist SR144528 (Tocris Bioscience, Bristol, UK), 1 μM was added 2 h before compound.

    Techniques: Incubation, Staining

    LV50 induce activation of caspase-3, caspase-8, and poly(ADP-ribose)-polymerase (PARP) as indicated by detection of cleaved proteins after Western blot analysis. Jurkat cells were treated with the compound, or alternatively, pretreated with CB2R antagonist SR144528 (1 μM) and then incubated with LV50 10 μM for indicated incubation times. Whole cell extracts were analyzed by antibodies specific for uncleaved caspase-3, cleaved caspase-3, uncleaved caspase-8, cleaved caspase-8, and PARP. The loading control was evaluated using anti-actin mAb. Densitometric cleaved caspase-3/uncleaved caspase-3, cleaved caspase-8/uncleaved caspase-8, and cleaved PARP/uncleaved PARP ratios are shown. The results are represented as the mean ± SD from three independent experiments. Statistical analysis indicated: *** p < 0.001 versus vehicle; §§ p < 0.01 versus pretreated with SR144528; § p < 0.05 versus pretreated with SR144528.

    Journal: International Journal of Molecular Sciences

    Article Title: Anti-Proliferative Properties and Proapoptotic Function of New CB2 Selective Cannabinoid Receptor Agonist in Jurkat Leukemia Cells

    doi: 10.3390/ijms19071958

    Figure Lengend Snippet: LV50 induce activation of caspase-3, caspase-8, and poly(ADP-ribose)-polymerase (PARP) as indicated by detection of cleaved proteins after Western blot analysis. Jurkat cells were treated with the compound, or alternatively, pretreated with CB2R antagonist SR144528 (1 μM) and then incubated with LV50 10 μM for indicated incubation times. Whole cell extracts were analyzed by antibodies specific for uncleaved caspase-3, cleaved caspase-3, uncleaved caspase-8, cleaved caspase-8, and PARP. The loading control was evaluated using anti-actin mAb. Densitometric cleaved caspase-3/uncleaved caspase-3, cleaved caspase-8/uncleaved caspase-8, and cleaved PARP/uncleaved PARP ratios are shown. The results are represented as the mean ± SD from three independent experiments. Statistical analysis indicated: *** p < 0.001 versus vehicle; §§ p < 0.01 versus pretreated with SR144528; § p < 0.05 versus pretreated with SR144528.

    Article Snippet: The incubation times used were in the range of 4–72 h. To investigate CB2R involvement, in some experiments, we pretreated Jurkat cells with a selective CB2R antagonist SR144528 (Tocris Bioscience, Bristol, UK), 1 μM was added 2 h before compound.

    Techniques: Activation Assay, Western Blot, Incubation, Control

    Molecular involvement during apoptotic execution. ( A ) Analysis of mitochondrial membrane permeabilization (MMP) of Jurkat cells (treated with 10 μM LV50 for 12 and 24 h) by biparametric flow cytometry after staining with 5,5′,6,6′tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Numbers reported represent the percentage of cells with depolarized mitochondria (green florescence, upper right quadrant). ( B ) Bar graphs show results obtained from three independent experiments and reported as mean ± SD. Histograms show JC-1 fluorescence intensity red/green ratio indicating an increase of green fluorescence in treated cells, represented as a lower ratio, compared with vehicle-treated cells. ** p < 0.01 (12 h); *** p < 0.001 (24 h). ( C ) Jurkat cells were treated with the compound, or alternatively, pretreated with CB2R antagonist SR144528 (1 μM) and then incubated with LV50 for indicated incubation times. Whole cell extracts were analyzed by Western blot using antibodies specific for Bid/t-Bid and cleaved caspase-9/uncleaved caspase-9. Furthermore, cytosolic fraction was analyzed by Western blot for cytochrome c release. The results are represented as the mean ± SD from three independent experiments. Statistical analysis indicated: * p < 0.05 versus vehicle; § p < 0.05 versus pretreated with SR144528; ** p < 0.01 versus vehicle; §§ p < 0.01 versus pretreated with SR144528.

    Journal: International Journal of Molecular Sciences

    Article Title: Anti-Proliferative Properties and Proapoptotic Function of New CB2 Selective Cannabinoid Receptor Agonist in Jurkat Leukemia Cells

    doi: 10.3390/ijms19071958

    Figure Lengend Snippet: Molecular involvement during apoptotic execution. ( A ) Analysis of mitochondrial membrane permeabilization (MMP) of Jurkat cells (treated with 10 μM LV50 for 12 and 24 h) by biparametric flow cytometry after staining with 5,5′,6,6′tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Numbers reported represent the percentage of cells with depolarized mitochondria (green florescence, upper right quadrant). ( B ) Bar graphs show results obtained from three independent experiments and reported as mean ± SD. Histograms show JC-1 fluorescence intensity red/green ratio indicating an increase of green fluorescence in treated cells, represented as a lower ratio, compared with vehicle-treated cells. ** p < 0.01 (12 h); *** p < 0.001 (24 h). ( C ) Jurkat cells were treated with the compound, or alternatively, pretreated with CB2R antagonist SR144528 (1 μM) and then incubated with LV50 for indicated incubation times. Whole cell extracts were analyzed by Western blot using antibodies specific for Bid/t-Bid and cleaved caspase-9/uncleaved caspase-9. Furthermore, cytosolic fraction was analyzed by Western blot for cytochrome c release. The results are represented as the mean ± SD from three independent experiments. Statistical analysis indicated: * p < 0.05 versus vehicle; § p < 0.05 versus pretreated with SR144528; ** p < 0.01 versus vehicle; §§ p < 0.01 versus pretreated with SR144528.

    Article Snippet: The incubation times used were in the range of 4–72 h. To investigate CB2R involvement, in some experiments, we pretreated Jurkat cells with a selective CB2R antagonist SR144528 (Tocris Bioscience, Bristol, UK), 1 μM was added 2 h before compound.

    Techniques: Membrane, Flow Cytometry, Staining, Fluorescence, Incubation, Western Blot