molt 3 cell lines  (ATCC)


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    ATCC molt 3 cell lines
    Molt 3 Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CEM Corporation molt 3 leukemia cell lines
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    CEM Corporation molt 3 leukemia cell lines
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    molt 3 cell lines  (ATCC)


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    ATCC molt 3 cell lines
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    molt3 cell line  (ATCC)


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    ATCC molt3 cell line
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    CEM Corporation cell lines molt 3
    Cell Lines Molt 3, supplied by CEM Corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CEM Corporation cell lines molt 3
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    molt 3 effector cell lines  (Thermo Fisher)


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    Thermo Fisher molt 3 effector cell lines
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    CEM Corporation fenps against molt 3 cell line
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    leukemic cell lines jurkat  (ATCC)


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    ATCC leukemic cell lines jurkat
    a Cell viability, evaluated by resazurin-based metabolic assay, as a function of CBD concentration in <t>human</t> <t>leukemic</t> cell lines of different lineages at 24 h of treatment. Cell lines derived from T-ALL <t>(Jurkat,</t> MOLT-3, and CEM), B-ALL (RS4;11 and Reh) and CML (K562) were tested. Data (resorufin fluorescence intensity, arbitrary units) were normalized to the vehicle-treated control and shown as mean ± SD ( n = 8; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant; one-way ANOVA). b Live cell count (trypan blue exclusion test) in long-lasting Jurkat cells cultures exposed to different CBD concentrations (0–100 μM). Left: cell count at 72 h after single CBD administration; right: fresh CBD was added, 50% of medium volume was changed and cells were counted every 24 h. Data are normalized to the initial point (0 h) and shown as mean ± SD ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA test). c Cell viability was evaluated by resazurin-based metabolic assay at 24 h of treatment with CBD (30 μM) in human tumor cell lines of different histogenesis, including CML (K562), B-ALL (Reh and RS4;11), T-ALL (CEM, MOLT-3, and Jurkat), cervical cancer (SiHa and HeLa), and breast cancer (MCF7-7 and MDA-MB-231). Data (resorufin fluorescence intensity) were normalized to vehicle-treated control and reported as mean ± SD ( n = 8 of four independent experiments; (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, Student’s t -test). d Cell viability was evaluated by resazurin-based metabolic assay at 24 h of treatment with CBD (30 μM) in non-cancerous cells. Human CD4 + cells were activated with anti-CD3/CD28 antibodies as described in Materials and Methods section. Data (resorufin fluorescence intensity) are normalized to the vehicle-treated control and reported as mean ± SD ( n = 8 in at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; Student’s t -test). e After preincubation with CBD (30 μM, 24 h), non-cancerous CD4 + cells were activated by anti-CD3/antiCD28 antibodies. Cell viability was evaluated by resazurin-based metabolic assay every 24 h. Data (resorufin fluorescence intensity) were normalized to 0 h time point and shown as mean ± SD ( n = 8 in at least three independent experiments). Statistical comparison between control and pretreated samples was undertaken at each time point (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, Student’s t -test). f Migration capacity of Jurkat cells pretreated with CBD (10 or 30 μM, 2 h) was evaluated by chemotactic migration assay, using a Transwell system. Cells were allowed to migrate for 4 h, CXCL12 was used as a chemoattractant. The percentage of migrated cells was determined by cells count in the lower chamber. Data are mean ± SD ( n = 4). Statistical comparison was made with respect to positive control (CXCL12) (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA test). g – i Cell death was evaluation by flow cytometry ( g , h ) and fluorescent microscopy ( I ) in Jurkat cells treated with different concentrations of CBD using Annexin V-AF488/PI double staining. Representative dot plots (2, 4, and 6 h) and images (12 h) are shown in g and i , correspondingly. Scale bar: 20 μm. In every dot plot lower left quadrant represents Annexin V − PI − (DN) live cells, in the lower right quadrant are Annexin V + PI − (early apoptotic) cells, Annexin V − PI + (primary necrotic) cells are in the upper left quadrant, whereas the double-stained population Annexin V + PI + (DP) in the upper right quadrant represents dead cells, which may include necrotic and late apoptotic ones. Statistical analysis of flow cytometry data is given in h ( n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA test)
    Leukemic Cell Lines Jurkat, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Cannabidiol directly targets mitochondria and disturbs calcium homeostasis in acute lymphoblastic leukemia"

    Article Title: Cannabidiol directly targets mitochondria and disturbs calcium homeostasis in acute lymphoblastic leukemia

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-2024-0

    a Cell viability, evaluated by resazurin-based metabolic assay, as a function of CBD concentration in human leukemic cell lines of different lineages at 24 h of treatment. Cell lines derived from T-ALL (Jurkat, MOLT-3, and CEM), B-ALL (RS4;11 and Reh) and CML (K562) were tested. Data (resorufin fluorescence intensity, arbitrary units) were normalized to the vehicle-treated control and shown as mean ± SD ( n = 8; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant; one-way ANOVA). b Live cell count (trypan blue exclusion test) in long-lasting Jurkat cells cultures exposed to different CBD concentrations (0–100 μM). Left: cell count at 72 h after single CBD administration; right: fresh CBD was added, 50% of medium volume was changed and cells were counted every 24 h. Data are normalized to the initial point (0 h) and shown as mean ± SD ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA test). c Cell viability was evaluated by resazurin-based metabolic assay at 24 h of treatment with CBD (30 μM) in human tumor cell lines of different histogenesis, including CML (K562), B-ALL (Reh and RS4;11), T-ALL (CEM, MOLT-3, and Jurkat), cervical cancer (SiHa and HeLa), and breast cancer (MCF7-7 and MDA-MB-231). Data (resorufin fluorescence intensity) were normalized to vehicle-treated control and reported as mean ± SD ( n = 8 of four independent experiments; (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, Student’s t -test). d Cell viability was evaluated by resazurin-based metabolic assay at 24 h of treatment with CBD (30 μM) in non-cancerous cells. Human CD4 + cells were activated with anti-CD3/CD28 antibodies as described in Materials and Methods section. Data (resorufin fluorescence intensity) are normalized to the vehicle-treated control and reported as mean ± SD ( n = 8 in at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; Student’s t -test). e After preincubation with CBD (30 μM, 24 h), non-cancerous CD4 + cells were activated by anti-CD3/antiCD28 antibodies. Cell viability was evaluated by resazurin-based metabolic assay every 24 h. Data (resorufin fluorescence intensity) were normalized to 0 h time point and shown as mean ± SD ( n = 8 in at least three independent experiments). Statistical comparison between control and pretreated samples was undertaken at each time point (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, Student’s t -test). f Migration capacity of Jurkat cells pretreated with CBD (10 or 30 μM, 2 h) was evaluated by chemotactic migration assay, using a Transwell system. Cells were allowed to migrate for 4 h, CXCL12 was used as a chemoattractant. The percentage of migrated cells was determined by cells count in the lower chamber. Data are mean ± SD ( n = 4). Statistical comparison was made with respect to positive control (CXCL12) (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA test). g – i Cell death was evaluation by flow cytometry ( g , h ) and fluorescent microscopy ( I ) in Jurkat cells treated with different concentrations of CBD using Annexin V-AF488/PI double staining. Representative dot plots (2, 4, and 6 h) and images (12 h) are shown in g and i , correspondingly. Scale bar: 20 μm. In every dot plot lower left quadrant represents Annexin V − PI − (DN) live cells, in the lower right quadrant are Annexin V + PI − (early apoptotic) cells, Annexin V − PI + (primary necrotic) cells are in the upper left quadrant, whereas the double-stained population Annexin V + PI + (DP) in the upper right quadrant represents dead cells, which may include necrotic and late apoptotic ones. Statistical analysis of flow cytometry data is given in h ( n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA test)
    Figure Legend Snippet: a Cell viability, evaluated by resazurin-based metabolic assay, as a function of CBD concentration in human leukemic cell lines of different lineages at 24 h of treatment. Cell lines derived from T-ALL (Jurkat, MOLT-3, and CEM), B-ALL (RS4;11 and Reh) and CML (K562) were tested. Data (resorufin fluorescence intensity, arbitrary units) were normalized to the vehicle-treated control and shown as mean ± SD ( n = 8; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant; one-way ANOVA). b Live cell count (trypan blue exclusion test) in long-lasting Jurkat cells cultures exposed to different CBD concentrations (0–100 μM). Left: cell count at 72 h after single CBD administration; right: fresh CBD was added, 50% of medium volume was changed and cells were counted every 24 h. Data are normalized to the initial point (0 h) and shown as mean ± SD ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA test). c Cell viability was evaluated by resazurin-based metabolic assay at 24 h of treatment with CBD (30 μM) in human tumor cell lines of different histogenesis, including CML (K562), B-ALL (Reh and RS4;11), T-ALL (CEM, MOLT-3, and Jurkat), cervical cancer (SiHa and HeLa), and breast cancer (MCF7-7 and MDA-MB-231). Data (resorufin fluorescence intensity) were normalized to vehicle-treated control and reported as mean ± SD ( n = 8 of four independent experiments; (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, Student’s t -test). d Cell viability was evaluated by resazurin-based metabolic assay at 24 h of treatment with CBD (30 μM) in non-cancerous cells. Human CD4 + cells were activated with anti-CD3/CD28 antibodies as described in Materials and Methods section. Data (resorufin fluorescence intensity) are normalized to the vehicle-treated control and reported as mean ± SD ( n = 8 in at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; Student’s t -test). e After preincubation with CBD (30 μM, 24 h), non-cancerous CD4 + cells were activated by anti-CD3/antiCD28 antibodies. Cell viability was evaluated by resazurin-based metabolic assay every 24 h. Data (resorufin fluorescence intensity) were normalized to 0 h time point and shown as mean ± SD ( n = 8 in at least three independent experiments). Statistical comparison between control and pretreated samples was undertaken at each time point (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, Student’s t -test). f Migration capacity of Jurkat cells pretreated with CBD (10 or 30 μM, 2 h) was evaluated by chemotactic migration assay, using a Transwell system. Cells were allowed to migrate for 4 h, CXCL12 was used as a chemoattractant. The percentage of migrated cells was determined by cells count in the lower chamber. Data are mean ± SD ( n = 4). Statistical comparison was made with respect to positive control (CXCL12) (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA test). g – i Cell death was evaluation by flow cytometry ( g , h ) and fluorescent microscopy ( I ) in Jurkat cells treated with different concentrations of CBD using Annexin V-AF488/PI double staining. Representative dot plots (2, 4, and 6 h) and images (12 h) are shown in g and i , correspondingly. Scale bar: 20 μm. In every dot plot lower left quadrant represents Annexin V − PI − (DN) live cells, in the lower right quadrant are Annexin V + PI − (early apoptotic) cells, Annexin V − PI + (primary necrotic) cells are in the upper left quadrant, whereas the double-stained population Annexin V + PI + (DP) in the upper right quadrant represents dead cells, which may include necrotic and late apoptotic ones. Statistical analysis of flow cytometry data is given in h ( n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA test)

    Techniques Used: Metabolic Assay, Concentration Assay, Derivative Assay, Fluorescence, Cell Counting, Migration, Positive Control, Flow Cytometry, Microscopy, Double Staining, Staining

    a Monitoring of Δψm loss in CBD-treated leukemic cells. Jurkat cells were double-stained with MtGreen and TMRE, mitochondria (ROI) were selected (upper panel) and intensity of fluorescence was monitored by confocal microscopy after CBD (30 μM) administration (lower panel, n = 36; see also Supplementary movie ). b Intensity of TMRE fluorescence as an indicator of Δψm in Jurkat cells, treated with different concentrations of CBD (0–100 μM) during 10 min. FCCP (10 μM) was used as a positive control. Data are mean ± SD ( n = 8 in three independent experiments). Statistical comparison with control was performed by means of one-way ANOVA test (* p < 0.05; **** p < 0.0001). c EYFP-Cyt-C is localized in mitochondria of Jurkat cells after 12 h of transfection. EYFP-Cyt-C and TMRE fluorescence are colocalized (200 nM TMRE, 200 ng EYFP-Cyt-C, pseudocolor). Images were acquired by confocal microscopy at 12 h after transfection, scale bar: 10 μm. d Representative images of five Jurkat cells, transfected with EYFP-Cyt-C (as in c ). Dense green fluorescent puncta (pseudocolor) reflect Cyt-C localization in intact mitochondria. Scale bar: 10 μm. e Representative images of five Jurkat cells, transfected with EYFP-Cyt-C (as in c ) and treated with CBD (30 μM, 1 h). Cyt-C was released from mitochondria as evidenced by a diffuse EYFP-CytC distribution. Scale bar: 10 μm. f EYFP-Cyt-C distribution in Jurkat cells, expressing EYFP-Cyt-C, at 0, 5, and 20 min with CBD (30 μM). Scale bar: 10 μm. g Caspase-9 and caspase-3 activity in vehicle- and CBD- treated (30 μM, 12 h) Jurkat cells. Staurosporine (STS, 5 μM) was used as a positive control. After incubation, cells were lysed, and caspase activity was determined by colorimetric assay (BioVision). Fold increase in activity compared to control was plotted as mean ± SE (four independent experiments). One-way ANOVA test was performed to compare CBD-treated to control group (* p < 0.05; ** p < 0.01). h Pretreatment with mPTP inhibitor CsA (10 μM) prevents Cyt-C release, induced by CBD (30 μM; 1 h). Compare these images with Fig. 4d, e (vehicle- and CBD-treated cells) and note the protective effect of CsA. Scale bar: 10 μm. i ROS production as evaluated by fluorescent microscopy in DCF-loaded (2 μM) Jurkat cells. CBD effect on ROS production was monitored after 1 h of incubation. PMA (1 μM) was used as a positive control
    Figure Legend Snippet: a Monitoring of Δψm loss in CBD-treated leukemic cells. Jurkat cells were double-stained with MtGreen and TMRE, mitochondria (ROI) were selected (upper panel) and intensity of fluorescence was monitored by confocal microscopy after CBD (30 μM) administration (lower panel, n = 36; see also Supplementary movie ). b Intensity of TMRE fluorescence as an indicator of Δψm in Jurkat cells, treated with different concentrations of CBD (0–100 μM) during 10 min. FCCP (10 μM) was used as a positive control. Data are mean ± SD ( n = 8 in three independent experiments). Statistical comparison with control was performed by means of one-way ANOVA test (* p < 0.05; **** p < 0.0001). c EYFP-Cyt-C is localized in mitochondria of Jurkat cells after 12 h of transfection. EYFP-Cyt-C and TMRE fluorescence are colocalized (200 nM TMRE, 200 ng EYFP-Cyt-C, pseudocolor). Images were acquired by confocal microscopy at 12 h after transfection, scale bar: 10 μm. d Representative images of five Jurkat cells, transfected with EYFP-Cyt-C (as in c ). Dense green fluorescent puncta (pseudocolor) reflect Cyt-C localization in intact mitochondria. Scale bar: 10 μm. e Representative images of five Jurkat cells, transfected with EYFP-Cyt-C (as in c ) and treated with CBD (30 μM, 1 h). Cyt-C was released from mitochondria as evidenced by a diffuse EYFP-CytC distribution. Scale bar: 10 μm. f EYFP-Cyt-C distribution in Jurkat cells, expressing EYFP-Cyt-C, at 0, 5, and 20 min with CBD (30 μM). Scale bar: 10 μm. g Caspase-9 and caspase-3 activity in vehicle- and CBD- treated (30 μM, 12 h) Jurkat cells. Staurosporine (STS, 5 μM) was used as a positive control. After incubation, cells were lysed, and caspase activity was determined by colorimetric assay (BioVision). Fold increase in activity compared to control was plotted as mean ± SE (four independent experiments). One-way ANOVA test was performed to compare CBD-treated to control group (* p < 0.05; ** p < 0.01). h Pretreatment with mPTP inhibitor CsA (10 μM) prevents Cyt-C release, induced by CBD (30 μM; 1 h). Compare these images with Fig. 4d, e (vehicle- and CBD-treated cells) and note the protective effect of CsA. Scale bar: 10 μm. i ROS production as evaluated by fluorescent microscopy in DCF-loaded (2 μM) Jurkat cells. CBD effect on ROS production was monitored after 1 h of incubation. PMA (1 μM) was used as a positive control

    Techniques Used: Staining, Fluorescence, Confocal Microscopy, Positive Control, Transfection, Expressing, Activity Assay, Incubation, Colorimetric Assay, Microscopy

    a – c [Ca 2+ ] i monitoring in leukemic cells, treated with CBD. Traces represent the mean ± SD of at least six samples from independent experiments. d Dose dependence of peak [Ca 2+ ] i values in T-ALL cell lines, exposed to CBD. Data are mean ± SD for at least six samples from independent experiments. e , f [Ca 2+ ] i monitoring in non-cancerous T cells in resting ( e ) and activated ( f ) states. Traces represent the mean ± SD of at least six samples from independent experiments. g Pharmacological analysis of [Ca 2+ ] i rise in response to CBD. Before CBD treatment, cells were preincubated over 20 min with either CB1 antagonist, rimonabant (1 μM), CB2 inverse agonist, AM630 (2 μM), or GPR55 antagonist, CID16020046 (3 μM); values Δ [Ca 2+ ] i were obtained by subtracting the [Ca 2+ ] i baseline level from [Ca 2+ ] i maximum increase. Data are mean ± SD of at least 6 samples from independent experiments. h [Ca 2+ ] i monitoring in Jurkat cells, suspended in Ca 2+ -free HBSS. Traces represent the mean ± SD of at least six samples from independent experiments. i , j Effect of Gd 3+ and RR, non-selective blockers of plasma membrane Ca 2+ - permeable channels, on [Ca 2+ ] i ( i and j , left) and cell viability evaluated by resazurin-based metabolic assay ( i and j , right). Gd 3+ (1 μM) or RR (1 μM) were added to Jurkat cells 20 min before the [Ca 2+ ] i measurement. i , j left: traces represent the mean ± SD of at least six samples from independent experiments. i , j right: data (resorufin fluorescence intensity) were normalized to vehicle-treated control and reported as mean ± SD ( n = 8 of four independent experiments (**** p < 0.0001, Student’s t -test)
    Figure Legend Snippet: a – c [Ca 2+ ] i monitoring in leukemic cells, treated with CBD. Traces represent the mean ± SD of at least six samples from independent experiments. d Dose dependence of peak [Ca 2+ ] i values in T-ALL cell lines, exposed to CBD. Data are mean ± SD for at least six samples from independent experiments. e , f [Ca 2+ ] i monitoring in non-cancerous T cells in resting ( e ) and activated ( f ) states. Traces represent the mean ± SD of at least six samples from independent experiments. g Pharmacological analysis of [Ca 2+ ] i rise in response to CBD. Before CBD treatment, cells were preincubated over 20 min with either CB1 antagonist, rimonabant (1 μM), CB2 inverse agonist, AM630 (2 μM), or GPR55 antagonist, CID16020046 (3 μM); values Δ [Ca 2+ ] i were obtained by subtracting the [Ca 2+ ] i baseline level from [Ca 2+ ] i maximum increase. Data are mean ± SD of at least 6 samples from independent experiments. h [Ca 2+ ] i monitoring in Jurkat cells, suspended in Ca 2+ -free HBSS. Traces represent the mean ± SD of at least six samples from independent experiments. i , j Effect of Gd 3+ and RR, non-selective blockers of plasma membrane Ca 2+ - permeable channels, on [Ca 2+ ] i ( i and j , left) and cell viability evaluated by resazurin-based metabolic assay ( i and j , right). Gd 3+ (1 μM) or RR (1 μM) were added to Jurkat cells 20 min before the [Ca 2+ ] i measurement. i , j left: traces represent the mean ± SD of at least six samples from independent experiments. i , j right: data (resorufin fluorescence intensity) were normalized to vehicle-treated control and reported as mean ± SD ( n = 8 of four independent experiments (**** p < 0.0001, Student’s t -test)

    Techniques Used: Metabolic Assay, Fluorescence

    cd8 t lymphocytic cell line molt 3  (ATCC)


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    ATCC cd8 t lymphocytic cell line molt 3
    Comparison of <t>CD8</t> + T‐cell infiltration and CCL5, CCND1, and PDE5 expression in tissues of finasteride‐treated and untreated BPH patients. A,B, Infiltration of CD8 + T cells and expression of CCL5, CCND1, and PDE5 were analyzed by IHC staining of serial paraffin sections from prostate tissues of BPH patients treated or not with finasteride for at least 6 months. C, Heat map showing the distribution of CD8 + T cells and CCL5, CCND1, and PDE5 expression in the finasteride‐treated and untreated groups. BPH, benign prostatic hyperplasia; IHC, immunohistochemistry; PDE5, phosphodiesterase type 5 [Color figure can be viewed at wileyonlinelibrary.com]
    Cd8 T Lymphocytic Cell Line Molt 3, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Activation of cGMP/PKG/p65 signaling associated with PDE5‐Is downregulates CCL5 secretion by CD8 + T cells in benign prostatic hyperplasia"

    Article Title: Activation of cGMP/PKG/p65 signaling associated with PDE5‐Is downregulates CCL5 secretion by CD8 + T cells in benign prostatic hyperplasia

    Journal: The Prostate

    doi: 10.1002/pros.23801

    Comparison of CD8 + T‐cell infiltration and CCL5, CCND1, and PDE5 expression in tissues of finasteride‐treated and untreated BPH patients. A,B, Infiltration of CD8 + T cells and expression of CCL5, CCND1, and PDE5 were analyzed by IHC staining of serial paraffin sections from prostate tissues of BPH patients treated or not with finasteride for at least 6 months. C, Heat map showing the distribution of CD8 + T cells and CCL5, CCND1, and PDE5 expression in the finasteride‐treated and untreated groups. BPH, benign prostatic hyperplasia; IHC, immunohistochemistry; PDE5, phosphodiesterase type 5 [Color figure can be viewed at wileyonlinelibrary.com]
    Figure Legend Snippet: Comparison of CD8 + T‐cell infiltration and CCL5, CCND1, and PDE5 expression in tissues of finasteride‐treated and untreated BPH patients. A,B, Infiltration of CD8 + T cells and expression of CCL5, CCND1, and PDE5 were analyzed by IHC staining of serial paraffin sections from prostate tissues of BPH patients treated or not with finasteride for at least 6 months. C, Heat map showing the distribution of CD8 + T cells and CCL5, CCND1, and PDE5 expression in the finasteride‐treated and untreated groups. BPH, benign prostatic hyperplasia; IHC, immunohistochemistry; PDE5, phosphodiesterase type 5 [Color figure can be viewed at wileyonlinelibrary.com]

    Techniques Used: Expressing, Immunohistochemistry

    Activation of the cGMP/PKG signaling pathway could reverse the induction of BEC proliferation by CD8 + T cells through suppressing the secretion of CCL5 by CD8 + T cells in low androgen conditions. BECs were cocultured with or without Molt‐3 cells in low androgen conditions for 4 days. A,C, BECs cultured with rhCCL5 (1 μg/mL) and tadalafil (100 nM) or Sp‐8‐Br‐PET‐cGMP (10 μM) were analyzed for proliferation at days 2, 4, and 6 using the CCK‐8 assay. The data are shown as the mean ± SD; * P < 0.05 and ** P < 0.01. B,D, BECs were harvested at day 4 and analyzed for CCND1 expression by Western blot analysis; β‐Tubulin was used as a loading control. E‐G, CCL5 mRNA expression in Molt‐3 cells at day 2 was analyzed by qPCR. CCL5 mRNA levels were downregulated by tadalafil (100 nM) or Sp‐8‐Br‐PET‐cGMP (10 μM) in Molt‐3 cells cocultured with BECs, but KT5823 (100 nM) reversed the effect. H, CCL5 secretion to conditioned medium of cocultures and monocultures treated or not with tadalafil and/or KT5823 at day 4 was assessed by ELISA. I,J, BPH‐1 cells treated or not with tadalafil or Sp‐8‐Br‐PET‐cGMP, anti‐CCL5 neutralizing antibody (2 μg/mL), and KT5823 were analyzed for proliferation at days 2, 4, and 6 using the CCK‐8 assay. Data are shown as the mean ± SD; * P < 0.05 and ** P < 0.01. BEC, BPH epithelial cell; BPH, benign prostatic hyperplasia; CCK‐8, cell counting kit‐8; cGMP, cyclic guanosine monophosphate; ELISA, enzyme‐linked immunosorbent assay; mRNA, messenger RNA; PKG, protein kinase G; qPCR, quantitative polymerase chain reaction, TDF, tadalafil [Color figure can be viewed at wileyonlinelibrary.com]
    Figure Legend Snippet: Activation of the cGMP/PKG signaling pathway could reverse the induction of BEC proliferation by CD8 + T cells through suppressing the secretion of CCL5 by CD8 + T cells in low androgen conditions. BECs were cocultured with or without Molt‐3 cells in low androgen conditions for 4 days. A,C, BECs cultured with rhCCL5 (1 μg/mL) and tadalafil (100 nM) or Sp‐8‐Br‐PET‐cGMP (10 μM) were analyzed for proliferation at days 2, 4, and 6 using the CCK‐8 assay. The data are shown as the mean ± SD; * P < 0.05 and ** P < 0.01. B,D, BECs were harvested at day 4 and analyzed for CCND1 expression by Western blot analysis; β‐Tubulin was used as a loading control. E‐G, CCL5 mRNA expression in Molt‐3 cells at day 2 was analyzed by qPCR. CCL5 mRNA levels were downregulated by tadalafil (100 nM) or Sp‐8‐Br‐PET‐cGMP (10 μM) in Molt‐3 cells cocultured with BECs, but KT5823 (100 nM) reversed the effect. H, CCL5 secretion to conditioned medium of cocultures and monocultures treated or not with tadalafil and/or KT5823 at day 4 was assessed by ELISA. I,J, BPH‐1 cells treated or not with tadalafil or Sp‐8‐Br‐PET‐cGMP, anti‐CCL5 neutralizing antibody (2 μg/mL), and KT5823 were analyzed for proliferation at days 2, 4, and 6 using the CCK‐8 assay. Data are shown as the mean ± SD; * P < 0.05 and ** P < 0.01. BEC, BPH epithelial cell; BPH, benign prostatic hyperplasia; CCK‐8, cell counting kit‐8; cGMP, cyclic guanosine monophosphate; ELISA, enzyme‐linked immunosorbent assay; mRNA, messenger RNA; PKG, protein kinase G; qPCR, quantitative polymerase chain reaction, TDF, tadalafil [Color figure can be viewed at wileyonlinelibrary.com]

    Techniques Used: Activation Assay, Cell Culture, CCK-8 Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Counting, Real-time Polymerase Chain Reaction

    Expression of signaling molecules downstream of the cGMP/PKG pathway involved in the inhibition of BEC proliferation in low androgen conditions. Cocultures of BECs and Molt‐3 cells were treated with tadalafil (A) or Sp‐8‐Br‐PET‐cGMP (B) with or without KT5823 for 1 and 2 hours. BECs and Molt‐3 cells were harvested separately and analyzed by Western blot analysis for total NF‐κB and phospho‐NF‐κB (Molt‐3 cells) and total STAT5, phospho‐STAT5, and CCND1 (BPH‐1 cells). β‐Tubulin and GAPDH were used as loading controls. BEC, BPH epithelial cell; BPH, benign prostatic hyperplasia; cGMP, cyclic guanosine monophosphate; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; NF‐κB, nuclear factor‐κB; PKG, protein kinase G; STAT5, signal transducer and activator of transcription 5; TDF, tadalafil
    Figure Legend Snippet: Expression of signaling molecules downstream of the cGMP/PKG pathway involved in the inhibition of BEC proliferation in low androgen conditions. Cocultures of BECs and Molt‐3 cells were treated with tadalafil (A) or Sp‐8‐Br‐PET‐cGMP (B) with or without KT5823 for 1 and 2 hours. BECs and Molt‐3 cells were harvested separately and analyzed by Western blot analysis for total NF‐κB and phospho‐NF‐κB (Molt‐3 cells) and total STAT5, phospho‐STAT5, and CCND1 (BPH‐1 cells). β‐Tubulin and GAPDH were used as loading controls. BEC, BPH epithelial cell; BPH, benign prostatic hyperplasia; cGMP, cyclic guanosine monophosphate; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; NF‐κB, nuclear factor‐κB; PKG, protein kinase G; STAT5, signal transducer and activator of transcription 5; TDF, tadalafil

    Techniques Used: Expressing, Inhibition, Western Blot

    Activation of cGMP/PKG signaling suppressed CCL5 secretion by CD8 + T cells and reversed the induction of BEC proliferation in vivo. A, The degree of prostatic hyperplasia in rats receiving regular diet (RD), HFD, or HFD + PDE5‐Is was evaluated by hematoxylin and eosin staining of rat prostate samples ( n = 6 rats per group). B, The serum testosterone levels of RD and HFD groups were measured using an automated chemiluminescence system at week 12; * P < 0.05, ** P < 0.01, and *** P < 0.001 (by t test). C, All rat prostate weight of three groups were tested at last; * P < 0.05, ** P < 0.01, and *** P < 0.001 (by one‐way ANOVA). D, CD8 + T‐cell infiltration and CCL5, CCND1, and PDE5 expression was analyzed by IHC staining in serial paraffin sections of the rat prostate. ANOVA, analysis of variance; BEC, BPH epithelial cell; cGMP, cyclic guanosine monophosphate; HFD, high‐fat diet; IHC, immunohistochemistry; PDE5, phosphodiesterase type 5; PKG, protein kinase G; RD, regular diet [Color figure can be viewed at wileyonlinelibrary.com]
    Figure Legend Snippet: Activation of cGMP/PKG signaling suppressed CCL5 secretion by CD8 + T cells and reversed the induction of BEC proliferation in vivo. A, The degree of prostatic hyperplasia in rats receiving regular diet (RD), HFD, or HFD + PDE5‐Is was evaluated by hematoxylin and eosin staining of rat prostate samples ( n = 6 rats per group). B, The serum testosterone levels of RD and HFD groups were measured using an automated chemiluminescence system at week 12; * P < 0.05, ** P < 0.01, and *** P < 0.001 (by t test). C, All rat prostate weight of three groups were tested at last; * P < 0.05, ** P < 0.01, and *** P < 0.001 (by one‐way ANOVA). D, CD8 + T‐cell infiltration and CCL5, CCND1, and PDE5 expression was analyzed by IHC staining in serial paraffin sections of the rat prostate. ANOVA, analysis of variance; BEC, BPH epithelial cell; cGMP, cyclic guanosine monophosphate; HFD, high‐fat diet; IHC, immunohistochemistry; PDE5, phosphodiesterase type 5; PKG, protein kinase G; RD, regular diet [Color figure can be viewed at wileyonlinelibrary.com]

    Techniques Used: Activation Assay, In Vivo, Staining, Expressing, Immunohistochemistry

    Schematic representation of the molecular mechanism underlying BEC proliferation in BPH. Activation of the cGMP/PKG signaling pathway in CD8 + T cells decreased NF‐κB phosphorylation and CCL5 secretion, resulting in the inhibition of CCL5/STAT5/CCND1 signaling in BECs and decrease of their proliferation in low androgen conditions. BEC, BPH epithelial cell; BPH, benign prostatic hyperplasia; cGMP, cyclic guanosine monophosphate; NF‐κB, nuclear factor‐κB; PDE5, phosphodiesterase type 5; PDE5‐I, PDE5 inhibitors; PKG, protein kinase G; STAT5, signal transducer and activator of transcription 5 [Color figure can be viewed at wileyonlinelibrary.com]
    Figure Legend Snippet: Schematic representation of the molecular mechanism underlying BEC proliferation in BPH. Activation of the cGMP/PKG signaling pathway in CD8 + T cells decreased NF‐κB phosphorylation and CCL5 secretion, resulting in the inhibition of CCL5/STAT5/CCND1 signaling in BECs and decrease of their proliferation in low androgen conditions. BEC, BPH epithelial cell; BPH, benign prostatic hyperplasia; cGMP, cyclic guanosine monophosphate; NF‐κB, nuclear factor‐κB; PDE5, phosphodiesterase type 5; PDE5‐I, PDE5 inhibitors; PKG, protein kinase G; STAT5, signal transducer and activator of transcription 5 [Color figure can be viewed at wileyonlinelibrary.com]

    Techniques Used: Activation Assay, Inhibition

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    a Cell viability, evaluated by resazurin-based metabolic assay, as a function of CBD concentration in human leukemic cell lines of different lineages at 24 h of treatment. Cell lines derived from T-ALL (Jurkat, MOLT-3, and CEM), B-ALL (RS4;11 and Reh) and CML (K562) were tested. Data (resorufin fluorescence intensity, arbitrary units) were normalized to the vehicle-treated control and shown as mean ± SD ( n = 8; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant; one-way ANOVA). b Live cell count (trypan blue exclusion test) in long-lasting Jurkat cells cultures exposed to different CBD concentrations (0–100 μM). Left: cell count at 72 h after single CBD administration; right: fresh CBD was added, 50% of medium volume was changed and cells were counted every 24 h. Data are normalized to the initial point (0 h) and shown as mean ± SD ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA test). c Cell viability was evaluated by resazurin-based metabolic assay at 24 h of treatment with CBD (30 μM) in human tumor cell lines of different histogenesis, including CML (K562), B-ALL (Reh and RS4;11), T-ALL (CEM, MOLT-3, and Jurkat), cervical cancer (SiHa and HeLa), and breast cancer (MCF7-7 and MDA-MB-231). Data (resorufin fluorescence intensity) were normalized to vehicle-treated control and reported as mean ± SD ( n = 8 of four independent experiments; (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, Student’s t -test). d Cell viability was evaluated by resazurin-based metabolic assay at 24 h of treatment with CBD (30 μM) in non-cancerous cells. Human CD4 + cells were activated with anti-CD3/CD28 antibodies as described in Materials and Methods section. Data (resorufin fluorescence intensity) are normalized to the vehicle-treated control and reported as mean ± SD ( n = 8 in at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; Student’s t -test). e After preincubation with CBD (30 μM, 24 h), non-cancerous CD4 + cells were activated by anti-CD3/antiCD28 antibodies. Cell viability was evaluated by resazurin-based metabolic assay every 24 h. Data (resorufin fluorescence intensity) were normalized to 0 h time point and shown as mean ± SD ( n = 8 in at least three independent experiments). Statistical comparison between control and pretreated samples was undertaken at each time point (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, Student’s t -test). f Migration capacity of Jurkat cells pretreated with CBD (10 or 30 μM, 2 h) was evaluated by chemotactic migration assay, using a Transwell system. Cells were allowed to migrate for 4 h, CXCL12 was used as a chemoattractant. The percentage of migrated cells was determined by cells count in the lower chamber. Data are mean ± SD ( n = 4). Statistical comparison was made with respect to positive control (CXCL12) (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA test). g – i Cell death was evaluation by flow cytometry ( g , h ) and fluorescent microscopy ( I ) in Jurkat cells treated with different concentrations of CBD using Annexin V-AF488/PI double staining. Representative dot plots (2, 4, and 6 h) and images (12 h) are shown in g and i , correspondingly. Scale bar: 20 μm. In every dot plot lower left quadrant represents Annexin V − PI − (DN) live cells, in the lower right quadrant are Annexin V + PI − (early apoptotic) cells, Annexin V − PI + (primary necrotic) cells are in the upper left quadrant, whereas the double-stained population Annexin V + PI + (DP) in the upper right quadrant represents dead cells, which may include necrotic and late apoptotic ones. Statistical analysis of flow cytometry data is given in h ( n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA test)

    Journal: Cell Death & Disease

    Article Title: Cannabidiol directly targets mitochondria and disturbs calcium homeostasis in acute lymphoblastic leukemia

    doi: 10.1038/s41419-019-2024-0

    Figure Lengend Snippet: a Cell viability, evaluated by resazurin-based metabolic assay, as a function of CBD concentration in human leukemic cell lines of different lineages at 24 h of treatment. Cell lines derived from T-ALL (Jurkat, MOLT-3, and CEM), B-ALL (RS4;11 and Reh) and CML (K562) were tested. Data (resorufin fluorescence intensity, arbitrary units) were normalized to the vehicle-treated control and shown as mean ± SD ( n = 8; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant; one-way ANOVA). b Live cell count (trypan blue exclusion test) in long-lasting Jurkat cells cultures exposed to different CBD concentrations (0–100 μM). Left: cell count at 72 h after single CBD administration; right: fresh CBD was added, 50% of medium volume was changed and cells were counted every 24 h. Data are normalized to the initial point (0 h) and shown as mean ± SD ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA test). c Cell viability was evaluated by resazurin-based metabolic assay at 24 h of treatment with CBD (30 μM) in human tumor cell lines of different histogenesis, including CML (K562), B-ALL (Reh and RS4;11), T-ALL (CEM, MOLT-3, and Jurkat), cervical cancer (SiHa and HeLa), and breast cancer (MCF7-7 and MDA-MB-231). Data (resorufin fluorescence intensity) were normalized to vehicle-treated control and reported as mean ± SD ( n = 8 of four independent experiments; (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, Student’s t -test). d Cell viability was evaluated by resazurin-based metabolic assay at 24 h of treatment with CBD (30 μM) in non-cancerous cells. Human CD4 + cells were activated with anti-CD3/CD28 antibodies as described in Materials and Methods section. Data (resorufin fluorescence intensity) are normalized to the vehicle-treated control and reported as mean ± SD ( n = 8 in at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; Student’s t -test). e After preincubation with CBD (30 μM, 24 h), non-cancerous CD4 + cells were activated by anti-CD3/antiCD28 antibodies. Cell viability was evaluated by resazurin-based metabolic assay every 24 h. Data (resorufin fluorescence intensity) were normalized to 0 h time point and shown as mean ± SD ( n = 8 in at least three independent experiments). Statistical comparison between control and pretreated samples was undertaken at each time point (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, Student’s t -test). f Migration capacity of Jurkat cells pretreated with CBD (10 or 30 μM, 2 h) was evaluated by chemotactic migration assay, using a Transwell system. Cells were allowed to migrate for 4 h, CXCL12 was used as a chemoattractant. The percentage of migrated cells was determined by cells count in the lower chamber. Data are mean ± SD ( n = 4). Statistical comparison was made with respect to positive control (CXCL12) (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA test). g – i Cell death was evaluation by flow cytometry ( g , h ) and fluorescent microscopy ( I ) in Jurkat cells treated with different concentrations of CBD using Annexin V-AF488/PI double staining. Representative dot plots (2, 4, and 6 h) and images (12 h) are shown in g and i , correspondingly. Scale bar: 20 μm. In every dot plot lower left quadrant represents Annexin V − PI − (DN) live cells, in the lower right quadrant are Annexin V + PI − (early apoptotic) cells, Annexin V − PI + (primary necrotic) cells are in the upper left quadrant, whereas the double-stained population Annexin V + PI + (DP) in the upper right quadrant represents dead cells, which may include necrotic and late apoptotic ones. Statistical analysis of flow cytometry data is given in h ( n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA test)

    Article Snippet: Leukemic cell lines Jurkat (ATCC®TIB™, Clone E6-1, male, 14 years), MOLT-3 (ATCC®CRL-1552™, male, 19 years), CCFR-CEM (ATCC®CCL-119™, T-ALL, female, 4 years), K562 (ATCC®CCL-243™, female, 53 years), Reh (ATCC®CRL-8286™, female) and RS4;11 (ATCC®CRL-1873™, female, 32 years) purchased from ATCC® (Manassas, VA, USA) were grown in suspension in Advanced RPMI 1640 medium, supplemented with 5% (v/v) of heat-inactivated fetal bovine serum (FBS), 100 U/mL of penicillin, 100 µg/mL streptomycin and 1% of GlutaMAX™ (all from Invitrogene, Carlsbad, CA, USA).

    Techniques: Metabolic Assay, Concentration Assay, Derivative Assay, Fluorescence, Cell Counting, Migration, Positive Control, Flow Cytometry, Microscopy, Double Staining, Staining

    a Monitoring of Δψm loss in CBD-treated leukemic cells. Jurkat cells were double-stained with MtGreen and TMRE, mitochondria (ROI) were selected (upper panel) and intensity of fluorescence was monitored by confocal microscopy after CBD (30 μM) administration (lower panel, n = 36; see also Supplementary movie ). b Intensity of TMRE fluorescence as an indicator of Δψm in Jurkat cells, treated with different concentrations of CBD (0–100 μM) during 10 min. FCCP (10 μM) was used as a positive control. Data are mean ± SD ( n = 8 in three independent experiments). Statistical comparison with control was performed by means of one-way ANOVA test (* p < 0.05; **** p < 0.0001). c EYFP-Cyt-C is localized in mitochondria of Jurkat cells after 12 h of transfection. EYFP-Cyt-C and TMRE fluorescence are colocalized (200 nM TMRE, 200 ng EYFP-Cyt-C, pseudocolor). Images were acquired by confocal microscopy at 12 h after transfection, scale bar: 10 μm. d Representative images of five Jurkat cells, transfected with EYFP-Cyt-C (as in c ). Dense green fluorescent puncta (pseudocolor) reflect Cyt-C localization in intact mitochondria. Scale bar: 10 μm. e Representative images of five Jurkat cells, transfected with EYFP-Cyt-C (as in c ) and treated with CBD (30 μM, 1 h). Cyt-C was released from mitochondria as evidenced by a diffuse EYFP-CytC distribution. Scale bar: 10 μm. f EYFP-Cyt-C distribution in Jurkat cells, expressing EYFP-Cyt-C, at 0, 5, and 20 min with CBD (30 μM). Scale bar: 10 μm. g Caspase-9 and caspase-3 activity in vehicle- and CBD- treated (30 μM, 12 h) Jurkat cells. Staurosporine (STS, 5 μM) was used as a positive control. After incubation, cells were lysed, and caspase activity was determined by colorimetric assay (BioVision). Fold increase in activity compared to control was plotted as mean ± SE (four independent experiments). One-way ANOVA test was performed to compare CBD-treated to control group (* p < 0.05; ** p < 0.01). h Pretreatment with mPTP inhibitor CsA (10 μM) prevents Cyt-C release, induced by CBD (30 μM; 1 h). Compare these images with Fig. 4d, e (vehicle- and CBD-treated cells) and note the protective effect of CsA. Scale bar: 10 μm. i ROS production as evaluated by fluorescent microscopy in DCF-loaded (2 μM) Jurkat cells. CBD effect on ROS production was monitored after 1 h of incubation. PMA (1 μM) was used as a positive control

    Journal: Cell Death & Disease

    Article Title: Cannabidiol directly targets mitochondria and disturbs calcium homeostasis in acute lymphoblastic leukemia

    doi: 10.1038/s41419-019-2024-0

    Figure Lengend Snippet: a Monitoring of Δψm loss in CBD-treated leukemic cells. Jurkat cells were double-stained with MtGreen and TMRE, mitochondria (ROI) were selected (upper panel) and intensity of fluorescence was monitored by confocal microscopy after CBD (30 μM) administration (lower panel, n = 36; see also Supplementary movie ). b Intensity of TMRE fluorescence as an indicator of Δψm in Jurkat cells, treated with different concentrations of CBD (0–100 μM) during 10 min. FCCP (10 μM) was used as a positive control. Data are mean ± SD ( n = 8 in three independent experiments). Statistical comparison with control was performed by means of one-way ANOVA test (* p < 0.05; **** p < 0.0001). c EYFP-Cyt-C is localized in mitochondria of Jurkat cells after 12 h of transfection. EYFP-Cyt-C and TMRE fluorescence are colocalized (200 nM TMRE, 200 ng EYFP-Cyt-C, pseudocolor). Images were acquired by confocal microscopy at 12 h after transfection, scale bar: 10 μm. d Representative images of five Jurkat cells, transfected with EYFP-Cyt-C (as in c ). Dense green fluorescent puncta (pseudocolor) reflect Cyt-C localization in intact mitochondria. Scale bar: 10 μm. e Representative images of five Jurkat cells, transfected with EYFP-Cyt-C (as in c ) and treated with CBD (30 μM, 1 h). Cyt-C was released from mitochondria as evidenced by a diffuse EYFP-CytC distribution. Scale bar: 10 μm. f EYFP-Cyt-C distribution in Jurkat cells, expressing EYFP-Cyt-C, at 0, 5, and 20 min with CBD (30 μM). Scale bar: 10 μm. g Caspase-9 and caspase-3 activity in vehicle- and CBD- treated (30 μM, 12 h) Jurkat cells. Staurosporine (STS, 5 μM) was used as a positive control. After incubation, cells were lysed, and caspase activity was determined by colorimetric assay (BioVision). Fold increase in activity compared to control was plotted as mean ± SE (four independent experiments). One-way ANOVA test was performed to compare CBD-treated to control group (* p < 0.05; ** p < 0.01). h Pretreatment with mPTP inhibitor CsA (10 μM) prevents Cyt-C release, induced by CBD (30 μM; 1 h). Compare these images with Fig. 4d, e (vehicle- and CBD-treated cells) and note the protective effect of CsA. Scale bar: 10 μm. i ROS production as evaluated by fluorescent microscopy in DCF-loaded (2 μM) Jurkat cells. CBD effect on ROS production was monitored after 1 h of incubation. PMA (1 μM) was used as a positive control

    Article Snippet: Leukemic cell lines Jurkat (ATCC®TIB™, Clone E6-1, male, 14 years), MOLT-3 (ATCC®CRL-1552™, male, 19 years), CCFR-CEM (ATCC®CCL-119™, T-ALL, female, 4 years), K562 (ATCC®CCL-243™, female, 53 years), Reh (ATCC®CRL-8286™, female) and RS4;11 (ATCC®CRL-1873™, female, 32 years) purchased from ATCC® (Manassas, VA, USA) were grown in suspension in Advanced RPMI 1640 medium, supplemented with 5% (v/v) of heat-inactivated fetal bovine serum (FBS), 100 U/mL of penicillin, 100 µg/mL streptomycin and 1% of GlutaMAX™ (all from Invitrogene, Carlsbad, CA, USA).

    Techniques: Staining, Fluorescence, Confocal Microscopy, Positive Control, Transfection, Expressing, Activity Assay, Incubation, Colorimetric Assay, Microscopy

    a – c [Ca 2+ ] i monitoring in leukemic cells, treated with CBD. Traces represent the mean ± SD of at least six samples from independent experiments. d Dose dependence of peak [Ca 2+ ] i values in T-ALL cell lines, exposed to CBD. Data are mean ± SD for at least six samples from independent experiments. e , f [Ca 2+ ] i monitoring in non-cancerous T cells in resting ( e ) and activated ( f ) states. Traces represent the mean ± SD of at least six samples from independent experiments. g Pharmacological analysis of [Ca 2+ ] i rise in response to CBD. Before CBD treatment, cells were preincubated over 20 min with either CB1 antagonist, rimonabant (1 μM), CB2 inverse agonist, AM630 (2 μM), or GPR55 antagonist, CID16020046 (3 μM); values Δ [Ca 2+ ] i were obtained by subtracting the [Ca 2+ ] i baseline level from [Ca 2+ ] i maximum increase. Data are mean ± SD of at least 6 samples from independent experiments. h [Ca 2+ ] i monitoring in Jurkat cells, suspended in Ca 2+ -free HBSS. Traces represent the mean ± SD of at least six samples from independent experiments. i , j Effect of Gd 3+ and RR, non-selective blockers of plasma membrane Ca 2+ - permeable channels, on [Ca 2+ ] i ( i and j , left) and cell viability evaluated by resazurin-based metabolic assay ( i and j , right). Gd 3+ (1 μM) or RR (1 μM) were added to Jurkat cells 20 min before the [Ca 2+ ] i measurement. i , j left: traces represent the mean ± SD of at least six samples from independent experiments. i , j right: data (resorufin fluorescence intensity) were normalized to vehicle-treated control and reported as mean ± SD ( n = 8 of four independent experiments (**** p < 0.0001, Student’s t -test)

    Journal: Cell Death & Disease

    Article Title: Cannabidiol directly targets mitochondria and disturbs calcium homeostasis in acute lymphoblastic leukemia

    doi: 10.1038/s41419-019-2024-0

    Figure Lengend Snippet: a – c [Ca 2+ ] i monitoring in leukemic cells, treated with CBD. Traces represent the mean ± SD of at least six samples from independent experiments. d Dose dependence of peak [Ca 2+ ] i values in T-ALL cell lines, exposed to CBD. Data are mean ± SD for at least six samples from independent experiments. e , f [Ca 2+ ] i monitoring in non-cancerous T cells in resting ( e ) and activated ( f ) states. Traces represent the mean ± SD of at least six samples from independent experiments. g Pharmacological analysis of [Ca 2+ ] i rise in response to CBD. Before CBD treatment, cells were preincubated over 20 min with either CB1 antagonist, rimonabant (1 μM), CB2 inverse agonist, AM630 (2 μM), or GPR55 antagonist, CID16020046 (3 μM); values Δ [Ca 2+ ] i were obtained by subtracting the [Ca 2+ ] i baseline level from [Ca 2+ ] i maximum increase. Data are mean ± SD of at least 6 samples from independent experiments. h [Ca 2+ ] i monitoring in Jurkat cells, suspended in Ca 2+ -free HBSS. Traces represent the mean ± SD of at least six samples from independent experiments. i , j Effect of Gd 3+ and RR, non-selective blockers of plasma membrane Ca 2+ - permeable channels, on [Ca 2+ ] i ( i and j , left) and cell viability evaluated by resazurin-based metabolic assay ( i and j , right). Gd 3+ (1 μM) or RR (1 μM) were added to Jurkat cells 20 min before the [Ca 2+ ] i measurement. i , j left: traces represent the mean ± SD of at least six samples from independent experiments. i , j right: data (resorufin fluorescence intensity) were normalized to vehicle-treated control and reported as mean ± SD ( n = 8 of four independent experiments (**** p < 0.0001, Student’s t -test)

    Article Snippet: Leukemic cell lines Jurkat (ATCC®TIB™, Clone E6-1, male, 14 years), MOLT-3 (ATCC®CRL-1552™, male, 19 years), CCFR-CEM (ATCC®CCL-119™, T-ALL, female, 4 years), K562 (ATCC®CCL-243™, female, 53 years), Reh (ATCC®CRL-8286™, female) and RS4;11 (ATCC®CRL-1873™, female, 32 years) purchased from ATCC® (Manassas, VA, USA) were grown in suspension in Advanced RPMI 1640 medium, supplemented with 5% (v/v) of heat-inactivated fetal bovine serum (FBS), 100 U/mL of penicillin, 100 µg/mL streptomycin and 1% of GlutaMAX™ (all from Invitrogene, Carlsbad, CA, USA).

    Techniques: Metabolic Assay, Fluorescence

    Comparison of CD8 + T‐cell infiltration and CCL5, CCND1, and PDE5 expression in tissues of finasteride‐treated and untreated BPH patients. A,B, Infiltration of CD8 + T cells and expression of CCL5, CCND1, and PDE5 were analyzed by IHC staining of serial paraffin sections from prostate tissues of BPH patients treated or not with finasteride for at least 6 months. C, Heat map showing the distribution of CD8 + T cells and CCL5, CCND1, and PDE5 expression in the finasteride‐treated and untreated groups. BPH, benign prostatic hyperplasia; IHC, immunohistochemistry; PDE5, phosphodiesterase type 5 [Color figure can be viewed at wileyonlinelibrary.com]

    Journal: The Prostate

    Article Title: Activation of cGMP/PKG/p65 signaling associated with PDE5‐Is downregulates CCL5 secretion by CD8 + T cells in benign prostatic hyperplasia

    doi: 10.1002/pros.23801

    Figure Lengend Snippet: Comparison of CD8 + T‐cell infiltration and CCL5, CCND1, and PDE5 expression in tissues of finasteride‐treated and untreated BPH patients. A,B, Infiltration of CD8 + T cells and expression of CCL5, CCND1, and PDE5 were analyzed by IHC staining of serial paraffin sections from prostate tissues of BPH patients treated or not with finasteride for at least 6 months. C, Heat map showing the distribution of CD8 + T cells and CCL5, CCND1, and PDE5 expression in the finasteride‐treated and untreated groups. BPH, benign prostatic hyperplasia; IHC, immunohistochemistry; PDE5, phosphodiesterase type 5 [Color figure can be viewed at wileyonlinelibrary.com]

    Article Snippet: BPH epithelial cell line BPH‐1 (KG1008; KeyGen Biotech Co, Ltd, Nanjing, China) and CD8 + T lymphocytic cell line Molt‐3 (CRL‐1552; American Type Culture Collection, Rockville, MD) were grown in Rosewell Park Memorial Institute (RPMI)‐1640 medium (SH30809.01B; HyClone, South Logan, UT) supplemented with 1% penicillin G, 1% streptomycin, and 10% fetal bovine serum (FBS) in a humidified incubator at the 5% CO 2 atmosphere and 37°C.

    Techniques: Expressing, Immunohistochemistry

    Activation of the cGMP/PKG signaling pathway could reverse the induction of BEC proliferation by CD8 + T cells through suppressing the secretion of CCL5 by CD8 + T cells in low androgen conditions. BECs were cocultured with or without Molt‐3 cells in low androgen conditions for 4 days. A,C, BECs cultured with rhCCL5 (1 μg/mL) and tadalafil (100 nM) or Sp‐8‐Br‐PET‐cGMP (10 μM) were analyzed for proliferation at days 2, 4, and 6 using the CCK‐8 assay. The data are shown as the mean ± SD; * P < 0.05 and ** P < 0.01. B,D, BECs were harvested at day 4 and analyzed for CCND1 expression by Western blot analysis; β‐Tubulin was used as a loading control. E‐G, CCL5 mRNA expression in Molt‐3 cells at day 2 was analyzed by qPCR. CCL5 mRNA levels were downregulated by tadalafil (100 nM) or Sp‐8‐Br‐PET‐cGMP (10 μM) in Molt‐3 cells cocultured with BECs, but KT5823 (100 nM) reversed the effect. H, CCL5 secretion to conditioned medium of cocultures and monocultures treated or not with tadalafil and/or KT5823 at day 4 was assessed by ELISA. I,J, BPH‐1 cells treated or not with tadalafil or Sp‐8‐Br‐PET‐cGMP, anti‐CCL5 neutralizing antibody (2 μg/mL), and KT5823 were analyzed for proliferation at days 2, 4, and 6 using the CCK‐8 assay. Data are shown as the mean ± SD; * P < 0.05 and ** P < 0.01. BEC, BPH epithelial cell; BPH, benign prostatic hyperplasia; CCK‐8, cell counting kit‐8; cGMP, cyclic guanosine monophosphate; ELISA, enzyme‐linked immunosorbent assay; mRNA, messenger RNA; PKG, protein kinase G; qPCR, quantitative polymerase chain reaction, TDF, tadalafil [Color figure can be viewed at wileyonlinelibrary.com]

    Journal: The Prostate

    Article Title: Activation of cGMP/PKG/p65 signaling associated with PDE5‐Is downregulates CCL5 secretion by CD8 + T cells in benign prostatic hyperplasia

    doi: 10.1002/pros.23801

    Figure Lengend Snippet: Activation of the cGMP/PKG signaling pathway could reverse the induction of BEC proliferation by CD8 + T cells through suppressing the secretion of CCL5 by CD8 + T cells in low androgen conditions. BECs were cocultured with or without Molt‐3 cells in low androgen conditions for 4 days. A,C, BECs cultured with rhCCL5 (1 μg/mL) and tadalafil (100 nM) or Sp‐8‐Br‐PET‐cGMP (10 μM) were analyzed for proliferation at days 2, 4, and 6 using the CCK‐8 assay. The data are shown as the mean ± SD; * P < 0.05 and ** P < 0.01. B,D, BECs were harvested at day 4 and analyzed for CCND1 expression by Western blot analysis; β‐Tubulin was used as a loading control. E‐G, CCL5 mRNA expression in Molt‐3 cells at day 2 was analyzed by qPCR. CCL5 mRNA levels were downregulated by tadalafil (100 nM) or Sp‐8‐Br‐PET‐cGMP (10 μM) in Molt‐3 cells cocultured with BECs, but KT5823 (100 nM) reversed the effect. H, CCL5 secretion to conditioned medium of cocultures and monocultures treated or not with tadalafil and/or KT5823 at day 4 was assessed by ELISA. I,J, BPH‐1 cells treated or not with tadalafil or Sp‐8‐Br‐PET‐cGMP, anti‐CCL5 neutralizing antibody (2 μg/mL), and KT5823 were analyzed for proliferation at days 2, 4, and 6 using the CCK‐8 assay. Data are shown as the mean ± SD; * P < 0.05 and ** P < 0.01. BEC, BPH epithelial cell; BPH, benign prostatic hyperplasia; CCK‐8, cell counting kit‐8; cGMP, cyclic guanosine monophosphate; ELISA, enzyme‐linked immunosorbent assay; mRNA, messenger RNA; PKG, protein kinase G; qPCR, quantitative polymerase chain reaction, TDF, tadalafil [Color figure can be viewed at wileyonlinelibrary.com]

    Article Snippet: BPH epithelial cell line BPH‐1 (KG1008; KeyGen Biotech Co, Ltd, Nanjing, China) and CD8 + T lymphocytic cell line Molt‐3 (CRL‐1552; American Type Culture Collection, Rockville, MD) were grown in Rosewell Park Memorial Institute (RPMI)‐1640 medium (SH30809.01B; HyClone, South Logan, UT) supplemented with 1% penicillin G, 1% streptomycin, and 10% fetal bovine serum (FBS) in a humidified incubator at the 5% CO 2 atmosphere and 37°C.

    Techniques: Activation Assay, Cell Culture, CCK-8 Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Counting, Real-time Polymerase Chain Reaction

    Expression of signaling molecules downstream of the cGMP/PKG pathway involved in the inhibition of BEC proliferation in low androgen conditions. Cocultures of BECs and Molt‐3 cells were treated with tadalafil (A) or Sp‐8‐Br‐PET‐cGMP (B) with or without KT5823 for 1 and 2 hours. BECs and Molt‐3 cells were harvested separately and analyzed by Western blot analysis for total NF‐κB and phospho‐NF‐κB (Molt‐3 cells) and total STAT5, phospho‐STAT5, and CCND1 (BPH‐1 cells). β‐Tubulin and GAPDH were used as loading controls. BEC, BPH epithelial cell; BPH, benign prostatic hyperplasia; cGMP, cyclic guanosine monophosphate; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; NF‐κB, nuclear factor‐κB; PKG, protein kinase G; STAT5, signal transducer and activator of transcription 5; TDF, tadalafil

    Journal: The Prostate

    Article Title: Activation of cGMP/PKG/p65 signaling associated with PDE5‐Is downregulates CCL5 secretion by CD8 + T cells in benign prostatic hyperplasia

    doi: 10.1002/pros.23801

    Figure Lengend Snippet: Expression of signaling molecules downstream of the cGMP/PKG pathway involved in the inhibition of BEC proliferation in low androgen conditions. Cocultures of BECs and Molt‐3 cells were treated with tadalafil (A) or Sp‐8‐Br‐PET‐cGMP (B) with or without KT5823 for 1 and 2 hours. BECs and Molt‐3 cells were harvested separately and analyzed by Western blot analysis for total NF‐κB and phospho‐NF‐κB (Molt‐3 cells) and total STAT5, phospho‐STAT5, and CCND1 (BPH‐1 cells). β‐Tubulin and GAPDH were used as loading controls. BEC, BPH epithelial cell; BPH, benign prostatic hyperplasia; cGMP, cyclic guanosine monophosphate; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; NF‐κB, nuclear factor‐κB; PKG, protein kinase G; STAT5, signal transducer and activator of transcription 5; TDF, tadalafil

    Article Snippet: BPH epithelial cell line BPH‐1 (KG1008; KeyGen Biotech Co, Ltd, Nanjing, China) and CD8 + T lymphocytic cell line Molt‐3 (CRL‐1552; American Type Culture Collection, Rockville, MD) were grown in Rosewell Park Memorial Institute (RPMI)‐1640 medium (SH30809.01B; HyClone, South Logan, UT) supplemented with 1% penicillin G, 1% streptomycin, and 10% fetal bovine serum (FBS) in a humidified incubator at the 5% CO 2 atmosphere and 37°C.

    Techniques: Expressing, Inhibition, Western Blot

    Activation of cGMP/PKG signaling suppressed CCL5 secretion by CD8 + T cells and reversed the induction of BEC proliferation in vivo. A, The degree of prostatic hyperplasia in rats receiving regular diet (RD), HFD, or HFD + PDE5‐Is was evaluated by hematoxylin and eosin staining of rat prostate samples ( n = 6 rats per group). B, The serum testosterone levels of RD and HFD groups were measured using an automated chemiluminescence system at week 12; * P < 0.05, ** P < 0.01, and *** P < 0.001 (by t test). C, All rat prostate weight of three groups were tested at last; * P < 0.05, ** P < 0.01, and *** P < 0.001 (by one‐way ANOVA). D, CD8 + T‐cell infiltration and CCL5, CCND1, and PDE5 expression was analyzed by IHC staining in serial paraffin sections of the rat prostate. ANOVA, analysis of variance; BEC, BPH epithelial cell; cGMP, cyclic guanosine monophosphate; HFD, high‐fat diet; IHC, immunohistochemistry; PDE5, phosphodiesterase type 5; PKG, protein kinase G; RD, regular diet [Color figure can be viewed at wileyonlinelibrary.com]

    Journal: The Prostate

    Article Title: Activation of cGMP/PKG/p65 signaling associated with PDE5‐Is downregulates CCL5 secretion by CD8 + T cells in benign prostatic hyperplasia

    doi: 10.1002/pros.23801

    Figure Lengend Snippet: Activation of cGMP/PKG signaling suppressed CCL5 secretion by CD8 + T cells and reversed the induction of BEC proliferation in vivo. A, The degree of prostatic hyperplasia in rats receiving regular diet (RD), HFD, or HFD + PDE5‐Is was evaluated by hematoxylin and eosin staining of rat prostate samples ( n = 6 rats per group). B, The serum testosterone levels of RD and HFD groups were measured using an automated chemiluminescence system at week 12; * P < 0.05, ** P < 0.01, and *** P < 0.001 (by t test). C, All rat prostate weight of three groups were tested at last; * P < 0.05, ** P < 0.01, and *** P < 0.001 (by one‐way ANOVA). D, CD8 + T‐cell infiltration and CCL5, CCND1, and PDE5 expression was analyzed by IHC staining in serial paraffin sections of the rat prostate. ANOVA, analysis of variance; BEC, BPH epithelial cell; cGMP, cyclic guanosine monophosphate; HFD, high‐fat diet; IHC, immunohistochemistry; PDE5, phosphodiesterase type 5; PKG, protein kinase G; RD, regular diet [Color figure can be viewed at wileyonlinelibrary.com]

    Article Snippet: BPH epithelial cell line BPH‐1 (KG1008; KeyGen Biotech Co, Ltd, Nanjing, China) and CD8 + T lymphocytic cell line Molt‐3 (CRL‐1552; American Type Culture Collection, Rockville, MD) were grown in Rosewell Park Memorial Institute (RPMI)‐1640 medium (SH30809.01B; HyClone, South Logan, UT) supplemented with 1% penicillin G, 1% streptomycin, and 10% fetal bovine serum (FBS) in a humidified incubator at the 5% CO 2 atmosphere and 37°C.

    Techniques: Activation Assay, In Vivo, Staining, Expressing, Immunohistochemistry

    Schematic representation of the molecular mechanism underlying BEC proliferation in BPH. Activation of the cGMP/PKG signaling pathway in CD8 + T cells decreased NF‐κB phosphorylation and CCL5 secretion, resulting in the inhibition of CCL5/STAT5/CCND1 signaling in BECs and decrease of their proliferation in low androgen conditions. BEC, BPH epithelial cell; BPH, benign prostatic hyperplasia; cGMP, cyclic guanosine monophosphate; NF‐κB, nuclear factor‐κB; PDE5, phosphodiesterase type 5; PDE5‐I, PDE5 inhibitors; PKG, protein kinase G; STAT5, signal transducer and activator of transcription 5 [Color figure can be viewed at wileyonlinelibrary.com]

    Journal: The Prostate

    Article Title: Activation of cGMP/PKG/p65 signaling associated with PDE5‐Is downregulates CCL5 secretion by CD8 + T cells in benign prostatic hyperplasia

    doi: 10.1002/pros.23801

    Figure Lengend Snippet: Schematic representation of the molecular mechanism underlying BEC proliferation in BPH. Activation of the cGMP/PKG signaling pathway in CD8 + T cells decreased NF‐κB phosphorylation and CCL5 secretion, resulting in the inhibition of CCL5/STAT5/CCND1 signaling in BECs and decrease of their proliferation in low androgen conditions. BEC, BPH epithelial cell; BPH, benign prostatic hyperplasia; cGMP, cyclic guanosine monophosphate; NF‐κB, nuclear factor‐κB; PDE5, phosphodiesterase type 5; PDE5‐I, PDE5 inhibitors; PKG, protein kinase G; STAT5, signal transducer and activator of transcription 5 [Color figure can be viewed at wileyonlinelibrary.com]

    Article Snippet: BPH epithelial cell line BPH‐1 (KG1008; KeyGen Biotech Co, Ltd, Nanjing, China) and CD8 + T lymphocytic cell line Molt‐3 (CRL‐1552; American Type Culture Collection, Rockville, MD) were grown in Rosewell Park Memorial Institute (RPMI)‐1640 medium (SH30809.01B; HyClone, South Logan, UT) supplemented with 1% penicillin G, 1% streptomycin, and 10% fetal bovine serum (FBS) in a humidified incubator at the 5% CO 2 atmosphere and 37°C.

    Techniques: Activation Assay, Inhibition