caspase-3 Search Results


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  • 93
    Millipore caspase 3
    ANDV nucleocapsid protein inhibits caspase 3-activity. Analysis of possible interactions between ANDV nucleocapsid protein and caspase 3. ( A ) Western blot analyses showing a cleaved fragment of the ANDV nucleocapsid protein (ANDV-N) after incubation with recombinant <t>caspase</t> 3 in the presence or absence of the caspase 3-inhibitor DEVD-CHO. Full-length and cleaved ANDV-N was visualized with the mAb 7B3/F7. One representative experiment out of three is shown. ( B ) ANDV-infected A549 cells were left untreated or treated with staurosporine, in the presence or absence of the caspase-inhibitor Z-VAD-fmk. Lysed cells were then subjected to Western blot analyses to visualize cleavage of the viral ANDV nucleocapsid protein (ANDV-N) after staurosporine-treatment. Full-length and cleaved ANDV-N was visualized with the mAb 7B3/F7. One representative experiment out of three is shown. ( C ) Caspase 3-activity was measured after pre-incubation of recombinant caspase 3 with recombinant ANDV nucleocapsid protein (rANDV-N) or with control protein (rDHFR) for 30 minutes. Level of caspase 3-activity after co-incubation with rDHFRS represent maximal caspase 3 activity. Level of caspase 3-activity after co-incubation of caspase 3 with rANDV-N was compared with caspase 3-activity after co-incubation with rDHFRS. Data shown are mean ± SEM of three independent experiments carried out in duplicate. Two-tailed Student's t test was used for statistical evaluation; * p
    Caspase 3, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 2538 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore caspase 3 casp3 antibody
    Lower levels of activated caspase 3 activity correlate with better discrimination learning. a ) Parallel relationship between cognitive scores (errors in reversal learning) and abundance of cells expressing activated caspase 3 in each animal  b ) Caspase 3 expression is directly correlated with errors in reversal learning. Pearson analysis r = 0.76, p
    Caspase 3 Casp3 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore caspase 3 7
    Src activity is required for survival and growth of 5637 cells in serum-free conditions. ( A ) 5637 cells were transfected with a control plasmid or plasmid expressing FLAG-tagged kinase-negative Src (KN-Src) or FLAG-tagged wild-type Src (WT-Src) as described in   Materials and Methods . After the transfection, cells were cultured in either serum-containing medium (FCS +) or serum-free medium (FCS −) for 24 h. Triton X-100-solubilized cell extracts were prepared and analyzed by immunoprecipitation (IP, 300 µg/lane) and/or immunoblotting (IB, 30 µg/lane) with the indicated antibodies as described in   Materials and Methods . The positions of the tyrosine-phosphorylated forms or the total proteins of p145 met , FLAG-tagged or endogenous Src, and tubulin are indicated. ( B ) 5637 cells (0.1×10 6  cells/dish) were cultured in serum-containing conditions for 24 h (○), transfected with a control plasmid ( ), KN-Src (▴), or WT-Src (▪) for 24 h as in panel A, and then cultured in serum-containing (FCS +) or serum-free medium (FCS −) for 48 h. Cell number was determined at 24, 48, 72 and 96 hours of incubation. ( C ) 5637 cells were treated as in panel B. After the treatments (96 h post incubation), cell death (black bars, shown as percentage of total cells) and caspase 3/7 protease activity of Triton X-100-solubilized cell extracts (20 µg/assay) (grey bars, shown as arbitrary unit) were determined by Trypan Blue exclusion and a synthetic substrate Ac-DEVD-AMC, respectively, as described in   Materials and Methods . Data shown are mean ± s.d. of three independent experiments. * P
    Caspase 3 7, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore caspase 3 activity
    Ablation of Mcl-1 does not result in activation of apoptosis. ( A ) Quantitation of TUNEL positive nuclei in heart sections. n = 3–4. ( B ) Quantitation of myocytes positive for cleaved <t>caspase-3</t> in heart sections. n = 3–4. ( C ) Caspase-3 activity
    Caspase 3 Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1989 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc casp3 caspase 3
    Increased apoptosis in KO myotubes. ( A ) Western blot of total lysates (top) and nuclear and mitochondrial fractions of WT and KO myotubes grown in differentiation medium for 6 to 7 d. No changes in the levels of activated (cleaved) <t>CASP3</t> products are
    Casp3 Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    WuXi AppTec caspase3
    C23 suppressed the transcriptional activity of p53 upon DNA damage and hypoxia A. pGL3-3×p53-BS-LUC and Renilla were co-transfected in HCT116 cells combined with and without C23 shRNA. 24 hours after transfection, cells were treated with Doxorubicin (100ng/ml) or upon 1% O 2 for indicated times and the cell lysates were analyzed by lucifease assay. B. HCT116 cells with and without stable overexpressing C23 were co-transfected with pGL3-3×p53-BS-LUC and Renilla. 24 hours after transfection, cells were treated with Doxorubicin (100ng/ml) or upon 1% O 2 for indicated times and then the cell lysates were subjected to lucifease assays. C. HCT116 cells with and without stable knockdown of C23 were treated with Doxorubicin (100ng/ml) for 24 hours, then p53 target gene mRNA expression levels were analyzed by qRT-PCR analysis. D-E. HCT116 cells with and without stable overexpressing C23 (a) or stable knockdown of C23(b) were treated with Doxorubicin (100ng/ml) or mock control for 24 hours. Cell lysates were then subjected to Western blot analysis with the indicated antibodies (D) and <t>caspase3/7</t> activity analysis (E), respectively. F. (a) HCT116 cells stably expressing the control shRNA, C23 shRNA, p53 shRNA,or C23 shRNA plus p53 shRNA. (b) HCT116 cells with and without p53 knockdown were introduced with C23 respectively. Protein level of C23 and p53 were evaluated by Western blot. GAPDH served as loading control. G. HCT116 cells stably expressing the control shRNA, C23 shRNA, p53 shRNA,or C23 shRNA plus p53 shRNA (a) and HCT116 cells stably knocked down p53 were additionally introduced with C23 respectively (b). Cell number was evaluated by cell counter. The data were represented as mean±S.D. from three independent experiments. H. (a) HCT116 cells stably expressing the control shRNA, C23 shRNA, p53 shRNA,or C23 shRNA plus p53 shRNA. (b) HCT116 cells stably knocked down p53 were co-transfected with C23 cDNA respectively. Cells were then treated with 100ng/ml Doxorubicin for 24 hours. Cell viability was determined by MTT assay. The data were shown as the mean±s.d. of three independent experiments.
    Caspase3, supplied by WuXi AppTec, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore caspase 3 enzyme
    C23 suppressed the transcriptional activity of p53 upon DNA damage and hypoxia A. pGL3-3×p53-BS-LUC and Renilla were co-transfected in HCT116 cells combined with and without C23 shRNA. 24 hours after transfection, cells were treated with Doxorubicin (100ng/ml) or upon 1% O 2 for indicated times and the cell lysates were analyzed by lucifease assay. B. HCT116 cells with and without stable overexpressing C23 were co-transfected with pGL3-3×p53-BS-LUC and Renilla. 24 hours after transfection, cells were treated with Doxorubicin (100ng/ml) or upon 1% O 2 for indicated times and then the cell lysates were subjected to lucifease assays. C. HCT116 cells with and without stable knockdown of C23 were treated with Doxorubicin (100ng/ml) for 24 hours, then p53 target gene mRNA expression levels were analyzed by qRT-PCR analysis. D-E. HCT116 cells with and without stable overexpressing C23 (a) or stable knockdown of C23(b) were treated with Doxorubicin (100ng/ml) or mock control for 24 hours. Cell lysates were then subjected to Western blot analysis with the indicated antibodies (D) and <t>caspase3/7</t> activity analysis (E), respectively. F. (a) HCT116 cells stably expressing the control shRNA, C23 shRNA, p53 shRNA,or C23 shRNA plus p53 shRNA. (b) HCT116 cells with and without p53 knockdown were introduced with C23 respectively. Protein level of C23 and p53 were evaluated by Western blot. GAPDH served as loading control. G. HCT116 cells stably expressing the control shRNA, C23 shRNA, p53 shRNA,or C23 shRNA plus p53 shRNA (a) and HCT116 cells stably knocked down p53 were additionally introduced with C23 respectively (b). Cell number was evaluated by cell counter. The data were represented as mean±S.D. from three independent experiments. H. (a) HCT116 cells stably expressing the control shRNA, C23 shRNA, p53 shRNA,or C23 shRNA plus p53 shRNA. (b) HCT116 cells stably knocked down p53 were co-transfected with C23 cDNA respectively. Cells were then treated with 100ng/ml Doxorubicin for 24 hours. Cell viability was determined by MTT assay. The data were shown as the mean±s.d. of three independent experiments.
    Caspase 3 Enzyme, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    WuXi AppTec caspase 3
    Effect of almorexant and suvorexant on intracellular Ca 2+ release, cell growth and <t>caspase-3</t> activity ( A ) Intracellular Ca 2+ production was detected in HEK-293 cells expressing recombinant native OX1R using Fluoforte Calcium Assay Kit (Enzo Life Sciences, NY, USA). Cells were challenged with 1 μM of OxA (top panel) or 1 μM OxA after preincubation with 10 μM suvorexant (middle panel) or 10 μM almorexant (bottom panel). ( B ) AsPC-1 cells were incubated for 48 h with OxA or almorexant or suvorexant in the presence (right panel) or in the absence (left panel) of 50 μM NSC87877. ( C ) colorometric Caspase-3 activity detection at 405 nm in AsPC-1 cells incubated with 1 μM OxA or 1 μM almorexant in the presence or in the absence of 50 μM NSC87877. * p
    Caspase 3, supplied by WuXi AppTec, used in various techniques. Bioz Stars score: 92/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc casp3
    Validation of protein and mRNA level dysregulations. (A) Quantitative immunoblots confirmed neuronal loss (marker NeuN), astrogliosis (marker GFAP) and microgliosis (marker IBA1) to occur in Atxn2 -CAG100-KIN spinal cord at the preterminal stage of 14 months age, but not at the early KIN stage of 3 months age and in the Atxn2 -KO at 6 months. Significantly increased levels in KIN at 14 months were also shown for TDP43 and the factor responsible for its cleavage, <t>CASP3.</t> (B) Quantitative RT-PCR analyses showed a significant deficit of NeuN transcript ( Rbfox3 ) already at incipient disease stage in 3-month-old KIN, whereas astrogliosis (marker Gfap ) and microgliosis (marker IBA1 transcript Aif1 ) became significant at late state. Protein abundance (C) and transcript levels (D) were also documented for PGRN (encoded by Grn mRNA) as molecular marker of lysosomal activation and atrophy, as well as RIPK1 as molecular marker of RNA-toxicity and necroptosis. Again, a significant elevation of Grn mRNA at the age of 3 months suggested atrophy and lysosomal breakdown to occur in parallel with first locomotor deficits, predating necroptotic cell death.
    Casp3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 437 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ANDV nucleocapsid protein inhibits caspase 3-activity. Analysis of possible interactions between ANDV nucleocapsid protein and caspase 3. ( A ) Western blot analyses showing a cleaved fragment of the ANDV nucleocapsid protein (ANDV-N) after incubation with recombinant caspase 3 in the presence or absence of the caspase 3-inhibitor DEVD-CHO. Full-length and cleaved ANDV-N was visualized with the mAb 7B3/F7. One representative experiment out of three is shown. ( B ) ANDV-infected A549 cells were left untreated or treated with staurosporine, in the presence or absence of the caspase-inhibitor Z-VAD-fmk. Lysed cells were then subjected to Western blot analyses to visualize cleavage of the viral ANDV nucleocapsid protein (ANDV-N) after staurosporine-treatment. Full-length and cleaved ANDV-N was visualized with the mAb 7B3/F7. One representative experiment out of three is shown. ( C ) Caspase 3-activity was measured after pre-incubation of recombinant caspase 3 with recombinant ANDV nucleocapsid protein (rANDV-N) or with control protein (rDHFR) for 30 minutes. Level of caspase 3-activity after co-incubation with rDHFRS represent maximal caspase 3 activity. Level of caspase 3-activity after co-incubation of caspase 3 with rANDV-N was compared with caspase 3-activity after co-incubation with rDHFRS. Data shown are mean ± SEM of three independent experiments carried out in duplicate. Two-tailed Student's t test was used for statistical evaluation; * p

    Journal: PLoS Pathogens

    Article Title: Hantavirus-infection Confers Resistance to Cytotoxic Lymphocyte-Mediated Apoptosis

    doi: 10.1371/journal.ppat.1003272

    Figure Lengend Snippet: ANDV nucleocapsid protein inhibits caspase 3-activity. Analysis of possible interactions between ANDV nucleocapsid protein and caspase 3. ( A ) Western blot analyses showing a cleaved fragment of the ANDV nucleocapsid protein (ANDV-N) after incubation with recombinant caspase 3 in the presence or absence of the caspase 3-inhibitor DEVD-CHO. Full-length and cleaved ANDV-N was visualized with the mAb 7B3/F7. One representative experiment out of three is shown. ( B ) ANDV-infected A549 cells were left untreated or treated with staurosporine, in the presence or absence of the caspase-inhibitor Z-VAD-fmk. Lysed cells were then subjected to Western blot analyses to visualize cleavage of the viral ANDV nucleocapsid protein (ANDV-N) after staurosporine-treatment. Full-length and cleaved ANDV-N was visualized with the mAb 7B3/F7. One representative experiment out of three is shown. ( C ) Caspase 3-activity was measured after pre-incubation of recombinant caspase 3 with recombinant ANDV nucleocapsid protein (rANDV-N) or with control protein (rDHFR) for 30 minutes. Level of caspase 3-activity after co-incubation with rDHFRS represent maximal caspase 3 activity. Level of caspase 3-activity after co-incubation of caspase 3 with rANDV-N was compared with caspase 3-activity after co-incubation with rDHFRS. Data shown are mean ± SEM of three independent experiments carried out in duplicate. Two-tailed Student's t test was used for statistical evaluation; * p

    Article Snippet: To analyze nucleocapsid protein-specific inhibition of caspase 3 and granzyme B activity, recombinant human caspase 3 (0.1 µg) or active recombinant granzyme B (0.1 µg) was incubated with 1 µg recombinant ANDV nucleocapsid protein or 1 µg rDHFR as control, for 30 minutes or as stated in the text.

    Techniques: Activity Assay, Western Blot, Incubation, Recombinant, Infection, Two Tailed Test

    Hantavirus-infection inhibits NK cell-mediated activation of caspase 3 in endothelial cells. Prevention of caspase 3 activation in infected cells exposed to IL-2-activated NK cells. ( A ) Representative flow cytometry histogram showing cellular caspase 3-activity in uninfected and HTNV-infected HLA class I blocked endothelial cells with and without co-incubation with IL-2-activated NK cells. Data shown is one representative donor out of six. ( B ) Percentage of caspase 3-positive uninfected and HTNV-infected endothelial cells after co-incubation with IL-2-activated NK cells analyzed by flow cytometry. Data shown represent two independent experiments from six donors. Two-tailed Student's t test was used for statistical evaluation; ** p

    Journal: PLoS Pathogens

    Article Title: Hantavirus-infection Confers Resistance to Cytotoxic Lymphocyte-Mediated Apoptosis

    doi: 10.1371/journal.ppat.1003272

    Figure Lengend Snippet: Hantavirus-infection inhibits NK cell-mediated activation of caspase 3 in endothelial cells. Prevention of caspase 3 activation in infected cells exposed to IL-2-activated NK cells. ( A ) Representative flow cytometry histogram showing cellular caspase 3-activity in uninfected and HTNV-infected HLA class I blocked endothelial cells with and without co-incubation with IL-2-activated NK cells. Data shown is one representative donor out of six. ( B ) Percentage of caspase 3-positive uninfected and HTNV-infected endothelial cells after co-incubation with IL-2-activated NK cells analyzed by flow cytometry. Data shown represent two independent experiments from six donors. Two-tailed Student's t test was used for statistical evaluation; ** p

    Article Snippet: To analyze nucleocapsid protein-specific inhibition of caspase 3 and granzyme B activity, recombinant human caspase 3 (0.1 µg) or active recombinant granzyme B (0.1 µg) was incubated with 1 µg recombinant ANDV nucleocapsid protein or 1 µg rDHFR as control, for 30 minutes or as stated in the text.

    Techniques: Infection, Activation Assay, Flow Cytometry, Cytometry, Activity Assay, Incubation, Two Tailed Test

    Lower levels of activated caspase 3 activity correlate with better discrimination learning. a ) Parallel relationship between cognitive scores (errors in reversal learning) and abundance of cells expressing activated caspase 3 in each animal  b ) Caspase 3 expression is directly correlated with errors in reversal learning. Pearson analysis r = 0.76, p

    Journal: PLoS ONE

    Article Title: Dietary and Behavioral Interventions Protect against Age Related Activation of Caspase Cascades in the Canine Brain

    doi: 10.1371/journal.pone.0024652

    Figure Lengend Snippet: Lower levels of activated caspase 3 activity correlate with better discrimination learning. a ) Parallel relationship between cognitive scores (errors in reversal learning) and abundance of cells expressing activated caspase 3 in each animal b ) Caspase 3 expression is directly correlated with errors in reversal learning. Pearson analysis r = 0.76, p

    Article Snippet: Specifically, wells were coated with 1 µl of active caspase-3 protease (Chemicon) and incubated with frontal cortex homogenates (1 µl, 4 ug/ul) from 4 aged beagles at 37°C for 1 hour.

    Techniques: Activity Assay, Expressing

    Double labeling with caspase 3 and neuronal or glial markers. a ) Double labeling of activated caspase 3 with the neuronal marker NeuN and b ) the glial marker GFAP revealed that the majority of cells positive for activated caspase-3 were also NeuN positive.

    Journal: PLoS ONE

    Article Title: Dietary and Behavioral Interventions Protect against Age Related Activation of Caspase Cascades in the Canine Brain

    doi: 10.1371/journal.pone.0024652

    Figure Lengend Snippet: Double labeling with caspase 3 and neuronal or glial markers. a ) Double labeling of activated caspase 3 with the neuronal marker NeuN and b ) the glial marker GFAP revealed that the majority of cells positive for activated caspase-3 were also NeuN positive.

    Article Snippet: Specifically, wells were coated with 1 µl of active caspase-3 protease (Chemicon) and incubated with frontal cortex homogenates (1 µl, 4 ug/ul) from 4 aged beagles at 37°C for 1 hour.

    Techniques: Labeling, Marker

    Activation of caspase 3 is associated with increased levels of caspase-3 cleavage products. a ) Fractin immunohistochemical staining in frontal cortices of aged dogs treated with AOX and/or ENR interventions showed significant reduction in expression of active caspase 3 in the E/A group *p

    Journal: PLoS ONE

    Article Title: Dietary and Behavioral Interventions Protect against Age Related Activation of Caspase Cascades in the Canine Brain

    doi: 10.1371/journal.pone.0024652

    Figure Lengend Snippet: Activation of caspase 3 is associated with increased levels of caspase-3 cleavage products. a ) Fractin immunohistochemical staining in frontal cortices of aged dogs treated with AOX and/or ENR interventions showed significant reduction in expression of active caspase 3 in the E/A group *p

    Article Snippet: Specifically, wells were coated with 1 µl of active caspase-3 protease (Chemicon) and incubated with frontal cortex homogenates (1 µl, 4 ug/ul) from 4 aged beagles at 37°C for 1 hour.

    Techniques: Activation Assay, Immunohistochemistry, Staining, Expressing

    Immunohistochemical staining for caspase 3 in aged canine brains. a ) Caspase 3 immunohistochemical staining in frontal cortices of aged dogs treated with AOX and/or ENR interventions showed significant reduction in expression of active caspase 3. **p

    Journal: PLoS ONE

    Article Title: Dietary and Behavioral Interventions Protect against Age Related Activation of Caspase Cascades in the Canine Brain

    doi: 10.1371/journal.pone.0024652

    Figure Lengend Snippet: Immunohistochemical staining for caspase 3 in aged canine brains. a ) Caspase 3 immunohistochemical staining in frontal cortices of aged dogs treated with AOX and/or ENR interventions showed significant reduction in expression of active caspase 3. **p

    Article Snippet: Specifically, wells were coated with 1 µl of active caspase-3 protease (Chemicon) and incubated with frontal cortex homogenates (1 µl, 4 ug/ul) from 4 aged beagles at 37°C for 1 hour.

    Techniques: Immunohistochemistry, Staining, Expressing

    Double-labeling detected by immunofluorescence revealed colocalization of fractin and active caspase 3 in several cells in the frontal cortex.

    Journal: PLoS ONE

    Article Title: Dietary and Behavioral Interventions Protect against Age Related Activation of Caspase Cascades in the Canine Brain

    doi: 10.1371/journal.pone.0024652

    Figure Lengend Snippet: Double-labeling detected by immunofluorescence revealed colocalization of fractin and active caspase 3 in several cells in the frontal cortex.

    Article Snippet: Specifically, wells were coated with 1 µl of active caspase-3 protease (Chemicon) and incubated with frontal cortex homogenates (1 µl, 4 ug/ul) from 4 aged beagles at 37°C for 1 hour.

    Techniques: Labeling, Immunofluorescence

    Increased caspase-3 activity is indicative of increased myocellular apoptosis and may be a mechanism contributing to cardiac dilatation in male HCM mice consuming the soy diet. Caspase-3 activity is significantly attenuated in HCM males consuming the casein diet. n = 5–13 in each group. * P

    Journal: Journal of Clinical Investigation

    Article Title: Soy diet worsens heart disease in mice

    doi: 10.1172/JCI24676

    Figure Lengend Snippet: Increased caspase-3 activity is indicative of increased myocellular apoptosis and may be a mechanism contributing to cardiac dilatation in male HCM mice consuming the soy diet. Caspase-3 activity is significantly attenuated in HCM males consuming the casein diet. n = 5–13 in each group. * P

    Article Snippet: Caspase-3 activity was determined by monitoring the rate of cleavage of a fluorogenic caspase-3 specific substrate (Acetyl-AspGluValAsp-AMC; Calbiochem).

    Techniques: Activity Assay, Mouse Assay

    Deletion of partial CASP-3 genomic DNA in MCF-7 cells. (A) Images of DNA electrophoresis of the polymerase chain reaction (PCR) products for the CASP-3 genomic DNA and cDNA, respectively. Both PCR products from MCF-7 cells are shorter than those from A431 cells, resulting from a 47-base pair deletion within exon 4 of the human CASP-3 genomic DNA. (B) Sequencing results of the PCR products from the two cell lines. The yellow underline indicates the sequence of the deleted fragment. (C) Results of Western blotting analysis show expression of pro-caspase-3 protein in A431 cells but not in MCF-7 cells. Tubulin was used as loading control.

    Journal: Journal of Breast Cancer

    Article Title: Cell-in-Cell Death Is Not Restricted by Caspase-3 Deficiency in MCF-7 Cells

    doi: 10.4048/jbc.2016.19.3.231

    Figure Lengend Snippet: Deletion of partial CASP-3 genomic DNA in MCF-7 cells. (A) Images of DNA electrophoresis of the polymerase chain reaction (PCR) products for the CASP-3 genomic DNA and cDNA, respectively. Both PCR products from MCF-7 cells are shorter than those from A431 cells, resulting from a 47-base pair deletion within exon 4 of the human CASP-3 genomic DNA. (B) Sequencing results of the PCR products from the two cell lines. The yellow underline indicates the sequence of the deleted fragment. (C) Results of Western blotting analysis show expression of pro-caspase-3 protein in A431 cells but not in MCF-7 cells. Tubulin was used as loading control.

    Article Snippet: Membranes were probed separately with anti-pro-caspase-3 (Millipore, Billerica, USA) and anti-activated caspase-3 (Millipore) antibodies to detect the expression of caspase-3.

    Techniques: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Sequencing, Western Blot, Expressing

    Lysosome-dependent cell-in-cell death of A431 and MCF-7 cell lines. (A) Kinetic quantification of internalized terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) positive cells, internalized cells containing cleaved caspase-3 and internalized cells demonstrating lysosome activation in cell-in-cell structures of A431 cells and MCF-7 cells. One representative experiment of three independent experiments is shown. Data are presented as means±SD. (B) Confocal images show TUNEL positive (green) and DNA fragmentation (blue) of internalized A431 cells (red) and MCF-7 cells (red) at 48 hours. Both internalized A431 and MCF-7 showed inconspicuous changes in cell size and gradual nuclei degradation after entosis. Nuclei were labeled with 4',6-diamidino-2-phenylindole (DAPI). The scale bars are 10 µm. (C) As early as 6 hours after cell engulfment, the release of active cathepsin B (red) from the lysosomes into the plasma of the internalized cells was detected and was also seen in the surrounding cytoplasm of outer cells. Cells were stained with CellTracker™ Green and cell nuclei were labeled with DAPI. The scale bars are 10 µm. (D) A431 cells (green) and MCF-7 (green) cells are stained for lysosomes using LysoTracker™ Red (red), which binds to acidified compartments after 30 hours of culture. Confocal images show positive staining for cathepsin B in the cytoplasm of internalized cells. Scale bars are 10 µm.

    Journal: Journal of Breast Cancer

    Article Title: Cell-in-Cell Death Is Not Restricted by Caspase-3 Deficiency in MCF-7 Cells

    doi: 10.4048/jbc.2016.19.3.231

    Figure Lengend Snippet: Lysosome-dependent cell-in-cell death of A431 and MCF-7 cell lines. (A) Kinetic quantification of internalized terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) positive cells, internalized cells containing cleaved caspase-3 and internalized cells demonstrating lysosome activation in cell-in-cell structures of A431 cells and MCF-7 cells. One representative experiment of three independent experiments is shown. Data are presented as means±SD. (B) Confocal images show TUNEL positive (green) and DNA fragmentation (blue) of internalized A431 cells (red) and MCF-7 cells (red) at 48 hours. Both internalized A431 and MCF-7 showed inconspicuous changes in cell size and gradual nuclei degradation after entosis. Nuclei were labeled with 4',6-diamidino-2-phenylindole (DAPI). The scale bars are 10 µm. (C) As early as 6 hours after cell engulfment, the release of active cathepsin B (red) from the lysosomes into the plasma of the internalized cells was detected and was also seen in the surrounding cytoplasm of outer cells. Cells were stained with CellTracker™ Green and cell nuclei were labeled with DAPI. The scale bars are 10 µm. (D) A431 cells (green) and MCF-7 (green) cells are stained for lysosomes using LysoTracker™ Red (red), which binds to acidified compartments after 30 hours of culture. Confocal images show positive staining for cathepsin B in the cytoplasm of internalized cells. Scale bars are 10 µm.

    Article Snippet: Membranes were probed separately with anti-pro-caspase-3 (Millipore, Billerica, USA) and anti-activated caspase-3 (Millipore) antibodies to detect the expression of caspase-3.

    Techniques: End Labeling, TUNEL Assay, Activation Assay, Labeling, Staining

    Absence of caspase-3 protein in MCF-7 cells leading to an atypical apoptosis. (A) Expression of cleaved caspase-3 protein in A431 cells but not in MCF-7 cells. Both tumor cell lines were treated with (+) or without (–) staurosporine (staurosp.) for 16 hours and the cell lysates were analyzed by Western blotting. β-Actin was used as loading control. (B) Cytotoxicity assays of A431 and MCF-7 cells using the LDH method after treatment with staurosporine for 16 hours. Cells treated with the solvent dimethyl sulphoxide (DMSO) were used as negative controls. (C) Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay showed similar mortalities of the two cell lines following treatment with staurosporine for 16 hours. (D) Confocal images show positive TUNEL staining in both A431 and MCF-7 cells after treatment with staurosporine. Nuclear pyknosis was obvious in dying A431 cells but not in dying MCF-7 cells. Cells were labeled with CellTracker™ Red, and cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). The scale bars are 10 µm. (E) Cell cycle analysis of A431 and MCF-7 cells treated with or without staurosporine for 8 hours. The sub-G1 apoptotic peak demonstrating nuclear pyknosis in apoptotic cells is seen before the G0/G1 peak in A431 cells but not in MCF-7 cells after apoptosis induction. (F) DNA was prepared from cells untreated or treated for 8 hours or 16 hours with staurosporine and analyzed using a 1.6% agarose gel. There was obvious DNA ladder formation in A431 but not in MCF-7 cells after apoptosis induction. Lane M: DNA ladder.

    Journal: Journal of Breast Cancer

    Article Title: Cell-in-Cell Death Is Not Restricted by Caspase-3 Deficiency in MCF-7 Cells

    doi: 10.4048/jbc.2016.19.3.231

    Figure Lengend Snippet: Absence of caspase-3 protein in MCF-7 cells leading to an atypical apoptosis. (A) Expression of cleaved caspase-3 protein in A431 cells but not in MCF-7 cells. Both tumor cell lines were treated with (+) or without (–) staurosporine (staurosp.) for 16 hours and the cell lysates were analyzed by Western blotting. β-Actin was used as loading control. (B) Cytotoxicity assays of A431 and MCF-7 cells using the LDH method after treatment with staurosporine for 16 hours. Cells treated with the solvent dimethyl sulphoxide (DMSO) were used as negative controls. (C) Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay showed similar mortalities of the two cell lines following treatment with staurosporine for 16 hours. (D) Confocal images show positive TUNEL staining in both A431 and MCF-7 cells after treatment with staurosporine. Nuclear pyknosis was obvious in dying A431 cells but not in dying MCF-7 cells. Cells were labeled with CellTracker™ Red, and cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). The scale bars are 10 µm. (E) Cell cycle analysis of A431 and MCF-7 cells treated with or without staurosporine for 8 hours. The sub-G1 apoptotic peak demonstrating nuclear pyknosis in apoptotic cells is seen before the G0/G1 peak in A431 cells but not in MCF-7 cells after apoptosis induction. (F) DNA was prepared from cells untreated or treated for 8 hours or 16 hours with staurosporine and analyzed using a 1.6% agarose gel. There was obvious DNA ladder formation in A431 but not in MCF-7 cells after apoptosis induction. Lane M: DNA ladder.

    Article Snippet: Membranes were probed separately with anti-pro-caspase-3 (Millipore, Billerica, USA) and anti-activated caspase-3 (Millipore) antibodies to detect the expression of caspase-3.

    Techniques: Expressing, Western Blot, End Labeling, TUNEL Assay, Staining, Labeling, Cell Cycle Assay, Agarose Gel Electrophoresis

    Entosis converting to apoptosis in the presence of caspase-3. (A) Immunofluorescence of cleaved caspase-3 activity in A431 cells, caspase-3 expressing MCF-7 cells and MCF-7 cells with or without concanamycin A (con A) treatment. The three kinds of cells showed typical lysosomal cell-in-cell death before treatment. After treatment we could see clear caspase-3 activation, nuclear shrinkage, nuclear pyknosis and other apoptotic forms in A431 and caspase-3 expressing MCF-7 cells. In contrast, no caspase-3 activity or other apoptosis characteristics was detected in MCF-7 cells after the same treatment. These pictures were taken after 24 hours of cell incubation. The scale bars are 10 µm. Arrows point to entosis cells. (B) Result of Western blot showed fusion protein caspase-3-green fluorescent protein (GFP) (arrows marked) expressed in caspase-3 expressing MCF-7 cells which was about 69 kDa. (C) Result of Western blotting showed caspase-3-GFP (69 kDa) and cleaved caspase-3 (17 kDa, arrow marked) were detected in caspase-3 expressing MCF-7 cells but not in MCF-7 cells. Both of the cells were treated with staurosporine for 16 hours. (D) Statistical analysis of cell-in-cell death of A431 cells, caspase-3 expressing MCF-7 cells and MCF-7 cells with or without Concanamycin A treatment for 48 hours determined by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay. Data are presented as means±SD.

    Journal: Journal of Breast Cancer

    Article Title: Cell-in-Cell Death Is Not Restricted by Caspase-3 Deficiency in MCF-7 Cells

    doi: 10.4048/jbc.2016.19.3.231

    Figure Lengend Snippet: Entosis converting to apoptosis in the presence of caspase-3. (A) Immunofluorescence of cleaved caspase-3 activity in A431 cells, caspase-3 expressing MCF-7 cells and MCF-7 cells with or without concanamycin A (con A) treatment. The three kinds of cells showed typical lysosomal cell-in-cell death before treatment. After treatment we could see clear caspase-3 activation, nuclear shrinkage, nuclear pyknosis and other apoptotic forms in A431 and caspase-3 expressing MCF-7 cells. In contrast, no caspase-3 activity or other apoptosis characteristics was detected in MCF-7 cells after the same treatment. These pictures were taken after 24 hours of cell incubation. The scale bars are 10 µm. Arrows point to entosis cells. (B) Result of Western blot showed fusion protein caspase-3-green fluorescent protein (GFP) (arrows marked) expressed in caspase-3 expressing MCF-7 cells which was about 69 kDa. (C) Result of Western blotting showed caspase-3-GFP (69 kDa) and cleaved caspase-3 (17 kDa, arrow marked) were detected in caspase-3 expressing MCF-7 cells but not in MCF-7 cells. Both of the cells were treated with staurosporine for 16 hours. (D) Statistical analysis of cell-in-cell death of A431 cells, caspase-3 expressing MCF-7 cells and MCF-7 cells with or without Concanamycin A treatment for 48 hours determined by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay. Data are presented as means±SD.

    Article Snippet: Membranes were probed separately with anti-pro-caspase-3 (Millipore, Billerica, USA) and anti-activated caspase-3 (Millipore) antibodies to detect the expression of caspase-3.

    Techniques: Immunofluorescence, Activity Assay, Expressing, Activation Assay, Incubation, Western Blot, End Labeling, TUNEL Assay

    Induction of apoptosis following depletion of SREBP in cancer cells is restricted to lipoprotein deplete conditions.  ( A ) RPE-myrAkt-ER cells were transfected with 25 nM siRNA oligonucleotides targeting SREBP1, SREBP2 or a combination of both. After 48 hours, cells were placed in medium containing 10% FCS or 1% LPDS for a further 48 hours in the presence of 100 nM 4-OHT or solvent (ethanol). Cell viability was determined by measuring caspase 3/7 activity (Apoptosis) normalized to total protein content (SRB). Graph shows mean ± SEM of three independent experiments. ( B ) The effect of SREBP depletion on cell viability in breast cancer cells. Cells were treated and analyzed as in A. Graphs show mean ± SEM of three independent experiments. Cell lines carry different mutations in components of the PI3-kinase pathway: MCF7 (PIK3CA E545K), T47D (PIK3CA L194F), HCC1954 (PIK3CA H1047R), BT549 (PTEN null ), MDA-MB-468 (PTEN null ), MDA-MB-231 (KRAS G13D) and SKBR3 (HER2 amplification). Information on cancer gene mutations was obtained from the Wellcome Trust Sanger Institute Cancer Genome Project ( http://www.sanger.ac.uk/genetics/CGP ). ( C ) Effect of depletion of SREBP1 or SREBP2 on viability of U87 glioblastoma cells. Graph shows mean ± SEM of three independent experiments. * P

    Journal: Cancer & Metabolism

    Article Title: Sterol regulatory element binding protein-dependent regulation of lipid synthesis supports cell survival and tumor growth

    doi: 10.1186/2049-3002-1-3

    Figure Lengend Snippet: Induction of apoptosis following depletion of SREBP in cancer cells is restricted to lipoprotein deplete conditions. ( A ) RPE-myrAkt-ER cells were transfected with 25 nM siRNA oligonucleotides targeting SREBP1, SREBP2 or a combination of both. After 48 hours, cells were placed in medium containing 10% FCS or 1% LPDS for a further 48 hours in the presence of 100 nM 4-OHT or solvent (ethanol). Cell viability was determined by measuring caspase 3/7 activity (Apoptosis) normalized to total protein content (SRB). Graph shows mean ± SEM of three independent experiments. ( B ) The effect of SREBP depletion on cell viability in breast cancer cells. Cells were treated and analyzed as in A. Graphs show mean ± SEM of three independent experiments. Cell lines carry different mutations in components of the PI3-kinase pathway: MCF7 (PIK3CA E545K), T47D (PIK3CA L194F), HCC1954 (PIK3CA H1047R), BT549 (PTEN null ), MDA-MB-468 (PTEN null ), MDA-MB-231 (KRAS G13D) and SKBR3 (HER2 amplification). Information on cancer gene mutations was obtained from the Wellcome Trust Sanger Institute Cancer Genome Project ( http://www.sanger.ac.uk/genetics/CGP ). ( C ) Effect of depletion of SREBP1 or SREBP2 on viability of U87 glioblastoma cells. Graph shows mean ± SEM of three independent experiments. * P

    Article Snippet: Thapsigargin and caspase 3/7 substrate were from Calbiochem.

    Techniques: Transfection, Activity Assay, Sulforhodamine B Assay, Multiple Displacement Amplification, Amplification

    Butyrate and SAHA induced apoptosis, caspase-3 activation, and cytochrome c release in HeLa cells. HeLa cells were harvested after 2 days of treatment with indicated concentrations of butyrate or SAHA. ( A ) Cell death was measured by Hoechst dye 33342

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Apoptotic and autophagic cell death induced by histone deacetylase inhibitors

    doi: 10.1073/pnas.0408345102

    Figure Lengend Snippet: Butyrate and SAHA induced apoptosis, caspase-3 activation, and cytochrome c release in HeLa cells. HeLa cells were harvested after 2 days of treatment with indicated concentrations of butyrate or SAHA. ( A ) Cell death was measured by Hoechst dye 33342

    Article Snippet: N -benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) and caspase-3 fluorogenic substrate were from Calbiochem.

    Techniques: Activation Assay

    Apaf-1 is required for butyrate and SAHA-induced caspase-3 activation but not cell death. Apaf-1 wild-type (WT, filled bars) and knockout (KO, open bars) MEFs were harvested after the treatment with butyrate ( A ) or SAHA ( B ) for 2 days or 3 days, and caspase-3

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Apoptotic and autophagic cell death induced by histone deacetylase inhibitors

    doi: 10.1073/pnas.0408345102

    Figure Lengend Snippet: Apaf-1 is required for butyrate and SAHA-induced caspase-3 activation but not cell death. Apaf-1 wild-type (WT, filled bars) and knockout (KO, open bars) MEFs were harvested after the treatment with butyrate ( A ) or SAHA ( B ) for 2 days or 3 days, and caspase-3

    Article Snippet: N -benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) and caspase-3 fluorogenic substrate were from Calbiochem.

    Techniques: Activation Assay, Knock-Out

    Overexpression of Bcl-XL blocked butyrate- and SAHA-induced cytochrome c release and caspase-3 activation, but not cell death. HeLa cell lines stably transfected with Bcl-XL (HeLa-Bcl-XL) or vector alone (HeLa) were treated for 2 days with various concentrations

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Apoptotic and autophagic cell death induced by histone deacetylase inhibitors

    doi: 10.1073/pnas.0408345102

    Figure Lengend Snippet: Overexpression of Bcl-XL blocked butyrate- and SAHA-induced cytochrome c release and caspase-3 activation, but not cell death. HeLa cell lines stably transfected with Bcl-XL (HeLa-Bcl-XL) or vector alone (HeLa) were treated for 2 days with various concentrations

    Article Snippet: N -benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) and caspase-3 fluorogenic substrate were from Calbiochem.

    Techniques: Over Expression, Activation Assay, Stable Transfection, Transfection, Plasmid Preparation

    The anticancer effects of AgNPs on the PC-3 cells, a cytotoxic effects and b effects of the level of phosphorylated stat 3, bcl-2, survivin, and caspase-3

    Journal: Nanoscale Research Letters

    Article Title: Biosynthesis, Antibacterial Activity and Anticancer Effects Against Prostate Cancer (PC-3) Cells of Silver Nanoparticles Using Dimocarpus Longan Lour. Peel Extract

    doi: 10.1186/s11671-016-1511-9

    Figure Lengend Snippet: The anticancer effects of AgNPs on the PC-3 cells, a cytotoxic effects and b effects of the level of phosphorylated stat 3, bcl-2, survivin, and caspase-3

    Article Snippet: The membranes were subsequently incubated with phosphor-stat 3, bcl-2, survivin, or caspase-3 primary antibodies (Millipore) at 4 °C overnight, respectively.

    Techniques:

    Cell death in the hippocampus involves activated caspase-3 and activated calpain. A: Hippocampi were freshly isolated from mice ( n = 3 at each time point) at the indicated days postinfection and caspase-3 and calpain-1 activity was measured using

    Journal: The American Journal of Pathology

    Article Title: Apoptosis of Hippocampal Pyramidal Neurons Is Virus Independent in a Mouse Model of Acute Neurovirulent Picornavirus Infection

    doi: 10.2353/ajpath.2009.081126

    Figure Lengend Snippet: Cell death in the hippocampus involves activated caspase-3 and activated calpain. A: Hippocampi were freshly isolated from mice ( n = 3 at each time point) at the indicated days postinfection and caspase-3 and calpain-1 activity was measured using

    Article Snippet: The activated caspase-3-specific antibody (Chemicon; Temecula, CA) was used at 1:100 dilution in PBS overnight at 4°C.

    Techniques: Isolation, Mouse Assay, Activity Assay

    Src activity is required for survival and growth of 5637 cells in serum-free conditions. ( A ) 5637 cells were transfected with a control plasmid or plasmid expressing FLAG-tagged kinase-negative Src (KN-Src) or FLAG-tagged wild-type Src (WT-Src) as described in   Materials and Methods . After the transfection, cells were cultured in either serum-containing medium (FCS +) or serum-free medium (FCS −) for 24 h. Triton X-100-solubilized cell extracts were prepared and analyzed by immunoprecipitation (IP, 300 µg/lane) and/or immunoblotting (IB, 30 µg/lane) with the indicated antibodies as described in   Materials and Methods . The positions of the tyrosine-phosphorylated forms or the total proteins of p145 met , FLAG-tagged or endogenous Src, and tubulin are indicated. ( B ) 5637 cells (0.1×10 6  cells/dish) were cultured in serum-containing conditions for 24 h (○), transfected with a control plasmid ( ), KN-Src (▴), or WT-Src (▪) for 24 h as in panel A, and then cultured in serum-containing (FCS +) or serum-free medium (FCS −) for 48 h. Cell number was determined at 24, 48, 72 and 96 hours of incubation. ( C ) 5637 cells were treated as in panel B. After the treatments (96 h post incubation), cell death (black bars, shown as percentage of total cells) and caspase 3/7 protease activity of Triton X-100-solubilized cell extracts (20 µg/assay) (grey bars, shown as arbitrary unit) were determined by Trypan Blue exclusion and a synthetic substrate Ac-DEVD-AMC, respectively, as described in   Materials and Methods . Data shown are mean ± s.d. of three independent experiments. * P

    Journal: Biology Open

    Article Title: Membrane microdomain-associated uroplakin IIIa contributes to Src-dependent mechanisms of anti-apoptotic proliferation in human bladder carcinoma cells

    doi: 10.1242/bio.20121115

    Figure Lengend Snippet: Src activity is required for survival and growth of 5637 cells in serum-free conditions. ( A ) 5637 cells were transfected with a control plasmid or plasmid expressing FLAG-tagged kinase-negative Src (KN-Src) or FLAG-tagged wild-type Src (WT-Src) as described in Materials and Methods . After the transfection, cells were cultured in either serum-containing medium (FCS +) or serum-free medium (FCS −) for 24 h. Triton X-100-solubilized cell extracts were prepared and analyzed by immunoprecipitation (IP, 300 µg/lane) and/or immunoblotting (IB, 30 µg/lane) with the indicated antibodies as described in Materials and Methods . The positions of the tyrosine-phosphorylated forms or the total proteins of p145 met , FLAG-tagged or endogenous Src, and tubulin are indicated. ( B ) 5637 cells (0.1×10 6 cells/dish) were cultured in serum-containing conditions for 24 h (○), transfected with a control plasmid ( ), KN-Src (▴), or WT-Src (▪) for 24 h as in panel A, and then cultured in serum-containing (FCS +) or serum-free medium (FCS −) for 48 h. Cell number was determined at 24, 48, 72 and 96 hours of incubation. ( C ) 5637 cells were treated as in panel B. After the treatments (96 h post incubation), cell death (black bars, shown as percentage of total cells) and caspase 3/7 protease activity of Triton X-100-solubilized cell extracts (20 µg/assay) (grey bars, shown as arbitrary unit) were determined by Trypan Blue exclusion and a synthetic substrate Ac-DEVD-AMC, respectively, as described in Materials and Methods . Data shown are mean ± s.d. of three independent experiments. * P

    Article Snippet: A synthetic substrate for caspase 3/7, Ac-Asp-Glu-Val-Asp-AMC (Ac-DEVD-AMC), was obtained from Calbiochem-EMD Chemicals USA.

    Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Cell Culture, Immunoprecipitation, Incubation

    Importance of HB-EGF secretion for survival and growth in serum-starved 5637 cells. ( A , B ) 5637 cells were cultured in serum-containing (FCS +) or serum-free (FCS −) medium in the presence of anti-HB-EGF antibody (10 µg/ml IgG) or control antibody (25 µg/ml IgG) as described in   Materials and Methods . After the treatments, cell samples were analyzed for (A) the proteolysis of UPIIIa (IB: UPIII) and the activation of Src (IB: pY418-Src), and for (B) cell death and caspase 3/7 activity. ( C ) 5637 cells were exposed to serum-free medium for 8 h in the presence of either GM6001 (10 µg/ml) or its inactive analog (GM6001 n.c., 10 µg/ml). Triton X-100-solubilized cell extracts were prepared and analyzed for tyrosine phosphorylation of 145 met  (upper panel) and p60 src  (lower panel) as in   Fig. 1A . Asterisks indicate the positions of phosphorylated forms of p145 met  (pp145 met ) and p60 src  (pp60 src ). Data obtained with the cell extracts prepared from cells grown in serum-containing medium (FCS +) are also shown. ( D ) Quantifications of HB-EGF in culture media were done under the indicated conditions as in   Fig. 7A .

    Journal: Biology Open

    Article Title: Membrane microdomain-associated uroplakin IIIa contributes to Src-dependent mechanisms of anti-apoptotic proliferation in human bladder carcinoma cells

    doi: 10.1242/bio.20121115

    Figure Lengend Snippet: Importance of HB-EGF secretion for survival and growth in serum-starved 5637 cells. ( A , B ) 5637 cells were cultured in serum-containing (FCS +) or serum-free (FCS −) medium in the presence of anti-HB-EGF antibody (10 µg/ml IgG) or control antibody (25 µg/ml IgG) as described in Materials and Methods . After the treatments, cell samples were analyzed for (A) the proteolysis of UPIIIa (IB: UPIII) and the activation of Src (IB: pY418-Src), and for (B) cell death and caspase 3/7 activity. ( C ) 5637 cells were exposed to serum-free medium for 8 h in the presence of either GM6001 (10 µg/ml) or its inactive analog (GM6001 n.c., 10 µg/ml). Triton X-100-solubilized cell extracts were prepared and analyzed for tyrosine phosphorylation of 145 met (upper panel) and p60 src (lower panel) as in Fig. 1A . Asterisks indicate the positions of phosphorylated forms of p145 met (pp145 met ) and p60 src (pp60 src ). Data obtained with the cell extracts prepared from cells grown in serum-containing medium (FCS +) are also shown. ( D ) Quantifications of HB-EGF in culture media were done under the indicated conditions as in Fig. 7A .

    Article Snippet: A synthetic substrate for caspase 3/7, Ac-Asp-Glu-Val-Asp-AMC (Ac-DEVD-AMC), was obtained from Calbiochem-EMD Chemicals USA.

    Techniques: Cell Culture, Activation Assay, Activity Assay

    GM6001 and anti-UPIII antibody inhibit Src-dependent signal transduction and proteolysis of UPIII, and promote apoptosis in serum-starved 5637 cells. ( A ) 5637 cells were cultured in serum-free (FCS −) or serum-containing (FCS +) conditions for 24 h in the absence or the presence of 100 µg/ml anti-UPIII-CT IgG (CT), 100 µg/ml anti-UPIII-ED IgG (ED), or 10 µM GM6001. Partial proteolysis of UPIII (IB: UPIII), and tyrosine phosphorylation of Src (IP: Src, IB: pY418) were analyzed by immunoblotting of the Triton X-100-solubilized cell extracts (20 µg/lane). ( B ) 5637 cells (0.1×10 6  cells/dish) were grown in serum-containing condition for 48 h and then exposed to serum-free medium in the absence ( ) or the presence of 100 µg/ml anti-UPIII-ED antibody (▴), 100 µg/ml anti-UPIII-CT antibody (▵), or 10 µM GM6001 (▪) for 48 h (total incubation time of 96 h). At 24, 48, 72, and 96 h of treatments, cell number was determined as in   Fig. 1B . ( C ) 5637 cells were cultured in serum-free medium (FCS −) in the absence or the presence of 100 µg/ml anti-UPIII-ED antibody, 100 µg/ml anti-UPIII-CT antibody, or 10 µM GM6001 for 8 h. After the treatments, cell death and caspase 3/7 activity were determined as in   Fig. 2C . * P

    Journal: Biology Open

    Article Title: Membrane microdomain-associated uroplakin IIIa contributes to Src-dependent mechanisms of anti-apoptotic proliferation in human bladder carcinoma cells

    doi: 10.1242/bio.20121115

    Figure Lengend Snippet: GM6001 and anti-UPIII antibody inhibit Src-dependent signal transduction and proteolysis of UPIII, and promote apoptosis in serum-starved 5637 cells. ( A ) 5637 cells were cultured in serum-free (FCS −) or serum-containing (FCS +) conditions for 24 h in the absence or the presence of 100 µg/ml anti-UPIII-CT IgG (CT), 100 µg/ml anti-UPIII-ED IgG (ED), or 10 µM GM6001. Partial proteolysis of UPIII (IB: UPIII), and tyrosine phosphorylation of Src (IP: Src, IB: pY418) were analyzed by immunoblotting of the Triton X-100-solubilized cell extracts (20 µg/lane). ( B ) 5637 cells (0.1×10 6 cells/dish) were grown in serum-containing condition for 48 h and then exposed to serum-free medium in the absence ( ) or the presence of 100 µg/ml anti-UPIII-ED antibody (▴), 100 µg/ml anti-UPIII-CT antibody (▵), or 10 µM GM6001 (▪) for 48 h (total incubation time of 96 h). At 24, 48, 72, and 96 h of treatments, cell number was determined as in Fig. 1B . ( C ) 5637 cells were cultured in serum-free medium (FCS −) in the absence or the presence of 100 µg/ml anti-UPIII-ED antibody, 100 µg/ml anti-UPIII-CT antibody, or 10 µM GM6001 for 8 h. After the treatments, cell death and caspase 3/7 activity were determined as in Fig. 2C . * P

    Article Snippet: A synthetic substrate for caspase 3/7, Ac-Asp-Glu-Val-Asp-AMC (Ac-DEVD-AMC), was obtained from Calbiochem-EMD Chemicals USA.

    Techniques: Transduction, Cell Culture, Incubation, Activity Assay

    Disruption of cholesterol-dependent MDs interferes with Src-dependent signal transduction and promotes apoptosis in serum-starved 5637 cells. ( A ) 5637 cells were grown in serum-free condition in the absence or the presence of methyl-β-cyclodextrin (MβCD, 15 µM) for 8 h. After the treatments, Triton X-100-solubilized cell extracts were prepared and analyzed for tyrosine phosphorylation of p145 met  and p60 src  by immunoprecipitation (300 µg/lane) and/or immunoblotting (30 µg/lane). Control data obtained with the extracts prepared from cells grown in serum-containing medium (FCS +) are also shown. ( B ) 5637 cells (0.1×10 7  cells/dish) were grown in serum-containing condition for 48 h and then exposed to serum-free medium with (▴) or without ( ) 15 µM MβCD for 48 h (total incubation time of 96 h). At 24, 48, 72, and 96 h of treatments, cell number was determined as in   Fig. 2B . ( C ) 5637 cells were cultured in serum-containing (FCS +) or serum-free (FCS −) medium with or without 15 µM MβCD as in panel B for 8 h. After the treatments, cell death and caspase 3/7 activity were determined as in   Fig. 2C . ( D ) 5637 cells were cultured in serum-containing medium (control, FCS +), serum-free medium in the absence (serum-starved) or the presence of 15 µM MβCD (serum-starved, + MβCD). Cell samples were subjected to subcellular fractionation by discontinuous sucrose density gradient ultracentrifugation as described in   Materials and Methods . Twelve fractions obtained were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. Rectangles indicate the positions of low density, detergent-insoluble MD fractions (fractions 4–6, denoted as “MD”) and non-MD fractions (fractions 10–12, denoted as “non-MD”). Asterisks indicate the positions of proteins of interest.

    Journal: Biology Open

    Article Title: Membrane microdomain-associated uroplakin IIIa contributes to Src-dependent mechanisms of anti-apoptotic proliferation in human bladder carcinoma cells

    doi: 10.1242/bio.20121115

    Figure Lengend Snippet: Disruption of cholesterol-dependent MDs interferes with Src-dependent signal transduction and promotes apoptosis in serum-starved 5637 cells. ( A ) 5637 cells were grown in serum-free condition in the absence or the presence of methyl-β-cyclodextrin (MβCD, 15 µM) for 8 h. After the treatments, Triton X-100-solubilized cell extracts were prepared and analyzed for tyrosine phosphorylation of p145 met and p60 src by immunoprecipitation (300 µg/lane) and/or immunoblotting (30 µg/lane). Control data obtained with the extracts prepared from cells grown in serum-containing medium (FCS +) are also shown. ( B ) 5637 cells (0.1×10 7 cells/dish) were grown in serum-containing condition for 48 h and then exposed to serum-free medium with (▴) or without ( ) 15 µM MβCD for 48 h (total incubation time of 96 h). At 24, 48, 72, and 96 h of treatments, cell number was determined as in Fig. 2B . ( C ) 5637 cells were cultured in serum-containing (FCS +) or serum-free (FCS −) medium with or without 15 µM MβCD as in panel B for 8 h. After the treatments, cell death and caspase 3/7 activity were determined as in Fig. 2C . ( D ) 5637 cells were cultured in serum-containing medium (control, FCS +), serum-free medium in the absence (serum-starved) or the presence of 15 µM MβCD (serum-starved, + MβCD). Cell samples were subjected to subcellular fractionation by discontinuous sucrose density gradient ultracentrifugation as described in Materials and Methods . Twelve fractions obtained were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. Rectangles indicate the positions of low density, detergent-insoluble MD fractions (fractions 4–6, denoted as “MD”) and non-MD fractions (fractions 10–12, denoted as “non-MD”). Asterisks indicate the positions of proteins of interest.

    Article Snippet: A synthetic substrate for caspase 3/7, Ac-Asp-Glu-Val-Asp-AMC (Ac-DEVD-AMC), was obtained from Calbiochem-EMD Chemicals USA.

    Techniques: Transduction, Immunoprecipitation, Incubation, Cell Culture, Activity Assay, Fractionation, SDS Page

    Compounds  2b  and  8b  prompted apoptotic death in melanoma cells.  (A) , UACC 903 cells (50,000 cells/well) were treated with  2b  and  8b  for 24 h then cells were subjected to Annexin-V activity assay using a Muse cell analyzer. Four different populations of cells were collected: healthy cells (lower left quadrant, Annexin-V negative), early apoptotic cells (lower right corner, positive for Annexin-V and 7-ADD negative), late apoptotic/dead cells (upper right quadrant, both Annexin 7-ADD positive), and necrotic cells (only 7-ADD positive, upper left quadrant).  (B)  Similarly, activity of caspase 3/7 was measured using muse cell analyzer as described under method and material section.

    Journal: European journal of medicinal chemistry

    Article Title: Design and synthesis of novel thiobarbituric acid derivatives targeting both wild-type and BRAF-mutated melanoma cells

    doi: 10.1016/j.ejmech.2017.11.006

    Figure Lengend Snippet: Compounds 2b and 8b prompted apoptotic death in melanoma cells. (A) , UACC 903 cells (50,000 cells/well) were treated with 2b and 8b for 24 h then cells were subjected to Annexin-V activity assay using a Muse cell analyzer. Four different populations of cells were collected: healthy cells (lower left quadrant, Annexin-V negative), early apoptotic cells (lower right corner, positive for Annexin-V and 7-ADD negative), late apoptotic/dead cells (upper right quadrant, both Annexin 7-ADD positive), and necrotic cells (only 7-ADD positive, upper left quadrant). (B) Similarly, activity of caspase 3/7 was measured using muse cell analyzer as described under method and material section.

    Article Snippet: To establish whether 2b and 8b induce caspase 3/7 activity, UACC903 and CHL-1 (5 × 105 ) cells were treated with 25 and 50 μM with 2b and 8b for 24 h. Caspase 3/7 activity was monitored using Muse Caspase-3/7 Assay kit with Muse cell analyzer (EMD Millipore, Billercia, MA, USA) as previously described.

    Techniques: Activity Assay

    Ablation of Mcl-1 does not result in activation of apoptosis. ( A ) Quantitation of TUNEL positive nuclei in heart sections. n = 3–4. ( B ) Quantitation of myocytes positive for cleaved caspase-3 in heart sections. n = 3–4. ( C ) Caspase-3 activity

    Journal: Genes & Development

    Article Title: Loss of MCL-1 leads to impaired autophagy and rapid development of heart failure

    doi: 10.1101/gad.215871.113

    Figure Lengend Snippet: Ablation of Mcl-1 does not result in activation of apoptosis. ( A ) Quantitation of TUNEL positive nuclei in heart sections. n = 3–4. ( B ) Quantitation of myocytes positive for cleaved caspase-3 in heart sections. n = 3–4. ( C ) Caspase-3 activity

    Article Snippet: Caspase-3 activity was determined in whole-heart homogenates using a fluorescent caspase-3 activity assay (EMD Biosciences).

    Techniques: Activation Assay, Quantitation Assay, TUNEL Assay, Activity Assay

    Apoptosis-related proteins were detected by Western blotting. ( A ) Western blot of Bcl-2, Bax and Caspase3 expressions (cropped gels). ( B ) The average relative quantitative expression of Bcl-2, Bax, Caspase-3 abundance in the three groups. n = 5, Mean ± SEM. ## P

    Journal: Scientific Reports

    Article Title: Cardiac shock wave therapy promotes arteriogenesis of coronary micrangium, and ILK is involved in the biomechanical effects by proteomic analysis

    doi: 10.1038/s41598-018-19393-z

    Figure Lengend Snippet: Apoptosis-related proteins were detected by Western blotting. ( A ) Western blot of Bcl-2, Bax and Caspase3 expressions (cropped gels). ( B ) The average relative quantitative expression of Bcl-2, Bax, Caspase-3 abundance in the three groups. n = 5, Mean ± SEM. ## P

    Article Snippet: Western blot The extracted samples were subjected to SDS-PAGE by using specific antibody for Bcl2 (1:1000), Bax (1:1000), Caspase3 (1:1000), GAPDH (1:1000) (Millipore, Germany).

    Techniques: Western Blot, Expressing

    Increased apoptosis in KO myotubes. ( A ) Western blot of total lysates (top) and nuclear and mitochondrial fractions of WT and KO myotubes grown in differentiation medium for 6 to 7 d. No changes in the levels of activated (cleaved) CASP3 products are

    Journal: Autophagy

    Article Title: Defects in calcium homeostasis and mitochondria can be reversed in Pompe disease

    doi: 10.1080/15548627.2015.1009779

    Figure Lengend Snippet: Increased apoptosis in KO myotubes. ( A ) Western blot of total lysates (top) and nuclear and mitochondrial fractions of WT and KO myotubes grown in differentiation medium for 6 to 7 d. No changes in the levels of activated (cleaved) CASP3 products are

    Article Snippet: The following antibodies were used for western blots: VCL/Vinculin (Sigma, V9131), LC3B (Sigma, L8918), CACNB1 (S7–18; Abcam, ab85020), MFN2/mitofusin 2 (Abcam, ab50830), COX4I1/COXIV (Abcam, ab14744), FIS1/TTC11/ Fission 1 (Abcam, ab71498), PARK2/Parkin (Abcam, ab15954), TUBA1A/α-tubulin (Abcam, ab18251), PINK1 (Abcam, ab23707), ubiquitin (linkage-specific K63; Millipore, 05–1308), SQSTM1 (Santa Cruz Biotechnology, sc-25575), CASP3/caspase 3 (Cell Signaling Technology, 9662), AIFM1/AIF (Cell Signaling Technology, 5318), HIST1H3A/ Histone H3 (D1H2; Cell Signaling Technology, 4499), OPA1 (BD Biosciences, 612606), DNM1L/DRP1 (DNM1L; BD Biosciences, 611738), Alexa Fluor-conjugated secondary anti-mouse (Life Technologies, A-21057) or anti-rabbit (Life Technologies, A-21076).

    Techniques: Western Blot

    IL-26 suppressed the apoptosis of HSCs by inhibition of caspase 3 (CASP3) and Bcl-2 associated X protein (BAX). A. Apoptosis of HSCs by annexin V-FITC/PI double staining after treatment with IL-26 at 0, 50, 100, and 200 pg/ml for 48 h. B. The mRNA expression levels of CASP3 and BAX were detected by quantitative real-time PCR (qRT-PCR) in the different concentrations of IL-26 groups. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the reference control. C and D. Western blotting analysis of the protein levels of CASP3, cleaved CASP3, and BAX in HSCs after IL-26 treatment. * P

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Interleukin-26 promotes the proliferation and activation of hepatic stellate cells to exacerbate liver fibrosis by the TGF-β1/Smad2 signaling pathway

    doi:

    Figure Lengend Snippet: IL-26 suppressed the apoptosis of HSCs by inhibition of caspase 3 (CASP3) and Bcl-2 associated X protein (BAX). A. Apoptosis of HSCs by annexin V-FITC/PI double staining after treatment with IL-26 at 0, 50, 100, and 200 pg/ml for 48 h. B. The mRNA expression levels of CASP3 and BAX were detected by quantitative real-time PCR (qRT-PCR) in the different concentrations of IL-26 groups. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the reference control. C and D. Western blotting analysis of the protein levels of CASP3, cleaved CASP3, and BAX in HSCs after IL-26 treatment. * P

    Article Snippet: Membranes were blocked with 5% non-fat milk at 37°C for 2 h and primary mouse monoclonal anti-TGF-β1 (#ab27969, 1:1000; Abcam, Cambridge, MA, USA) and anti-Bcl-2 associated X protein (BAX) (#ab77566, 1:500; Abcam) antibodies, and primary rabbit monoclonal anti-SMAD family member 2 (Smad2) (#ab40855, 1:500; Abcam), anti-caspase 3 (CASP3) (#9662, 1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-cleaved CASP3 (#9661, 1:1500; Cell Signaling Technology) antibodies were prepared in blocking solution and incubated overnight at 4°C.

    Techniques: Inhibition, Double Staining, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

    C23 suppressed the transcriptional activity of p53 upon DNA damage and hypoxia A. pGL3-3×p53-BS-LUC and Renilla were co-transfected in HCT116 cells combined with and without C23 shRNA. 24 hours after transfection, cells were treated with Doxorubicin (100ng/ml) or upon 1% O 2 for indicated times and the cell lysates were analyzed by lucifease assay. B. HCT116 cells with and without stable overexpressing C23 were co-transfected with pGL3-3×p53-BS-LUC and Renilla. 24 hours after transfection, cells were treated with Doxorubicin (100ng/ml) or upon 1% O 2 for indicated times and then the cell lysates were subjected to lucifease assays. C. HCT116 cells with and without stable knockdown of C23 were treated with Doxorubicin (100ng/ml) for 24 hours, then p53 target gene mRNA expression levels were analyzed by qRT-PCR analysis. D-E. HCT116 cells with and without stable overexpressing C23 (a) or stable knockdown of C23(b) were treated with Doxorubicin (100ng/ml) or mock control for 24 hours. Cell lysates were then subjected to Western blot analysis with the indicated antibodies (D) and caspase3/7 activity analysis (E), respectively. F. (a) HCT116 cells stably expressing the control shRNA, C23 shRNA, p53 shRNA,or C23 shRNA plus p53 shRNA. (b) HCT116 cells with and without p53 knockdown were introduced with C23 respectively. Protein level of C23 and p53 were evaluated by Western blot. GAPDH served as loading control. G. HCT116 cells stably expressing the control shRNA, C23 shRNA, p53 shRNA,or C23 shRNA plus p53 shRNA (a) and HCT116 cells stably knocked down p53 were additionally introduced with C23 respectively (b). Cell number was evaluated by cell counter. The data were represented as mean±S.D. from three independent experiments. H. (a) HCT116 cells stably expressing the control shRNA, C23 shRNA, p53 shRNA,or C23 shRNA plus p53 shRNA. (b) HCT116 cells stably knocked down p53 were co-transfected with C23 cDNA respectively. Cells were then treated with 100ng/ml Doxorubicin for 24 hours. Cell viability was determined by MTT assay. The data were shown as the mean±s.d. of three independent experiments.

    Journal: Oncotarget

    Article Title: C23 promotes tumorigenesis via suppressing p53 activity

    doi: 10.18632/oncotarget.11071

    Figure Lengend Snippet: C23 suppressed the transcriptional activity of p53 upon DNA damage and hypoxia A. pGL3-3×p53-BS-LUC and Renilla were co-transfected in HCT116 cells combined with and without C23 shRNA. 24 hours after transfection, cells were treated with Doxorubicin (100ng/ml) or upon 1% O 2 for indicated times and the cell lysates were analyzed by lucifease assay. B. HCT116 cells with and without stable overexpressing C23 were co-transfected with pGL3-3×p53-BS-LUC and Renilla. 24 hours after transfection, cells were treated with Doxorubicin (100ng/ml) or upon 1% O 2 for indicated times and then the cell lysates were subjected to lucifease assays. C. HCT116 cells with and without stable knockdown of C23 were treated with Doxorubicin (100ng/ml) for 24 hours, then p53 target gene mRNA expression levels were analyzed by qRT-PCR analysis. D-E. HCT116 cells with and without stable overexpressing C23 (a) or stable knockdown of C23(b) were treated with Doxorubicin (100ng/ml) or mock control for 24 hours. Cell lysates were then subjected to Western blot analysis with the indicated antibodies (D) and caspase3/7 activity analysis (E), respectively. F. (a) HCT116 cells stably expressing the control shRNA, C23 shRNA, p53 shRNA,or C23 shRNA plus p53 shRNA. (b) HCT116 cells with and without p53 knockdown were introduced with C23 respectively. Protein level of C23 and p53 were evaluated by Western blot. GAPDH served as loading control. G. HCT116 cells stably expressing the control shRNA, C23 shRNA, p53 shRNA,or C23 shRNA plus p53 shRNA (a) and HCT116 cells stably knocked down p53 were additionally introduced with C23 respectively (b). Cell number was evaluated by cell counter. The data were represented as mean±S.D. from three independent experiments. H. (a) HCT116 cells stably expressing the control shRNA, C23 shRNA, p53 shRNA,or C23 shRNA plus p53 shRNA. (b) HCT116 cells stably knocked down p53 were co-transfected with C23 cDNA respectively. Cells were then treated with 100ng/ml Doxorubicin for 24 hours. Cell viability was determined by MTT assay. The data were shown as the mean±s.d. of three independent experiments.

    Article Snippet: The following antibodies were used in this study: GFP (Life, A11122 and Clonetech 632381), Flag (Sigma, F3165), GAPDH (Cell Signaling, 5174S), C23 (Santa Cruz Biotechnology, SC-8031 and Cell Signaling, 14574S), Caspase3 (Stressgene, AAP-113) and p53 (ABGENT, AJ1573a and Santa Cruz Biotechnology, SC-126).

    Techniques: Activity Assay, Transfection, shRNA, Expressing, Quantitative RT-PCR, Western Blot, Stable Transfection, MTT Assay

    Effect of almorexant and suvorexant on intracellular Ca 2+ release, cell growth and caspase-3 activity ( A ) Intracellular Ca 2+ production was detected in HEK-293 cells expressing recombinant native OX1R using Fluoforte Calcium Assay Kit (Enzo Life Sciences, NY, USA). Cells were challenged with 1 μM of OxA (top panel) or 1 μM OxA after preincubation with 10 μM suvorexant (middle panel) or 10 μM almorexant (bottom panel). ( B ) AsPC-1 cells were incubated for 48 h with OxA or almorexant or suvorexant in the presence (right panel) or in the absence (left panel) of 50 μM NSC87877. ( C ) colorometric Caspase-3 activity detection at 405 nm in AsPC-1 cells incubated with 1 μM OxA or 1 μM almorexant in the presence or in the absence of 50 μM NSC87877. * p

    Journal: Oncotarget

    Article Title: In vitro, in vivo and ex vivo demonstration of the antitumoral role of hypocretin-1/orexin-A and almorexant in pancreatic ductal adenocarcinoma

    doi: 10.18632/oncotarget.24084

    Figure Lengend Snippet: Effect of almorexant and suvorexant on intracellular Ca 2+ release, cell growth and caspase-3 activity ( A ) Intracellular Ca 2+ production was detected in HEK-293 cells expressing recombinant native OX1R using Fluoforte Calcium Assay Kit (Enzo Life Sciences, NY, USA). Cells were challenged with 1 μM of OxA (top panel) or 1 μM OxA after preincubation with 10 μM suvorexant (middle panel) or 10 μM almorexant (bottom panel). ( B ) AsPC-1 cells were incubated for 48 h with OxA or almorexant or suvorexant in the presence (right panel) or in the absence (left panel) of 50 μM NSC87877. ( C ) colorometric Caspase-3 activity detection at 405 nm in AsPC-1 cells incubated with 1 μM OxA or 1 μM almorexant in the presence or in the absence of 50 μM NSC87877. * p

    Article Snippet: Slides were immunolabeled with antibodies against OX1R (Life Technology, PA5-33837, polyclonal rabbit, 1/100), activated caspase-3 (Abgent, E87-77, polyclonal rabbit, 1/100) or Ki-67 (DAKO, clone MIB-1, monoclonal mouse, 1/100).

    Techniques: Activity Assay, Expressing, Recombinant, Calcium Assay, Incubation

    Effect of orexin-A on apoptosis in AsPC-1 cells - ( A ), SHP-2 protein tyrosine phosphatase inhibitor, NSC-87877, blocks orexin-induced apoptosis. AsPC-1 cells were challenged with (black bars) or without (white bars) 1 μM orexin-A for 48 hr in the absence or presence of NSC-87877 (50 μM). Apoptosis was measured by determination of annexin V-PE binding, and results are expressed as the percentage of apoptotic cells; ( B ) and ( C ). Indirect immunostaining of activated caspase-3 in AsPC-1 cells in the presence or absence of orexin-A. Paraformaldehyde-fixed AsPC-1 cells were challenged with (orexin-A) or without (basal) 1 μM orexin-A for 48 hr. Activated caspase-3 immunostaining is shown in B and scored in C. Results are means ± SE of three separate experiments. *** p

    Journal: Oncotarget

    Article Title: In vitro, in vivo and ex vivo demonstration of the antitumoral role of hypocretin-1/orexin-A and almorexant in pancreatic ductal adenocarcinoma

    doi: 10.18632/oncotarget.24084

    Figure Lengend Snippet: Effect of orexin-A on apoptosis in AsPC-1 cells - ( A ), SHP-2 protein tyrosine phosphatase inhibitor, NSC-87877, blocks orexin-induced apoptosis. AsPC-1 cells were challenged with (black bars) or without (white bars) 1 μM orexin-A for 48 hr in the absence or presence of NSC-87877 (50 μM). Apoptosis was measured by determination of annexin V-PE binding, and results are expressed as the percentage of apoptotic cells; ( B ) and ( C ). Indirect immunostaining of activated caspase-3 in AsPC-1 cells in the presence or absence of orexin-A. Paraformaldehyde-fixed AsPC-1 cells were challenged with (orexin-A) or without (basal) 1 μM orexin-A for 48 hr. Activated caspase-3 immunostaining is shown in B and scored in C. Results are means ± SE of three separate experiments. *** p

    Article Snippet: Slides were immunolabeled with antibodies against OX1R (Life Technology, PA5-33837, polyclonal rabbit, 1/100), activated caspase-3 (Abgent, E87-77, polyclonal rabbit, 1/100) or Ki-67 (DAKO, clone MIB-1, monoclonal mouse, 1/100).

    Techniques: Binding Assay, Immunostaining

    Validation of protein and mRNA level dysregulations. (A) Quantitative immunoblots confirmed neuronal loss (marker NeuN), astrogliosis (marker GFAP) and microgliosis (marker IBA1) to occur in Atxn2 -CAG100-KIN spinal cord at the preterminal stage of 14 months age, but not at the early KIN stage of 3 months age and in the Atxn2 -KO at 6 months. Significantly increased levels in KIN at 14 months were also shown for TDP43 and the factor responsible for its cleavage, CASP3. (B) Quantitative RT-PCR analyses showed a significant deficit of NeuN transcript ( Rbfox3 ) already at incipient disease stage in 3-month-old KIN, whereas astrogliosis (marker Gfap ) and microgliosis (marker IBA1 transcript Aif1 ) became significant at late state. Protein abundance (C) and transcript levels (D) were also documented for PGRN (encoded by Grn mRNA) as molecular marker of lysosomal activation and atrophy, as well as RIPK1 as molecular marker of RNA-toxicity and necroptosis. Again, a significant elevation of Grn mRNA at the age of 3 months suggested atrophy and lysosomal breakdown to occur in parallel with first locomotor deficits, predating necroptotic cell death.

    Journal: bioRxiv

    Article Title: Atxn2-CAG100-KnockIn mouse spinal cord shows progressive TDP43 pathology associated with cholesterol biosynthesis suppression

    doi: 10.1101/838177

    Figure Lengend Snippet: Validation of protein and mRNA level dysregulations. (A) Quantitative immunoblots confirmed neuronal loss (marker NeuN), astrogliosis (marker GFAP) and microgliosis (marker IBA1) to occur in Atxn2 -CAG100-KIN spinal cord at the preterminal stage of 14 months age, but not at the early KIN stage of 3 months age and in the Atxn2 -KO at 6 months. Significantly increased levels in KIN at 14 months were also shown for TDP43 and the factor responsible for its cleavage, CASP3. (B) Quantitative RT-PCR analyses showed a significant deficit of NeuN transcript ( Rbfox3 ) already at incipient disease stage in 3-month-old KIN, whereas astrogliosis (marker Gfap ) and microgliosis (marker IBA1 transcript Aif1 ) became significant at late state. Protein abundance (C) and transcript levels (D) were also documented for PGRN (encoded by Grn mRNA) as molecular marker of lysosomal activation and atrophy, as well as RIPK1 as molecular marker of RNA-toxicity and necroptosis. Again, a significant elevation of Grn mRNA at the age of 3 months suggested atrophy and lysosomal breakdown to occur in parallel with first locomotor deficits, predating necroptotic cell death.

    Article Snippet: The following antibodies were used: ACTB (Sigma A5441, 1:10000), ATXN2 (Proteintech 21776-1-AP, 1:500), CASP3 (Cell Signaling 9665, 1:1000), GFAP (Dako ZO334, 1:2000), GPNMB (Biotechne AF 2330, 1:500), IBA1 (Wako 019-19741, 1:2000), NeuN (Millipore ABN78, 1:1000), PGRN (Biotechne AF 2557, 1:250), PQBP1 (Biomol A302-802A-M, 1:500), RIPK1 (Cell Signaling 3493S, 1:500), TDP43 (Abcam ab41881, 1:1000).

    Techniques: Western Blot, Marker, Quantitative RT-PCR, Activation Assay