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    Becton Dickinson annexin v fitc apoptosis detection kit becton dickinson
    Annexin V Fitc Apoptosis Detection Kit Becton Dickinson, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson annexin v apoptosis detection kit
    UBXN2A overexpression induces <t>apoptosis</t> in colon cancer cell lines but not in normal, non-transformed cells. CCD-18Co, HCT-116, and SW48 were transfected with GFP-empty or GFP-UBXN2A. 48 h after transfection, cells were stained with <t>Annexin</t> V and early apoptosis was determined using flow cytometry as described in Materials and Methods. ( a ) Representative flow-cytometry analysis data from an Annexin V assay. The histograms show a comparison of the distribution of Annexin V positive cells (M1) after transient transfection of cells with GFP-empty vector or GFP-UBXN2A. The data were gated on GFP-UBXN2A positive cells prior to Annexin V analysis. ( b ) UBXN2A expression for 48 h significantly increased the number of apoptotic cells in HCT-116 and SW48 cell lines, while CCD-18Co normal colon cells were unaffected. ( c ) HCT-116 were transfected with scrambled shRNA or shRNA against UBXN2A (clone I and II). 48 h after silencing, superconfluent cultures of HCT-116 were analyzed by Annexin V assay using flow cytometry. Expression of GFP containing shRNA against UBXN2A resulted in 50% less apoptosis in comparison with cells transfected with scrambled shRNA. Values are expressed as mean (±S.E.M.) from three independent experiments (** P
    Annexin V Apoptosis Detection Kit, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 2158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson annexin v 7aad apoptosis detection kit annexin v
    UBXN2A overexpression induces <t>apoptosis</t> in colon cancer cell lines but not in normal, non-transformed cells. CCD-18Co, HCT-116, and SW48 were transfected with GFP-empty or GFP-UBXN2A. 48 h after transfection, cells were stained with <t>Annexin</t> V and early apoptosis was determined using flow cytometry as described in Materials and Methods. ( a ) Representative flow-cytometry analysis data from an Annexin V assay. The histograms show a comparison of the distribution of Annexin V positive cells (M1) after transient transfection of cells with GFP-empty vector or GFP-UBXN2A. The data were gated on GFP-UBXN2A positive cells prior to Annexin V analysis. ( b ) UBXN2A expression for 48 h significantly increased the number of apoptotic cells in HCT-116 and SW48 cell lines, while CCD-18Co normal colon cells were unaffected. ( c ) HCT-116 were transfected with scrambled shRNA or shRNA against UBXN2A (clone I and II). 48 h after silencing, superconfluent cultures of HCT-116 were analyzed by Annexin V assay using flow cytometry. Expression of GFP containing shRNA against UBXN2A resulted in 50% less apoptosis in comparison with cells transfected with scrambled shRNA. Values are expressed as mean (±S.E.M.) from three independent experiments (** P
    Annexin V 7aad Apoptosis Detection Kit Annexin V, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson annexin v staining annexin v apoptosis detection kit
    Pterostilbene induces cell <t>apoptosis.</t> OVCAR-8 and Caov-3 cells were treated with vehicle and PTE (25–300 μm) for 48 h. Apoptosis was determined by flow cytometry using <t>annexin</t> V and PI staining ( A , B ) or by Western blot for the expression of cleaved poly-ADP ribose polymerase (PARP) ( C ). Results are representative of 3 or more preparations. *, p
    Annexin V Staining Annexin V Apoptosis Detection Kit, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson apoptosis analysis pe annexin v apoptosis detection kit
    Pterostilbene induces cell <t>apoptosis.</t> OVCAR-8 and Caov-3 cells were treated with vehicle and PTE (25–300 μm) for 48 h. Apoptosis was determined by flow cytometry using <t>annexin</t> V and PI staining ( A , B ) or by Western blot for the expression of cleaved poly-ADP ribose polymerase (PARP) ( C ). Results are representative of 3 or more preparations. *, p
    Apoptosis Analysis Pe Annexin V Apoptosis Detection Kit, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    UBXN2A overexpression induces apoptosis in colon cancer cell lines but not in normal, non-transformed cells. CCD-18Co, HCT-116, and SW48 were transfected with GFP-empty or GFP-UBXN2A. 48 h after transfection, cells were stained with Annexin V and early apoptosis was determined using flow cytometry as described in Materials and Methods. ( a ) Representative flow-cytometry analysis data from an Annexin V assay. The histograms show a comparison of the distribution of Annexin V positive cells (M1) after transient transfection of cells with GFP-empty vector or GFP-UBXN2A. The data were gated on GFP-UBXN2A positive cells prior to Annexin V analysis. ( b ) UBXN2A expression for 48 h significantly increased the number of apoptotic cells in HCT-116 and SW48 cell lines, while CCD-18Co normal colon cells were unaffected. ( c ) HCT-116 were transfected with scrambled shRNA or shRNA against UBXN2A (clone I and II). 48 h after silencing, superconfluent cultures of HCT-116 were analyzed by Annexin V assay using flow cytometry. Expression of GFP containing shRNA against UBXN2A resulted in 50% less apoptosis in comparison with cells transfected with scrambled shRNA. Values are expressed as mean (±S.E.M.) from three independent experiments (** P

    Journal: Cell Death & Disease

    Article Title: Ubiquitin-like (UBX)-domain-containing protein, UBXN2A, promotes cell death by interfering with the p53-Mortalin interactions in colon cancer cells

    doi: 10.1038/cddis.2014.100

    Figure Lengend Snippet: UBXN2A overexpression induces apoptosis in colon cancer cell lines but not in normal, non-transformed cells. CCD-18Co, HCT-116, and SW48 were transfected with GFP-empty or GFP-UBXN2A. 48 h after transfection, cells were stained with Annexin V and early apoptosis was determined using flow cytometry as described in Materials and Methods. ( a ) Representative flow-cytometry analysis data from an Annexin V assay. The histograms show a comparison of the distribution of Annexin V positive cells (M1) after transient transfection of cells with GFP-empty vector or GFP-UBXN2A. The data were gated on GFP-UBXN2A positive cells prior to Annexin V analysis. ( b ) UBXN2A expression for 48 h significantly increased the number of apoptotic cells in HCT-116 and SW48 cell lines, while CCD-18Co normal colon cells were unaffected. ( c ) HCT-116 were transfected with scrambled shRNA or shRNA against UBXN2A (clone I and II). 48 h after silencing, superconfluent cultures of HCT-116 were analyzed by Annexin V assay using flow cytometry. Expression of GFP containing shRNA against UBXN2A resulted in 50% less apoptosis in comparison with cells transfected with scrambled shRNA. Values are expressed as mean (±S.E.M.) from three independent experiments (** P

    Article Snippet: Assessment of apoptosis Apoptosis in cells was assessed using an Annexin V Apoptosis Detection Kit (BD Pharmingen, San Jose, CA, USA) analyzed by using a BD Accuri C6 flow cytometer according to the manufacturer's instructions.

    Techniques: Over Expression, Transformation Assay, Transfection, Staining, Flow Cytometry, Cytometry, Annexin V Assay, Plasmid Preparation, Expressing, shRNA

    Interaction of the SEP domain of UBXN2A with the p53-binding site (SBD domain) of mot-2 is sufficient to induce apoptosis. ( a , b ) Schematic diagram of UBXN2A and mot-2 protein domain structures. ( c – f ) Comprehensive mapping of protein–protein interaction sites by Y2H method using α-galactosidase activity and nutritional selection verified that (i) WT-UBXN2A interacts with WT-mot-2, (ii) the SEP domain of UBXN2A is sufficient to interact with WT-mot-2, and (iii) a partial section of p53 binding site on the SBD domain of mot-2 (aa:438-506) is sufficient for binding to WT-UBXN2A. ( g ) An apoptosis assay using Annexin V staining followed by flow cytometry analysis confirmed that only the SEP domain of UBXN2A is required to induce apoptosis in HCT-116 cells, similar to full WT-UBXN2A. No increase in apoptosis was seen with the GFP-UBX domain. ( h ) HCT-116 cells were transfected with GFP-UBXN2A (WT) or its truncated forms (GFP-SEP or GFP-UBX domains). After 48 h, cells were subjected to a crystal violet cell cytotoxicity assay. Counting the remaining colonies showed both WT-UBXN2A and GFP-SEP domains significantly induce cell cytotoxicity (* P

    Journal: Cell Death & Disease

    Article Title: Ubiquitin-like (UBX)-domain-containing protein, UBXN2A, promotes cell death by interfering with the p53-Mortalin interactions in colon cancer cells

    doi: 10.1038/cddis.2014.100

    Figure Lengend Snippet: Interaction of the SEP domain of UBXN2A with the p53-binding site (SBD domain) of mot-2 is sufficient to induce apoptosis. ( a , b ) Schematic diagram of UBXN2A and mot-2 protein domain structures. ( c – f ) Comprehensive mapping of protein–protein interaction sites by Y2H method using α-galactosidase activity and nutritional selection verified that (i) WT-UBXN2A interacts with WT-mot-2, (ii) the SEP domain of UBXN2A is sufficient to interact with WT-mot-2, and (iii) a partial section of p53 binding site on the SBD domain of mot-2 (aa:438-506) is sufficient for binding to WT-UBXN2A. ( g ) An apoptosis assay using Annexin V staining followed by flow cytometry analysis confirmed that only the SEP domain of UBXN2A is required to induce apoptosis in HCT-116 cells, similar to full WT-UBXN2A. No increase in apoptosis was seen with the GFP-UBX domain. ( h ) HCT-116 cells were transfected with GFP-UBXN2A (WT) or its truncated forms (GFP-SEP or GFP-UBX domains). After 48 h, cells were subjected to a crystal violet cell cytotoxicity assay. Counting the remaining colonies showed both WT-UBXN2A and GFP-SEP domains significantly induce cell cytotoxicity (* P

    Article Snippet: Assessment of apoptosis Apoptosis in cells was assessed using an Annexin V Apoptosis Detection Kit (BD Pharmingen, San Jose, CA, USA) analyzed by using a BD Accuri C6 flow cytometer according to the manufacturer's instructions.

    Techniques: Binding Assay, Activity Assay, Selection, Apoptosis Assay, Staining, Flow Cytometry, Cytometry, Transfection, Cytotoxicity Assay

    Induction of apoptosis by UBXN2A is p53 dependent and caspase-mediated in colon cancer cell lines. ( a , b ) HCT-116 (p53+/+) or HCT-116 (p53−/−) cells were transiently transfected with GFP-empty or GFP-UBXN2A for 48 h. An Annexin V apoptosis assay ( a ) and Prestoblue cell viability ( b ) assay show that overexpression of UBXN2A leads to a significant increase in cell apoptosis ( c ) and reduction of cell viability ( d ) in HCT-116 with WT-p53 (p53+/+). There was not a significant change between GFP-empty and GFP-UBXN2A in p53-KO cells (* P

    Journal: Cell Death & Disease

    Article Title: Ubiquitin-like (UBX)-domain-containing protein, UBXN2A, promotes cell death by interfering with the p53-Mortalin interactions in colon cancer cells

    doi: 10.1038/cddis.2014.100

    Figure Lengend Snippet: Induction of apoptosis by UBXN2A is p53 dependent and caspase-mediated in colon cancer cell lines. ( a , b ) HCT-116 (p53+/+) or HCT-116 (p53−/−) cells were transiently transfected with GFP-empty or GFP-UBXN2A for 48 h. An Annexin V apoptosis assay ( a ) and Prestoblue cell viability ( b ) assay show that overexpression of UBXN2A leads to a significant increase in cell apoptosis ( c ) and reduction of cell viability ( d ) in HCT-116 with WT-p53 (p53+/+). There was not a significant change between GFP-empty and GFP-UBXN2A in p53-KO cells (* P

    Article Snippet: Assessment of apoptosis Apoptosis in cells was assessed using an Annexin V Apoptosis Detection Kit (BD Pharmingen, San Jose, CA, USA) analyzed by using a BD Accuri C6 flow cytometer according to the manufacturer's instructions.

    Techniques: Transfection, Apoptosis Assay, Over Expression

    ADC failed to protect HOG-induced HUVEC apoptosis under Nrf2 silenced conditions A. HUVECs were transfected with specific siRNA against Nrf2 or scrambled siRNA (control). After transfection for 24 h, cells were incubated with HOG in the presence or absence of ADC or RES for 24 h. HUVEC apoptosis was determined by Annexin V/PI staining using a flow cytometer. B. HUVECs were pre-treated with ZnPP for 2 h and then incubated with HOG in the presence or absence of ADC or RES for 24 h. Apoptotic cell death was quantified by flow cytometry. Values represent the mean ± SD of three independent experiments. Statistical significance was set at Ф P

    Journal: Oncotarget

    Article Title: Activation of Nrf2-mediated anti-oxidant genes by antrodin C prevents hyperglycemia-induced senescence and apoptosis in human endothelial cells

    doi: 10.18632/oncotarget.19951

    Figure Lengend Snippet: ADC failed to protect HOG-induced HUVEC apoptosis under Nrf2 silenced conditions A. HUVECs were transfected with specific siRNA against Nrf2 or scrambled siRNA (control). After transfection for 24 h, cells were incubated with HOG in the presence or absence of ADC or RES for 24 h. HUVEC apoptosis was determined by Annexin V/PI staining using a flow cytometer. B. HUVECs were pre-treated with ZnPP for 2 h and then incubated with HOG in the presence or absence of ADC or RES for 24 h. Apoptotic cell death was quantified by flow cytometry. Values represent the mean ± SD of three independent experiments. Statistical significance was set at Ф P

    Article Snippet: Quantification of apoptosis Apoptotic cell death was measured according to the percentage of cells with hypodiploid DNA by using Annexin V and PI binding staining with an Annexin V/PI Apoptosis Detection Kit (BD Bioscences, San Jose, CA).

    Techniques: Transfection, Incubation, Staining, Flow Cytometry, Cytometry

    Protective effect of ADC on HG-induced HUVEC apoptosis and growth arrest A. HUVECs were incubated with HG in the presence or absence of ADC or RES for 72 h. Apoptotic cell death was performed with Annexin V/PI staining and the hypodiploid DNA was determined by flow cytometry. B. HUVECs were treated with HG in the presence or absence of ADC or RES for 72 h. LDH release in the supernatant of HUVEC cultures was measured by LDH assay kit as described in Materials and Methods. C. - E. Protein expression levels of cytochrome C, caspase-9 and caspase-3 levels were determined by western blot analysis. The relative protein expression of cytochrome C was normalized with β-actin, whereas cleaved capase-9 and cleaved caspase-3 levels were normalized with pro-caspase-9 and pro-caspase-3, respectively. F. HUVECs proliferation index was determined by flow cytometry. G. HUVECs were incubated with HG in the presence of ADC or RES for 72 h. Cell-cycle distribution was measured by flow cytometer using PI. Percentage of cell population in each transition phase is shown in the histogram. Values represent the mean ± SD of three independent experiments. Statistical significance was set at Ф P

    Journal: Oncotarget

    Article Title: Activation of Nrf2-mediated anti-oxidant genes by antrodin C prevents hyperglycemia-induced senescence and apoptosis in human endothelial cells

    doi: 10.18632/oncotarget.19951

    Figure Lengend Snippet: Protective effect of ADC on HG-induced HUVEC apoptosis and growth arrest A. HUVECs were incubated with HG in the presence or absence of ADC or RES for 72 h. Apoptotic cell death was performed with Annexin V/PI staining and the hypodiploid DNA was determined by flow cytometry. B. HUVECs were treated with HG in the presence or absence of ADC or RES for 72 h. LDH release in the supernatant of HUVEC cultures was measured by LDH assay kit as described in Materials and Methods. C. - E. Protein expression levels of cytochrome C, caspase-9 and caspase-3 levels were determined by western blot analysis. The relative protein expression of cytochrome C was normalized with β-actin, whereas cleaved capase-9 and cleaved caspase-3 levels were normalized with pro-caspase-9 and pro-caspase-3, respectively. F. HUVECs proliferation index was determined by flow cytometry. G. HUVECs were incubated with HG in the presence of ADC or RES for 72 h. Cell-cycle distribution was measured by flow cytometer using PI. Percentage of cell population in each transition phase is shown in the histogram. Values represent the mean ± SD of three independent experiments. Statistical significance was set at Ф P

    Article Snippet: Quantification of apoptosis Apoptotic cell death was measured according to the percentage of cells with hypodiploid DNA by using Annexin V and PI binding staining with an Annexin V/PI Apoptosis Detection Kit (BD Bioscences, San Jose, CA).

    Techniques: Incubation, Staining, Flow Cytometry, Cytometry, Lactate Dehydrogenase Assay, Expressing, Western Blot

    ADC protects HUVECs from hyperosmotic glucose (HOG)-induced cell death HUVECs were incubated with HOG in the presence or absence of ADC or RES for 24 h. A. Cell viability was determined by MTT colorimetric assay. B. HUVEC injury was determined by LDH release into the culture media. C. Intracellular ROS generation was quantified by DCFH 2 -DA flurogenic assay. D. Apoptosis was determined by Annexin V/PI staining assay. E. , F. Protein levels of Bax and cytochrome C levels were measured by western blot analysis. The relative protein levels of Bax and cytochrome C were normalized with β-actin. G. , H. The activation (cleaved form) of caspase-9, caspase-3 and PARP were determined by western blot analysis and the protein levels were normalized with their corresponding pro-form. I. HUVECs were pre-incubated with caspase-3 inhibitor (Z-VAD-FMK, 30 µM) for 2 h and then incubated with HG in the presence or absence of ADC or RES for 24 h. The cell viability was determined by MTT assay. Values represent the mean ± SD of three independent experiments. Statistical significance was set at Ф P

    Journal: Oncotarget

    Article Title: Activation of Nrf2-mediated anti-oxidant genes by antrodin C prevents hyperglycemia-induced senescence and apoptosis in human endothelial cells

    doi: 10.18632/oncotarget.19951

    Figure Lengend Snippet: ADC protects HUVECs from hyperosmotic glucose (HOG)-induced cell death HUVECs were incubated with HOG in the presence or absence of ADC or RES for 24 h. A. Cell viability was determined by MTT colorimetric assay. B. HUVEC injury was determined by LDH release into the culture media. C. Intracellular ROS generation was quantified by DCFH 2 -DA flurogenic assay. D. Apoptosis was determined by Annexin V/PI staining assay. E. , F. Protein levels of Bax and cytochrome C levels were measured by western blot analysis. The relative protein levels of Bax and cytochrome C were normalized with β-actin. G. , H. The activation (cleaved form) of caspase-9, caspase-3 and PARP were determined by western blot analysis and the protein levels were normalized with their corresponding pro-form. I. HUVECs were pre-incubated with caspase-3 inhibitor (Z-VAD-FMK, 30 µM) for 2 h and then incubated with HG in the presence or absence of ADC or RES for 24 h. The cell viability was determined by MTT assay. Values represent the mean ± SD of three independent experiments. Statistical significance was set at Ф P

    Article Snippet: Quantification of apoptosis Apoptotic cell death was measured according to the percentage of cells with hypodiploid DNA by using Annexin V and PI binding staining with an Annexin V/PI Apoptosis Detection Kit (BD Bioscences, San Jose, CA).

    Techniques: Incubation, MTT Assay, Colorimetric Assay, Staining, Western Blot, Activation Assay

    Effects of silencing the expression of 3‐OST2, HVEM and nectin‐2 on the anti‐apoptotic properties of HSV‐1 gD and CyPB. The contribution of 3‐OST2 (as a 3‐ O ‐sulfated HS‐generating enzyme) and of HVEM and nectin‐2 to the protective effect of HSV‐1 gD and CyPB was analysed by measuring staurosporin‐induced apoptosis in siRNA‐treated macrophages. Following treatment with siRNA for 48 h, cells were incubated or not with HSV‐1 gD and CyPB (1 μg·mL −1 ) for 8 h, after which they were exposed to staurosporin (0.5 μ m ) for 4 h. (A) The percentage of apoptotic cell population was evaluated by analysing the binding of fluorescein‐conjugated annexin‐V. Each bar of histogram represents mean ± SD of the rate of apoptotic cells (annexin‐V + /PI − ) obtained from five distinct experiments. (B) In parallel experiments, the activation of caspase‐3 was analysed in siRNA‐treated cells after exposure to staurosporin. Results are expressed as fold increase in caspase‐3 activity by comparison with untreated cells and correspond to means ± SD from five independent experiments (** P

    Journal: FEBS Open Bio

    Article Title: Participation of 3‐O‐sulfated heparan sulfates in the protection of macrophages by herpes simplex virus‐1 glycoprotein D and cyclophilin B against apoptosis

    doi: 10.1002/2211-5463.12145

    Figure Lengend Snippet: Effects of silencing the expression of 3‐OST2, HVEM and nectin‐2 on the anti‐apoptotic properties of HSV‐1 gD and CyPB. The contribution of 3‐OST2 (as a 3‐ O ‐sulfated HS‐generating enzyme) and of HVEM and nectin‐2 to the protective effect of HSV‐1 gD and CyPB was analysed by measuring staurosporin‐induced apoptosis in siRNA‐treated macrophages. Following treatment with siRNA for 48 h, cells were incubated or not with HSV‐1 gD and CyPB (1 μg·mL −1 ) for 8 h, after which they were exposed to staurosporin (0.5 μ m ) for 4 h. (A) The percentage of apoptotic cell population was evaluated by analysing the binding of fluorescein‐conjugated annexin‐V. Each bar of histogram represents mean ± SD of the rate of apoptotic cells (annexin‐V + /PI − ) obtained from five distinct experiments. (B) In parallel experiments, the activation of caspase‐3 was analysed in siRNA‐treated cells after exposure to staurosporin. Results are expressed as fold increase in caspase‐3 activity by comparison with untreated cells and correspond to means ± SD from five independent experiments (** P

    Article Snippet: Measurement of apoptosis Following treatment of macrophages with recombinant HSV‐1 gD or CyPB (both at 1 μg·mL−1 ) for 8 h, apoptosis was induced by the addition of 0.5 μm staurosporin (Merck Millipore, Darmstadt, Germany) for 4 h. For apoptosis analysis, macrophages (2 × 105 cells per point) were stained with annexin‐V and PI, using the Annexin‐V Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer's instructions.

    Techniques: Expressing, Incubation, Binding Assay, Activation Assay, Activity Assay

    Protective effects of HSV‐1 gD and CyPB against apoptosis in primary macrophages. (A, B) Flow cytometry analysis of staurosporin‐induced apoptosis. Macrophages were either untreated or treated with HSV‐1 gD or CyPB (both at 1 μg·mL −1 ) for 8 h and subsequently exposed to staurosporin (0.5 μ m ) for 4 h. At the end of incubation, cells were stained with fluorescein‐conjugated annexin‐V (FL1) and PI (FL2) for flow cytometry analysis. (A) Representative dot‐blots showing the distribution of viable (annexin‐V − /PI − ), early apoptotic (annexin‐V + /PI − ), late apoptotic (annexin‐V + /PI + ) and necrotic (annexin‐V − /PI + ) cell populations. (B) Percentages of early apoptotic cells (annexin‐V + /PI − ). Values are means ± SD from five experiments conducted with macrophages from distinct donors. (C) Analysis of caspase‐3 activation. Following incubation in the absence or presence of HSV‐1 gD or CyPB, macrophages were exposed to staurosporin for 4 h, after which they were lysed. Caspase‐3 activity was then measured in cell lysates using the fluorescent Ac‐DEVD‐AMC substrate, as described in “ Materials and methods ”. Data are expressed as fold increase in caspase‐3 activity by comparison with cells cultured in the absence of staurosporin. Results are means ± SD of five independent experiments performed with cells isolated from distinct donors (*** P

    Journal: FEBS Open Bio

    Article Title: Participation of 3‐O‐sulfated heparan sulfates in the protection of macrophages by herpes simplex virus‐1 glycoprotein D and cyclophilin B against apoptosis

    doi: 10.1002/2211-5463.12145

    Figure Lengend Snippet: Protective effects of HSV‐1 gD and CyPB against apoptosis in primary macrophages. (A, B) Flow cytometry analysis of staurosporin‐induced apoptosis. Macrophages were either untreated or treated with HSV‐1 gD or CyPB (both at 1 μg·mL −1 ) for 8 h and subsequently exposed to staurosporin (0.5 μ m ) for 4 h. At the end of incubation, cells were stained with fluorescein‐conjugated annexin‐V (FL1) and PI (FL2) for flow cytometry analysis. (A) Representative dot‐blots showing the distribution of viable (annexin‐V − /PI − ), early apoptotic (annexin‐V + /PI − ), late apoptotic (annexin‐V + /PI + ) and necrotic (annexin‐V − /PI + ) cell populations. (B) Percentages of early apoptotic cells (annexin‐V + /PI − ). Values are means ± SD from five experiments conducted with macrophages from distinct donors. (C) Analysis of caspase‐3 activation. Following incubation in the absence or presence of HSV‐1 gD or CyPB, macrophages were exposed to staurosporin for 4 h, after which they were lysed. Caspase‐3 activity was then measured in cell lysates using the fluorescent Ac‐DEVD‐AMC substrate, as described in “ Materials and methods ”. Data are expressed as fold increase in caspase‐3 activity by comparison with cells cultured in the absence of staurosporin. Results are means ± SD of five independent experiments performed with cells isolated from distinct donors (*** P

    Article Snippet: Measurement of apoptosis Following treatment of macrophages with recombinant HSV‐1 gD or CyPB (both at 1 μg·mL−1 ) for 8 h, apoptosis was induced by the addition of 0.5 μm staurosporin (Merck Millipore, Darmstadt, Germany) for 4 h. For apoptosis analysis, macrophages (2 × 105 cells per point) were stained with annexin‐V and PI, using the Annexin‐V Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer's instructions.

    Techniques: Flow Cytometry, Cytometry, Incubation, Staining, Activation Assay, Activity Assay, Cell Culture, Isolation

    Theophylline induced cell death and apoptosis in MCF-7 cells ( A ) MCF-7 cells were treated with indicated amount of theophylline, caffeine, or both combinations for 24 h. Cell viability were measured with the MTS assay. ( B ) MCF-7 cells were treated with 10 mM theophylline and caffeine for 24 h. The apoptosis analysis was used with Annexin V using the flow cytometry analysis. MCF-7 cells were treated with indicated concentration of theophylline for 24 h. Cell lysates were subject to ( C ) the RT-PCR analysis with primers for p53, p53 beta, SRSF3. GAPDH as a loading control; ( D ) Western blotting analysis with antibodies against p53 and SRSF3. ACTN served as a loading control. The results are representative of two independent experiments.

    Journal: Oncotarget

    Article Title: Theophylline exhibits anti-cancer activity via suppressing SRSF3 in cervical and breast cancer cell lines

    doi: 10.18632/oncotarget.21464

    Figure Lengend Snippet: Theophylline induced cell death and apoptosis in MCF-7 cells ( A ) MCF-7 cells were treated with indicated amount of theophylline, caffeine, or both combinations for 24 h. Cell viability were measured with the MTS assay. ( B ) MCF-7 cells were treated with 10 mM theophylline and caffeine for 24 h. The apoptosis analysis was used with Annexin V using the flow cytometry analysis. MCF-7 cells were treated with indicated concentration of theophylline for 24 h. Cell lysates were subject to ( C ) the RT-PCR analysis with primers for p53, p53 beta, SRSF3. GAPDH as a loading control; ( D ) Western blotting analysis with antibodies against p53 and SRSF3. ACTN served as a loading control. The results are representative of two independent experiments.

    Article Snippet: For early and late apoptosis analysis, the cells were measured by PE Annexin V Apoptosis Detection Kit (BD Biosciences) and APO-DIRECT Kit (BD Biosciences), respectively.

    Techniques: MTS Assay, Flow Cytometry, Cytometry, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

    APA induced apoptosis by increasing expression of Casp-3,-8,-9 and PARPin MCF-7 and MDA-MB-435 cells ( A – B ) Effect of APA on apoptosis of MCF-7 cells. Cells were stained with Annexin V-PE and the apoptotic cells were analyzed with flow cytometry. (A) is the representative data in MCF-7 cells and (B) is quantitation data for MCF-7 cells and MDA-MB-435 cells; ( C ) Effect of APA on cleaved Casp3,-8,-9 and PARP by western blot; ( D – E ) Quantitation of the western data for MCF-7 (D) and MDA-MB-435 (E) cells. * p

    Journal: Oncotarget

    Article Title: Amplexicaule A exerts anti-tumor effects by inducing apoptosis in human breast cancer

    doi: 10.18632/oncotarget.7848

    Figure Lengend Snippet: APA induced apoptosis by increasing expression of Casp-3,-8,-9 and PARPin MCF-7 and MDA-MB-435 cells ( A – B ) Effect of APA on apoptosis of MCF-7 cells. Cells were stained with Annexin V-PE and the apoptotic cells were analyzed with flow cytometry. (A) is the representative data in MCF-7 cells and (B) is quantitation data for MCF-7 cells and MDA-MB-435 cells; ( C ) Effect of APA on cleaved Casp3,-8,-9 and PARP by western blot; ( D – E ) Quantitation of the western data for MCF-7 (D) and MDA-MB-435 (E) cells. * p

    Article Snippet: Apoptosis assay APA-induced apoptosis in MCF-7 and MDB-MA-435 cells was determined by flow cytometry using the Annexin V-PE Apoptosis Detection Kit following the manufacturer's instructions (BD Bioscience) (28–29).

    Techniques: Expressing, Multiple Displacement Amplification, Staining, Flow Cytometry, Cytometry, Quantitation Assay, Western Blot

    SPOCK1 exerts an anti-apoptotic effect via the PI3K/Akt pathway. (A) Apoptosis was determined by flow cytometry. Cells stained with annexin-V-APC were considered as apoptotic. The apoptotic index was defined as the percentage of apoptotic cells. (B) Apoptotic changes in the nuclear morphology of GBC-SD and NOZ cells as indicated by Hoechst 44322 staining (blue). The apoptotic index, defined as the percentage of apoptotic cells, was calculated and is summarized in the bar chart (* P

    Journal: Molecular Cancer

    Article Title: SPOCK1 as a potential cancer prognostic marker promotes the proliferation and metastasis of gallbladder cancer cells by activating the PI3K/AKT pathway

    doi: 10.1186/s12943-014-0276-y

    Figure Lengend Snippet: SPOCK1 exerts an anti-apoptotic effect via the PI3K/Akt pathway. (A) Apoptosis was determined by flow cytometry. Cells stained with annexin-V-APC were considered as apoptotic. The apoptotic index was defined as the percentage of apoptotic cells. (B) Apoptotic changes in the nuclear morphology of GBC-SD and NOZ cells as indicated by Hoechst 44322 staining (blue). The apoptotic index, defined as the percentage of apoptotic cells, was calculated and is summarized in the bar chart (* P

    Article Snippet: Flow cytometric analysis of cell apoptosis The extent of apoptosis was measured with an Annexin V-APC Apoptosis Detection kit (BD Biosciences) according to the manufacturer′s instructions.

    Techniques: Flow Cytometry, Cytometry, Staining

    Enz-ATRA treatment-triggered apoptosis is mitochondria-dependent but caspase-independent. NB4-R1 (A) and NB4-R2 (B) cells were treated with 2 μM enzastaurin (EN) and/or 1 μM ATRA (RA) for four days. One representative scatter plots of flow-cytometric analysis of mitochondrial transmembrane potential (assessed by uptake of rhodamine 123 [Rh123]) is shown. The percentages of Rh123- cells are shown in the corresponding panels. Western-blotting analysis of caspase-3, caspase-6 and caspase-7 in NB4-R1 (C) and NB4-R2 (D) cells treated with 2 μM enzastaurin and/or 1 μM ATRA for 24 h. Expression of β-actin was assessed as internal control. Similar results were obtained in three independent experiments. NB4-R1 and NB4-R2 cells were pretreated with different concentrations of (1, 2, 4 μM) DEVD (caspase-3/7 inhibitor) or different concentrations of (1, 5, 10 μM) VEID (caspase-6 inhibitor) for 1 h prior to enz-ATRA treatment for four days. The effect of DEVD or VEID on enz-ATRA treatment-triggered apoptosis in NB4-R1 (E) and NB4-R2 (F) cells was determined by Annexin V analysis. The percentages of Annexin V + cells are shown in the corresponding panels. Results were representative among three independent experiments.

    Journal: American Journal of Cancer Research

    Article Title: Combination of enzastaurin and ATRA exerts dose-dependent dual effects on ATRA-resistant acute promyelocytic leukemia cells

    doi:

    Figure Lengend Snippet: Enz-ATRA treatment-triggered apoptosis is mitochondria-dependent but caspase-independent. NB4-R1 (A) and NB4-R2 (B) cells were treated with 2 μM enzastaurin (EN) and/or 1 μM ATRA (RA) for four days. One representative scatter plots of flow-cytometric analysis of mitochondrial transmembrane potential (assessed by uptake of rhodamine 123 [Rh123]) is shown. The percentages of Rh123- cells are shown in the corresponding panels. Western-blotting analysis of caspase-3, caspase-6 and caspase-7 in NB4-R1 (C) and NB4-R2 (D) cells treated with 2 μM enzastaurin and/or 1 μM ATRA for 24 h. Expression of β-actin was assessed as internal control. Similar results were obtained in three independent experiments. NB4-R1 and NB4-R2 cells were pretreated with different concentrations of (1, 2, 4 μM) DEVD (caspase-3/7 inhibitor) or different concentrations of (1, 5, 10 μM) VEID (caspase-6 inhibitor) for 1 h prior to enz-ATRA treatment for four days. The effect of DEVD or VEID on enz-ATRA treatment-triggered apoptosis in NB4-R1 (E) and NB4-R2 (F) cells was determined by Annexin V analysis. The percentages of Annexin V + cells are shown in the corresponding panels. Results were representative among three independent experiments.

    Article Snippet: Annexin-V assay was performed according to instructions provided in the Annexin V-7AAD Apoptosis Detection Kit (BD Biosciences Pharmingen, San Diego, CA, USA).

    Techniques: Flow Cytometry, Western Blot, Expressing

    Effects of enz-ATRA treatment on cell growth, survival, apoptosis and differentiation in NB4-R1 cells. NB4-R1 cells were treated with 1 μM (1EN), 2 μM enzastaurin (2EN), 1 μM ATRA (RA) and in enz-ATRA combination (EN+RA) for four days. One representative experiment of cell growth (A) and cell viability (B) is shown. Each value represents the mean ± SD of triplicate samples. Similar results were obtained in three independent experiments. Representative morphology of NB4-R1 cells treated with the indicated drugs for four days (C). Scale bar represents 5 μm and the magnification is 1,000. Similar results were obtained in three independent experiments. Annexin-V assay of NB4-R1 cells treated with enzastaurin or/and ATRA for four days (D). The percentages of Annexin V + cells are shown in the corresponding panels. Results were representative among three independent experiments. Differentiation was also evaluated by NBT-reduction assay (E) and flow-cytometric analysis of CD11b expression in NB4-R1 cells (F) with the indicated treatment for four days. For NBT-reduction assay, one representative experiment is shown. Each value represents the mean ± SD of triplicate samples. Similar results were obtained in three independent experiments. For flow-cytometric analysis of CD11b expression, each value represents the mean ± SD of three independent measurements. * P

    Journal: American Journal of Cancer Research

    Article Title: Combination of enzastaurin and ATRA exerts dose-dependent dual effects on ATRA-resistant acute promyelocytic leukemia cells

    doi:

    Figure Lengend Snippet: Effects of enz-ATRA treatment on cell growth, survival, apoptosis and differentiation in NB4-R1 cells. NB4-R1 cells were treated with 1 μM (1EN), 2 μM enzastaurin (2EN), 1 μM ATRA (RA) and in enz-ATRA combination (EN+RA) for four days. One representative experiment of cell growth (A) and cell viability (B) is shown. Each value represents the mean ± SD of triplicate samples. Similar results were obtained in three independent experiments. Representative morphology of NB4-R1 cells treated with the indicated drugs for four days (C). Scale bar represents 5 μm and the magnification is 1,000. Similar results were obtained in three independent experiments. Annexin-V assay of NB4-R1 cells treated with enzastaurin or/and ATRA for four days (D). The percentages of Annexin V + cells are shown in the corresponding panels. Results were representative among three independent experiments. Differentiation was also evaluated by NBT-reduction assay (E) and flow-cytometric analysis of CD11b expression in NB4-R1 cells (F) with the indicated treatment for four days. For NBT-reduction assay, one representative experiment is shown. Each value represents the mean ± SD of triplicate samples. Similar results were obtained in three independent experiments. For flow-cytometric analysis of CD11b expression, each value represents the mean ± SD of three independent measurements. * P

    Article Snippet: Annexin-V assay was performed according to instructions provided in the Annexin V-7AAD Apoptosis Detection Kit (BD Biosciences Pharmingen, San Diego, CA, USA).

    Techniques: Annexin V Assay, Flow Cytometry, Expressing

    MEK inhibition primarily suppresses differentiation and restores the protein levels of C/EBPβ or PU.1. Cells were exposed to 0.5 μM U0126 for 1 h prior to other treatment. The attenuation of MEK activation by U0126 (U) was detected by Western-blotting analysis of phosphorylated ERK1/2 in NB4-R1 (A) and NB4-R2 cells (B) with indicated treatments for 12 h and 36 h, respectively. The same membrane incubated with the antibody to phosphorylated Erk1/2 was stripped and followed by detection of ERK1/2. Similar results were obtained in three independent experiments. Effect of U0126 on morphology in NB4-R1 (C) and NB4-R2 cells (D) incubated with the indicated drugs for four days. Scale bar represents 5 μm and the magnification is 1,000. One representative experiment among three independent assays is shown. Similar results were obtained in three independent experiments. The effect of U0126 on apoptosis and differentiation was also confirmed by Annexin-V assay (E) and flow-cytometric analysis of CD11b expression (F) in NB4-R1 and NB4-R2 cells with the indicated treatments for four days. Each value represented the mean ± SD of three independent measurements. ### P

    Journal: American Journal of Cancer Research

    Article Title: Combination of enzastaurin and ATRA exerts dose-dependent dual effects on ATRA-resistant acute promyelocytic leukemia cells

    doi:

    Figure Lengend Snippet: MEK inhibition primarily suppresses differentiation and restores the protein levels of C/EBPβ or PU.1. Cells were exposed to 0.5 μM U0126 for 1 h prior to other treatment. The attenuation of MEK activation by U0126 (U) was detected by Western-blotting analysis of phosphorylated ERK1/2 in NB4-R1 (A) and NB4-R2 cells (B) with indicated treatments for 12 h and 36 h, respectively. The same membrane incubated with the antibody to phosphorylated Erk1/2 was stripped and followed by detection of ERK1/2. Similar results were obtained in three independent experiments. Effect of U0126 on morphology in NB4-R1 (C) and NB4-R2 cells (D) incubated with the indicated drugs for four days. Scale bar represents 5 μm and the magnification is 1,000. One representative experiment among three independent assays is shown. Similar results were obtained in three independent experiments. The effect of U0126 on apoptosis and differentiation was also confirmed by Annexin-V assay (E) and flow-cytometric analysis of CD11b expression (F) in NB4-R1 and NB4-R2 cells with the indicated treatments for four days. Each value represented the mean ± SD of three independent measurements. ### P

    Article Snippet: Annexin-V assay was performed according to instructions provided in the Annexin V-7AAD Apoptosis Detection Kit (BD Biosciences Pharmingen, San Diego, CA, USA).

    Techniques: Inhibition, Activation Assay, Western Blot, Incubation, Annexin V Assay, Flow Cytometry, Expressing

    Effects of enz-ATRA treatment on cell growth, survival, apoptosis and differentiation in NB4-R2 cells. NB4-R2 cells were treated with 1 μM (1EN), 2 μM enzastaurin (2EN), 1 μM ATRA (RA) and in enz-ATRA combination (EN+RA) for four days. One representative experiment of cell growth (A) and cell viability (B) is shown. Each value represents the mean ± SD of triplicate samples. Similar results were obtained in three independent experiments. Representative morphology of NB4-R2 cells treated with the indicated drugs for four days (C). Scale bar represents 5 μm and the magnification is 1,000. Similar results were obtained in three independent experiments. Annexin-V assay of NB4-R2 cells treated with enzastaurin or/and ATRA for four days (D). The percentages of Annexin V + cells are shown in the corresponding panels. Results were representative among three independent experiments. Differentiation was also assessed by NBT-reduction assay (E) and flow-cytometric analysis of CD11b expression in NB4-R2 cells (F) with the indicated treatment for four days. For NBT-reduction assay, one representative experiment is shown. Each value represents the mean ± SD of triplicate samples. Similar results were obtained in three independent experiments. For flow-cytometric analysis of CD11b expression, each value represents the mean ± SD of three independent measurements. ** P

    Journal: American Journal of Cancer Research

    Article Title: Combination of enzastaurin and ATRA exerts dose-dependent dual effects on ATRA-resistant acute promyelocytic leukemia cells

    doi:

    Figure Lengend Snippet: Effects of enz-ATRA treatment on cell growth, survival, apoptosis and differentiation in NB4-R2 cells. NB4-R2 cells were treated with 1 μM (1EN), 2 μM enzastaurin (2EN), 1 μM ATRA (RA) and in enz-ATRA combination (EN+RA) for four days. One representative experiment of cell growth (A) and cell viability (B) is shown. Each value represents the mean ± SD of triplicate samples. Similar results were obtained in three independent experiments. Representative morphology of NB4-R2 cells treated with the indicated drugs for four days (C). Scale bar represents 5 μm and the magnification is 1,000. Similar results were obtained in three independent experiments. Annexin-V assay of NB4-R2 cells treated with enzastaurin or/and ATRA for four days (D). The percentages of Annexin V + cells are shown in the corresponding panels. Results were representative among three independent experiments. Differentiation was also assessed by NBT-reduction assay (E) and flow-cytometric analysis of CD11b expression in NB4-R2 cells (F) with the indicated treatment for four days. For NBT-reduction assay, one representative experiment is shown. Each value represents the mean ± SD of triplicate samples. Similar results were obtained in three independent experiments. For flow-cytometric analysis of CD11b expression, each value represents the mean ± SD of three independent measurements. ** P

    Article Snippet: Annexin-V assay was performed according to instructions provided in the Annexin V-7AAD Apoptosis Detection Kit (BD Biosciences Pharmingen, San Diego, CA, USA).

    Techniques: Annexin V Assay, Flow Cytometry, Expressing

    siRNA against the CD44 3′-UTR enhances proliferation and decreases CD44 and CDC42 protein levels and apoptosis. ( a ) Total RNA was collected from CD44 3′-UTR cells transfected with different siRNAs targeting CD44 3′-UTR. These samples were used in RT–PCR to measure CD44 mRNA levels. All siRNAs were found to decrease the levels of CD44 mRNA. ( b ) CD44 3′-UTR and control cells were transfected with different siRNAs targeting the CD44 3′-UTR. Cell lysates were subjected to western-blot analysis. Both CD44 and CDC42 levels decreased as a result of siRNA targeting CD44 3′-UTR. ( c ) Cell-cycle distribution was analyzed. There was an increase in the S and G2 phases of the siRNA treated cells compared with the control, which had an increased G1 population. n = 3. ( d ) Apoptosis of the siRNA treatment was analyzed using Annexin V staining. Treatments with siRNA-187, siRNA-541 and siRNA-650 decreased apoptosis compared with the control. n = 5.

    Journal: Nucleic Acids Research

    Article Title: Expression of CD44 3?-untranslated region regulates endogenous microRNA functions in tumorigenesis and angiogenesis

    doi: 10.1093/nar/gkq1003

    Figure Lengend Snippet: siRNA against the CD44 3′-UTR enhances proliferation and decreases CD44 and CDC42 protein levels and apoptosis. ( a ) Total RNA was collected from CD44 3′-UTR cells transfected with different siRNAs targeting CD44 3′-UTR. These samples were used in RT–PCR to measure CD44 mRNA levels. All siRNAs were found to decrease the levels of CD44 mRNA. ( b ) CD44 3′-UTR and control cells were transfected with different siRNAs targeting the CD44 3′-UTR. Cell lysates were subjected to western-blot analysis. Both CD44 and CDC42 levels decreased as a result of siRNA targeting CD44 3′-UTR. ( c ) Cell-cycle distribution was analyzed. There was an increase in the S and G2 phases of the siRNA treated cells compared with the control, which had an increased G1 population. n = 3. ( d ) Apoptosis of the siRNA treatment was analyzed using Annexin V staining. Treatments with siRNA-187, siRNA-541 and siRNA-650 decreased apoptosis compared with the control. n = 5.

    Article Snippet: The cells were separately washed twice with cold PBS and stained with binding buffer containing fluorescein-labeled annexin V and with PI (APC Annexin V Apoptosis Detection Kit I, BD Pharmingen) following manufacturer’s instructions.

    Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining

    NB-ZSG or ZSG expression does not affect CD4 + T cell viability, proliferation or induce apoptosis. a An MTS assay was used to measure the viability of CD4 + T cells expressing NB-ZSG1 or ZSG1 or NT. b The proliferation of CD4 + T cells expressing NB-ZSG1 or ZSG1 or NT was measured using VPD450 dye assay. c An apoptosis assay using PE Annexin V was used to monitor levels of apoptosis in CD4 + T cells expressing NB-ZSG1 or ZSG1 or NT. All assays were performed in triplicate and mean values and standard deviations are shown

    Journal: Virology Journal

    Article Title: A mutant Tat protein inhibits infection of human cells by strains from diverse HIV-1 subtypes

    doi: 10.1186/s12985-017-0705-9

    Figure Lengend Snippet: NB-ZSG or ZSG expression does not affect CD4 + T cell viability, proliferation or induce apoptosis. a An MTS assay was used to measure the viability of CD4 + T cells expressing NB-ZSG1 or ZSG1 or NT. b The proliferation of CD4 + T cells expressing NB-ZSG1 or ZSG1 or NT was measured using VPD450 dye assay. c An apoptosis assay using PE Annexin V was used to monitor levels of apoptosis in CD4 + T cells expressing NB-ZSG1 or ZSG1 or NT. All assays were performed in triplicate and mean values and standard deviations are shown

    Article Snippet: Apoptosis events were quantified using a PE Annexin V apoptosis detection kits (BD PharmingenTM ) as per the manufacturer’s instructions.

    Techniques: Expressing, MTS Assay, Apoptosis Assay

    Expression of NE G185R induces accelerated apoptosis in 32D/GR cells. A , 32D/GR cells were transfected with the empty pBabe-puro vector (32D/Ctr) or expression constructs for NE (32D/NE) or NE G185R (32D/G185R) and examined for expression of NE proteins by Western blotting. B and C , 32D/GR clones as indicated were cultured in G-CSF. The numbers of viable cells and cell viability were determined on different days by exclusion of trypan blue staining. Error bars represent S.D. D , cells as indicated were cultured in G-CSF for 4 days and stained with annexin V and 7-aminoactinomycin ( 7AAD ). The percentages of annexin V-positive (early and late apoptotic) cells are indicated. E , morphological features of 32D/Ctr, 32D/NE, and 32D/G185R cells following culture in G-CSF for 5 days. F , 32D/NE and 32D/G185R cells were cultured in G-CSF for 5 days prior to evaluation of surface expression of Gr-1 by flow cytometry. ** denotes p

    Journal: The Journal of Biological Chemistry

    Article Title: A Truncated Granulocyte Colony-stimulating Factor Receptor (G-CSFR) Inhibits Apoptosis Induced by Neutrophil Elastase G185R Mutant

    doi: 10.1074/jbc.M116.755157

    Figure Lengend Snippet: Expression of NE G185R induces accelerated apoptosis in 32D/GR cells. A , 32D/GR cells were transfected with the empty pBabe-puro vector (32D/Ctr) or expression constructs for NE (32D/NE) or NE G185R (32D/G185R) and examined for expression of NE proteins by Western blotting. B and C , 32D/GR clones as indicated were cultured in G-CSF. The numbers of viable cells and cell viability were determined on different days by exclusion of trypan blue staining. Error bars represent S.D. D , cells as indicated were cultured in G-CSF for 4 days and stained with annexin V and 7-aminoactinomycin ( 7AAD ). The percentages of annexin V-positive (early and late apoptotic) cells are indicated. E , morphological features of 32D/Ctr, 32D/NE, and 32D/G185R cells following culture in G-CSF for 5 days. F , 32D/NE and 32D/G185R cells were cultured in G-CSF for 5 days prior to evaluation of surface expression of Gr-1 by flow cytometry. ** denotes p

    Article Snippet: Apoptosis was examined using the Annexin V-phycoerythrin apoptosis detection kit (BD Biosciences).

    Techniques: Expressing, Transfection, Plasmid Preparation, Construct, Western Blot, Clone Assay, Cell Culture, Staining, Flow Cytometry, Cytometry

    G-CSFR d715 blocks apoptosis induced by NE G185R in FDCP-mix A4 cells. A , surface expression of human WT G-CSFR ( GR ) or G-CSFR d715 was examined by flow cytometry. B , the expression of NE G185R in FDCP/GR and FDCP/d715 cells was examined by Western blotting analysis. Cells were subsequently cultured in G-CSF, and the numbers of viable cells ( C ) and cell viabilities ( D ) were determined daily. Error bars represent S.D. E , cells as indicated were cultured in G-CSF for 4 days. Apoptotic cell death was evaluated by annexin V assay. Numbers indicate the percentages of annexin V-positive cells. ** denotes p

    Journal: The Journal of Biological Chemistry

    Article Title: A Truncated Granulocyte Colony-stimulating Factor Receptor (G-CSFR) Inhibits Apoptosis Induced by Neutrophil Elastase G185R Mutant

    doi: 10.1074/jbc.M116.755157

    Figure Lengend Snippet: G-CSFR d715 blocks apoptosis induced by NE G185R in FDCP-mix A4 cells. A , surface expression of human WT G-CSFR ( GR ) or G-CSFR d715 was examined by flow cytometry. B , the expression of NE G185R in FDCP/GR and FDCP/d715 cells was examined by Western blotting analysis. Cells were subsequently cultured in G-CSF, and the numbers of viable cells ( C ) and cell viabilities ( D ) were determined daily. Error bars represent S.D. E , cells as indicated were cultured in G-CSF for 4 days. Apoptotic cell death was evaluated by annexin V assay. Numbers indicate the percentages of annexin V-positive cells. ** denotes p

    Article Snippet: Apoptosis was examined using the Annexin V-phycoerythrin apoptosis detection kit (BD Biosciences).

    Techniques: Expressing, Flow Cytometry, Cytometry, Western Blot, Cell Culture, Annexin V Assay

    Effect of SH on ESCC cell apoptosis. ESCC cells were pretreated with SH and exposed to 8 Gy X-ray. Annexin V-PE/7AAD staining and flow cytometry were used to measure and analyze the cell apoptosis ratio. SH combined with radiation therapy significantly increased ESCC cell apoptosis (*P

    Journal: Oncology Reports

    Article Title: Effect of sinomenine hydrochloride on radiosensitivity of esophageal squamous cell carcinoma cells

    doi: 10.3892/or.2018.6228

    Figure Lengend Snippet: Effect of SH on ESCC cell apoptosis. ESCC cells were pretreated with SH and exposed to 8 Gy X-ray. Annexin V-PE/7AAD staining and flow cytometry were used to measure and analyze the cell apoptosis ratio. SH combined with radiation therapy significantly increased ESCC cell apoptosis (*P

    Article Snippet: Annexin V-7AAD apoptosis-detection kits were purchased from BD Biosciences (Franklin Lakes, NJ, USA).

    Techniques: Staining, Flow Cytometry, Cytometry

    Knockdown of GOLIM4 induced apoptosis in head and neck cancer cells ( A ) Flow cytometry detected the expression of annexin V after knockdown of GOLIM4 in FaDu cells. ( B ) Quantitation of cell apoptosis induced by knockdown of GOLIM4 in (A). * P

    Journal: Bioscience Reports

    Article Title: Golgi integral membrane protein 4 manipulates cellular proliferation, apoptosis, and cell cycle in human head and neck cancer

    doi: 10.1042/BSR20180454

    Figure Lengend Snippet: Knockdown of GOLIM4 induced apoptosis in head and neck cancer cells ( A ) Flow cytometry detected the expression of annexin V after knockdown of GOLIM4 in FaDu cells. ( B ) Quantitation of cell apoptosis induced by knockdown of GOLIM4 in (A). * P

    Article Snippet: Cell apoptosis was evaluated by Annexin V Apoptosis Detection Kit APC (88-8007, BD Bioscience ) .

    Techniques: Flow Cytometry, Cytometry, Expressing, Quantitation Assay

    JQ1 and Bortezomib exhibit synergistic cytotoxicity in BETi-resistant cells. a Representative phase contrast microscope images of LoVo and HCT116 cells treated with JQ1 (1 μM), Bortezomib (5 nM), or JQ1+Bortezomib for 72 h. b LoVo and HCT116 cells were treated with JQ1 (1 μM), Bortezomib (5 nM), or JQ1+Bortezomib for 48 h. Cell apoptosis was analyzed by Annexin V/PI staining. c Western blot of cleaved PARP1 in the HCT116 and LoVo cells treated with indicated compounds. d LoVo and HCT116 cells were treated with JQ1 (1 μM), Bortezomib (5 nM), or JQ1+Bortezomib for 48 h. Cell cycles were analyzed by PI staining. e Immunohistochemical staining of Ki67, cleaved PARP in xenograft tumor sections of HCT116 cells. Bar, 100 μm

    Journal: Cell Death & Disease

    Article Title: Co-inhibition of BET proteins and NF-κB as a potential therapy for colorectal cancer through synergistic inhibiting MYC and FOXM1 expressions

    doi: 10.1038/s41419-018-0354-y

    Figure Lengend Snippet: JQ1 and Bortezomib exhibit synergistic cytotoxicity in BETi-resistant cells. a Representative phase contrast microscope images of LoVo and HCT116 cells treated with JQ1 (1 μM), Bortezomib (5 nM), or JQ1+Bortezomib for 72 h. b LoVo and HCT116 cells were treated with JQ1 (1 μM), Bortezomib (5 nM), or JQ1+Bortezomib for 48 h. Cell apoptosis was analyzed by Annexin V/PI staining. c Western blot of cleaved PARP1 in the HCT116 and LoVo cells treated with indicated compounds. d LoVo and HCT116 cells were treated with JQ1 (1 μM), Bortezomib (5 nM), or JQ1+Bortezomib for 48 h. Cell cycles were analyzed by PI staining. e Immunohistochemical staining of Ki67, cleaved PARP in xenograft tumor sections of HCT116 cells. Bar, 100 μm

    Article Snippet: Apoptosis was measured by PharmingenTM Annexin V Apoptosis Detection Kit (BD Biosciences, Rockville, USA) according to the manufacturer’s instructions, and analyzed by flow cytometry after incubation for 20 min.

    Techniques: Microscopy, Staining, Western Blot, Immunohistochemistry

    DCA inhibits cell proliferation, arrests the cell cycle, and induces apoptosis. (A-B) HepG2 and HepG3B cells were treated with dichloroacetate (DCA) at 0, 20, 40, 60, or 80 mM for 24 h and 48 h and then tested by MTT assay. Cells exposed to DCA at the indicated doses for 24 h were stained with propidium iodide (C) and Annexin V/PI labeling (D), then subjected to flow cytometry analysis. Total cell death and the cell cycle were quantified by flow cytometry (E-F). Data are the mean ±SD, n=3, * P

    Journal: Journal of Cancer

    Article Title: Pyruvate dehydrogenase kinase 1 interferes with glucose metabolism reprogramming and mitochondrial quality control to aggravate stress damage in cancer

    doi: 10.7150/jca.34330

    Figure Lengend Snippet: DCA inhibits cell proliferation, arrests the cell cycle, and induces apoptosis. (A-B) HepG2 and HepG3B cells were treated with dichloroacetate (DCA) at 0, 20, 40, 60, or 80 mM for 24 h and 48 h and then tested by MTT assay. Cells exposed to DCA at the indicated doses for 24 h were stained with propidium iodide (C) and Annexin V/PI labeling (D), then subjected to flow cytometry analysis. Total cell death and the cell cycle were quantified by flow cytometry (E-F). Data are the mean ±SD, n=3, * P

    Article Snippet: Flow cytometry Cell were harvested and stained with Annexin V-FITC and propidium iodide (PI) (Annexin V Apoptosis Detection Kit, BD Pharmingen, USA) to measure cellular apoptosis.

    Techniques: MTT Assay, Staining, Labeling, Flow Cytometry, Cytometry

    Apoptosis analysis after lenti-X4R5-Cas9 modification in primary CD4 + T cells. a Annexin V and 7-AAD were utilized to stain modified CD4 + T at 1,3 and 5 days post nucleofection with flow cytometry. Necrotic cells (Annexin V-/7AAD +), necrotic or late apoptotic cells (Annexin V +/7AAD +); early apoptotic cells (Annexin V +/7AAD-); viable cells (Annexin V-/7AAD-). b Early apoptosis ratio at day 1,3,5 post gene disruption. c relative early apoptosis ratio (Q3/total none viable cells). The total none viable cells = Q2 + Q3. The data shown were the mean ± SD of three independent experiments. *P

    Journal: Cell & Bioscience

    Article Title: Genome editing of the HIV co-receptors CCR5 and CXCR4 by CRISPR-Cas9 protects CD4+ T cells from HIV-1 infection

    doi: 10.1186/s13578-017-0174-2

    Figure Lengend Snippet: Apoptosis analysis after lenti-X4R5-Cas9 modification in primary CD4 + T cells. a Annexin V and 7-AAD were utilized to stain modified CD4 + T at 1,3 and 5 days post nucleofection with flow cytometry. Necrotic cells (Annexin V-/7AAD +), necrotic or late apoptotic cells (Annexin V +/7AAD +); early apoptotic cells (Annexin V +/7AAD-); viable cells (Annexin V-/7AAD-). b Early apoptosis ratio at day 1,3,5 post gene disruption. c relative early apoptosis ratio (Q3/total none viable cells). The total none viable cells = Q2 + Q3. The data shown were the mean ± SD of three independent experiments. *P

    Article Snippet: To assess the effect of both CXCR4 and CCR5 editing on cell apoptosis, the Annexin V Apoptosis detection kit 1 (BD Pharmingin) was used to evaluate early apoptosis.

    Techniques: Modification, Staining, Flow Cytometry, Cytometry

    Dense granule protein 15 (GRA15 II )-induced loss of cell viability and apoptosis. Choriocarcinoma JEG-3 cells were transfected with either empty vector (pEGFP, encoding enhanced green fluorescent protein) or pEGFP-GRA15 II for 24 h. Untransfected cells served as the negative control, and staurosporine (STS) treated cells (1 μM, 6 h) served as the positive control. a Cell viability was measured using the MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium) assay. b Cell apoptosis was determined by the phycoerythrin-annexin V/7-AAD flow cytometry assay. * P

    Journal: Parasites & Vectors

    Article Title: Toxoplasma gondii dense granule protein 15 induces apoptosis in choriocarcinoma JEG-3 cells through endoplasmic reticulum stress

    doi: 10.1186/s13071-018-2835-3

    Figure Lengend Snippet: Dense granule protein 15 (GRA15 II )-induced loss of cell viability and apoptosis. Choriocarcinoma JEG-3 cells were transfected with either empty vector (pEGFP, encoding enhanced green fluorescent protein) or pEGFP-GRA15 II for 24 h. Untransfected cells served as the negative control, and staurosporine (STS) treated cells (1 μM, 6 h) served as the positive control. a Cell viability was measured using the MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium) assay. b Cell apoptosis was determined by the phycoerythrin-annexin V/7-AAD flow cytometry assay. * P

    Article Snippet: A phycoerythrin-annexin V apoptosis detection kit (flow cytometry based assay, BD Biosciences, Franklin Lakes, NJ, USA) was used to determine cell apoptosis.

    Techniques: Transfection, Plasmid Preparation, Negative Control, Positive Control, Flow Cytometry, Cytometry

    Effects of IRE1α inhibitor 4μ8C and JNK inhibitor SP6000125 on loss of cell viability and apoptosis of pEGFP-GRA15 II -transfected cells. Choriocarcinoma JEG-3 cells were transfected with either the empty vector (pEGFP, encoding enhanced green fluorescent protein) or pEGFP-GRA15 II for 24 h. Tunicamycin (TM) treated (1 μM, 24 h) cells served as the control. Cells were treated with either 4μ8C (100 nM, 12 h) or SP6000125 (20 μM, 12 h) 12 h after pEGFP-GRA15 II transfection. a Cell viability was measured using the MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium) assay. b Cell apoptosis was determined by the phycoerythrin-annexin V/7-AAD flow cytometry assay. * P

    Journal: Parasites & Vectors

    Article Title: Toxoplasma gondii dense granule protein 15 induces apoptosis in choriocarcinoma JEG-3 cells through endoplasmic reticulum stress

    doi: 10.1186/s13071-018-2835-3

    Figure Lengend Snippet: Effects of IRE1α inhibitor 4μ8C and JNK inhibitor SP6000125 on loss of cell viability and apoptosis of pEGFP-GRA15 II -transfected cells. Choriocarcinoma JEG-3 cells were transfected with either the empty vector (pEGFP, encoding enhanced green fluorescent protein) or pEGFP-GRA15 II for 24 h. Tunicamycin (TM) treated (1 μM, 24 h) cells served as the control. Cells were treated with either 4μ8C (100 nM, 12 h) or SP6000125 (20 μM, 12 h) 12 h after pEGFP-GRA15 II transfection. a Cell viability was measured using the MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium) assay. b Cell apoptosis was determined by the phycoerythrin-annexin V/7-AAD flow cytometry assay. * P

    Article Snippet: A phycoerythrin-annexin V apoptosis detection kit (flow cytometry based assay, BD Biosciences, Franklin Lakes, NJ, USA) was used to determine cell apoptosis.

    Techniques: Transfection, Plasmid Preparation, Flow Cytometry, Cytometry

    Annexin V-PI cytofluorimetric analyses were performed to observe apoptosis. hTERT-hNOF cells were treated with 15-second (a) air or (b) air APPJ. After treatment, 3 hr of incubation was performed in supplemented media before analysis. 10,000 cells were counted per each running.

    Journal: BioMed Research International

    Article Title: Effects of a Nonthermal Atmospheric Pressure Plasma Jet on Human Gingival Fibroblasts for Biomedical Application

    doi: 10.1155/2016/2876916

    Figure Lengend Snippet: Annexin V-PI cytofluorimetric analyses were performed to observe apoptosis. hTERT-hNOF cells were treated with 15-second (a) air or (b) air APPJ. After treatment, 3 hr of incubation was performed in supplemented media before analysis. 10,000 cells were counted per each running.

    Article Snippet: Annexin V-PI Cytofluorimetric Analyses Cell apoptosis and necrosis were analyzed with the annexin V-fluoroisothiocyanate apoptosis detection kit (BD Biosciences Pharmingen, CA, USA) following the manufacturer's instructions. hTERT-hNOF cells incubated for 3 days as described in . were treated with 15-second air or air APPJ.

    Techniques: Incubation

    S-phase arrest induced by CPT or LMP-400 is abrogated by VE-821 ( A ) Experimental protocol. ( B ) Cell cycle effects were determined by propidium iodide (PI) staining. Vertical dashed lines correspond to 2N and 4N DNA contents. ( C ) S-phase progression was monitored by following the cells that were replicating (EdU-labeled) at the time of Top1 inhibitor treatment. ( D ) Effect of VE-821 on apoptotic cells48 h after Top1 inhibitors removal. Annexin V/PI double staining was used to monitor apoptosis.

    Journal: Cancer research

    Article Title: ATR inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase I inhibitors by disabling DNA replication initiation and fork elongation responses

    doi: 10.1158/0008-5472.CAN-13-3369

    Figure Lengend Snippet: S-phase arrest induced by CPT or LMP-400 is abrogated by VE-821 ( A ) Experimental protocol. ( B ) Cell cycle effects were determined by propidium iodide (PI) staining. Vertical dashed lines correspond to 2N and 4N DNA contents. ( C ) S-phase progression was monitored by following the cells that were replicating (EdU-labeled) at the time of Top1 inhibitor treatment. ( D ) Effect of VE-821 on apoptotic cells48 h after Top1 inhibitors removal. Annexin V/PI double staining was used to monitor apoptosis.

    Article Snippet: Apoptotic cells were detected 48 hour-post Top1 inhibitors using Annexin V/PI co-staining (FITC Annexin V Apoptosis kit, BD Biosciences).

    Techniques: Cycling Probe Technology, Staining, Labeling, Double Staining

    RA190 triggers apoptosis and cell surface presentation of HSP90 (A,B) HeLa cells treated with 1 μM RA190 (190) or botezomib (Bz) for 12 hr (A) or MM.1S cells treated with 0.5 μM RA190 or bortezomib for 6 hr (B) were analyzed by flow cytometry after staining with fluorescein-labeled Annexin-V (C) HeLa cells treated as in (A) were analyzed by flow cytometry after staining for active caspase-3 and the percent of positive cells ±SD was determined. (D) A representative flow cytometry analysis of MM.1S cells treated as in (B) and stained for active caspase-3. (E) Lysates of HeLa cells either untreated (C) or treated with 1 μM RA190 (190) or bortezomib (Bz) after the periods of treatment indicated were immunobloted with PARP-specific antibody. (F) MM.1S cells were untreated (C) or treated with 0.5 μM RA190 or botezomib for 6 hr, then lysed for immunoblot analysis with PARP-specific antibody. (G) HeLa cells were treated with 1 μM RA190, botezomib or cisplatin, and stained at the indicated time points for cell surface HSP90 and analyzed by flow cytometry.

    Journal: Cancer cell

    Article Title: A bis-Benzylidine Piperidone Targeting Proteasome Ubiquitin Receptor RPN13/ADRM1 as a therapy for cancer

    doi: 10.1016/j.ccr.2013.11.001

    Figure Lengend Snippet: RA190 triggers apoptosis and cell surface presentation of HSP90 (A,B) HeLa cells treated with 1 μM RA190 (190) or botezomib (Bz) for 12 hr (A) or MM.1S cells treated with 0.5 μM RA190 or bortezomib for 6 hr (B) were analyzed by flow cytometry after staining with fluorescein-labeled Annexin-V (C) HeLa cells treated as in (A) were analyzed by flow cytometry after staining for active caspase-3 and the percent of positive cells ±SD was determined. (D) A representative flow cytometry analysis of MM.1S cells treated as in (B) and stained for active caspase-3. (E) Lysates of HeLa cells either untreated (C) or treated with 1 μM RA190 (190) or bortezomib (Bz) after the periods of treatment indicated were immunobloted with PARP-specific antibody. (F) MM.1S cells were untreated (C) or treated with 0.5 μM RA190 or botezomib for 6 hr, then lysed for immunoblot analysis with PARP-specific antibody. (G) HeLa cells were treated with 1 μM RA190, botezomib or cisplatin, and stained at the indicated time points for cell surface HSP90 and analyzed by flow cytometry.

    Article Snippet: Annexin V-PE Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA) was used according to the manufacturer’s protocol.

    Techniques: Flow Cytometry, Cytometry, Staining, Labeling

    PPD and metformin cooperate in induction of ECC’s apoptosis. A-C: Ishikawa and RL95-2 cells were treated with PPD (40 uM) and or metformin (20 mM) for 24 h, and then the Annexin-V/7-AAD assay was used to analyze the apoptosis of Ishikawa and RL95-2 cells. Early apoptotic cells: Annexin V + /7-AAD - cells; late apoptotic or necrotic cells: Annexin V + /7-AAD + cells. The data were expressed as means ± SEM. ** P

    Journal: American Journal of Translational Research

    Article Title: Protopanaxadiol and metformin synergistically inhibit estrogen-mediated proliferation and anti-autophagy effects in endometrial cancer cells

    doi:

    Figure Lengend Snippet: PPD and metformin cooperate in induction of ECC’s apoptosis. A-C: Ishikawa and RL95-2 cells were treated with PPD (40 uM) and or metformin (20 mM) for 24 h, and then the Annexin-V/7-AAD assay was used to analyze the apoptosis of Ishikawa and RL95-2 cells. Early apoptotic cells: Annexin V + /7-AAD - cells; late apoptotic or necrotic cells: Annexin V + /7-AAD + cells. The data were expressed as means ± SEM. ** P

    Article Snippet: The anti-human Beclin-1, P62, LC3B and Actin antibodies (Abs) were purchased from R & D Systems (USA); the Annexin V/7-AAD Apoptosis Detection Kit was purchased from BD Biosciences (San Jose, CA, USA); the anti-human ERα Abs were purchased from Abcam (USA); the Cell Counting Kit-8 (CCK-8) were purchased from Dojindo (Japan), the peridininchlorophylla protein cyanine 5.5 (PerCP-Cy™5.5)-conjugated anti-human Ki-67 and allophycocyanin (APC)-conjugated anti-human Bcl-2 Abs were purchased from BD Biosciences; PE-conjugated Bcl-xL Abs were purchased from Cell Signaling Technology; APC-conjugated Fas, PE-conjugated Fas Ligand (FasL) and fluorescein isothiocyanate (FITC)-conjugated anti-human Cytokeratin (CK7) Abs were purchased from Biolegend (San Diego, CA, USA); and 17β-estrogen (E2 ), PPD and metformin were purchased from Sigma-Aldrich Co.

    Techniques:

    The anti-tumor mechanisms of afatinib in ESCC cell lines and PDXs. a Six ESCC cell lines were treated with 0, 10 nM, 100 nM, and 1 μM afatinib and harvested after 48 h. Immunoblots show the response of EGFR downstream signaling molecules to afatinib. b IHC staining for pERK, pS6, and Ki-67 in seven PDXs tumors after 21 days treatment. Representative images and interpretation (by two independent pathologists) are shown (× 200 magnification; scale bars = 100 μM). c–h KYSE450, KYSE140, and KYSE510 cells were treated with 0, 10 nM, 100 nM, and 1 μM afatinib after serum-starvation for 12 h. After 48 h of treatment, the cells were harvested and assayed as described below. The effects of afatinib on cell cycle distribution were assessed using flow cytometry after PI/RNase staining ( c ). The distribution of cells in the cell cycle is depicted ( d ). G1 phase-associated proteins (P21, P27, CDK4, CDK6, and CCND1) were assessed using western blotting ( e ). Flow cytometry showed the apoptosis induced by afatinib treatment using PE-annexin V and 7-AAD staining ( f ). The percentage of cells in early apoptosis (Q3) and late apoptosis (Q2) was calculated as the total apoptosis ratio ( g ). Apoptosis-related proteins (c-PARP, c-caspase8, BCL2, and BAX) were measured by western blotting after afatinib treatment ( h ). Data are presented as means ± SDs of three independent assays. P values were calculated using one-way ANOVA or unpaired two-tailed t tests.* P

    Journal: Journal of Hematology & Oncology

    Article Title: Mouse avatar models of esophageal squamous cell carcinoma proved the potential for EGFR-TKI afatinib and uncovered Src family kinases involved in acquired resistance

    doi: 10.1186/s13045-018-0651-z

    Figure Lengend Snippet: The anti-tumor mechanisms of afatinib in ESCC cell lines and PDXs. a Six ESCC cell lines were treated with 0, 10 nM, 100 nM, and 1 μM afatinib and harvested after 48 h. Immunoblots show the response of EGFR downstream signaling molecules to afatinib. b IHC staining for pERK, pS6, and Ki-67 in seven PDXs tumors after 21 days treatment. Representative images and interpretation (by two independent pathologists) are shown (× 200 magnification; scale bars = 100 μM). c–h KYSE450, KYSE140, and KYSE510 cells were treated with 0, 10 nM, 100 nM, and 1 μM afatinib after serum-starvation for 12 h. After 48 h of treatment, the cells were harvested and assayed as described below. The effects of afatinib on cell cycle distribution were assessed using flow cytometry after PI/RNase staining ( c ). The distribution of cells in the cell cycle is depicted ( d ). G1 phase-associated proteins (P21, P27, CDK4, CDK6, and CCND1) were assessed using western blotting ( e ). Flow cytometry showed the apoptosis induced by afatinib treatment using PE-annexin V and 7-AAD staining ( f ). The percentage of cells in early apoptosis (Q3) and late apoptosis (Q2) was calculated as the total apoptosis ratio ( g ). Apoptosis-related proteins (c-PARP, c-caspase8, BCL2, and BAX) were measured by western blotting after afatinib treatment ( h ). Data are presented as means ± SDs of three independent assays. P values were calculated using one-way ANOVA or unpaired two-tailed t tests.* P

    Article Snippet: Cell cycle and apoptosis analysis Cells were treated with afatinib or vehicle for 48 h. Apoptosis was measured by PE Annexin V Apoptosis Detection Kit I (#559763, BD Pharmingen), and cell cycle distribution was performed using PI/RNase staining buffer solution (#550825, BD Pharmingen) according to manufacturer’s instructions.

    Techniques: Western Blot, Immunohistochemistry, Staining, Flow Cytometry, Cytometry, Two Tailed Test

    : cytotoxicity of envelope glycoprotein 2 (E2) proteins on human umbilical vein endothelial cells. Results presented as mean and standard deviation of percentage obtained in the assay. In each run, 30,000 cells were analysed and all experiments were performed in triplicate. c-: negative control; early apoptosis: annexin V stained cells; late apoptosis: cells double-positive for annexin V and propidium iodide (PI); LPS: lipopolysaccharide (1.0 µg/mL); necrosis: cells stained with PI; Sup: culture supernatant Escherichia coli BL21; TNF-α: tumour necrosis factor alpha (10 ng/mL); ***: p

    Journal: Memórias do Instituto Oswaldo Cruz

    Article Title: Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure

    doi: 10.1590/0074-0276140090

    Figure Lengend Snippet: : cytotoxicity of envelope glycoprotein 2 (E2) proteins on human umbilical vein endothelial cells. Results presented as mean and standard deviation of percentage obtained in the assay. In each run, 30,000 cells were analysed and all experiments were performed in triplicate. c-: negative control; early apoptosis: annexin V stained cells; late apoptosis: cells double-positive for annexin V and propidium iodide (PI); LPS: lipopolysaccharide (1.0 µg/mL); necrosis: cells stained with PI; Sup: culture supernatant Escherichia coli BL21; TNF-α: tumour necrosis factor alpha (10 ng/mL); ***: p

    Article Snippet: The evaluation of cell death was performed using the Annexin V-FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, USA) according to the manufacturer’s protocol.

    Techniques: Standard Deviation, Negative Control, Staining

    hmiR- let-7b Conjugates Increase Apoptosis In Vitro (A) Mean of annexin V + PI + from at least three independent experiments at 72 h post-treatment with hmiR let-7b conjugate (DCA, EPA, and Chol) in the HCC827 cell line. *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Hydrophobically Modified let-7b miRNA Enhances Biodistribution to NSCLC and Downregulates HMGA2 In Vivo

    doi: 10.1016/j.omtn.2019.11.008

    Figure Lengend Snippet: hmiR- let-7b Conjugates Increase Apoptosis In Vitro (A) Mean of annexin V + PI + from at least three independent experiments at 72 h post-treatment with hmiR let-7b conjugate (DCA, EPA, and Chol) in the HCC827 cell line. *p

    Article Snippet: For each sample, 1 × 106 cells were stained with the annexin V‐APC/PI apoptosis detection kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions, and then analyzed using fluorescence-activated cell sorting (FACS; BD FACSCanto II, BD Biosciences, CA, USA).

    Techniques: In Vitro

    LINC00460 Regulates CRC Cell Apoptosis In Vitro (A) Flow cytometry assays were performed to analyze the cell apoptosis in si-LINC00460-transfected HCT116, SW480, and HT-29 cells. LL, dead cells; LR, early apoptotic cells; UL, viable cells; UR, terminal apoptotic cells. (B) Tunel staining assays were performed to analyze the cell apoptosis when LINC00460 was knocked down. Green, apoptotic cell; blue, DAPI staining for nuclear. (C) Cleaved caspase-3 was detected by annexin V+-PI staining and analyzed by flow cytometry. The data are presented as the mean ± SD of three independent experiments; *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: A Novel lncRNA, LINC00460, Affects Cell Proliferation and Apoptosis by Regulating KLF2 and CUL4A Expression in Colorectal Cancer

    doi: 10.1016/j.omtn.2018.06.012

    Figure Lengend Snippet: LINC00460 Regulates CRC Cell Apoptosis In Vitro (A) Flow cytometry assays were performed to analyze the cell apoptosis in si-LINC00460-transfected HCT116, SW480, and HT-29 cells. LL, dead cells; LR, early apoptotic cells; UL, viable cells; UR, terminal apoptotic cells. (B) Tunel staining assays were performed to analyze the cell apoptosis when LINC00460 was knocked down. Green, apoptotic cell; blue, DAPI staining for nuclear. (C) Cleaved caspase-3 was detected by annexin V+-PI staining and analyzed by flow cytometry. The data are presented as the mean ± SD of three independent experiments; *p

    Article Snippet: Double staining with fluorescein isothiocyanate (FITC)-annexin V and propidium iodide was performed using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s recommendations.

    Techniques: In Vitro, Flow Cytometry, Cytometry, Transfection, TUNEL Assay, Staining

    Capsaicin induces apoptosis of cholangiocarcinoma cells. SZ-1 (A) and TFK-1 (B) cells were treated with capsaicin or DMSO as control for 24 h, 48 and 96 h. Viable cells were collected and were stained by Annexin V/PI dye. The stained cells were determined by flow cytometry. Data are representing the results of two independent experiments. Data are expressed as mean ± SD of triplicates.

    Journal: PLoS ONE

    Article Title: Capsaicin Treatment Attenuates Cholangiocarcinoma Carcinogenesis

    doi: 10.1371/journal.pone.0095605

    Figure Lengend Snippet: Capsaicin induces apoptosis of cholangiocarcinoma cells. SZ-1 (A) and TFK-1 (B) cells were treated with capsaicin or DMSO as control for 24 h, 48 and 96 h. Viable cells were collected and were stained by Annexin V/PI dye. The stained cells were determined by flow cytometry. Data are representing the results of two independent experiments. Data are expressed as mean ± SD of triplicates.

    Article Snippet: The cells were then resuspended in 1 ml of 1X binding buffer and were stained with Annexin V-FITC and PI according to the manufacture's instruction using Annexin V Apoptosis Detection Kit II (BD Biosciences, San Diego, USA).

    Techniques: Staining, Flow Cytometry, Cytometry

    Tumor growth and apoptosis following ectopic expression of IκBαM in the ovarian cancer cell lines OVCAR3 and OVCA433. A, EMSA showing that the binding of NF-κB to its promoter DNA consensus sequence was reduced in IκBαM-transfected cells. B, luciferase reporter assays showing the reduced transcriptional activity of NF-κB. Error bars, 95% CIs. Mt, mutant; Wt, wild type. C, tumor growth in nude mice injected subcutaneously with OVCAR3 and OVCA433 cells. Mice injected with IκBαM-transfected cells displayed enhanced tumor burden. Error bars, 95% CIs. D, average number of tumor nodules in mice injected with ovarian cancer cells in the presence and absence of IκBαM. The number of nodules was higher in mice injected with IκBαM-treated cells than in those injected with control cells. Error bars, 95% CIs. E and F, Annexin V staining showing a reduction in the percentage of apoptotic cells after knockdown of NF-κB. Error bars, 95% CIs. G and H, analysis of apoptosis in xenograft mouse tumor tissues using a TUNEL assay with Apo-BrdU (green) in situ fragmentation (×400). Error bars, 95% CIs. PI, red.

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: The Biphasic Role of NF-?B in Progression and Chemoresistance of Ovarian Cancer

    doi: 10.1158/1078-0432.CCR-10-3265

    Figure Lengend Snippet: Tumor growth and apoptosis following ectopic expression of IκBαM in the ovarian cancer cell lines OVCAR3 and OVCA433. A, EMSA showing that the binding of NF-κB to its promoter DNA consensus sequence was reduced in IκBαM-transfected cells. B, luciferase reporter assays showing the reduced transcriptional activity of NF-κB. Error bars, 95% CIs. Mt, mutant; Wt, wild type. C, tumor growth in nude mice injected subcutaneously with OVCAR3 and OVCA433 cells. Mice injected with IκBαM-transfected cells displayed enhanced tumor burden. Error bars, 95% CIs. D, average number of tumor nodules in mice injected with ovarian cancer cells in the presence and absence of IκBαM. The number of nodules was higher in mice injected with IκBαM-treated cells than in those injected with control cells. Error bars, 95% CIs. E and F, Annexin V staining showing a reduction in the percentage of apoptotic cells after knockdown of NF-κB. Error bars, 95% CIs. G and H, analysis of apoptosis in xenograft mouse tumor tissues using a TUNEL assay with Apo-BrdU (green) in situ fragmentation (×400). Error bars, 95% CIs. PI, red.

    Article Snippet: The cells were then fixed with 75% alcohol at −20°C for 3 days and stained with Annexin V and propidium iodide (PI) according to the manufacturer’s instructions in the Annexin V-fluorescence apoptosis detection kit I (BD Biosciences Pharmingen).

    Techniques: Expressing, Binding Assay, Sequencing, Transfection, Luciferase, Activity Assay, Mutagenesis, Mouse Assay, Injection, Staining, TUNEL Assay, In Situ

    miR-145 suppresses tumor progression through targeting FLI-1 Upon transfection with FLI-1 construct, we rescued the expression of FLI-1 in U2OS and MG-63 cells. The expression of FLI-1 protein was validated by western blot assays; ( A ) CCK-8 assays were used to detect in U2OS and MG-63 cells co-transfected with miR-145 mimic and pcDNA- FLI-1 -plasmid; ( B ) Cell apoptosis of U2OS and MG-63 cells treated as described was detected by Annexin V-PI staining; and ( C ) Transwell assays were performed to detect the effects on cell migration of U2OS and MG-63 cells treated as described. Upon transfection with the FLI-1 plasmid, miR-145-mediated suppression of cell proliferation and cell migration was abolished, and promotion of cell apoptosis was inhibited. (×200); ( D ) We detected the effect of FLI-1 siRNA in MG63 and U2OS cells by CCK8 assay. * p

    Journal: Oncotarget

    Article Title: miR-145 promotes osteosarcoma growth by reducing expression of the transcription factor friend leukemia virus integration 1

    doi: 10.18632/oncotarget.9948

    Figure Lengend Snippet: miR-145 suppresses tumor progression through targeting FLI-1 Upon transfection with FLI-1 construct, we rescued the expression of FLI-1 in U2OS and MG-63 cells. The expression of FLI-1 protein was validated by western blot assays; ( A ) CCK-8 assays were used to detect in U2OS and MG-63 cells co-transfected with miR-145 mimic and pcDNA- FLI-1 -plasmid; ( B ) Cell apoptosis of U2OS and MG-63 cells treated as described was detected by Annexin V-PI staining; and ( C ) Transwell assays were performed to detect the effects on cell migration of U2OS and MG-63 cells treated as described. Upon transfection with the FLI-1 plasmid, miR-145-mediated suppression of cell proliferation and cell migration was abolished, and promotion of cell apoptosis was inhibited. (×200); ( D ) We detected the effect of FLI-1 siRNA in MG63 and U2OS cells by CCK8 assay. * p

    Article Snippet: Apoptosis assay Cells were transfected with miR-145 mimics or control mimics for 24 h. Forty-eight hours after transient transfection, according to the Annexin V-PI Apoptosis Detection Kit I (BD Pharmingen, CA, USA) protocol, cells were harvested and re-suspended with 500 μL of binding buffer.

    Techniques: Transfection, Construct, Expressing, Western Blot, CCK-8 Assay, Plasmid Preparation, Staining, Migration

    Anti-apoptotic effect of anti-p75 NTR Selective immunologic blockade of the cell death receptor p75 NTR inhibited TiO 2 -NP induced apoptosis. Lower respiratory tract bronchial (A) and alveolar (B) epithelial cells were pretreated anti-human p75 NTR antibody for 24 h before exposure to 10 μg/ml of TiO 2 -NP. Control group was treated with human IgG antibody. Cells were harvested, stained with annexin V-FITC and propidium iodide (PI), and viability was analyzed by FACS. Data shown are the means ± SDs of 3 independent experiments. *p

    Journal: Journal of toxicology and environmental health. Part A

    Article Title: Nanoparticles-Induced Apoptosis of Human Airway Epithelium is mediated by ProNGF/p75NTR Signaling

    doi: 10.1080/15287394.2016.1238329

    Figure Lengend Snippet: Anti-apoptotic effect of anti-p75 NTR Selective immunologic blockade of the cell death receptor p75 NTR inhibited TiO 2 -NP induced apoptosis. Lower respiratory tract bronchial (A) and alveolar (B) epithelial cells were pretreated anti-human p75 NTR antibody for 24 h before exposure to 10 μg/ml of TiO 2 -NP. Control group was treated with human IgG antibody. Cells were harvested, stained with annexin V-FITC and propidium iodide (PI), and viability was analyzed by FACS. Data shown are the means ± SDs of 3 independent experiments. *p

    Article Snippet: MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay kits were purchased from Cayman Chemical (Ann Arbor, MI); FITC Annexin-V Apoptosis Detection kits from BD Biosciences (San Jose, CA); and RNeasy kits for total RNA isolation from Qiagen (Valencia, CA).

    Techniques: Staining, FACS

    Nano vs. fine particles-induced cell death FACS data for nasal (A) , tracheal (B) , bronchial (C) and alveolar (D) epithelial cells are shown. Airway epithelial cells were exposed to 10 μg/ml of TiO 2 -NP or TiO 2 -FP, then stained with annexin V-FITC and propidium iodide (PI), and cytotoxicity was measured by FACS. Bronchial and alveolar epithelial cells showed increased fractions of early apoptotic cells after TiO 2 -NP exposure, while TiO 2 -FP diverted cells to necrosis. Data shown are representative of 3 independent experiments.

    Journal: Journal of toxicology and environmental health. Part A

    Article Title: Nanoparticles-Induced Apoptosis of Human Airway Epithelium is mediated by ProNGF/p75NTR Signaling

    doi: 10.1080/15287394.2016.1238329

    Figure Lengend Snippet: Nano vs. fine particles-induced cell death FACS data for nasal (A) , tracheal (B) , bronchial (C) and alveolar (D) epithelial cells are shown. Airway epithelial cells were exposed to 10 μg/ml of TiO 2 -NP or TiO 2 -FP, then stained with annexin V-FITC and propidium iodide (PI), and cytotoxicity was measured by FACS. Bronchial and alveolar epithelial cells showed increased fractions of early apoptotic cells after TiO 2 -NP exposure, while TiO 2 -FP diverted cells to necrosis. Data shown are representative of 3 independent experiments.

    Article Snippet: MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay kits were purchased from Cayman Chemical (Ann Arbor, MI); FITC Annexin-V Apoptosis Detection kits from BD Biosciences (San Jose, CA); and RNeasy kits for total RNA isolation from Qiagen (Valencia, CA).

    Techniques: FACS, Staining

    Anti-apoptotic effect of NGF gene silencing NGF gene knockdown by siRNA reduced TiO 2 -NP mediated apoptosis. After transfection with either scrambled siRNA (scRNA) or NGF-specific siRNA (NGFsiRNA) for 48 hr, bronchial epithelial cells were exposed to 10 μg/ml TiO 2 -NP, stained with annexin V-FITC and propidium iodide (PI), and analyzed by FACS. Each experiment included: unexposed cells treated with scRNA (A) ; unexposed cells treated with NGFsiRNA (B) ; cells transfected with scRNA before exposure to TiO 2 -NP (C) ; and cells transfected with NGFsiRNA before exposure to TiO 2 -NP (D) . Data shown are the means ± SDs of 3 independent experiments. *p

    Journal: Journal of toxicology and environmental health. Part A

    Article Title: Nanoparticles-Induced Apoptosis of Human Airway Epithelium is mediated by ProNGF/p75NTR Signaling

    doi: 10.1080/15287394.2016.1238329

    Figure Lengend Snippet: Anti-apoptotic effect of NGF gene silencing NGF gene knockdown by siRNA reduced TiO 2 -NP mediated apoptosis. After transfection with either scrambled siRNA (scRNA) or NGF-specific siRNA (NGFsiRNA) for 48 hr, bronchial epithelial cells were exposed to 10 μg/ml TiO 2 -NP, stained with annexin V-FITC and propidium iodide (PI), and analyzed by FACS. Each experiment included: unexposed cells treated with scRNA (A) ; unexposed cells treated with NGFsiRNA (B) ; cells transfected with scRNA before exposure to TiO 2 -NP (C) ; and cells transfected with NGFsiRNA before exposure to TiO 2 -NP (D) . Data shown are the means ± SDs of 3 independent experiments. *p

    Article Snippet: MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay kits were purchased from Cayman Chemical (Ann Arbor, MI); FITC Annexin-V Apoptosis Detection kits from BD Biosciences (San Jose, CA); and RNeasy kits for total RNA isolation from Qiagen (Valencia, CA).

    Techniques: Transfection, Staining, FACS

    PERK inhibitor GSK2606414 decreased UPR activation and partially abolished chemosensitizing effect of siKRT8 but did not reverse the blockage of autophagy flux in vitro. Chordoma cell line CM319 and UCH1 were transfected with siKRT8 followed by treatment of doxorubicin (0.5 μM), irinotecan (50 μM), and GSK2606414 (2 μM) for 24 h. a Western blotting analysis and quantification of p-PERK, p-eIF2α, Cleaved PARP, SQSTM1, and LC3B protein expression (p-PERK and p-eIF2α were normalized to PERK and eIF2α expression, respectively; Cleaved PARP, LC3B, and SQSTM1 were normalized to GAPDH expression; quantification data of p-PERK, p-eIF2α, Cleaved PARP, SQSTM1, and LC3B in “si + Doxo 24 h” group and “si + Irino 24 h” group were derived from the same data set in Figs. 4b and 5b ). b Apoptosis of chordoma cells was determined by Annexin V-PE/PI staining measured by flow cytometry (quantification data of apoptosis in “si + Doxo 24 h” group and “si + Irino 24 h” group were derived from the same data set in Fig. 4d ) ( n = 3, * p

    Journal: Cell Death & Disease

    Article Title: Knockdown of cytokeratin 8 overcomes chemoresistance of chordoma cells by aggravating endoplasmic reticulum stress through PERK/eIF2α arm of unfolded protein response and blocking autophagy

    doi: 10.1038/s41419-019-2125-9

    Figure Lengend Snippet: PERK inhibitor GSK2606414 decreased UPR activation and partially abolished chemosensitizing effect of siKRT8 but did not reverse the blockage of autophagy flux in vitro. Chordoma cell line CM319 and UCH1 were transfected with siKRT8 followed by treatment of doxorubicin (0.5 μM), irinotecan (50 μM), and GSK2606414 (2 μM) for 24 h. a Western blotting analysis and quantification of p-PERK, p-eIF2α, Cleaved PARP, SQSTM1, and LC3B protein expression (p-PERK and p-eIF2α were normalized to PERK and eIF2α expression, respectively; Cleaved PARP, LC3B, and SQSTM1 were normalized to GAPDH expression; quantification data of p-PERK, p-eIF2α, Cleaved PARP, SQSTM1, and LC3B in “si + Doxo 24 h” group and “si + Irino 24 h” group were derived from the same data set in Figs. 4b and 5b ). b Apoptosis of chordoma cells was determined by Annexin V-PE/PI staining measured by flow cytometry (quantification data of apoptosis in “si + Doxo 24 h” group and “si + Irino 24 h” group were derived from the same data set in Fig. 4d ) ( n = 3, * p

    Article Snippet: The apoptosis of chordoma cells was evaluated by Annexin V-PE/PI apoptosis detection kit (BD Biosciences, USA) by flow cytometry.

    Techniques: Activation Assay, In Vitro, Transfection, Western Blot, Expressing, Derivative Assay, Staining, Flow Cytometry, Cytometry

    Knockdown of KRT8 in chordoma cell increased its’ sensitivity to chemotherapy by promoting its apoptosis in vitro. Chordoma cell line CM319 and UCH1 were transfected with siKRT8 followed by treatment of doxorubicin (0.5 μM) or irinotecan (50 μM) for 24 h. a Cell viability of chordoma cells were determined by CCK8 assay. b Western blotting analysis and quantification of KRT8, cleaved PARP, and Caspase 4 protein expression (normalized to GAPDH expression, quantification data of KRT8 in the “Doxo 24 h” group and “Irino 24h” group were derived from the same data set in Fig. 1b ). c Immunofluorescence staining of KRT8 of CM319 and UCH1 cell line. d Apoptosis of chordoma cells was determined by Annexin V-PE/PI staining measured by flow cytometry ( n = 3, * p

    Journal: Cell Death & Disease

    Article Title: Knockdown of cytokeratin 8 overcomes chemoresistance of chordoma cells by aggravating endoplasmic reticulum stress through PERK/eIF2α arm of unfolded protein response and blocking autophagy

    doi: 10.1038/s41419-019-2125-9

    Figure Lengend Snippet: Knockdown of KRT8 in chordoma cell increased its’ sensitivity to chemotherapy by promoting its apoptosis in vitro. Chordoma cell line CM319 and UCH1 were transfected with siKRT8 followed by treatment of doxorubicin (0.5 μM) or irinotecan (50 μM) for 24 h. a Cell viability of chordoma cells were determined by CCK8 assay. b Western blotting analysis and quantification of KRT8, cleaved PARP, and Caspase 4 protein expression (normalized to GAPDH expression, quantification data of KRT8 in the “Doxo 24 h” group and “Irino 24h” group were derived from the same data set in Fig. 1b ). c Immunofluorescence staining of KRT8 of CM319 and UCH1 cell line. d Apoptosis of chordoma cells was determined by Annexin V-PE/PI staining measured by flow cytometry ( n = 3, * p

    Article Snippet: The apoptosis of chordoma cells was evaluated by Annexin V-PE/PI apoptosis detection kit (BD Biosciences, USA) by flow cytometry.

    Techniques: In Vitro, Transfection, CCK-8 Assay, Western Blot, Expressing, Derivative Assay, Immunofluorescence, Staining, Flow Cytometry, Cytometry

    HDLs, but not LDLs, promote Treg survival. A: Bar graphs show median and range of the baseline survival of Tregs, naïve cells, and memory cells in X-VIVO medium. B, C: Dot line graphs represent the absolute number of cells cultured for 24 h in the absence (medium) or in the presence of HDL (300 μg/ml): Tregs (B), naïveCD4 + T cells (C), and memory CD4 + T cells (D). E: Absolute number of Tregs cultured for 24 h in the presence or absence of LDL (300 μg/ml). F: Tregs were stained for apoptosis with annexin V and 7AAD. Normalized survival was calculated based on cells cultured in medium alone. Annexin V − 7AAD − cells were considered as live. G: Tregs were stained intracellularly for the cell cycle marker with Ki67. H: Absolute number of Tregs cultured for 24 h in medium or in the presence of oleic acid bound to albumin, oleic acid alone, albumin, or HDL (all at 300 μg/ml). Comparisons between groups were done with U Mann-Whitney or Wilcoxon tests. Each line represents a single donor.

    Journal: Journal of Lipid Research

    Article Title: High density lipoproteins selectively promote the survival of human regulatory T cells [S]

    doi: 10.1194/jlr.M072835

    Figure Lengend Snippet: HDLs, but not LDLs, promote Treg survival. A: Bar graphs show median and range of the baseline survival of Tregs, naïve cells, and memory cells in X-VIVO medium. B, C: Dot line graphs represent the absolute number of cells cultured for 24 h in the absence (medium) or in the presence of HDL (300 μg/ml): Tregs (B), naïveCD4 + T cells (C), and memory CD4 + T cells (D). E: Absolute number of Tregs cultured for 24 h in the presence or absence of LDL (300 μg/ml). F: Tregs were stained for apoptosis with annexin V and 7AAD. Normalized survival was calculated based on cells cultured in medium alone. Annexin V − 7AAD − cells were considered as live. G: Tregs were stained intracellularly for the cell cycle marker with Ki67. H: Absolute number of Tregs cultured for 24 h in medium or in the presence of oleic acid bound to albumin, oleic acid alone, albumin, or HDL (all at 300 μg/ml). Comparisons between groups were done with U Mann-Whitney or Wilcoxon tests. Each line represents a single donor.

    Article Snippet: Apoptosis was quantified using the Annexin V/7AAD Apoptosis Detection Kit I (BD).

    Techniques: Cell Culture, Staining, Marker, MANN-WHITNEY

    Knockdown of UXT sensitizes 293T cells to TNF-α–induced apoptosis. (A) 293T cells were transfected with the indicated siRNAs. 48 h after transfection, cells were treated with 50 ng/ml TNF-α plus 5 μg/ml CHX or were left untreated for 18 h. Representative microscopic images are shown. (B and C) Cells were prepared as in A. After that, cells were analyzed using the annexin V (B) or TUNEL assay (C) to monitor cell apoptosis. The percentages indicate the fractions of positive annexin V cells in total cells. Data represent means ± SD (error bars) of at least three independent experiments. Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: UXT is a novel and essential cofactor in the NF-?B transcriptional enhanceosome

    doi: 10.1083/jcb.200611081

    Figure Lengend Snippet: Knockdown of UXT sensitizes 293T cells to TNF-α–induced apoptosis. (A) 293T cells were transfected with the indicated siRNAs. 48 h after transfection, cells were treated with 50 ng/ml TNF-α plus 5 μg/ml CHX or were left untreated for 18 h. Representative microscopic images are shown. (B and C) Cells were prepared as in A. After that, cells were analyzed using the annexin V (B) or TUNEL assay (C) to monitor cell apoptosis. The percentages indicate the fractions of positive annexin V cells in total cells. Data represent means ± SD (error bars) of at least three independent experiments. Bar, 10 μm.

    Article Snippet: 48 h after transfection, cells were treated with 50 ng/ml TNF-α and 5 μg/ml CHX or left untreated for 18 h. Floating and adherent cells were collected and analyzed using the annexin V–phycoerythrin Apoptosis Detection kit I (BD Biosciences) and In Situ Cell Death Detection kit (TUNEL; Roche) according to the manufacturer's instructions.

    Techniques: Transfection, TUNEL Assay

    Biological effects of KX-01 on mucinous ovarian carcinoma cells in vitro. ( A ) MTT assay results showing the effect of KX-01 on cell survival at various concentrations in a panel of mucinous ovarian carcinoma cells. Cell survival was calculated as the number of cells surviving relative to the number of cells surviving in the control group. IC50 indicates median inhibitory rate. ( B ) EdU assay results showing inhibition of cell proliferation in cells treated with KX-01 compared with control cells in RMUG-S and RMUG-L cells. ( C ) Annexin V and 7-aminoactinomycin D assay results showing cell apoptosis in cells treated with KX-01 compared with control cells in RMUG-S and RMUG-L cells. ( D ) Flow cytometry results showing induction of mitotic arrest in cells treated with KX-01 compared with control cells in RMUG-S and RMUG-L cells. ( E ) Cell cytotoxicity in RMUG-S cells treated with various concentrations of oxaliplatin with or without 100nM KX-01 for 72 hours. Results are presented as in A . ( F ) Isobologram analysis for the combination of KX-01 and oxaliplatin in RMUG-S cells. The interaction index was

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: Targeting Src and tubulin in mucinous ovarian carcinoma

    doi: 10.1158/1078-0432.CCR-13-1305

    Figure Lengend Snippet: Biological effects of KX-01 on mucinous ovarian carcinoma cells in vitro. ( A ) MTT assay results showing the effect of KX-01 on cell survival at various concentrations in a panel of mucinous ovarian carcinoma cells. Cell survival was calculated as the number of cells surviving relative to the number of cells surviving in the control group. IC50 indicates median inhibitory rate. ( B ) EdU assay results showing inhibition of cell proliferation in cells treated with KX-01 compared with control cells in RMUG-S and RMUG-L cells. ( C ) Annexin V and 7-aminoactinomycin D assay results showing cell apoptosis in cells treated with KX-01 compared with control cells in RMUG-S and RMUG-L cells. ( D ) Flow cytometry results showing induction of mitotic arrest in cells treated with KX-01 compared with control cells in RMUG-S and RMUG-L cells. ( E ) Cell cytotoxicity in RMUG-S cells treated with various concentrations of oxaliplatin with or without 100nM KX-01 for 72 hours. Results are presented as in A . ( F ) Isobologram analysis for the combination of KX-01 and oxaliplatin in RMUG-S cells. The interaction index was

    Article Snippet: Apoptosis was evaluated using the Annexin V-phycoerythrin (PE) apoptosis detection kit (BD Biosciences).

    Techniques: In Vitro, MTT Assay, EdU Assay, Inhibition, Flow Cytometry, Cytometry

    Flow cytometric analysis by Annexin V staining of Melanoma B16 cells treated for 24 h with 0.2 mg/mL crude fucose-containing sulfated polysaccharide (FCSP) products extracted from S. henslowianum C. Agardh (FSAR) and F. vesiculosus (FVES), respectively. ( a ) Apoptosis induced by FSAR (41 ± 3%, n = 2), FVES (30 ± 5%, n = 2); control 11.66 % (data not shown) ( b ) FSAR data and ( c ) FVES data for FACS scans of FCSP treated Melanoma 16 cells that were viable and not undergoing apoptosis (Annexin V − and 7-AAD − ); undergoing early apoptosis, with membrane integrity intact (Annexin V + and 7-AAD − ); in the latest stage apoptosis and dead (Annexin V + and 7-AAD + ), respectively.

    Journal: Marine Drugs

    Article Title: Fucose-Containing Sulfated Polysaccharides from Brown Seaweeds Inhibit Proliferation of Melanoma Cells and Induce Apoptosis by Activation of Caspase-3 in Vitro

    doi: 10.3390/md9122605

    Figure Lengend Snippet: Flow cytometric analysis by Annexin V staining of Melanoma B16 cells treated for 24 h with 0.2 mg/mL crude fucose-containing sulfated polysaccharide (FCSP) products extracted from S. henslowianum C. Agardh (FSAR) and F. vesiculosus (FVES), respectively. ( a ) Apoptosis induced by FSAR (41 ± 3%, n = 2), FVES (30 ± 5%, n = 2); control 11.66 % (data not shown) ( b ) FSAR data and ( c ) FVES data for FACS scans of FCSP treated Melanoma 16 cells that were viable and not undergoing apoptosis (Annexin V − and 7-AAD − ); undergoing early apoptosis, with membrane integrity intact (Annexin V + and 7-AAD − ); in the latest stage apoptosis and dead (Annexin V + and 7-AAD + ), respectively.

    Article Snippet: The PE Annexin V Apoptosis Detection Kit 1 was obtained from BD Biosciences (Franklin Lakes, NJ, USA).

    Techniques: Flow Cytometry, Staining, FACS

    Loss of USP22 in cancer cells results in G1-phase cell cycle arrest. H1299 human lung cancer cells depleted of USP22 via infection with an shRNA-encoding lentivirus (or a luciferase shRNA as a control). After selection, cells counted and plated at 80,000 cells/mL on day 2 postinfection. ( A ) Cell number determined by direct counting of triplicate wells in a six-well plate via hemocytometer at the indicated time points. ( B ) Colony growth assessed via fixation and methylene blue staining of foci at day 7 postinfection. ( C ) Efficient knockdown of USP22 confirmed by both qRT-PCR and immunoblot (IB). ( D ) Cell viability quantified via flow cytometry. ( E ) Quantification of three experimental replicates representing average and SD of percent cell death, based on permeability. ( F ) Apoptosis measured by Annexin V-PE and 7AAD DNA staining, quantified by flow cytometry. ( G ) Quantification of three experimental replicates representing average and SD of percent apoptosis based on population of Annexin V-PE + cells. NS, not significant. ( H ) PARP and CASPASE-3 (CAS-3) cleavage demonstrated by IB. The black arrowhead indicates cleaved species. ( I ) Progression through the cell cycle determined by EdU incorporation and PI staining followed by flow cytometry; the percent of cells in G1 phase is represented in blue. ( J ) Quantification of three experimental replicates of cell cycle phase distribution. Error bars indicate SD based on three independent experiments. * P

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

    Article Title: Control of CCND1 ubiquitylation by the catalytic SAGA subunit USP22 is essential for cell cycle progression through G1 in cancer cells

    doi: 10.1073/pnas.1807704115

    Figure Lengend Snippet: Loss of USP22 in cancer cells results in G1-phase cell cycle arrest. H1299 human lung cancer cells depleted of USP22 via infection with an shRNA-encoding lentivirus (or a luciferase shRNA as a control). After selection, cells counted and plated at 80,000 cells/mL on day 2 postinfection. ( A ) Cell number determined by direct counting of triplicate wells in a six-well plate via hemocytometer at the indicated time points. ( B ) Colony growth assessed via fixation and methylene blue staining of foci at day 7 postinfection. ( C ) Efficient knockdown of USP22 confirmed by both qRT-PCR and immunoblot (IB). ( D ) Cell viability quantified via flow cytometry. ( E ) Quantification of three experimental replicates representing average and SD of percent cell death, based on permeability. ( F ) Apoptosis measured by Annexin V-PE and 7AAD DNA staining, quantified by flow cytometry. ( G ) Quantification of three experimental replicates representing average and SD of percent apoptosis based on population of Annexin V-PE + cells. NS, not significant. ( H ) PARP and CASPASE-3 (CAS-3) cleavage demonstrated by IB. The black arrowhead indicates cleaved species. ( I ) Progression through the cell cycle determined by EdU incorporation and PI staining followed by flow cytometry; the percent of cells in G1 phase is represented in blue. ( J ) Quantification of three experimental replicates of cell cycle phase distribution. Error bars indicate SD based on three independent experiments. * P

    Article Snippet: To quantify apoptotic cell death, cells were collected by trypsinization and stained using the Annexin V PE-7AAD apoptosis detection kit (BD Pharmingen).

    Techniques: Infection, shRNA, Luciferase, Selection, Staining, Quantitative RT-PCR, Flow Cytometry, Cytometry, Permeability

    Inhibition of autophagy promotes apoptosis of RSV-infected HEp-2 cells at 48 hpi. (A) HEp-2 cells were uninfected or infected with RSV in the presence or absence of 3-MA for indicated times. Cell viability was determined by MTS assay as described in Materials and Methods. The optical density at 490 nm (OD 490 ) values of sample wells were normalized to that of the blank well. (B, C) HEp-2-sh- ATG5 (B), HEp-2-sh- BECN1 (C), or HEp-2-sh- luciferase cells were uninfected or infected with RSV for indicated times. Cell viability was determined by MTS assay as described in Materials and Methods. The OD 490 values of sample wells were normalized to that of the blank well. (D) HEp-2 cells were mock infected or infected with RSV (MOI = 5) for 48 h in the presence or absence of autophagy inhibitor 3-MA. The apoptosis rate was determined by flow cytometry assay using annexin V-PE and 7-aminoactinomycin D (7-AAD) staining as described in Materials and Methods. (E) HEp-2 cells were treated as described for panel D, and the expressions of total CASP3, cleaved CASP3, total PARP, and cleaved PARP were analyzed with their specific antibodies. The relative levels of targeted proteins were quantitated by densitometry and normalized to the GAPDH control. (F) HEp-2 cells were transfected with negative-control siRNA, ATG5 siRNA, or BECN1 siRNA for 24 h and then uninfected or infected with RSV for another 48 h. The apoptosis rate was determined by flow cytometry assay using annexin V-FITC and PI staining as described in Materials and Methods. (G and H) HEp-2-sh- ATG5 (G), HEp-2-sh- BECN 1 (H), HEp-2-sh- luciferase , or HEp-2 cells were mock infected or infected with RSV (MOI = 5) for 48 h. The expressions of total CASP3, cleaved-CASP3, PARP, and cleaved PARP were analyzed with specific antibodies. The relative levels of targeted proteins were quantitated by densitometry and normalized to the GAPDH control. *, P

    Journal: Journal of Virology

    Article Title: Respiratory Syncytial Virus Replication Is Promoted by Autophagy-Mediated Inhibition of Apoptosis

    doi: 10.1128/JVI.02193-17

    Figure Lengend Snippet: Inhibition of autophagy promotes apoptosis of RSV-infected HEp-2 cells at 48 hpi. (A) HEp-2 cells were uninfected or infected with RSV in the presence or absence of 3-MA for indicated times. Cell viability was determined by MTS assay as described in Materials and Methods. The optical density at 490 nm (OD 490 ) values of sample wells were normalized to that of the blank well. (B, C) HEp-2-sh- ATG5 (B), HEp-2-sh- BECN1 (C), or HEp-2-sh- luciferase cells were uninfected or infected with RSV for indicated times. Cell viability was determined by MTS assay as described in Materials and Methods. The OD 490 values of sample wells were normalized to that of the blank well. (D) HEp-2 cells were mock infected or infected with RSV (MOI = 5) for 48 h in the presence or absence of autophagy inhibitor 3-MA. The apoptosis rate was determined by flow cytometry assay using annexin V-PE and 7-aminoactinomycin D (7-AAD) staining as described in Materials and Methods. (E) HEp-2 cells were treated as described for panel D, and the expressions of total CASP3, cleaved CASP3, total PARP, and cleaved PARP were analyzed with their specific antibodies. The relative levels of targeted proteins were quantitated by densitometry and normalized to the GAPDH control. (F) HEp-2 cells were transfected with negative-control siRNA, ATG5 siRNA, or BECN1 siRNA for 24 h and then uninfected or infected with RSV for another 48 h. The apoptosis rate was determined by flow cytometry assay using annexin V-FITC and PI staining as described in Materials and Methods. (G and H) HEp-2-sh- ATG5 (G), HEp-2-sh- BECN 1 (H), HEp-2-sh- luciferase , or HEp-2 cells were mock infected or infected with RSV (MOI = 5) for 48 h. The expressions of total CASP3, cleaved-CASP3, PARP, and cleaved PARP were analyzed with specific antibodies. The relative levels of targeted proteins were quantitated by densitometry and normalized to the GAPDH control. *, P

    Article Snippet: Apoptosis was determined by the phycoerythrin (PE)-annexin V apoptosis detection kit (559763; BD Biosciences) or the FITC-annexin V apoptosis detection kit (556547; BD Biosciences) according to the kit guidelines.

    Techniques: Inhibition, Infection, MTS Assay, Luciferase, Flow Cytometry, Cytometry, Staining, Transfection, Negative Control

    SIRT1 is important for the survival of cancer cells with neural stemness. (A) The relationship between the mRNA levels of SIRT1 and Sox2 was determined in a variety of cells via real-time PCR and analyzed as a 2D scatter plot. (B) F3.Ras.CNSCs and U87 cells were treated with SRT and harvested at the indicated times. The level of SIRT1 and active caspase-3 was determined by immunoblotting. Alpha-tubulin was loading control. (C) For apoptosis validation, F3.Ras.CNSCs and U87 cells were treated with SRT for 72 h, stained with 7-aminoactinomycin (7-AAD) and annexin V–phycoerythrin (PE), and then analyzed on a conventional flow cytometer (left panel). A percentage of annexin V–positive cells is graphically presented (right panel) ( n = 3). (D) F3.Ras.CNSCs and U87 cells were infected with shCont or shSIRT1 virus supernatant, stained with 7-AAD and annexin V-PE, and then analyzed on a conventional flow cytometer (left panel). A percentage of annexin V–positive cells is graphically presented (right panel) ( n = 3).

    Journal: Neuro-Oncology

    Article Title: SIRT1 is required for oncogenic transformation of neural stem cells and for the survival of “cancer cells with neural stemness” in a p53-dependent manner

    doi: 10.1093/neuonc/nou145

    Figure Lengend Snippet: SIRT1 is important for the survival of cancer cells with neural stemness. (A) The relationship between the mRNA levels of SIRT1 and Sox2 was determined in a variety of cells via real-time PCR and analyzed as a 2D scatter plot. (B) F3.Ras.CNSCs and U87 cells were treated with SRT and harvested at the indicated times. The level of SIRT1 and active caspase-3 was determined by immunoblotting. Alpha-tubulin was loading control. (C) For apoptosis validation, F3.Ras.CNSCs and U87 cells were treated with SRT for 72 h, stained with 7-aminoactinomycin (7-AAD) and annexin V–phycoerythrin (PE), and then analyzed on a conventional flow cytometer (left panel). A percentage of annexin V–positive cells is graphically presented (right panel) ( n = 3). (D) F3.Ras.CNSCs and U87 cells were infected with shCont or shSIRT1 virus supernatant, stained with 7-AAD and annexin V-PE, and then analyzed on a conventional flow cytometer (left panel). A percentage of annexin V–positive cells is graphically presented (right panel) ( n = 3).

    Article Snippet: The apoptotic cell population was analyzed using the Phycoerythrin Annexin V Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer's instructions.

    Techniques: Real-time Polymerase Chain Reaction, Staining, Flow Cytometry, Cytometry, Infection

    Mechanism of α-TEA and trastuzumab-mediated tumor cell inhibition . (A) MDA-MB-453 tumor cells were treated with Vα-TEA and trastuzumab (or isotype antibody) for 72 hours. Cells were collected and analyzed for apoptosis by PE-Annexin-V stain. Mean frequency ± SD of apoptotic cells (PE-Annexin-V positive) is shown from three independent experiments. (B) MDA-MB-453 tumor cells were treated with Vα-TEA and trastuzumab (or isotype antibody) for 24 hours. Phosphorylated-(Ser473)-AKT, total AKT and GAPDH levels were determined by western immunoblotting. Numbers indicate fold-change over untreated (isotype-treated) cells.

    Journal: BMC Cancer

    Article Title: The vitamin E analog, alpha-tocopheryloxyacetic acid enhances the anti-tumor activity of trastuzumab against HER2/neu-expressing breast cancer

    doi: 10.1186/1471-2407-11-471

    Figure Lengend Snippet: Mechanism of α-TEA and trastuzumab-mediated tumor cell inhibition . (A) MDA-MB-453 tumor cells were treated with Vα-TEA and trastuzumab (or isotype antibody) for 72 hours. Cells were collected and analyzed for apoptosis by PE-Annexin-V stain. Mean frequency ± SD of apoptotic cells (PE-Annexin-V positive) is shown from three independent experiments. (B) MDA-MB-453 tumor cells were treated with Vα-TEA and trastuzumab (or isotype antibody) for 24 hours. Phosphorylated-(Ser473)-AKT, total AKT and GAPDH levels were determined by western immunoblotting. Numbers indicate fold-change over untreated (isotype-treated) cells.

    Article Snippet: Cells were collected using 10 mM EDTA and stained using the PE-Annexin-V/7AAD apoptosis detection kit (BD Pharmingen, San Diego, CA) according to the manufacturer's instructions and analyzed by flow-cytometry using a LSR-II flow cytometer (BD Bioscience) with data acquisition using DIVA software (BD Bioscience).

    Techniques: Inhibition, Multiple Displacement Amplification, Staining, Western Blot

    USP4 participates in antiviral immunity against EV71 infection. ( A ) Analysis of phase contrast microscopy of RD cells transfected with either an empty vector or USP4, and then infected with EV71 (MOI = 0.5) at the indicated time (magnification 20x). ( B ) Apoptosis assay of EV71-infected RD cells transfected with empty vector or USP4. The apoptosis assay was conducted using annexin V/FITC and PI double staining and flow cytometry. Q3, Q4, and Q2 represent normal cells, early apoptotic cells, and late apoptotic/necrotic cells, respectively. ( C , F ) q-PCR and western blot analysis of EV71 protein expression in RD cells transfected with control vector or HA-USP4 plasmids, followed by treatment with EV71 for variable lengths of time. ( E ) Viral titre measurement of EV71 propagating in RD cells transiently transfected with control vector or HA-USP4 plasmids. Data in ( D , E ) are presented as the mean ± SD. * P

    Journal: Scientific Reports

    Article Title: USP4 positively regulates RLR-induced NF-κB activation by targeting TRAF6 for K48-linked deubiquitination and inhibits enterovirus 71 replication

    doi: 10.1038/s41598-018-31734-6

    Figure Lengend Snippet: USP4 participates in antiviral immunity against EV71 infection. ( A ) Analysis of phase contrast microscopy of RD cells transfected with either an empty vector or USP4, and then infected with EV71 (MOI = 0.5) at the indicated time (magnification 20x). ( B ) Apoptosis assay of EV71-infected RD cells transfected with empty vector or USP4. The apoptosis assay was conducted using annexin V/FITC and PI double staining and flow cytometry. Q3, Q4, and Q2 represent normal cells, early apoptotic cells, and late apoptotic/necrotic cells, respectively. ( C , F ) q-PCR and western blot analysis of EV71 protein expression in RD cells transfected with control vector or HA-USP4 plasmids, followed by treatment with EV71 for variable lengths of time. ( E ) Viral titre measurement of EV71 propagating in RD cells transiently transfected with control vector or HA-USP4 plasmids. Data in ( D , E ) are presented as the mean ± SD. * P

    Article Snippet: Cell apoptosis was subsequently determined using an annexin V/PI-based apoptosis detection kit (BD Biosciences) and analysed using a flow cytometer (BD FACSCanto II).

    Techniques: Infection, Microscopy, Transfection, Plasmid Preparation, Apoptosis Assay, Double Staining, Flow Cytometry, Cytometry, Polymerase Chain Reaction, Western Blot, Expressing

    MAVS Y9F is dispensable for MAVS-induced apoptosis. (A) HEK293T cells were transfected with increasing amount of Flag-vector, Flag-WT or Flag-Y9F mutant. Cells were harvested for Trypan Blue counting 48 h post-transfection. Data are mean of three independent experiments ± SEM done in triplicate. (B) HEK293T cells were transfected with 1.0 µg expression vector encoding Flag-vector, Flag-WT or Flag-Y9F mutant. Annexin V assays were performed 48 hours post-transfection.

    Journal: PLoS ONE

    Article Title: Identification of Tyrosine-9 of MAVS as Critical Target for Inducible Phosphorylation That Determines Activation

    doi: 10.1371/journal.pone.0041687

    Figure Lengend Snippet: MAVS Y9F is dispensable for MAVS-induced apoptosis. (A) HEK293T cells were transfected with increasing amount of Flag-vector, Flag-WT or Flag-Y9F mutant. Cells were harvested for Trypan Blue counting 48 h post-transfection. Data are mean of three independent experiments ± SEM done in triplicate. (B) HEK293T cells were transfected with 1.0 µg expression vector encoding Flag-vector, Flag-WT or Flag-Y9F mutant. Annexin V assays were performed 48 hours post-transfection.

    Article Snippet: Annexin V staining was performed as indicated with the Annexin V-Biotin Apoptosis Detection Kit (BD Bioscience).

    Techniques: Transfection, Plasmid Preparation, Mutagenesis, Expressing

    Pterostilbene induces cell apoptosis. OVCAR-8 and Caov-3 cells were treated with vehicle and PTE (25–300 μm) for 48 h. Apoptosis was determined by flow cytometry using annexin V and PI staining ( A , B ) or by Western blot for the expression of cleaved poly-ADP ribose polymerase (PARP) ( C ). Results are representative of 3 or more preparations. *, p

    Journal: International Journal of Molecular Sciences

    Article Title: Pterostilbene Suppresses Ovarian Cancer Growth via Induction of Apoptosis and Blockade of Cell Cycle Progression Involving Inhibition of the STAT3 Pathway

    doi: 10.3390/ijms19071983

    Figure Lengend Snippet: Pterostilbene induces cell apoptosis. OVCAR-8 and Caov-3 cells were treated with vehicle and PTE (25–300 μm) for 48 h. Apoptosis was determined by flow cytometry using annexin V and PI staining ( A , B ) or by Western blot for the expression of cleaved poly-ADP ribose polymerase (PARP) ( C ). Results are representative of 3 or more preparations. *, p

    Article Snippet: Annexin V Staining Annexin V apoptosis detection kit (BD biosciences, Franklin Lakes, NJ, USA) was used to measure apoptosis.

    Techniques: Flow Cytometry, Cytometry, Staining, Western Blot, Expressing