annexin v-fitc Search Results


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  • 99
    Thermo Fisher annexin v fitc
    Annexin V Fitc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4336 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore annexin v fitc
    Annexin V Fitc, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson annexin v fitc
    Effects of DL-buthionin-(S,R)-sulfoximine on Taurolidine induced cell death in HT29, Chang Liver and HT1080 cells . HT29 (a-c), Chang Liver (d-f) and HT1080 cells (g-i) were incubated with either the glutathione depleting agent DL-buthionin-(S,R)-sulfoximine(BSO) (1 mM), Taurolidine (TRD) (250 μM) or the combination of both agents (TRD 250 μM + BSO 1 mM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d, g), apoptotic (b, e, h) and necrotic cells (c, f, i) were determined by FACS-analysis for Annexin <t>V-FITC</t> and Propidiumiodide. Values are means ± SEM of 9 (HT29 and HT1080) and 4 (Chang Liver) independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).
    Annexin V Fitc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 20718 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson annexin v fitc apoptosis detection kit
    The reverse signaling of tmTNF-α promotes AICD through upregulating FasL/Fas and TRAIL/DR4 (A, B, C, D) Jurkat cells were simultaneously stimulated with PHA-P (5 μg/ml) and TNF-α pAb (1:100) for 24 h. PHA-preactivated primary T cells were restimulated with αCD3 (10 μg/ml) and TNF-α pAb for 24 h. Expression of FasL/Fas (A, B) and TRAIL/DR4/DR5 (C, D) was detected by flow cytometry. (E, F) Jurkat cells transfected with 100 nM siRNA for FasL or TRAIL for 48 h were activated with PHA-P (5 μg/ml) in the absence or presence of TNF-α pAb for 24 h. The mRNA levels of FasL and TRAIL were determined by Real-time qPCR (E) and the <t>apoptosis</t> was detected by Annexin V/PI (F). All the quantitative data represent means ± S.D. of at least three independent experiments. * p
    Annexin V Fitc Apoptosis Detection Kit, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 8914 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore annexin v fitc apoptosis detection kit
    The reverse signaling of tmTNF-α promotes AICD through upregulating FasL/Fas and TRAIL/DR4 (A, B, C, D) Jurkat cells were simultaneously stimulated with PHA-P (5 μg/ml) and TNF-α pAb (1:100) for 24 h. PHA-preactivated primary T cells were restimulated with αCD3 (10 μg/ml) and TNF-α pAb for 24 h. Expression of FasL/Fas (A, B) and TRAIL/DR4/DR5 (C, D) was detected by flow cytometry. (E, F) Jurkat cells transfected with 100 nM siRNA for FasL or TRAIL for 48 h were activated with PHA-P (5 μg/ml) in the absence or presence of TNF-α pAb for 24 h. The mRNA levels of FasL and TRAIL were determined by Real-time qPCR (E) and the <t>apoptosis</t> was detected by Annexin V/PI (F). All the quantitative data represent means ± S.D. of at least three independent experiments. * p
    Annexin V Fitc Apoptosis Detection Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Keygen Biotech annexin v fitc apoptosis detection kit
    Curcumin alone or combined with irinotecan induces cell <t>apoptosis</t> through ER stress-mediated CHOP expression in human CRC cell lines ( A , B ) The inhibition efficiency of siRNAs against CHOP. LoVo cells (A) or HT-29 cells (B) were transfected with siRNAs targeting CHOP, followed by treatments with curcumin alone or with irinotecan for 24 h, and the protein levels of CHOP was assessed by western blotting. ( C , D ) The effects of CHOP siRNA on cell apoptosis induced by curcumin alone or with irinotecan. LoVo cells (C) or HT-29 cells (D) were treated as described above, and then cell apoptosis was measured by Annexin <t>V-FITC/PI</t> staining. Values are means ± SEM. *
    Annexin V Fitc Apoptosis Detection Kit, supplied by Keygen Biotech, used in various techniques. Bioz Stars score: 93/100, based on 2044 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Beyotime annexin v fitc apoptosis detection kit
    Effects of miR-522 on human NSCLC cells <t>apoptosis.</t> ( a ) Annexin <t>V-FITC/propidium</t> iodide staining and flow cytometry were performed to detect apoptosis. ( b ) The quantitative presentation of apoptotic cell populations. * P
    Annexin V Fitc Apoptosis Detection Kit, supplied by Beyotime, used in various techniques. Bioz Stars score: 99/100, based on 1893 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioVision annexin v fitc
    Effects of miR-522 on human NSCLC cells <t>apoptosis.</t> ( a ) Annexin <t>V-FITC/propidium</t> iodide staining and flow cytometry were performed to detect apoptosis. ( b ) The quantitative presentation of apoptotic cell populations. * P
    Annexin V Fitc, supplied by BioVision, used in various techniques. Bioz Stars score: 99/100, based on 1261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher annexin v fitc apoptosis detection kit
    Effects of miR-522 on human NSCLC cells <t>apoptosis.</t> ( a ) Annexin <t>V-FITC/propidium</t> iodide staining and flow cytometry were performed to detect apoptosis. ( b ) The quantitative presentation of apoptotic cell populations. * P
    Annexin V Fitc Apoptosis Detection Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2344 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Beyotime annexin v fitc
    (A) Confocal microscopy analysis of the colocalization of 2B-GFP and Bak-GFP within the mitochondria in HeLa cells. HeLa cells were transfected with pGFP, pGFP-2B, or pGFP-Bak. At 24 h posttransfection, they were stained with MitoTracker Red and subjected to confocal microscopy analysis. An overlay of the MitoTracker Red (red) and GFP florescence (green) is also shown. Mito, mitochondria. (B) Colocalization index estimation for cells transfected with pGFP, pGFP-2B, or pGFP-Bak and labeled for MitoTracker Red as for panel A. To determine the percentage of colocalization, green and merged images were loaded into the Leica TCS SP8 platform, and the ratio of GFP-positive cells to counted cells (in a total of 100 cells) was determined. Cells with GFP and mitochondrion colocalization (Manders overlap coefficient of > 0.8) were counted. (C) Immunoblotting analysis of 2B-Flag in the cytosol and mitochondrial fractions of HeLa cells infected with EV71 or transfected with pCMV-2B-Flag. Cytosolic and heavy membrane fractions were separated as described in Materials and Methods. Mitochondrial fractions were processed for immunoblotting with antibodies specific to EV71 2B, Bak, and Cox IV. Cytosolic fractions were immunoblotted with antibodies to EV71 2B and β-actin (internal control). Control, cells transfected with Tris-acetate-EDTA (TAE) buffer. Mock, cells with no EV71 infection. (D) Western blot analysis of the sublocation of 2B. Cells were transfected with pCMV-2B-Flag or pCMV-Flag. The mitochondrial fractions were isolated at 48 h posttransfection. The inner and outer mitochondrial membranes were isolated by sucrose gradient centrifugation and immunoblotted with an antibody to the Flag tag, Cox IV, and TSPO (internal control of outer mitochondrial membrane fraction). M, mitochondria of cells transfected with pCMV-Flag. I, inner mitochondrial membrane fraction. O, outer mitochondrial membrane fraction. (E) Analysis of cell apoptosis induced by 2B. Cells were treated with 30 nM staurosporine (positive control) or transfected with increasing amounts of pCMV-2B-Flag, pCMV-Flag, or pCMV-Bak. At 24 h posttransfection, cells were harvested and stained with annexin <t>V-FITC.</t> The percentages of apoptotic cells corresponding to the respective fluorescence intensities were calculated. (F) Analysis of the antiapoptosis effects of caspase inhibitors on 2B-induced apoptosis. Cells either infected with EV71 or transfected with pCMV-2B-Flag or pCMV-Bak-Flag (positive control) were treated with dimethyl sulfoxide (DMSO), zDEVD.fmk, or zLEHD.fmk for 24 h and then stained with annexin V-FITC and subjected to flow cytometry. NT, cells with no treatment. Control, cells transfected with pCMV-Flag.
    Annexin V Fitc, supplied by Beyotime, used in various techniques. Bioz Stars score: 93/100, based on 960 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson annexin v fitc kit
    25- epi Ritterostatin G N 1 N is cytotoxic at nanomolar concentrations in melanoma cells. (A and B) MTT assay results showing A375, WM35, A375SM, and UCSD-354L melanoma cell viability after treatment with various concentrations of 25- epi Ritterostatin G N 1 N for 72 h. IC 50 , 50% inhibitory concentration. (C) Percentage of viable WM35 and A375 cells after treatment with 0.5 µ M 25- epi Ritterostatin G N 1 N for 72 h, determined according to Annexin <t>V-FITC/propidium</t> iodide staining and subsequent flow cytometry analysis. (D) The bar graphs represent the percentage of viable A375, WM35, A375SM, and UCSD-354L cells (mean ± standard error) after treatment with 0.5 µ M 25- epi Ritterostatin G N 1 N for 48 and 72 h, for 3 independent measurements, according to Annexin V-FITC/propidium iodide staining analysis. (E and F) Giemsa staining for colony formation assessment in A375 and WM35 cells treated with varying concentrations of 25- epi Ritterostatin G N 1 N [or dimethyl sulfoxide (DMSO) only] for 14 days. The bar graphs represent the mean (± standard error of the mean) of three independent measurements. * p
    Annexin V Fitc Kit, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 949 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    BioLegend annexin v fitc
    MLL -translocation cell lines are sensitive to inhibition of pan-Cullin-dependent and CKS1-dependent protein degradation. (A) Western blots for THP-1 ( MLL-AF9 ) and KOPN-8 ( MLL-ENL ) cells treated with vehicle control (DMSO), 0.1 μM MLN4924 or 1 μM C1 for 16 h and 24 h. Histone H3 was used as a loading control. Western blots are representative of 3 independent experiments. (B) Cell viability was assessed for THP-1, KOPN-8, healthy PBMC and CD34 + control cells after 3 days treatment with indicated concentrations of the pan-Cullin inhibitor (MLN4924) or the SKP2-CKS1 inhibitor (C1). Results represent the mean of 3 independent experiments with standard deviation bars. (C) Proportion of apoptotic CD34 + , THP-1 and KOPN-8 cells, in response to two different concentrations of C1 or MLN4924, was measured by PI and Annexin <t>V-FITC</t> staining using flow cytometry. Significance was tested using the Student's t -test versus DMSO treated cells (N = 3). (D) Cell cycle status was measured for KOPN-8 cells in response to DMSO vehicle control or MLN4924 (1 μM) using the Click-iT EdU incorporation kit. Graphs are representative of 3 independent experiments.
    Annexin V Fitc, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 766 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Keygen Biotech annexin v fitc
    GA-TAT induces apoptosis in EJ cells. ( A ) EJ cells were exposed to 1.0 μM TAT, GA, or GA-TAT for 24 h and then collected and mixed with 200 μL of dye mixture containing 100 μg/mL AO and EB. Cellular morphological changes were observed by using fluorescence microscopy (200× magnification). ( B ) After exposure to 1.0 μM TAT, GA, or GA-TAT for 24 h, EJ cells were harvested and stained with Annexin <t>V-FITC</t> and PI, followed by flow cytometric analysis of apoptosis. Arrows indicate apoptotic cells. * P
    Annexin V Fitc, supplied by Keygen Biotech, used in various techniques. Bioz Stars score: 94/100, based on 1117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioVision annexin v fluorescein isothiocyanate apoptosis detection kit
    <t>Apoptosis</t> assay of mouse spleen cells before and after irreversible electroporation treatment using double-staining with annexin V-fluorescein <t>isothiocyanate/propidium</t> iodide. Apoptosis was quantified by flow cytometry. A: Control group; B: 1 d post-IRE; C: 3 d post-IRE; D: 7 d post-IRE; E: Percentages of apoptotic cells before and after the IRE intervention. Data are mean ± SD ( n = 8). IRE: irreversible electroporation.
    Annexin V Fluorescein Isothiocyanate Apoptosis Detection Kit, supplied by BioVision, used in various techniques. Bioz Stars score: 99/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pharmingen annexin v fitc
    Holotoxin A 1 activations of acid SMase and neutral SMase induce apoptosis in human primary leukemia cells. ( A , B ) Holotoxin A 1 (6 h) induced apoptosis in ( A ) human primary leukemia cells, but not in ( B ) human hematopoietic progenitor CD34 + cells. Upper panels: Representative flow cytometry results show the percentage of apoptotic cells measured with annexin <t>V-FITC/PI</t> staining. Lower panels: Mean ± SD of three independent experiments. *** p
    Annexin V Fitc, supplied by Pharmingen, used in various techniques. Bioz Stars score: 93/100, based on 859 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effects of DL-buthionin-(S,R)-sulfoximine on Taurolidine induced cell death in HT29, Chang Liver and HT1080 cells . HT29 (a-c), Chang Liver (d-f) and HT1080 cells (g-i) were incubated with either the glutathione depleting agent DL-buthionin-(S,R)-sulfoximine(BSO) (1 mM), Taurolidine (TRD) (250 μM) or the combination of both agents (TRD 250 μM + BSO 1 mM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d, g), apoptotic (b, e, h) and necrotic cells (c, f, i) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 9 (HT29 and HT1080) and 4 (Chang Liver) independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines

    doi: 10.1186/1756-9966-29-21

    Figure Lengend Snippet: Effects of DL-buthionin-(S,R)-sulfoximine on Taurolidine induced cell death in HT29, Chang Liver and HT1080 cells . HT29 (a-c), Chang Liver (d-f) and HT1080 cells (g-i) were incubated with either the glutathione depleting agent DL-buthionin-(S,R)-sulfoximine(BSO) (1 mM), Taurolidine (TRD) (250 μM) or the combination of both agents (TRD 250 μM + BSO 1 mM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d, g), apoptotic (b, e, h) and necrotic cells (c, f, i) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 9 (HT29 and HT1080) and 4 (Chang Liver) independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).

    Article Snippet: Subsequently, 5 μl Annexin V-FITC (BD Biosciences, Heidelberg, Germany) was added to the cell suspension followed by gently vortexing and incubation for 10 min at room temperature in the dark.

    Techniques: Incubation, FACS

    Effects of caspase-inhibition on Taurolidine induced cell death in HT29, Chang Liver and HT1080 cells . HT29 (a-c), Chang Liver (d-f) and HT1080 cells (g-i) were incubated with either z-VAD.fmk (1 μM), Taurolidine (TRD) (250 μM) or the combination of both agents (TRD 250 μM + zVAD.fmk 1 μM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d, g), apoptotic (b, e, h) and necrotic cells (c, f, i) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 5 (HT29), 6 (Chang Liver) and 4 (HT1080) independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines

    doi: 10.1186/1756-9966-29-21

    Figure Lengend Snippet: Effects of caspase-inhibition on Taurolidine induced cell death in HT29, Chang Liver and HT1080 cells . HT29 (a-c), Chang Liver (d-f) and HT1080 cells (g-i) were incubated with either z-VAD.fmk (1 μM), Taurolidine (TRD) (250 μM) or the combination of both agents (TRD 250 μM + zVAD.fmk 1 μM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d, g), apoptotic (b, e, h) and necrotic cells (c, f, i) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 5 (HT29), 6 (Chang Liver) and 4 (HT1080) independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).

    Article Snippet: Subsequently, 5 μl Annexin V-FITC (BD Biosciences, Heidelberg, Germany) was added to the cell suspension followed by gently vortexing and incubation for 10 min at room temperature in the dark.

    Techniques: Inhibition, Incubation, FACS

    Effects of DL-buthionin-(S,R)-sulfoximine on Taurolidine induced cell death in AsPC-1 and BxPC-3 cells . AsPC-1 (a-c) and BxPC-3 cells (d-f) were incubated with either the glutathione depleting agent DL-buthionin-(S,R)-sulfoximine(BSO) (1 mM), Taurolidine (TRD) (250 μM for BxPC-3 and 1000 μM for AsPC-1) or the combination of both agents (TRD 250 μM/1000 μM + BSO 1 mM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d), apoptotic (b, e) and necrotic cells (c, f) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 4 independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines

    doi: 10.1186/1756-9966-29-21

    Figure Lengend Snippet: Effects of DL-buthionin-(S,R)-sulfoximine on Taurolidine induced cell death in AsPC-1 and BxPC-3 cells . AsPC-1 (a-c) and BxPC-3 cells (d-f) were incubated with either the glutathione depleting agent DL-buthionin-(S,R)-sulfoximine(BSO) (1 mM), Taurolidine (TRD) (250 μM for BxPC-3 and 1000 μM for AsPC-1) or the combination of both agents (TRD 250 μM/1000 μM + BSO 1 mM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d), apoptotic (b, e) and necrotic cells (c, f) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 4 independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).

    Article Snippet: Subsequently, 5 μl Annexin V-FITC (BD Biosciences, Heidelberg, Germany) was added to the cell suspension followed by gently vortexing and incubation for 10 min at room temperature in the dark.

    Techniques: Incubation, FACS

    Effects of caspase-inhibition on Taurolidine induced cell death in AsPC-1 and BxPC-3 cells . AsPC-1 (a-c) and BxPC-3 cells (d-f) were incubated with either z-VAD.fmk (1 μM), Taurolidine (TRD) (250 μM for BxPC-3 and 1000 μM for AsPC-1) or the combination of both agents (TRD 250 μM/1000 μM + zVAD.fmk 1 μM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d), apoptotic (b, e) and necrotic cells (c, f) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 3 (AsPC-1) and 6 (BxPC-3) independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines

    doi: 10.1186/1756-9966-29-21

    Figure Lengend Snippet: Effects of caspase-inhibition on Taurolidine induced cell death in AsPC-1 and BxPC-3 cells . AsPC-1 (a-c) and BxPC-3 cells (d-f) were incubated with either z-VAD.fmk (1 μM), Taurolidine (TRD) (250 μM for BxPC-3 and 1000 μM for AsPC-1) or the combination of both agents (TRD 250 μM/1000 μM + zVAD.fmk 1 μM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d), apoptotic (b, e) and necrotic cells (c, f) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 3 (AsPC-1) and 6 (BxPC-3) independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).

    Article Snippet: Subsequently, 5 μl Annexin V-FITC (BD Biosciences, Heidelberg, Germany) was added to the cell suspension followed by gently vortexing and incubation for 10 min at room temperature in the dark.

    Techniques: Inhibition, Incubation, FACS

    Effects of Taurolidine on viability, apoptosis and necrosis in AsPC-1 and BxPC-3 cells . AsPC-1 (a-c) and BxPC-3 cells (d-f) were incubated with Taurolidine (TRD) (100 μM, 250 μM and 1000 μM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d), apoptotic (b, d) and necrotic cells (c, f) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 4 independent experiments with consecutive passages. Asterisk symbols on columns indicate differences between control and TRD treatment. Asterisk symbols on brackets indicate differences between TRD groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines

    doi: 10.1186/1756-9966-29-21

    Figure Lengend Snippet: Effects of Taurolidine on viability, apoptosis and necrosis in AsPC-1 and BxPC-3 cells . AsPC-1 (a-c) and BxPC-3 cells (d-f) were incubated with Taurolidine (TRD) (100 μM, 250 μM and 1000 μM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d), apoptotic (b, d) and necrotic cells (c, f) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 4 independent experiments with consecutive passages. Asterisk symbols on columns indicate differences between control and TRD treatment. Asterisk symbols on brackets indicate differences between TRD groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).

    Article Snippet: Subsequently, 5 μl Annexin V-FITC (BD Biosciences, Heidelberg, Germany) was added to the cell suspension followed by gently vortexing and incubation for 10 min at room temperature in the dark.

    Techniques: Incubation, FACS

    Representative dot plots obtained by FACS-anaylsis after incubation of different cell lines with Taurolidine . Chang Liver, HT1080 and BxPC-3 cells were incubated with Taurolidine (TRD) (100 μM, 250 μM and 1000 μM) and with Povidon 5% (control) for 24 h. FACS-analysis was performed for Annexin V-FITC (x-axis) and Propidiumiodide (y-axis). Lower left quadrant: Annexin V and propidium iodide negative (viable), lower right quadrant: Annexin V positive and propidium iodide negative (apoptotic), upper right quadrant: Annexin V and propidium iodide positive (necrotic).

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines

    doi: 10.1186/1756-9966-29-21

    Figure Lengend Snippet: Representative dot plots obtained by FACS-anaylsis after incubation of different cell lines with Taurolidine . Chang Liver, HT1080 and BxPC-3 cells were incubated with Taurolidine (TRD) (100 μM, 250 μM and 1000 μM) and with Povidon 5% (control) for 24 h. FACS-analysis was performed for Annexin V-FITC (x-axis) and Propidiumiodide (y-axis). Lower left quadrant: Annexin V and propidium iodide negative (viable), lower right quadrant: Annexin V positive and propidium iodide negative (apoptotic), upper right quadrant: Annexin V and propidium iodide positive (necrotic).

    Article Snippet: Subsequently, 5 μl Annexin V-FITC (BD Biosciences, Heidelberg, Germany) was added to the cell suspension followed by gently vortexing and incubation for 10 min at room temperature in the dark.

    Techniques: FACS, Incubation

    Effects of N-acetylcysteine on Taurolidine induced cell death in HT29, Chang Liver and HT1080 cells . HT29 (a-c), Chang Liver (d-f) and HT1080 cells (g-i) were incubated with either the radical scavenger N-acetylcysteine (NAC) (5 mM), Taurolidine (TRD) (250 μM) or the combination of both agents (TRD 250 μM + NAC 5 mM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d, g), apoptotic (b, e, h) and necrotic cells (c, f, i) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 4 (HT29 and Chang Liver) and 12 (HT1080) independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines

    doi: 10.1186/1756-9966-29-21

    Figure Lengend Snippet: Effects of N-acetylcysteine on Taurolidine induced cell death in HT29, Chang Liver and HT1080 cells . HT29 (a-c), Chang Liver (d-f) and HT1080 cells (g-i) were incubated with either the radical scavenger N-acetylcysteine (NAC) (5 mM), Taurolidine (TRD) (250 μM) or the combination of both agents (TRD 250 μM + NAC 5 mM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d, g), apoptotic (b, e, h) and necrotic cells (c, f, i) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 4 (HT29 and Chang Liver) and 12 (HT1080) independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).

    Article Snippet: Subsequently, 5 μl Annexin V-FITC (BD Biosciences, Heidelberg, Germany) was added to the cell suspension followed by gently vortexing and incubation for 10 min at room temperature in the dark.

    Techniques: Incubation, FACS

    Effects of N-acetylcysteine on Taurolidine induced cell death in AsPC-1 and BxPC-3 cells . AsPC-1 (a-c) and BxPC-3 cells (d-f) were incubated with either the radical scavenger N-acetylcysteine (NAC) (5 mM), Taurolidine (TRD) (250 μM for BxPC-3 and 1000 μM for AsPC-1) or the combination of both agents (TRD 250 μM/1000 μM + NAC 5 mM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d), apoptotic (b, e) and necrotic cells (c, f) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 4 independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines

    doi: 10.1186/1756-9966-29-21

    Figure Lengend Snippet: Effects of N-acetylcysteine on Taurolidine induced cell death in AsPC-1 and BxPC-3 cells . AsPC-1 (a-c) and BxPC-3 cells (d-f) were incubated with either the radical scavenger N-acetylcysteine (NAC) (5 mM), Taurolidine (TRD) (250 μM for BxPC-3 and 1000 μM for AsPC-1) or the combination of both agents (TRD 250 μM/1000 μM + NAC 5 mM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d), apoptotic (b, e) and necrotic cells (c, f) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 4 independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).

    Article Snippet: Subsequently, 5 μl Annexin V-FITC (BD Biosciences, Heidelberg, Germany) was added to the cell suspension followed by gently vortexing and incubation for 10 min at room temperature in the dark.

    Techniques: Incubation, FACS

    Effects of Taurolidine on viability, apoptosis and necrosis in HT29, Chang Liver and HT1080 cells . HT29 (a-c), Chang Liver (d-f) and HT1080 cells (g-i) were incubated with Taurolidine (TRD) (100 μM, 250 μM and 1000 μM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d, g), apoptotic (b, e, h) and necrotic cells (c, f, i) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 5 (HT29), 4 (Chang Liver) and 9 (HT1080) independent experiments with consecutive passages. Asterisk symbols on columns indicate differences between control and TRD treatment. Asterisk symbols on brackets indicate differences between TRD groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines

    doi: 10.1186/1756-9966-29-21

    Figure Lengend Snippet: Effects of Taurolidine on viability, apoptosis and necrosis in HT29, Chang Liver and HT1080 cells . HT29 (a-c), Chang Liver (d-f) and HT1080 cells (g-i) were incubated with Taurolidine (TRD) (100 μM, 250 μM and 1000 μM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d, g), apoptotic (b, e, h) and necrotic cells (c, f, i) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 5 (HT29), 4 (Chang Liver) and 9 (HT1080) independent experiments with consecutive passages. Asterisk symbols on columns indicate differences between control and TRD treatment. Asterisk symbols on brackets indicate differences between TRD groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).

    Article Snippet: Subsequently, 5 μl Annexin V-FITC (BD Biosciences, Heidelberg, Germany) was added to the cell suspension followed by gently vortexing and incubation for 10 min at room temperature in the dark.

    Techniques: Incubation, FACS

    Co-culture with Hs-5 cells protected against combination treatment in 3 cell lines, while addition of Hs-5 conditioned medium was protective in only RPMI-8226. Hs-5 cells were plated at a density of 0.05 × 10 6 cells/mL and allowed to grow for 48 h. MM cells were added to the Hs-5 cells after 48 h, 30 min prior to drug treatment. Conditioned medium was collected and filtered then diluted to 50% and added to MM cells. 24 h cell death was assessed via Annexin-V-FITC/PI (control and conditioned medium treated cells) or Annexin-V-FITC/anti-CD38-PE (Hs-5 co-cultured cells) flow cytometry. Data in are presented as mean +/− SEM of at least 3 independent experiments.

    Journal: Cancer Biology & Therapy

    Article Title: Dual inhibition of Mcl-1 by the combination of carfilzomib and TG02 in multiple myeloma

    doi: 10.1080/15384047.2016.1192086

    Figure Lengend Snippet: Co-culture with Hs-5 cells protected against combination treatment in 3 cell lines, while addition of Hs-5 conditioned medium was protective in only RPMI-8226. Hs-5 cells were plated at a density of 0.05 × 10 6 cells/mL and allowed to grow for 48 h. MM cells were added to the Hs-5 cells after 48 h, 30 min prior to drug treatment. Conditioned medium was collected and filtered then diluted to 50% and added to MM cells. 24 h cell death was assessed via Annexin-V-FITC/PI (control and conditioned medium treated cells) or Annexin-V-FITC/anti-CD38-PE (Hs-5 co-cultured cells) flow cytometry. Data in are presented as mean +/− SEM of at least 3 independent experiments.

    Article Snippet: 24 h cell death was assessed via Annexin-V-FITC/PI (control and conditioned medium treated cells) or Annexin-V-FITC/anti-CD38-PE (BD Biosciences 347687) (Hs-5 co-cultured cells) flow cytometry.

    Techniques: Co-Culture Assay, Cell Culture, Flow Cytometry, Cytometry

    Continuous carfilzomib and TG02 co-treatment results in at least additive cell death. Cells were plated at a density of 0.25 × 10 6 cells/mL and treated with the indicated concentrations of (A) carfilzomib, (B) TG02 or the (C) combination for 24 hours. (D) Cells were co-treated with carfilzomib (IC10 and IC50 concentrations) and TG02 for 1 hour, washed and then treated with TG02 for 23 hours. Cell death was determined via Annexin–V–FITC/PI flow cytometry. Data are presented as mean +/− SEM of at least 3 independent experiments. IC50 and IC10 significance are indicated above and below the mean values, respectively. (E) Ficoll isolated buffy coat from a myeloma patient BM aspirate was collected and washed with PBS. Plasma cells were either treated in the presence of buffy coat cells (MM53 and MM55) or CD138+ plasma cells were isolated and treated (MM54). Twenty-four hour apoptosis was determined by staining with anti-CD38, anti-CD45, and Annexin V-FITC. *p > 0.05, **p > 0.01, ***p > 0.001.

    Journal: Cancer Biology & Therapy

    Article Title: Dual inhibition of Mcl-1 by the combination of carfilzomib and TG02 in multiple myeloma

    doi: 10.1080/15384047.2016.1192086

    Figure Lengend Snippet: Continuous carfilzomib and TG02 co-treatment results in at least additive cell death. Cells were plated at a density of 0.25 × 10 6 cells/mL and treated with the indicated concentrations of (A) carfilzomib, (B) TG02 or the (C) combination for 24 hours. (D) Cells were co-treated with carfilzomib (IC10 and IC50 concentrations) and TG02 for 1 hour, washed and then treated with TG02 for 23 hours. Cell death was determined via Annexin–V–FITC/PI flow cytometry. Data are presented as mean +/− SEM of at least 3 independent experiments. IC50 and IC10 significance are indicated above and below the mean values, respectively. (E) Ficoll isolated buffy coat from a myeloma patient BM aspirate was collected and washed with PBS. Plasma cells were either treated in the presence of buffy coat cells (MM53 and MM55) or CD138+ plasma cells were isolated and treated (MM54). Twenty-four hour apoptosis was determined by staining with anti-CD38, anti-CD45, and Annexin V-FITC. *p > 0.05, **p > 0.01, ***p > 0.001.

    Article Snippet: 24 h cell death was assessed via Annexin-V-FITC/PI (control and conditioned medium treated cells) or Annexin-V-FITC/anti-CD38-PE (BD Biosciences 347687) (Hs-5 co-cultured cells) flow cytometry.

    Techniques: Flow Cytometry, Cytometry, Isolation, Staining

    The decreased in Mcl-1 protein expression is not due to CDK9 inhibition. MM.1s cells were treated with siCDK9 or si (control) for 24 hours and then treated with carfilzomib, TG02 or the combination for an additional 24 hours. (A) Knockdown of siCDK9 was determined using protein gel blot and densitometry analysis. Relative expression, normalized to actin, of the indicated proteins are shown below the corresponding bands. (B) Cell death was determined via Annexin-V-FITC/PtdIns flow cytometry. Data are presented as mean +/− SEM of at least 3 independent experiments.

    Journal: Cancer Biology & Therapy

    Article Title: Dual inhibition of Mcl-1 by the combination of carfilzomib and TG02 in multiple myeloma

    doi: 10.1080/15384047.2016.1192086

    Figure Lengend Snippet: The decreased in Mcl-1 protein expression is not due to CDK9 inhibition. MM.1s cells were treated with siCDK9 or si (control) for 24 hours and then treated with carfilzomib, TG02 or the combination for an additional 24 hours. (A) Knockdown of siCDK9 was determined using protein gel blot and densitometry analysis. Relative expression, normalized to actin, of the indicated proteins are shown below the corresponding bands. (B) Cell death was determined via Annexin-V-FITC/PtdIns flow cytometry. Data are presented as mean +/− SEM of at least 3 independent experiments.

    Article Snippet: 24 h cell death was assessed via Annexin-V-FITC/PI (control and conditioned medium treated cells) or Annexin-V-FITC/anti-CD38-PE (BD Biosciences 347687) (Hs-5 co-cultured cells) flow cytometry.

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

    Overexpression of Mcl-1 in RPMI-8226 cells confers significantly less protection than over expression of Bcl-2 or Bcl-x L. RPMI-8226 cells overexpressing the anti-apoptotic proteins shown were treated with increasing doses of (A) carfilzomib (B) TG02 or (C) the combination for 24 hours. Cell death was determined via Annexin-V-FITC/PI flow cytometry. Data are presented as mean +/− SEM of at least 3 independent experiments. Statistical analysis for RPMI-8226 Mcl-1 over expressing cells only shown. *p > 0.05, **p > 0.01, ***p > 0.001.

    Journal: Cancer Biology & Therapy

    Article Title: Dual inhibition of Mcl-1 by the combination of carfilzomib and TG02 in multiple myeloma

    doi: 10.1080/15384047.2016.1192086

    Figure Lengend Snippet: Overexpression of Mcl-1 in RPMI-8226 cells confers significantly less protection than over expression of Bcl-2 or Bcl-x L. RPMI-8226 cells overexpressing the anti-apoptotic proteins shown were treated with increasing doses of (A) carfilzomib (B) TG02 or (C) the combination for 24 hours. Cell death was determined via Annexin-V-FITC/PI flow cytometry. Data are presented as mean +/− SEM of at least 3 independent experiments. Statistical analysis for RPMI-8226 Mcl-1 over expressing cells only shown. *p > 0.05, **p > 0.01, ***p > 0.001.

    Article Snippet: 24 h cell death was assessed via Annexin-V-FITC/PI (control and conditioned medium treated cells) or Annexin-V-FITC/anti-CD38-PE (BD Biosciences 347687) (Hs-5 co-cultured cells) flow cytometry.

    Techniques: Over Expression, Flow Cytometry, Cytometry, Expressing

    The reverse signaling of tmTNF-α promotes AICD through upregulating FasL/Fas and TRAIL/DR4 (A, B, C, D) Jurkat cells were simultaneously stimulated with PHA-P (5 μg/ml) and TNF-α pAb (1:100) for 24 h. PHA-preactivated primary T cells were restimulated with αCD3 (10 μg/ml) and TNF-α pAb for 24 h. Expression of FasL/Fas (A, B) and TRAIL/DR4/DR5 (C, D) was detected by flow cytometry. (E, F) Jurkat cells transfected with 100 nM siRNA for FasL or TRAIL for 48 h were activated with PHA-P (5 μg/ml) in the absence or presence of TNF-α pAb for 24 h. The mRNA levels of FasL and TRAIL were determined by Real-time qPCR (E) and the apoptosis was detected by Annexin V/PI (F). All the quantitative data represent means ± S.D. of at least three independent experiments. * p

    Journal: Oncotarget

    Article Title: Transmembrane TNF-α promotes activation-induced cell death by forward and reverse signaling

    doi: 10.18632/oncotarget.19124

    Figure Lengend Snippet: The reverse signaling of tmTNF-α promotes AICD through upregulating FasL/Fas and TRAIL/DR4 (A, B, C, D) Jurkat cells were simultaneously stimulated with PHA-P (5 μg/ml) and TNF-α pAb (1:100) for 24 h. PHA-preactivated primary T cells were restimulated with αCD3 (10 μg/ml) and TNF-α pAb for 24 h. Expression of FasL/Fas (A, B) and TRAIL/DR4/DR5 (C, D) was detected by flow cytometry. (E, F) Jurkat cells transfected with 100 nM siRNA for FasL or TRAIL for 48 h were activated with PHA-P (5 μg/ml) in the absence or presence of TNF-α pAb for 24 h. The mRNA levels of FasL and TRAIL were determined by Real-time qPCR (E) and the apoptosis was detected by Annexin V/PI (F). All the quantitative data represent means ± S.D. of at least three independent experiments. * p

    Article Snippet: Apoptosis detection The apoptosis was evaluated by an Annexin V-FITC Apoptosis Detection Kit (BD biosciences), according to the manufacturer's instructions.

    Techniques: Expressing, Flow Cytometry, Cytometry, Transfection, Real-time Polymerase Chain Reaction

    tmTNF-α is involved in AICD (A, B, C, D) Jurkat cells transfected with 10 μM of TNF-α AS, TACE AS or NS for 48 h were treated with PHA-P (5 μg/ml) for 24 h. Apoptosis rate (A,C) was evaluated by Annexin V/PI and expression of tmTNF-α (A,C) and TACE (B) on the cell surface was analyzed by flow cytometry. Relative levels of TACE mRNA was detected by Real time qPCR (B). Western blot analysis of tmTNF-α expression (D). (E, F) PHA-preactivated primary T cells were transfected with 10 μM of TNF-α AS or NS for 48 h, and then treated with αCD3 (10 μg/ml) for 24 h. A TACE inhibitor TAPI-1 (10 μM) was added simultaneously with αCD3 treatment, and vehicle DMSO served as a control. Apoptosis was evaluated by Annexin V/PI and tmTNF-α expression was detected by flow cytometry. (G, H) tmTNF-α overexpressing 24 h-PHA activated and fixed Jurkat cells were cocultured with 3 h-PHA activated Jurkat cells at an effector/target ratio of 10:1 for 48 h (G). For primary T cells, tmTNF-α overexpressing, αCD3-restimulated and fixed T cells were co-cultured with PHA-preactivated T cells (preactivated T) at an effector/target ratio of 10:1 for 48h (H). For neutralization of tmTNF-α, effector cells were treated with TNF-α pAb for 30 min and then washed prior to the addition to the target cells. Apoptosis was determined by the Annexin V/PI. All the quantitative data represent means ± S.D. of at least three independent experiments. * p

    Journal: Oncotarget

    Article Title: Transmembrane TNF-α promotes activation-induced cell death by forward and reverse signaling

    doi: 10.18632/oncotarget.19124

    Figure Lengend Snippet: tmTNF-α is involved in AICD (A, B, C, D) Jurkat cells transfected with 10 μM of TNF-α AS, TACE AS or NS for 48 h were treated with PHA-P (5 μg/ml) for 24 h. Apoptosis rate (A,C) was evaluated by Annexin V/PI and expression of tmTNF-α (A,C) and TACE (B) on the cell surface was analyzed by flow cytometry. Relative levels of TACE mRNA was detected by Real time qPCR (B). Western blot analysis of tmTNF-α expression (D). (E, F) PHA-preactivated primary T cells were transfected with 10 μM of TNF-α AS or NS for 48 h, and then treated with αCD3 (10 μg/ml) for 24 h. A TACE inhibitor TAPI-1 (10 μM) was added simultaneously with αCD3 treatment, and vehicle DMSO served as a control. Apoptosis was evaluated by Annexin V/PI and tmTNF-α expression was detected by flow cytometry. (G, H) tmTNF-α overexpressing 24 h-PHA activated and fixed Jurkat cells were cocultured with 3 h-PHA activated Jurkat cells at an effector/target ratio of 10:1 for 48 h (G). For primary T cells, tmTNF-α overexpressing, αCD3-restimulated and fixed T cells were co-cultured with PHA-preactivated T cells (preactivated T) at an effector/target ratio of 10:1 for 48h (H). For neutralization of tmTNF-α, effector cells were treated with TNF-α pAb for 30 min and then washed prior to the addition to the target cells. Apoptosis was determined by the Annexin V/PI. All the quantitative data represent means ± S.D. of at least three independent experiments. * p

    Article Snippet: Apoptosis detection The apoptosis was evaluated by an Annexin V-FITC Apoptosis Detection Kit (BD biosciences), according to the manufacturer's instructions.

    Techniques: Transfection, Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Neutralization

    sTNF-α participates in AICD (A, B) Jurkat cells transfected with 10 μM of TNF-α AS or TACE AS for 48 h were stimulated with PHA-P (5 μg/ml) for 24 h. For primary T cells, PHA-preactivated T cells transfected with TNF-α AS for 48 h were restimulated with αCD3 (10 μg/ml) for 24 h; or PHA-preactivated T cells simultaneously treated with TAPI-1 (10 μM) and αCD3 (10 μg/ml) for 24 h. Concentration of sTNF-α in supernatants was detected by ELISA. (C, D) Recombinant human sTNF-α (50U/ml) was incubated with PHA-P activated Jurkat cells or αCD3-restimulated primary T cells for 24 h and the apoptosis was detected by Annexin V/PI. Non-stimulated Jurkat T cells or preactivated primary T cells served as a control. All the quantitative data represent means ± S.D. of at least three independent experiments. ** p

    Journal: Oncotarget

    Article Title: Transmembrane TNF-α promotes activation-induced cell death by forward and reverse signaling

    doi: 10.18632/oncotarget.19124

    Figure Lengend Snippet: sTNF-α participates in AICD (A, B) Jurkat cells transfected with 10 μM of TNF-α AS or TACE AS for 48 h were stimulated with PHA-P (5 μg/ml) for 24 h. For primary T cells, PHA-preactivated T cells transfected with TNF-α AS for 48 h were restimulated with αCD3 (10 μg/ml) for 24 h; or PHA-preactivated T cells simultaneously treated with TAPI-1 (10 μM) and αCD3 (10 μg/ml) for 24 h. Concentration of sTNF-α in supernatants was detected by ELISA. (C, D) Recombinant human sTNF-α (50U/ml) was incubated with PHA-P activated Jurkat cells or αCD3-restimulated primary T cells for 24 h and the apoptosis was detected by Annexin V/PI. Non-stimulated Jurkat T cells or preactivated primary T cells served as a control. All the quantitative data represent means ± S.D. of at least three independent experiments. ** p

    Article Snippet: Apoptosis detection The apoptosis was evaluated by an Annexin V-FITC Apoptosis Detection Kit (BD biosciences), according to the manufacturer's instructions.

    Techniques: Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation

    The reverse signaling of tmTNF-α enhances AICD (A, C) Jurkat cells were incubated with PHA-P (5 μg/ml), TNF-α pAb (1:100), sTNFR1 or with both PHA-P and TNF-α pAb, or PHA-P and sTNFR1 in indicated concentrations for 24 h. Apoptosis was detected by Annexin V/PI. (B) PHA preactivated primary T cells were stimulated with αCD3 (10 μg/ml), TNF-α pAb, or both for 24 h. Apoptosis was detected by Annexin V/PI. Rabbit serum served as a control. All the quantitative data represent means ± S.D. of at least three independent experiments. ** p

    Journal: Oncotarget

    Article Title: Transmembrane TNF-α promotes activation-induced cell death by forward and reverse signaling

    doi: 10.18632/oncotarget.19124

    Figure Lengend Snippet: The reverse signaling of tmTNF-α enhances AICD (A, C) Jurkat cells were incubated with PHA-P (5 μg/ml), TNF-α pAb (1:100), sTNFR1 or with both PHA-P and TNF-α pAb, or PHA-P and sTNFR1 in indicated concentrations for 24 h. Apoptosis was detected by Annexin V/PI. (B) PHA preactivated primary T cells were stimulated with αCD3 (10 μg/ml), TNF-α pAb, or both for 24 h. Apoptosis was detected by Annexin V/PI. Rabbit serum served as a control. All the quantitative data represent means ± S.D. of at least three independent experiments. ** p

    Article Snippet: Apoptosis detection The apoptosis was evaluated by an Annexin V-FITC Apoptosis Detection Kit (BD biosciences), according to the manufacturer's instructions.

    Techniques: Incubation

    The reverse signaling of tmTNF-α enhances the sensitivity of T cells to TNF-α-induced AICD through upregulating TNFR (A, B, C, D) Jurkat cells were stimulated with PHA-P (5 μg/ml) in the absence or presence of TNF-α pAb (1:100) for 24 h. PHA preactivated T cells were reactivated by αCD3 (10 μg/ml) with or without TNF-α pAb (1:100) for 24 h. The expression of tmTNF-α (A, B) and two types of TNFR (C, D) was detected by flow cytometry. (E, F) Jurkat cells activated for 3 h by PHA-P (5 μg/ml) with or without TNF-α pAb were incubated for 4 h with sTNF-α (50 U/ml) or tmTNF-α stably transfected and fixed NIH3T3 cells at an effector/target ratio of 10:1. The apoptosis was detected by Annexin V/PI. (G, H, I) Jurkat cells transfected with 100 nM siRNA for TNFR1 or TNFR2 for 48 h were activated by PHA-P (5 μg/ml) with TNF-α pAb for 3 h, followed by incubation for 4 h with sTNF-α (50 U/ml) or tmTNF-α stably transfected and fixed NIH3T3 cells at an effector/target ratio of 10:1. The expression of TNFR1 and TNFR2 was analyzed by flow cytometry (G) and the apoptosis was detected by Annexin V/PI (H, I). For neutralization of tmTNF-α, the effector cells were treated with TNF-α pAb for 30 min prior to the addition to the target cells. All the quantitative data represent means ± S.D. of at least three independent experiments. * p

    Journal: Oncotarget

    Article Title: Transmembrane TNF-α promotes activation-induced cell death by forward and reverse signaling

    doi: 10.18632/oncotarget.19124

    Figure Lengend Snippet: The reverse signaling of tmTNF-α enhances the sensitivity of T cells to TNF-α-induced AICD through upregulating TNFR (A, B, C, D) Jurkat cells were stimulated with PHA-P (5 μg/ml) in the absence or presence of TNF-α pAb (1:100) for 24 h. PHA preactivated T cells were reactivated by αCD3 (10 μg/ml) with or without TNF-α pAb (1:100) for 24 h. The expression of tmTNF-α (A, B) and two types of TNFR (C, D) was detected by flow cytometry. (E, F) Jurkat cells activated for 3 h by PHA-P (5 μg/ml) with or without TNF-α pAb were incubated for 4 h with sTNF-α (50 U/ml) or tmTNF-α stably transfected and fixed NIH3T3 cells at an effector/target ratio of 10:1. The apoptosis was detected by Annexin V/PI. (G, H, I) Jurkat cells transfected with 100 nM siRNA for TNFR1 or TNFR2 for 48 h were activated by PHA-P (5 μg/ml) with TNF-α pAb for 3 h, followed by incubation for 4 h with sTNF-α (50 U/ml) or tmTNF-α stably transfected and fixed NIH3T3 cells at an effector/target ratio of 10:1. The expression of TNFR1 and TNFR2 was analyzed by flow cytometry (G) and the apoptosis was detected by Annexin V/PI (H, I). For neutralization of tmTNF-α, the effector cells were treated with TNF-α pAb for 30 min prior to the addition to the target cells. All the quantitative data represent means ± S.D. of at least three independent experiments. * p

    Article Snippet: Apoptosis detection The apoptosis was evaluated by an Annexin V-FITC Apoptosis Detection Kit (BD biosciences), according to the manufacturer's instructions.

    Techniques: Expressing, Flow Cytometry, Cytometry, Incubation, Stable Transfection, Transfection, Neutralization

    Elevation of tmTNF-α expression in AICD (A) Jurkat cells were activated with PHA-P for 24 h in indicated doses and the apoptosis was evaluated by Annexin V/PI. (B, C, D) Jurkat cells were stimulated with 5 μg/ml PHA for indicated time points. The apoptosis (B) and tmTNF-α expression (C) were analyzed by flow cytometry. The kinetic curves of apoptosis rate and tmTNF-α expression were shown in the panel D. (E, F, G) Primary human T cells were preactivated by PHA (5 μg/ml) for 3 days and cultured in the presence of IL-2 (50 U/ml) for 7 days. The preactivated T cells were restimulated for 24 h with plate-coated αCD3 (OKT3, 10 μg/ml). The apoptosis was measured by Annexin V/PI (E, left) and its quantitative analysis was shown in the histogram (E, right). Hochest/PI double staining in situ for apoptosis (F). The tmTNF-α expression was determined by flow cytometry (G, left) and its quantitative analysis was shown in the histogram (G, right). All the quantitative data represent means ± S.D. of at least three independent experiments. * p

    Journal: Oncotarget

    Article Title: Transmembrane TNF-α promotes activation-induced cell death by forward and reverse signaling

    doi: 10.18632/oncotarget.19124

    Figure Lengend Snippet: Elevation of tmTNF-α expression in AICD (A) Jurkat cells were activated with PHA-P for 24 h in indicated doses and the apoptosis was evaluated by Annexin V/PI. (B, C, D) Jurkat cells were stimulated with 5 μg/ml PHA for indicated time points. The apoptosis (B) and tmTNF-α expression (C) were analyzed by flow cytometry. The kinetic curves of apoptosis rate and tmTNF-α expression were shown in the panel D. (E, F, G) Primary human T cells were preactivated by PHA (5 μg/ml) for 3 days and cultured in the presence of IL-2 (50 U/ml) for 7 days. The preactivated T cells were restimulated for 24 h with plate-coated αCD3 (OKT3, 10 μg/ml). The apoptosis was measured by Annexin V/PI (E, left) and its quantitative analysis was shown in the histogram (E, right). Hochest/PI double staining in situ for apoptosis (F). The tmTNF-α expression was determined by flow cytometry (G, left) and its quantitative analysis was shown in the histogram (G, right). All the quantitative data represent means ± S.D. of at least three independent experiments. * p

    Article Snippet: Apoptosis detection The apoptosis was evaluated by an Annexin V-FITC Apoptosis Detection Kit (BD biosciences), according to the manufacturer's instructions.

    Techniques: Expressing, Flow Cytometry, Cytometry, Cell Culture, Double Staining, In Situ

    Inhibitors of other K + channels have minimal effect on TRAIL-induced apoptosis. A375 cells were treated with TRAIL and U37883A (U37), α-dendrotoxin (DTX), charybdotoxin (CTX) and 5-hydroxydecanoate (5-HD) alone or in combination for 24 h, and apoptotic cell death was measured by flow cytometry using annexin V-FITC and PI staining. The data are representative of three independent experiments.

    Journal: International Journal of Oncology

    Article Title: Depolarization potentiates TRAIL-induced apoptosis in human melanoma cells: Role for ATP-sensitive K+ channels and endoplasmic reticulum stress

    doi: 10.3892/ijo.2012.1483

    Figure Lengend Snippet: Inhibitors of other K + channels have minimal effect on TRAIL-induced apoptosis. A375 cells were treated with TRAIL and U37883A (U37), α-dendrotoxin (DTX), charybdotoxin (CTX) and 5-hydroxydecanoate (5-HD) alone or in combination for 24 h, and apoptotic cell death was measured by flow cytometry using annexin V-FITC and PI staining. The data are representative of three independent experiments.

    Article Snippet: Subsequently, the cells were stained with FITC-conjugated annexin V and PI using a commercially available kit (Annexin V FITC Apoptosis Detection Kit I; BD Biosciences) to label dead or damaged cells.

    Techniques: Flow Cytometry, Cytometry, Staining

    TRAIL induces membrane depolarization during apoptosis in human melanoma cells. (A) Human A375 melanoma cells were treated with TRAIL at the indicated concentrations for 24 h, stained with annexin V-FITC and PI, and analyzed by flow cytometry. Annexin V + cells were considered to be apoptotic cells. The data represent the means ± SE from four independent experiments. ** P

    Journal: International Journal of Oncology

    Article Title: Depolarization potentiates TRAIL-induced apoptosis in human melanoma cells: Role for ATP-sensitive K+ channels and endoplasmic reticulum stress

    doi: 10.3892/ijo.2012.1483

    Figure Lengend Snippet: TRAIL induces membrane depolarization during apoptosis in human melanoma cells. (A) Human A375 melanoma cells were treated with TRAIL at the indicated concentrations for 24 h, stained with annexin V-FITC and PI, and analyzed by flow cytometry. Annexin V + cells were considered to be apoptotic cells. The data represent the means ± SE from four independent experiments. ** P

    Article Snippet: Subsequently, the cells were stained with FITC-conjugated annexin V and PI using a commercially available kit (Annexin V FITC Apoptosis Detection Kit I; BD Biosciences) to label dead or damaged cells.

    Techniques: Staining, Flow Cytometry, Cytometry

    K ATP channel inhibitors sensitize human melanoma cells to TRAIL-induced apoptosis. (A–D) A375 (A), A2058 (B) and SK-MEL-2 (C,D) cells were treated with TRAIL and U37883A (U37) or glibenclamide (GLB) alone or in combination for 24 h, stained with calcein-AM and ethidium bromide homodimer and observed under a fluorescence microscope (×100). The results shown are representative of three independent experiments. (E,F) After treatment of A375 cells with TRAIL and U37 or GLB alone or in combination for 24 h (E) or 72 h (F), apoptotic cell death was evaluated by flow cytometry using annexin V-FITC and PI staining. The data represent the means ± SE from four independent experiments. * P

    Journal: International Journal of Oncology

    Article Title: Depolarization potentiates TRAIL-induced apoptosis in human melanoma cells: Role for ATP-sensitive K+ channels and endoplasmic reticulum stress

    doi: 10.3892/ijo.2012.1483

    Figure Lengend Snippet: K ATP channel inhibitors sensitize human melanoma cells to TRAIL-induced apoptosis. (A–D) A375 (A), A2058 (B) and SK-MEL-2 (C,D) cells were treated with TRAIL and U37883A (U37) or glibenclamide (GLB) alone or in combination for 24 h, stained with calcein-AM and ethidium bromide homodimer and observed under a fluorescence microscope (×100). The results shown are representative of three independent experiments. (E,F) After treatment of A375 cells with TRAIL and U37 or GLB alone or in combination for 24 h (E) or 72 h (F), apoptotic cell death was evaluated by flow cytometry using annexin V-FITC and PI staining. The data represent the means ± SE from four independent experiments. * P

    Article Snippet: Subsequently, the cells were stained with FITC-conjugated annexin V and PI using a commercially available kit (Annexin V FITC Apoptosis Detection Kit I; BD Biosciences) to label dead or damaged cells.

    Techniques: Staining, Fluorescence, Microscopy, Flow Cytometry, Cytometry

    The intrinsic apoptotic pathway is involved in, but is not sufficient for, the sensitization to TRAIL-induced apoptosis in melanoma cells. (A,B) A375 cells were treated with TRAIL and U37883A (U37), glibenclamide (GLB) or KCl alone or in combination for 24 h. Caspase-3/7 activation and ΔΨ m depolarization were determined by flow cytometry. The data are representative of three independent experiments. (C) A375 cells were treated with 25 ng/ml TRAIL and KCl alone or in combination in the presence or absence of z-VAD-fmk (VAD), z-DEVD-fmk (DEVD) or z-ATAD-fmk (ATAD) for 24 h and apoptotic cells were evaluated by flow cytometry using annexin V-FITC and PI staining. The data represent the means ± SE from five independent experiments. * P

    Journal: International Journal of Oncology

    Article Title: Depolarization potentiates TRAIL-induced apoptosis in human melanoma cells: Role for ATP-sensitive K+ channels and endoplasmic reticulum stress

    doi: 10.3892/ijo.2012.1483

    Figure Lengend Snippet: The intrinsic apoptotic pathway is involved in, but is not sufficient for, the sensitization to TRAIL-induced apoptosis in melanoma cells. (A,B) A375 cells were treated with TRAIL and U37883A (U37), glibenclamide (GLB) or KCl alone or in combination for 24 h. Caspase-3/7 activation and ΔΨ m depolarization were determined by flow cytometry. The data are representative of three independent experiments. (C) A375 cells were treated with 25 ng/ml TRAIL and KCl alone or in combination in the presence or absence of z-VAD-fmk (VAD), z-DEVD-fmk (DEVD) or z-ATAD-fmk (ATAD) for 24 h and apoptotic cells were evaluated by flow cytometry using annexin V-FITC and PI staining. The data represent the means ± SE from five independent experiments. * P

    Article Snippet: Subsequently, the cells were stained with FITC-conjugated annexin V and PI using a commercially available kit (Annexin V FITC Apoptosis Detection Kit I; BD Biosciences) to label dead or damaged cells.

    Techniques: Activation Assay, Flow Cytometry, Cytometry, Staining

    K + loading sensitizes human melanoma cells to TRAIL-induced apoptosis. (A–C) A375 (A), SK-MEL-2 (B) and A2058 (C) cells were treated with 25 ng/ml TRAIL and 50 mM KCl alone or in combination for 24 h. After removal of the medium, the cells were stained with calcein-AM and ethidium bromide homodimer to label live cells (green) and dead cells with compromised cell membranes (red), respectively. Images were obtained with a fluorescence microscope (×100). The results shown are representative of four independent experiments. (D) After treatment with 6.3 and 25 ng/ml TRAIL and KCl alone or in combination for 24 h, A375 cells were stained with annexin V-FITC and PI and analyzed by flow cytometry. The data represent means ± SE from four independent experiments. * P

    Journal: International Journal of Oncology

    Article Title: Depolarization potentiates TRAIL-induced apoptosis in human melanoma cells: Role for ATP-sensitive K+ channels and endoplasmic reticulum stress

    doi: 10.3892/ijo.2012.1483

    Figure Lengend Snippet: K + loading sensitizes human melanoma cells to TRAIL-induced apoptosis. (A–C) A375 (A), SK-MEL-2 (B) and A2058 (C) cells were treated with 25 ng/ml TRAIL and 50 mM KCl alone or in combination for 24 h. After removal of the medium, the cells were stained with calcein-AM and ethidium bromide homodimer to label live cells (green) and dead cells with compromised cell membranes (red), respectively. Images were obtained with a fluorescence microscope (×100). The results shown are representative of four independent experiments. (D) After treatment with 6.3 and 25 ng/ml TRAIL and KCl alone or in combination for 24 h, A375 cells were stained with annexin V-FITC and PI and analyzed by flow cytometry. The data represent means ± SE from four independent experiments. * P

    Article Snippet: Subsequently, the cells were stained with FITC-conjugated annexin V and PI using a commercially available kit (Annexin V FITC Apoptosis Detection Kit I; BD Biosciences) to label dead or damaged cells.

    Techniques: Staining, Fluorescence, Microscopy, Flow Cytometry, Cytometry

    Concurrent suppression of PKC α and β is essential for the induction of PKC δ-mediated apoptosis. (a) After being transfected a wild-type PKCδ ( WT-PKCδ ) or constitutively-active PKC δ ( CAT-PKCδ ), NIH3T3/ Hras cells were lysed and then subjected to immunoblotting with anti-PKC δ antibody. β-actin was used as a loading control. (b) After introduced PKC δ or CAT-PKC δ , NIH3T3/ Hras cells, in the presence or absence of PKC α and β, were subjected to Annexin V-FITC analysis. The percentages of apoptotic cells were plotted. Error bars represent SD over 3 independent experiments (n = 3, *, p

    Journal: Oncogene

    Article Title: Roles of PKC Isoforms in the Induction of Apoptosis Elicited by Aberrant Ras

    doi: 10.1038/onc.2009.344

    Figure Lengend Snippet: Concurrent suppression of PKC α and β is essential for the induction of PKC δ-mediated apoptosis. (a) After being transfected a wild-type PKCδ ( WT-PKCδ ) or constitutively-active PKC δ ( CAT-PKCδ ), NIH3T3/ Hras cells were lysed and then subjected to immunoblotting with anti-PKC δ antibody. β-actin was used as a loading control. (b) After introduced PKC δ or CAT-PKC δ , NIH3T3/ Hras cells, in the presence or absence of PKC α and β, were subjected to Annexin V-FITC analysis. The percentages of apoptotic cells were plotted. Error bars represent SD over 3 independent experiments (n = 3, *, p

    Article Snippet: After treatments, cells were prepared and stained with Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences) according to manufacturer’s instructions.

    Techniques: Transfection

    Concurrent knockdown of PKC α and β by the shRNAs triggers apoptosis in cells expressing v-ras . (a) After being infected with shRNA-PKC α , β , or scrambled shRNA s, NIH3T3 and NIH3T3/ Hras cells were lysed and subjected to immunoblotting (left panels). The relative expression level of PKC α and β was measured and plotted (right panel). The error bars are SD of 3 independent experiments. (b) After being infected with shRNA-PKCα, β or the combination of both, NIH3T3 or NIH3T3/ Hras cells were subjected to DNA fragmentation analysis (left panel). The percentage of the cells with sub-G 1 DNA content was then plotted. Annexin V-FITC analysis was also performed after infecting cells with the shRNAs (right panel). The percentage of the cells stained positively with AnnexinV-FITC was plotted accordingly. The error bars are SD over 5 independent experiments (n = 5, *, p

    Journal: Oncogene

    Article Title: Roles of PKC Isoforms in the Induction of Apoptosis Elicited by Aberrant Ras

    doi: 10.1038/onc.2009.344

    Figure Lengend Snippet: Concurrent knockdown of PKC α and β by the shRNAs triggers apoptosis in cells expressing v-ras . (a) After being infected with shRNA-PKC α , β , or scrambled shRNA s, NIH3T3 and NIH3T3/ Hras cells were lysed and subjected to immunoblotting (left panels). The relative expression level of PKC α and β was measured and plotted (right panel). The error bars are SD of 3 independent experiments. (b) After being infected with shRNA-PKCα, β or the combination of both, NIH3T3 or NIH3T3/ Hras cells were subjected to DNA fragmentation analysis (left panel). The percentage of the cells with sub-G 1 DNA content was then plotted. Annexin V-FITC analysis was also performed after infecting cells with the shRNAs (right panel). The percentage of the cells stained positively with AnnexinV-FITC was plotted accordingly. The error bars are SD over 5 independent experiments (n = 5, *, p

    Article Snippet: After treatments, cells were prepared and stained with Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences) according to manufacturer’s instructions.

    Techniques: Expressing, Infection, shRNA, Staining

    Nuclear p73 is phosphorylated and required for the induction of apoptosis. (a) The nuclear fractions from NIH3T3 and NIH3T3/ Hras cells with or without co-knocking down PKC α and β were prepared and immunoprecipitated with anti-p73 antibody. The immuno-complexes were then subjected to immunoblotting with the anti-phospho-serine antibody. The input was judged by probing the same amount of immunoprecipitates with anti-p73 antibody. (b) After being infected with shRNAs (shRNA-PKC α, β plus p73 ) or sc shRNAs (mixed three scrambled shRNAs ), NIH3T3/ Hras cells were lysed and subjected to immunoblotting with corresponding antibodies. The relative expression level of proteins was normalized by β-actin and presented as n-fold. (c) After triple-infection, NIH3T3/ Hras cells were subjected to Annexin V-FITC assay and the apoptotic cell population was plotted. Error bars represent SD from 5 independent experiments (n = 5, *, p

    Journal: Oncogene

    Article Title: Roles of PKC Isoforms in the Induction of Apoptosis Elicited by Aberrant Ras

    doi: 10.1038/onc.2009.344

    Figure Lengend Snippet: Nuclear p73 is phosphorylated and required for the induction of apoptosis. (a) The nuclear fractions from NIH3T3 and NIH3T3/ Hras cells with or without co-knocking down PKC α and β were prepared and immunoprecipitated with anti-p73 antibody. The immuno-complexes were then subjected to immunoblotting with the anti-phospho-serine antibody. The input was judged by probing the same amount of immunoprecipitates with anti-p73 antibody. (b) After being infected with shRNAs (shRNA-PKC α, β plus p73 ) or sc shRNAs (mixed three scrambled shRNAs ), NIH3T3/ Hras cells were lysed and subjected to immunoblotting with corresponding antibodies. The relative expression level of proteins was normalized by β-actin and presented as n-fold. (c) After triple-infection, NIH3T3/ Hras cells were subjected to Annexin V-FITC assay and the apoptotic cell population was plotted. Error bars represent SD from 5 independent experiments (n = 5, *, p

    Article Snippet: After treatments, cells were prepared and stained with Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences) according to manufacturer’s instructions.

    Techniques: Immunoprecipitation, Infection, shRNA, Expressing

    Induction of apoptosis in NIH3T3/ Hras cells after suppression of PKC. (a) Ras expression and activation status in NIH3T3 and NIH3T3/ Hras cells. Left panels: the cells, with or without transiently co-infected with shRNA-PKCα and β or scrambled shRNAs for 48 h, were lysed and immunoblotted with anti-Ras antibody. The relative expression level of proteins was normalized by β-actin and presented as n-fold. Right panel: cell lysates were also assayed for Ras activity. GTPγS-treated lysate was a positive control. (b) Cells were treated with PKC inhibitor GO6976 (1 μM) or DMSO (control) for 48 h and then assayed by DNA fragmentation and Annexin V-FITC assays. Subsequently, the percentages of the cells with sub-G 1 DNA content or stained positively with AnnexinV-FITC were plotted. The error bars are SD (standard deviation) of 5 independent experiments (n = 5, *, p

    Journal: Oncogene

    Article Title: Roles of PKC Isoforms in the Induction of Apoptosis Elicited by Aberrant Ras

    doi: 10.1038/onc.2009.344

    Figure Lengend Snippet: Induction of apoptosis in NIH3T3/ Hras cells after suppression of PKC. (a) Ras expression and activation status in NIH3T3 and NIH3T3/ Hras cells. Left panels: the cells, with or without transiently co-infected with shRNA-PKCα and β or scrambled shRNAs for 48 h, were lysed and immunoblotted with anti-Ras antibody. The relative expression level of proteins was normalized by β-actin and presented as n-fold. Right panel: cell lysates were also assayed for Ras activity. GTPγS-treated lysate was a positive control. (b) Cells were treated with PKC inhibitor GO6976 (1 μM) or DMSO (control) for 48 h and then assayed by DNA fragmentation and Annexin V-FITC assays. Subsequently, the percentages of the cells with sub-G 1 DNA content or stained positively with AnnexinV-FITC were plotted. The error bars are SD (standard deviation) of 5 independent experiments (n = 5, *, p

    Article Snippet: After treatments, cells were prepared and stained with Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences) according to manufacturer’s instructions.

    Techniques: Expressing, Activation Assay, Infection, shRNA, Activity Assay, Positive Control, Staining, Standard Deviation

    XPD is required for triplex-induced apoptosis. ( A ) Monolayer growth studies demonstrate that XPD-deficient cells are resistant to triplex-induced decrease in cell growth. ( B ) Knockdown of XPD results in significant reduction of induced apoptosis as measured by Annexin V staining (*** P

    Journal: Nucleic Acids Research

    Article Title: XPD-dependent activation of apoptosis in response to triplex-induced DNA damage

    doi: 10.1093/nar/gkt670

    Figure Lengend Snippet: XPD is required for triplex-induced apoptosis. ( A ) Monolayer growth studies demonstrate that XPD-deficient cells are resistant to triplex-induced decrease in cell growth. ( B ) Knockdown of XPD results in significant reduction of induced apoptosis as measured by Annexin V staining (*** P

    Article Snippet: Apoptosis analysis Cells were analyzed by flow cytometry 24 h posttreatment using the Annexin V-FITC/PI apoptosis detection kit (BD Pharmingen) according to the manufacturer's protocol.

    Techniques: Staining

    Role of XPA in the activation of triplex-induced apoptosis. ( A ) Analysis of γH2AX expression levels as a measure of triplex-induced DSBs in XPA-proficient and XPA-deficient cells 24 h post AG30 treatment. ( B ) Detection of Annexin V binding indicates an increase in apoptotic cell death in the absence of XPA 24 h posttreatment with 2 μg of oligonucleotides. ( C ) Western blot analysis of caspase-mediated cleavage of PARP as a measure of triplex-induced apoptosis.

    Journal: Nucleic Acids Research

    Article Title: XPD-dependent activation of apoptosis in response to triplex-induced DNA damage

    doi: 10.1093/nar/gkt670

    Figure Lengend Snippet: Role of XPA in the activation of triplex-induced apoptosis. ( A ) Analysis of γH2AX expression levels as a measure of triplex-induced DSBs in XPA-proficient and XPA-deficient cells 24 h post AG30 treatment. ( B ) Detection of Annexin V binding indicates an increase in apoptotic cell death in the absence of XPA 24 h posttreatment with 2 μg of oligonucleotides. ( C ) Western blot analysis of caspase-mediated cleavage of PARP as a measure of triplex-induced apoptosis.

    Article Snippet: Apoptosis analysis Cells were analyzed by flow cytometry 24 h posttreatment using the Annexin V-FITC/PI apoptosis detection kit (BD Pharmingen) according to the manufacturer's protocol.

    Techniques: Activation Assay, Expressing, Binding Assay, Western Blot

    Activation of apoptosis minimizes triplex-induced genomic instability. ( A ) Western blot analysis of H2AX phosphorylation at serine 139 and tyrosine 142 in XPA-proficient and -deficient cells 24 h following AG30 treatment. ( B ) Analysis of triplex-induced apoptosis in XPD+/+ and XPD−/− cells as measured by Annexin V staining. ( C ) Western blot analysis of the phosphorylation status of H2AX at serine 139 and tyrosine 142 in XPD-proficient and -deficient cell 24 h following TFO treatment. ( D and E ) Quantification of the relative S139 and Y142 phosphorylation levels in XPD-deficient cells compared with XPD-proficient cells in response to triplex-induced DNA DSBs. ( F ) Schematic of XPD-dependent triplex-induced apoptosis. ( G ) Triplex-induced genomic instability as determined by mutation frequencies in the supFG1 reporter gene in XPD +/+ and XPD −/− cells treated with TFOs. The frequency of mutations was calculated by dividing the number of colorless mutant plaques by the total number of plaques counted. Each experiment was performed in triplicate, and the standard errors were calculated for the mutation frequency. (mean ± SEM, n = 3).

    Journal: Nucleic Acids Research

    Article Title: XPD-dependent activation of apoptosis in response to triplex-induced DNA damage

    doi: 10.1093/nar/gkt670

    Figure Lengend Snippet: Activation of apoptosis minimizes triplex-induced genomic instability. ( A ) Western blot analysis of H2AX phosphorylation at serine 139 and tyrosine 142 in XPA-proficient and -deficient cells 24 h following AG30 treatment. ( B ) Analysis of triplex-induced apoptosis in XPD+/+ and XPD−/− cells as measured by Annexin V staining. ( C ) Western blot analysis of the phosphorylation status of H2AX at serine 139 and tyrosine 142 in XPD-proficient and -deficient cell 24 h following TFO treatment. ( D and E ) Quantification of the relative S139 and Y142 phosphorylation levels in XPD-deficient cells compared with XPD-proficient cells in response to triplex-induced DNA DSBs. ( F ) Schematic of XPD-dependent triplex-induced apoptosis. ( G ) Triplex-induced genomic instability as determined by mutation frequencies in the supFG1 reporter gene in XPD +/+ and XPD −/− cells treated with TFOs. The frequency of mutations was calculated by dividing the number of colorless mutant plaques by the total number of plaques counted. Each experiment was performed in triplicate, and the standard errors were calculated for the mutation frequency. (mean ± SEM, n = 3).

    Article Snippet: Apoptosis analysis Cells were analyzed by flow cytometry 24 h posttreatment using the Annexin V-FITC/PI apoptosis detection kit (BD Pharmingen) according to the manufacturer's protocol.

    Techniques: Activation Assay, Western Blot, Staining, Mutagenesis

    Induction of apoptosis via formation of triplex structures in AV16 cells. ( A ) Annexin V binding to exposed phosphatidylserine residues 24 h after treatment with 2 μg of oligonucleotides. ( B ) Western blot analysis of caspase-mediated cleavage of PARP. Cells were collected and lysates prepared 24 h posttreatment. ( C ) Activation of apoptosis can be attributed to triplex formation rather than G-quadruplex formation as determined by Western blot analysis of cleaved PARP following 2 μg treatment with AG30 or A8 30. ( D ) Time course of induced apoptosis by Western blot analysis of cleaved PARP. ( E ) Dose response of increasing concentrations of AG30 and its effect on apoptotic cell death 48 h posttreatment. ( F ) Clonogenic survival after 48 h exposure of AV16 cells to AG30 or MIX30.

    Journal: Nucleic Acids Research

    Article Title: XPD-dependent activation of apoptosis in response to triplex-induced DNA damage

    doi: 10.1093/nar/gkt670

    Figure Lengend Snippet: Induction of apoptosis via formation of triplex structures in AV16 cells. ( A ) Annexin V binding to exposed phosphatidylserine residues 24 h after treatment with 2 μg of oligonucleotides. ( B ) Western blot analysis of caspase-mediated cleavage of PARP. Cells were collected and lysates prepared 24 h posttreatment. ( C ) Activation of apoptosis can be attributed to triplex formation rather than G-quadruplex formation as determined by Western blot analysis of cleaved PARP following 2 μg treatment with AG30 or A8 30. ( D ) Time course of induced apoptosis by Western blot analysis of cleaved PARP. ( E ) Dose response of increasing concentrations of AG30 and its effect on apoptotic cell death 48 h posttreatment. ( F ) Clonogenic survival after 48 h exposure of AV16 cells to AG30 or MIX30.

    Article Snippet: Apoptosis analysis Cells were analyzed by flow cytometry 24 h posttreatment using the Annexin V-FITC/PI apoptosis detection kit (BD Pharmingen) according to the manufacturer's protocol.

    Techniques: Binding Assay, Western Blot, Activation Assay

    Mitofilin overexpression in rat H9c2 myoblasts reduces cell death. A , top : flow cytometry with unstained (blank, purple cells), Annexin V only (early apoptosis, orange cells), and 7-AAD only (dead cells and/or late apoptosis, green cells) used for calibration.

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Mitochondrial inner membrane protein (mitofilin) knockdown induces cell death by apoptosis via an AIF-PARP-dependent mechanism and cell cycle arrest

    doi: 10.1152/ajpcell.00230.2017

    Figure Lengend Snippet: Mitofilin overexpression in rat H9c2 myoblasts reduces cell death. A , top : flow cytometry with unstained (blank, purple cells), Annexin V only (early apoptosis, orange cells), and 7-AAD only (dead cells and/or late apoptosis, green cells) used for calibration.

    Article Snippet: Cell apoptosis was determined using Annexin V-FITC/PI Apoptosis Detection Kit (BD Bioscience, BD PharMingen, catalog no. 556547) according to the manufacturer's instructions.

    Techniques: Over Expression, Flow Cytometry, Cytometry

    Mitofilin knockdown in human embryonic kidney (HEK) 293 cells increases cell death. A , top : flow cytometry with unstained (blank, purple cells), Annexin V only (early apoptosis, brown cells), and 7-aminoactinomycin-D (7-AAD) only (dead cells and/or later

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Mitochondrial inner membrane protein (mitofilin) knockdown induces cell death by apoptosis via an AIF-PARP-dependent mechanism and cell cycle arrest

    doi: 10.1152/ajpcell.00230.2017

    Figure Lengend Snippet: Mitofilin knockdown in human embryonic kidney (HEK) 293 cells increases cell death. A , top : flow cytometry with unstained (blank, purple cells), Annexin V only (early apoptosis, brown cells), and 7-aminoactinomycin-D (7-AAD) only (dead cells and/or later

    Article Snippet: Cell apoptosis was determined using Annexin V-FITC/PI Apoptosis Detection Kit (BD Bioscience, BD PharMingen, catalog no. 556547) according to the manufacturer's instructions.

    Techniques: Flow Cytometry, Cytometry

    Knockdown of mitofilin in rat H9c2 myoblasts increases cell apoptosis and reduces viability. A , top : flow cytometry with unstained (blank), Annexin V only (early apoptosis, pink cells), and propidium iodide (PI) only (dead cells and/or late apoptosis,

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Mitochondrial inner membrane protein (mitofilin) knockdown induces cell death by apoptosis via an AIF-PARP-dependent mechanism and cell cycle arrest

    doi: 10.1152/ajpcell.00230.2017

    Figure Lengend Snippet: Knockdown of mitofilin in rat H9c2 myoblasts increases cell apoptosis and reduces viability. A , top : flow cytometry with unstained (blank), Annexin V only (early apoptosis, pink cells), and propidium iodide (PI) only (dead cells and/or late apoptosis,

    Article Snippet: Cell apoptosis was determined using Annexin V-FITC/PI Apoptosis Detection Kit (BD Bioscience, BD PharMingen, catalog no. 556547) according to the manufacturer's instructions.

    Techniques: Flow Cytometry, Cytometry

    Curcumin alone or combined with irinotecan induces cell apoptosis through ER stress-mediated CHOP expression in human CRC cell lines ( A , B ) The inhibition efficiency of siRNAs against CHOP. LoVo cells (A) or HT-29 cells (B) were transfected with siRNAs targeting CHOP, followed by treatments with curcumin alone or with irinotecan for 24 h, and the protein levels of CHOP was assessed by western blotting. ( C , D ) The effects of CHOP siRNA on cell apoptosis induced by curcumin alone or with irinotecan. LoVo cells (C) or HT-29 cells (D) were treated as described above, and then cell apoptosis was measured by Annexin V-FITC/PI staining. Values are means ± SEM. *

    Journal: Oncotarget

    Article Title: Curcumin enhances the effects of irinotecan on colorectal cancer cells through the generation of reactive oxygen species and activation of the endoplasmic reticulum stress pathway

    doi: 10.18632/oncotarget.16828

    Figure Lengend Snippet: Curcumin alone or combined with irinotecan induces cell apoptosis through ER stress-mediated CHOP expression in human CRC cell lines ( A , B ) The inhibition efficiency of siRNAs against CHOP. LoVo cells (A) or HT-29 cells (B) were transfected with siRNAs targeting CHOP, followed by treatments with curcumin alone or with irinotecan for 24 h, and the protein levels of CHOP was assessed by western blotting. ( C , D ) The effects of CHOP siRNA on cell apoptosis induced by curcumin alone or with irinotecan. LoVo cells (C) or HT-29 cells (D) were treated as described above, and then cell apoptosis was measured by Annexin V-FITC/PI staining. Values are means ± SEM. *

    Article Snippet: Quantification of apoptotic cells Annexin V/P staining was used to quantify the effect of irinotecan, curcumin and their combination on apoptosis with Annexin V-FITC Apoptosis Detection kit (KeyGEN, Nanjing, China).

    Techniques: Expressing, Inhibition, Transfection, Western Blot, Staining

    Effects of curcumin and/or irinotecan on intracellular calcium, apoptosis, and cell cycle arrest in human CRC cell lines ( A , B ) LoVo cells or HT-29 cells were treated with curcumin and/or irinotecan at the optimal concentrations for 24 h, and then cells were stained with Fluo-3/AM probes and analyzed by flow cytometry. ( C ) Graphs show the geometric mean values of fluorescence intensity. ( D ) LoVo cells or HT-29 cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. ( E , F ) The number of cells in G2/M phase was determined via flow cytometry. ( G , H ) Distribution of MCF 7 cells in various phases of the cell cycle. Values are means ± SEM. **

    Journal: Oncotarget

    Article Title: Curcumin enhances the effects of irinotecan on colorectal cancer cells through the generation of reactive oxygen species and activation of the endoplasmic reticulum stress pathway

    doi: 10.18632/oncotarget.16828

    Figure Lengend Snippet: Effects of curcumin and/or irinotecan on intracellular calcium, apoptosis, and cell cycle arrest in human CRC cell lines ( A , B ) LoVo cells or HT-29 cells were treated with curcumin and/or irinotecan at the optimal concentrations for 24 h, and then cells were stained with Fluo-3/AM probes and analyzed by flow cytometry. ( C ) Graphs show the geometric mean values of fluorescence intensity. ( D ) LoVo cells or HT-29 cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. ( E , F ) The number of cells in G2/M phase was determined via flow cytometry. ( G , H ) Distribution of MCF 7 cells in various phases of the cell cycle. Values are means ± SEM. **

    Article Snippet: Quantification of apoptotic cells Annexin V/P staining was used to quantify the effect of irinotecan, curcumin and their combination on apoptosis with Annexin V-FITC Apoptosis Detection kit (KeyGEN, Nanjing, China).

    Techniques: Staining, Flow Cytometry, Cytometry, Fluorescence

    Anti-colorectal cancer effects of curcumin and/or irinotecan are dependent on ROS ( A , B ) The effects of NAC on cell growth inhibition induced by curcumin and/or irinotecan. After pretreatment with 5 mM NAC for 2 h, LoVo cells (A) or HT-29 cells (B) were treated with curcumin and/or irinotecan for 24 h, then cell viability was assessed by CCK-8 assay. ( C , D ) The effects of NAC on apoptosis induced by curcumin and/or irinotecan. After cells were treated as described above, cell apoptosis was measured by Annexin V-FITC/PI staining. Values are means ± SEM. *

    Journal: Oncotarget

    Article Title: Curcumin enhances the effects of irinotecan on colorectal cancer cells through the generation of reactive oxygen species and activation of the endoplasmic reticulum stress pathway

    doi: 10.18632/oncotarget.16828

    Figure Lengend Snippet: Anti-colorectal cancer effects of curcumin and/or irinotecan are dependent on ROS ( A , B ) The effects of NAC on cell growth inhibition induced by curcumin and/or irinotecan. After pretreatment with 5 mM NAC for 2 h, LoVo cells (A) or HT-29 cells (B) were treated with curcumin and/or irinotecan for 24 h, then cell viability was assessed by CCK-8 assay. ( C , D ) The effects of NAC on apoptosis induced by curcumin and/or irinotecan. After cells were treated as described above, cell apoptosis was measured by Annexin V-FITC/PI staining. Values are means ± SEM. *

    Article Snippet: Quantification of apoptotic cells Annexin V/P staining was used to quantify the effect of irinotecan, curcumin and their combination on apoptosis with Annexin V-FITC Apoptosis Detection kit (KeyGEN, Nanjing, China).

    Techniques: Inhibition, CCK-8 Assay, Staining

    ER Stress is mediates the anti-colorectal cancer effects of curcumin alone or combined with irinotecan ( A , B ) The effects of an ER stress inhibitor on cell growth inhibition induced by curcumin alone or with irinotecan. After pretreatment with 0.1 μM mithramycin (MTM) for 30 min, LoVo cells (A) or HT-29 cells (B) were treated with curcumin alone or with irinotecan for 24 h, then cell viability was assessed by CCK-8 assay. ( C , D ) The effects of an ER stress inhibitor on apoptosis induced by curcumin alone or with irinotecan. After cells were treated as described above, cell apoptosis was measured by Annexin V-FITC/PI staining. Values are means ± SEM. *

    Journal: Oncotarget

    Article Title: Curcumin enhances the effects of irinotecan on colorectal cancer cells through the generation of reactive oxygen species and activation of the endoplasmic reticulum stress pathway

    doi: 10.18632/oncotarget.16828

    Figure Lengend Snippet: ER Stress is mediates the anti-colorectal cancer effects of curcumin alone or combined with irinotecan ( A , B ) The effects of an ER stress inhibitor on cell growth inhibition induced by curcumin alone or with irinotecan. After pretreatment with 0.1 μM mithramycin (MTM) for 30 min, LoVo cells (A) or HT-29 cells (B) were treated with curcumin alone or with irinotecan for 24 h, then cell viability was assessed by CCK-8 assay. ( C , D ) The effects of an ER stress inhibitor on apoptosis induced by curcumin alone or with irinotecan. After cells were treated as described above, cell apoptosis was measured by Annexin V-FITC/PI staining. Values are means ± SEM. *

    Article Snippet: Quantification of apoptotic cells Annexin V/P staining was used to quantify the effect of irinotecan, curcumin and their combination on apoptosis with Annexin V-FITC Apoptosis Detection kit (KeyGEN, Nanjing, China).

    Techniques: Inhibition, CCK-8 Assay, Staining

    Enhancing TRAIL induced apoptosis through blocking NF-κB pathway is effective in multiple BLCA cell lines. (A) 5637 cells were treated with various concentrations of PDTC for 24-h, cell viability and IC 50 values were measured by MTS assays. (B) Effects of TRAIL and PDTC treatment on 5637 cell apoptosis. 5637 cells were identified by Annexin V-FITC detection after DMSO (Mock), TRAIL (2 ng/mL), or PDTC (20 μM) treatment and combined TRAIL (2 ng/mL) and PDTC (20 μM) treatment for 24-h. (C) Histogram showing the apoptosis rates of 5637 cells. (D) UM-UC 3 cells were treated with various concentrations of PDTC for 24-h, cell viability and IC 50 values were measured by MTS assays. (E) Effects of TRAIL and PDTC treatment on UM-UC 3 cell apoptosis. UM-UC 3 cells were identified by Annexin V-FITC detection after DMSO (control), TRAIL (10 ng/mL), or PDTC (40 μM) treatment and combined TRAIL (10 ng/mL) with PDTC (40 μM) treatment for 24-h. (F) Histogram showing the apoptosis rates of UM-UC 3 cells. Data represent the mean ± SD. *P

    Journal: International Journal of Biological Sciences

    Article Title: Andrographolide Enhances TRAIL-Induced Apoptosis via p53-Mediated Death Receptors Up-Regulation and Suppression of the NF-кB Pathway in Bladder Cancer Cells

    doi: 10.7150/ijbs.30847

    Figure Lengend Snippet: Enhancing TRAIL induced apoptosis through blocking NF-κB pathway is effective in multiple BLCA cell lines. (A) 5637 cells were treated with various concentrations of PDTC for 24-h, cell viability and IC 50 values were measured by MTS assays. (B) Effects of TRAIL and PDTC treatment on 5637 cell apoptosis. 5637 cells were identified by Annexin V-FITC detection after DMSO (Mock), TRAIL (2 ng/mL), or PDTC (20 μM) treatment and combined TRAIL (2 ng/mL) and PDTC (20 μM) treatment for 24-h. (C) Histogram showing the apoptosis rates of 5637 cells. (D) UM-UC 3 cells were treated with various concentrations of PDTC for 24-h, cell viability and IC 50 values were measured by MTS assays. (E) Effects of TRAIL and PDTC treatment on UM-UC 3 cell apoptosis. UM-UC 3 cells were identified by Annexin V-FITC detection after DMSO (control), TRAIL (10 ng/mL), or PDTC (40 μM) treatment and combined TRAIL (10 ng/mL) with PDTC (40 μM) treatment for 24-h. (F) Histogram showing the apoptosis rates of UM-UC 3 cells. Data represent the mean ± SD. *P

    Article Snippet: Cells treated under different conditions for 24-h were resuspended in 6-well culture plates, washed twice with cold PBS, and pelleted by centrifugation at 5000g for 5 min. Apoptotic cells were quantified using an Annexin V-FITC apoptosis detection kit (KeyGEN BioTECH, Nanjing, China).

    Techniques: Blocking Assay

    Andro enhances TRAIL-mediated suppression of cell viability and apoptosis rates in other BLCA cell lines. (A, B) 5637 cells were treated with various concentrations of TRAIL or/and Andro for 24-h, and cell viability and IC 50 values were measured by MTS assays. (C, D) UM-UC 3 cells were treated with various concentrations of TRAIL or/and Andro for 24-h, and cell viability and IC 50 values were measured by MTS assays. (E) Effects of TRAIL and Andro treatment on 5637 and UM-UC 3 cell apoptosis. 5637 cells were identified by Annexin V-FITC detection following DMSO (control), TRAIL (2 ng/mL), or Andro (10 μM) treatment and combined TRAIL (2 ng/mL) and Andro (10 μM) treatment for 24-h. UM-UC 3 cells were identified by Annexin V-FITC detection following DMSO (control), TRAIL (10 ng/mL), or Andro (20 μM) treatment and combined TRAIL (10 ng/mL) with Andro (20 μM) treatment for 24-h. Histogram showing the apoptosis rates of 5637 and UM-UC3 cell lines. Data represent the mean ± SD. *P

    Journal: International Journal of Biological Sciences

    Article Title: Andrographolide Enhances TRAIL-Induced Apoptosis via p53-Mediated Death Receptors Up-Regulation and Suppression of the NF-кB Pathway in Bladder Cancer Cells

    doi: 10.7150/ijbs.30847

    Figure Lengend Snippet: Andro enhances TRAIL-mediated suppression of cell viability and apoptosis rates in other BLCA cell lines. (A, B) 5637 cells were treated with various concentrations of TRAIL or/and Andro for 24-h, and cell viability and IC 50 values were measured by MTS assays. (C, D) UM-UC 3 cells were treated with various concentrations of TRAIL or/and Andro for 24-h, and cell viability and IC 50 values were measured by MTS assays. (E) Effects of TRAIL and Andro treatment on 5637 and UM-UC 3 cell apoptosis. 5637 cells were identified by Annexin V-FITC detection following DMSO (control), TRAIL (2 ng/mL), or Andro (10 μM) treatment and combined TRAIL (2 ng/mL) and Andro (10 μM) treatment for 24-h. UM-UC 3 cells were identified by Annexin V-FITC detection following DMSO (control), TRAIL (10 ng/mL), or Andro (20 μM) treatment and combined TRAIL (10 ng/mL) with Andro (20 μM) treatment for 24-h. Histogram showing the apoptosis rates of 5637 and UM-UC3 cell lines. Data represent the mean ± SD. *P

    Article Snippet: Cells treated under different conditions for 24-h were resuspended in 6-well culture plates, washed twice with cold PBS, and pelleted by centrifugation at 5000g for 5 min. Apoptotic cells were quantified using an Annexin V-FITC apoptosis detection kit (KeyGEN BioTECH, Nanjing, China).

    Techniques:

    Andro sensitizes TRAIL-induced apoptosis but not necrosis in T24 cells. (A) TRAIL combined with Andro treatment inhibited the cell viability of BLCA cell lines. T24 cells were treated with various concentrations of TRAIL and/or Andro [2 (+) or 4 (++) or 8 (+++) μM] for 24-h, and cell viability was examined by MTS assay. (B) Protein expression of hallmarks of apoptosis in T24 cells treated with 2 ng/mL TRAIL or/and 8 µM of Andro for 24-h. (C) Effects of TRAIL and Andro treatment on apoptosis in T24 cell lines. Cells were identified by Annexin V-FITC detection following DMSO (control), TRAIL (2 ng/mL), and/or Andro (8 μM) treatment for 24-h. Histogram showing the apoptosis rates of all cell lines. (D) Images (200×) showing apoptotic cells after treatment under different conditions. Histograms of T24 cells treated with DMSO, TRAIL (2 ng/mL), and/or Andro (8 μM) and Z-AVD-FMK (0.05 μM) or Nec-1 (0.05 μM). Cell viability was determined by MTS assay after 24-h. Data represent the mean ± SD. *P

    Journal: International Journal of Biological Sciences

    Article Title: Andrographolide Enhances TRAIL-Induced Apoptosis via p53-Mediated Death Receptors Up-Regulation and Suppression of the NF-кB Pathway in Bladder Cancer Cells

    doi: 10.7150/ijbs.30847

    Figure Lengend Snippet: Andro sensitizes TRAIL-induced apoptosis but not necrosis in T24 cells. (A) TRAIL combined with Andro treatment inhibited the cell viability of BLCA cell lines. T24 cells were treated with various concentrations of TRAIL and/or Andro [2 (+) or 4 (++) or 8 (+++) μM] for 24-h, and cell viability was examined by MTS assay. (B) Protein expression of hallmarks of apoptosis in T24 cells treated with 2 ng/mL TRAIL or/and 8 µM of Andro for 24-h. (C) Effects of TRAIL and Andro treatment on apoptosis in T24 cell lines. Cells were identified by Annexin V-FITC detection following DMSO (control), TRAIL (2 ng/mL), and/or Andro (8 μM) treatment for 24-h. Histogram showing the apoptosis rates of all cell lines. (D) Images (200×) showing apoptotic cells after treatment under different conditions. Histograms of T24 cells treated with DMSO, TRAIL (2 ng/mL), and/or Andro (8 μM) and Z-AVD-FMK (0.05 μM) or Nec-1 (0.05 μM). Cell viability was determined by MTS assay after 24-h. Data represent the mean ± SD. *P

    Article Snippet: Cells treated under different conditions for 24-h were resuspended in 6-well culture plates, washed twice with cold PBS, and pelleted by centrifugation at 5000g for 5 min. Apoptotic cells were quantified using an Annexin V-FITC apoptosis detection kit (KeyGEN BioTECH, Nanjing, China).

    Techniques: MTS Assay, Expressing

    Combination treatment with TRAIL and Andro enhances cell apoptosis rates through inhibiting the NF-кB pathway in T24 cells. (A, B) T24 cells were treated with 2 ng/mL TRAIL and/or 8 µM Andro for 24-h, RelA/cIAP2 protein levels and its quantification were assessed by immunoblot. (C) Histograms showing the relative mRNA levels of RelA , Bcl-2 , and XIAP in T24 cells treated with TRAIL or/and Andro. (D) T24 cells were treated with PDTC at various concentrations for 24-h, and cell viability and IC 50 values were measured using MTS assays. (E) T24 cells were treated with 2 ng/mL TRAIL or 5 μM PDTC or 8 μM Andro or with a combination of TRAIL and Andro/PDTC for 24-h. Cell viability was measured by MTS assay. (F, G) Cell apoptosis rates of T24 cells according to Annexin V-FITC detection. T24 cells were treated with 2 ng/mL TRAIL or 5 μM PDTC or 8 μM Andro or with a combination of TRAIL and Andro/PDTC for 24-h. Histograms showing the apoptosis rates. Data represent the mean ± SD. *P

    Journal: International Journal of Biological Sciences

    Article Title: Andrographolide Enhances TRAIL-Induced Apoptosis via p53-Mediated Death Receptors Up-Regulation and Suppression of the NF-кB Pathway in Bladder Cancer Cells

    doi: 10.7150/ijbs.30847

    Figure Lengend Snippet: Combination treatment with TRAIL and Andro enhances cell apoptosis rates through inhibiting the NF-кB pathway in T24 cells. (A, B) T24 cells were treated with 2 ng/mL TRAIL and/or 8 µM Andro for 24-h, RelA/cIAP2 protein levels and its quantification were assessed by immunoblot. (C) Histograms showing the relative mRNA levels of RelA , Bcl-2 , and XIAP in T24 cells treated with TRAIL or/and Andro. (D) T24 cells were treated with PDTC at various concentrations for 24-h, and cell viability and IC 50 values were measured using MTS assays. (E) T24 cells were treated with 2 ng/mL TRAIL or 5 μM PDTC or 8 μM Andro or with a combination of TRAIL and Andro/PDTC for 24-h. Cell viability was measured by MTS assay. (F, G) Cell apoptosis rates of T24 cells according to Annexin V-FITC detection. T24 cells were treated with 2 ng/mL TRAIL or 5 μM PDTC or 8 μM Andro or with a combination of TRAIL and Andro/PDTC for 24-h. Histograms showing the apoptosis rates. Data represent the mean ± SD. *P

    Article Snippet: Cells treated under different conditions for 24-h were resuspended in 6-well culture plates, washed twice with cold PBS, and pelleted by centrifugation at 5000g for 5 min. Apoptotic cells were quantified using an Annexin V-FITC apoptosis detection kit (KeyGEN BioTECH, Nanjing, China).

    Techniques: MTS Assay

    Effects of miR-522 on human NSCLC cells apoptosis. ( a ) Annexin V-FITC/propidium iodide staining and flow cytometry were performed to detect apoptosis. ( b ) The quantitative presentation of apoptotic cell populations. * P

    Journal: Scientific Reports

    Article Title: Downregulation of miR-522 suppresses proliferation and metastasis of non-small cell lung cancer cells by directly targeting DENN/MADD domain containing 2D

    doi: 10.1038/srep19346

    Figure Lengend Snippet: Effects of miR-522 on human NSCLC cells apoptosis. ( a ) Annexin V-FITC/propidium iodide staining and flow cytometry were performed to detect apoptosis. ( b ) The quantitative presentation of apoptotic cell populations. * P

    Article Snippet: Annexin V-FITC Apoptosis Detection kit An Annexin V-FITC Apoptosis Detection kit was used according to the manufacturer’s instructions to detect apoptosis following treatment with miR-522 or miR-522 inhibitor (Beyotime, Shanghai, China).

    Techniques: Staining, Flow Cytometry, Cytometry

    Overexpression of BCL2L2 promotes proliferation and inhibits apoptosis in SCC‐25 cells. (a) Protein expression of BCL2L2 was measured by western blot in SCC‐25 cells after cotransfection with pcDNA3.1‐ BCL2L2 and miR‐16. (b) Cell proliferation was measured at 24, 48, 72, 96, and 120 hr after transfection by CCK‐8. (c) Annexin V/PI staining analysis was performed to investigate the apoptosis of SCC‐25 cells. (d) The histogram showed the apoptosis rate of SCC‐25 cells. Data were presented as mean ± standard deviation for three independent experiments. * p

    Journal: Journal of Cellular Physiology

    Article Title: MicroRNA‐16 functions as a tumor‐suppressor gene in oral squamous cell carcinoma by targeting AKT3 and BCL2L2. MicroRNA‐16 functions as a tumor‐suppressor gene in oral squamous cell carcinoma by targeting AKT3 and BCL2L2

    doi: 10.1002/jcp.26833

    Figure Lengend Snippet: Overexpression of BCL2L2 promotes proliferation and inhibits apoptosis in SCC‐25 cells. (a) Protein expression of BCL2L2 was measured by western blot in SCC‐25 cells after cotransfection with pcDNA3.1‐ BCL2L2 and miR‐16. (b) Cell proliferation was measured at 24, 48, 72, 96, and 120 hr after transfection by CCK‐8. (c) Annexin V/PI staining analysis was performed to investigate the apoptosis of SCC‐25 cells. (d) The histogram showed the apoptosis rate of SCC‐25 cells. Data were presented as mean ± standard deviation for three independent experiments. * p

    Article Snippet: For the apoptosis assay, transfected cells were stained with FITC‐conjugated Annexin V and PI using an Annexin V‐FITC Apoptosis Detection Kit (Beyotime) according to the manufacturer’s instructions.

    Techniques: Over Expression, Expressing, Western Blot, Cotransfection, Transfection, CCK-8 Assay, Staining, Standard Deviation

    miR‐16 induces apoptosis of SCC‐25 and CAL‐27 cell lines. The miR‐16 mimic/inhibitor and the corresponding negative control were transfected into SCC‐25 and CAL‐27 cell lines, respectively. Annexin V/PI staining analysis was performed to investigate the apoptosis rate of SCC‐25 (a) and CAL‐27 (b) cells. Data were presented as mean ± standard deviation for three independent experiments. * p

    Journal: Journal of Cellular Physiology

    Article Title: MicroRNA‐16 functions as a tumor‐suppressor gene in oral squamous cell carcinoma by targeting AKT3 and BCL2L2. MicroRNA‐16 functions as a tumor‐suppressor gene in oral squamous cell carcinoma by targeting AKT3 and BCL2L2

    doi: 10.1002/jcp.26833

    Figure Lengend Snippet: miR‐16 induces apoptosis of SCC‐25 and CAL‐27 cell lines. The miR‐16 mimic/inhibitor and the corresponding negative control were transfected into SCC‐25 and CAL‐27 cell lines, respectively. Annexin V/PI staining analysis was performed to investigate the apoptosis rate of SCC‐25 (a) and CAL‐27 (b) cells. Data were presented as mean ± standard deviation for three independent experiments. * p

    Article Snippet: For the apoptosis assay, transfected cells were stained with FITC‐conjugated Annexin V and PI using an Annexin V‐FITC Apoptosis Detection Kit (Beyotime) according to the manufacturer’s instructions.

    Techniques: Negative Control, Transfection, Staining, Standard Deviation

    Overexpression of AKT3 promotes proliferation and inhibits apoptosis in SCC‐25 cells. (a) Protein expression of AKT3 was measured by western blot in SCC‐25 cells after cotransfection with pcDNA3.1‐AKT3 and miR‐16. (b) Cell proliferation was measured at 24, 48, 72, 96, and 120 hr after transfection by CCK‐8. (c) Annexin V/PI staining analysis was performed to investigate the apoptosis of SCC‐25 cells. (d) The histogram showed the apoptosis rate of SCC‐25 cells. Data were presented as mean ± standard deviation for three independent experiments. * p

    Journal: Journal of Cellular Physiology

    Article Title: MicroRNA‐16 functions as a tumor‐suppressor gene in oral squamous cell carcinoma by targeting AKT3 and BCL2L2. MicroRNA‐16 functions as a tumor‐suppressor gene in oral squamous cell carcinoma by targeting AKT3 and BCL2L2

    doi: 10.1002/jcp.26833

    Figure Lengend Snippet: Overexpression of AKT3 promotes proliferation and inhibits apoptosis in SCC‐25 cells. (a) Protein expression of AKT3 was measured by western blot in SCC‐25 cells after cotransfection with pcDNA3.1‐AKT3 and miR‐16. (b) Cell proliferation was measured at 24, 48, 72, 96, and 120 hr after transfection by CCK‐8. (c) Annexin V/PI staining analysis was performed to investigate the apoptosis of SCC‐25 cells. (d) The histogram showed the apoptosis rate of SCC‐25 cells. Data were presented as mean ± standard deviation for three independent experiments. * p

    Article Snippet: For the apoptosis assay, transfected cells were stained with FITC‐conjugated Annexin V and PI using an Annexin V‐FITC Apoptosis Detection Kit (Beyotime) according to the manufacturer’s instructions.

    Techniques: Over Expression, Expressing, Western Blot, Cotransfection, Transfection, CCK-8 Assay, Staining, Standard Deviation

    (A) Confocal microscopy analysis of the colocalization of 2B-GFP and Bak-GFP within the mitochondria in HeLa cells. HeLa cells were transfected with pGFP, pGFP-2B, or pGFP-Bak. At 24 h posttransfection, they were stained with MitoTracker Red and subjected to confocal microscopy analysis. An overlay of the MitoTracker Red (red) and GFP florescence (green) is also shown. Mito, mitochondria. (B) Colocalization index estimation for cells transfected with pGFP, pGFP-2B, or pGFP-Bak and labeled for MitoTracker Red as for panel A. To determine the percentage of colocalization, green and merged images were loaded into the Leica TCS SP8 platform, and the ratio of GFP-positive cells to counted cells (in a total of 100 cells) was determined. Cells with GFP and mitochondrion colocalization (Manders overlap coefficient of > 0.8) were counted. (C) Immunoblotting analysis of 2B-Flag in the cytosol and mitochondrial fractions of HeLa cells infected with EV71 or transfected with pCMV-2B-Flag. Cytosolic and heavy membrane fractions were separated as described in Materials and Methods. Mitochondrial fractions were processed for immunoblotting with antibodies specific to EV71 2B, Bak, and Cox IV. Cytosolic fractions were immunoblotted with antibodies to EV71 2B and β-actin (internal control). Control, cells transfected with Tris-acetate-EDTA (TAE) buffer. Mock, cells with no EV71 infection. (D) Western blot analysis of the sublocation of 2B. Cells were transfected with pCMV-2B-Flag or pCMV-Flag. The mitochondrial fractions were isolated at 48 h posttransfection. The inner and outer mitochondrial membranes were isolated by sucrose gradient centrifugation and immunoblotted with an antibody to the Flag tag, Cox IV, and TSPO (internal control of outer mitochondrial membrane fraction). M, mitochondria of cells transfected with pCMV-Flag. I, inner mitochondrial membrane fraction. O, outer mitochondrial membrane fraction. (E) Analysis of cell apoptosis induced by 2B. Cells were treated with 30 nM staurosporine (positive control) or transfected with increasing amounts of pCMV-2B-Flag, pCMV-Flag, or pCMV-Bak. At 24 h posttransfection, cells were harvested and stained with annexin V-FITC. The percentages of apoptotic cells corresponding to the respective fluorescence intensities were calculated. (F) Analysis of the antiapoptosis effects of caspase inhibitors on 2B-induced apoptosis. Cells either infected with EV71 or transfected with pCMV-2B-Flag or pCMV-Bak-Flag (positive control) were treated with dimethyl sulfoxide (DMSO), zDEVD.fmk, or zLEHD.fmk for 24 h and then stained with annexin V-FITC and subjected to flow cytometry. NT, cells with no treatment. Control, cells transfected with pCMV-Flag.

    Journal: Journal of Virology

    Article Title: Enterovirus 71 2B Induces Cell Apoptosis by Directly Inducing the Conformational Activation of the Proapoptotic Protein Bax

    doi: 10.1128/JVI.01499-16

    Figure Lengend Snippet: (A) Confocal microscopy analysis of the colocalization of 2B-GFP and Bak-GFP within the mitochondria in HeLa cells. HeLa cells were transfected with pGFP, pGFP-2B, or pGFP-Bak. At 24 h posttransfection, they were stained with MitoTracker Red and subjected to confocal microscopy analysis. An overlay of the MitoTracker Red (red) and GFP florescence (green) is also shown. Mito, mitochondria. (B) Colocalization index estimation for cells transfected with pGFP, pGFP-2B, or pGFP-Bak and labeled for MitoTracker Red as for panel A. To determine the percentage of colocalization, green and merged images were loaded into the Leica TCS SP8 platform, and the ratio of GFP-positive cells to counted cells (in a total of 100 cells) was determined. Cells with GFP and mitochondrion colocalization (Manders overlap coefficient of > 0.8) were counted. (C) Immunoblotting analysis of 2B-Flag in the cytosol and mitochondrial fractions of HeLa cells infected with EV71 or transfected with pCMV-2B-Flag. Cytosolic and heavy membrane fractions were separated as described in Materials and Methods. Mitochondrial fractions were processed for immunoblotting with antibodies specific to EV71 2B, Bak, and Cox IV. Cytosolic fractions were immunoblotted with antibodies to EV71 2B and β-actin (internal control). Control, cells transfected with Tris-acetate-EDTA (TAE) buffer. Mock, cells with no EV71 infection. (D) Western blot analysis of the sublocation of 2B. Cells were transfected with pCMV-2B-Flag or pCMV-Flag. The mitochondrial fractions were isolated at 48 h posttransfection. The inner and outer mitochondrial membranes were isolated by sucrose gradient centrifugation and immunoblotted with an antibody to the Flag tag, Cox IV, and TSPO (internal control of outer mitochondrial membrane fraction). M, mitochondria of cells transfected with pCMV-Flag. I, inner mitochondrial membrane fraction. O, outer mitochondrial membrane fraction. (E) Analysis of cell apoptosis induced by 2B. Cells were treated with 30 nM staurosporine (positive control) or transfected with increasing amounts of pCMV-2B-Flag, pCMV-Flag, or pCMV-Bak. At 24 h posttransfection, cells were harvested and stained with annexin V-FITC. The percentages of apoptotic cells corresponding to the respective fluorescence intensities were calculated. (F) Analysis of the antiapoptosis effects of caspase inhibitors on 2B-induced apoptosis. Cells either infected with EV71 or transfected with pCMV-2B-Flag or pCMV-Bak-Flag (positive control) were treated with dimethyl sulfoxide (DMSO), zDEVD.fmk, or zLEHD.fmk for 24 h and then stained with annexin V-FITC and subjected to flow cytometry. NT, cells with no treatment. Control, cells transfected with pCMV-Flag.

    Article Snippet: Annexin V-FITC was obtained from Biyotime, China.

    Techniques: Confocal Microscopy, Transfection, Staining, Labeling, Infection, Western Blot, Isolation, Gradient Centrifugation, FLAG-tag, Positive Control, Fluorescence, Flow Cytometry, Cytometry

    Experiments indicating that Bcl-X L prevents 2B-induced apoptosis and Cyt c release in HeLa cells. (A) Analysis of 2B- or EV71-induced apoptosis in HeLa-Bcl-X L cells by annexin V-FITC staining and flow cytometry. Staurosporine (control), cells treated with staurosporine for 12 h. EV71, cells infected for 24 h with EV71 (MOI = 0.5). HeLa, HeLa cells transduced with pcDNA3.0-HA. (B) Western blot analysis of Cyt c in the cytosol fraction of HeLa (HeLa cells transduced with the pcDNA3.0-HA) or HeLa-Bcl-X L cells transfected with pCMV-Flag (control) or pCMV-2B-Flag. Stau, cells treated with staurosporine (30 nM) for 12 h. (C) Analysis of caspase-3-like activity in cell lysates of HeLa-Bcl-X L or HeLa cells (HeLa cells transduced with the pcDNA3.0-HA) transfected with pCMV-Flag or pCMV-2B-Flag or infected with EV71. Caspase-3-like activity was determined as described in Materials and Methods. Staurosporine (positive control), cells treated with staurosporine for 12 h. (D) Quantification of Cyt c release and Bax binding in isolated mitochondria from siBax HeLa cells infected with EV71 (MOI = 1) and siBax HeLa cells transfected with pCMV-2B-Flag or pCMV-Flag by Western blotting. Mitochondria were isolated and incubated for 1 h with 1 μM recombinant Bax or/and 10 μM Bcl-X L at RT. After a short centrifugation at 4°C, the resulting supernatants were immunoblotted with antibody to Cyt c . Equal amounts of each pellet fraction were lysed, boiled, and immunoblotted with antibody to His tag.

    Journal: Journal of Virology

    Article Title: Enterovirus 71 2B Induces Cell Apoptosis by Directly Inducing the Conformational Activation of the Proapoptotic Protein Bax

    doi: 10.1128/JVI.01499-16

    Figure Lengend Snippet: Experiments indicating that Bcl-X L prevents 2B-induced apoptosis and Cyt c release in HeLa cells. (A) Analysis of 2B- or EV71-induced apoptosis in HeLa-Bcl-X L cells by annexin V-FITC staining and flow cytometry. Staurosporine (control), cells treated with staurosporine for 12 h. EV71, cells infected for 24 h with EV71 (MOI = 0.5). HeLa, HeLa cells transduced with pcDNA3.0-HA. (B) Western blot analysis of Cyt c in the cytosol fraction of HeLa (HeLa cells transduced with the pcDNA3.0-HA) or HeLa-Bcl-X L cells transfected with pCMV-Flag (control) or pCMV-2B-Flag. Stau, cells treated with staurosporine (30 nM) for 12 h. (C) Analysis of caspase-3-like activity in cell lysates of HeLa-Bcl-X L or HeLa cells (HeLa cells transduced with the pcDNA3.0-HA) transfected with pCMV-Flag or pCMV-2B-Flag or infected with EV71. Caspase-3-like activity was determined as described in Materials and Methods. Staurosporine (positive control), cells treated with staurosporine for 12 h. (D) Quantification of Cyt c release and Bax binding in isolated mitochondria from siBax HeLa cells infected with EV71 (MOI = 1) and siBax HeLa cells transfected with pCMV-2B-Flag or pCMV-Flag by Western blotting. Mitochondria were isolated and incubated for 1 h with 1 μM recombinant Bax or/and 10 μM Bcl-X L at RT. After a short centrifugation at 4°C, the resulting supernatants were immunoblotted with antibody to Cyt c . Equal amounts of each pellet fraction were lysed, boiled, and immunoblotted with antibody to His tag.

    Article Snippet: Annexin V-FITC was obtained from Biyotime, China.

    Techniques: Staining, Flow Cytometry, Cytometry, Infection, Transduction, Western Blot, Transfection, Activity Assay, Positive Control, Binding Assay, Isolation, Incubation, Recombinant, Centrifugation

    Experiments indicating that the N-terminal domain from aa 22 to 35 of 2B is crucial for Bax activation and the interaction between Bax and 2B. (A) Coimmunoprecipitation analysis of the region of 2B that is responsible for Bax interaction. HeLa cells were transfected with vectors expressing full-length 2B, 2B(Δ1–22) (2B with deletion of aa 1 to 22), 2B(Δ1–35) (2B with deletion of aa 1 to 35), or 2B(Δ22–35) (2B with deletion of aa 22 to 35). At 24 h posttransfection, the cells were harvested, and coimmunoprecipitation assay was performed using Flag antibody. The precipitated proteins were immunoblotted with antibodies to Bax and Flag. Control, cells transfected with pCMV-Flag. (B) Western blot analysis of Bax activation in cells expressing 2B, 2B(Δ1–22), 2B(Δ1–35), or 2B(Δ22–35). Activated Bax was immunoprecipitated from cell lysates by using anti-Bax6A7 antibody. The eluates were probed with antibody to Bax. Control, cells transfected with pCMV-Flag. (C) Analysis of cell apoptosis in cells expressing 2B, 2B(Δ1–22), 2B(Δ1–35), or 2B(Δ22–35). Cells were harvested and stained with annexin V-FITC as described in Materials and Methods. Cell apoptosis was then determined by flow cytometry. Control, cells transfected with pCMV-Flag. Staurosporine (positive control), = cells treated with staurosporine for 12 h. (D) Western blot analysis of Cyt c in the cytoplasmic fractions of cells expressing 2B, 2B(Δ1–22), 2B(Δ1–35), or 2B(Δ22–35). Equal amounts of each cytosol fraction were immunoblotted with antibody to Cyt c . (E) Analysis of the caspase-3-like activity in cells expressing 2B, 2B(Δ1–22), 2B(Δ1–35), or 2B(Δ22–35). Control, cells transfected with pCMV-Flag. Staurosporine (positive control), cells treated with staurosporine for 12 h.

    Journal: Journal of Virology

    Article Title: Enterovirus 71 2B Induces Cell Apoptosis by Directly Inducing the Conformational Activation of the Proapoptotic Protein Bax

    doi: 10.1128/JVI.01499-16

    Figure Lengend Snippet: Experiments indicating that the N-terminal domain from aa 22 to 35 of 2B is crucial for Bax activation and the interaction between Bax and 2B. (A) Coimmunoprecipitation analysis of the region of 2B that is responsible for Bax interaction. HeLa cells were transfected with vectors expressing full-length 2B, 2B(Δ1–22) (2B with deletion of aa 1 to 22), 2B(Δ1–35) (2B with deletion of aa 1 to 35), or 2B(Δ22–35) (2B with deletion of aa 22 to 35). At 24 h posttransfection, the cells were harvested, and coimmunoprecipitation assay was performed using Flag antibody. The precipitated proteins were immunoblotted with antibodies to Bax and Flag. Control, cells transfected with pCMV-Flag. (B) Western blot analysis of Bax activation in cells expressing 2B, 2B(Δ1–22), 2B(Δ1–35), or 2B(Δ22–35). Activated Bax was immunoprecipitated from cell lysates by using anti-Bax6A7 antibody. The eluates were probed with antibody to Bax. Control, cells transfected with pCMV-Flag. (C) Analysis of cell apoptosis in cells expressing 2B, 2B(Δ1–22), 2B(Δ1–35), or 2B(Δ22–35). Cells were harvested and stained with annexin V-FITC as described in Materials and Methods. Cell apoptosis was then determined by flow cytometry. Control, cells transfected with pCMV-Flag. Staurosporine (positive control), = cells treated with staurosporine for 12 h. (D) Western blot analysis of Cyt c in the cytoplasmic fractions of cells expressing 2B, 2B(Δ1–22), 2B(Δ1–35), or 2B(Δ22–35). Equal amounts of each cytosol fraction were immunoblotted with antibody to Cyt c . (E) Analysis of the caspase-3-like activity in cells expressing 2B, 2B(Δ1–22), 2B(Δ1–35), or 2B(Δ22–35). Control, cells transfected with pCMV-Flag. Staurosporine (positive control), cells treated with staurosporine for 12 h.

    Article Snippet: Annexin V-FITC was obtained from Biyotime, China.

    Techniques: Activation Assay, Transfection, Expressing, Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation, Staining, Flow Cytometry, Cytometry, Positive Control, Activity Assay

    25- epi Ritterostatin G N 1 N is cytotoxic at nanomolar concentrations in melanoma cells. (A and B) MTT assay results showing A375, WM35, A375SM, and UCSD-354L melanoma cell viability after treatment with various concentrations of 25- epi Ritterostatin G N 1 N for 72 h. IC 50 , 50% inhibitory concentration. (C) Percentage of viable WM35 and A375 cells after treatment with 0.5 µ M 25- epi Ritterostatin G N 1 N for 72 h, determined according to Annexin V-FITC/propidium iodide staining and subsequent flow cytometry analysis. (D) The bar graphs represent the percentage of viable A375, WM35, A375SM, and UCSD-354L cells (mean ± standard error) after treatment with 0.5 µ M 25- epi Ritterostatin G N 1 N for 48 and 72 h, for 3 independent measurements, according to Annexin V-FITC/propidium iodide staining analysis. (E and F) Giemsa staining for colony formation assessment in A375 and WM35 cells treated with varying concentrations of 25- epi Ritterostatin G N 1 N [or dimethyl sulfoxide (DMSO) only] for 14 days. The bar graphs represent the mean (± standard error of the mean) of three independent measurements. * p

    Journal: International Journal of Oncology

    Article Title: Antitumor agent 25-epi Ritterostatin GN1N induces endoplasmic reticulum stress and autophagy mediated cell death in melanoma cells

    doi: 10.3892/ijo.2017.3944

    Figure Lengend Snippet: 25- epi Ritterostatin G N 1 N is cytotoxic at nanomolar concentrations in melanoma cells. (A and B) MTT assay results showing A375, WM35, A375SM, and UCSD-354L melanoma cell viability after treatment with various concentrations of 25- epi Ritterostatin G N 1 N for 72 h. IC 50 , 50% inhibitory concentration. (C) Percentage of viable WM35 and A375 cells after treatment with 0.5 µ M 25- epi Ritterostatin G N 1 N for 72 h, determined according to Annexin V-FITC/propidium iodide staining and subsequent flow cytometry analysis. (D) The bar graphs represent the percentage of viable A375, WM35, A375SM, and UCSD-354L cells (mean ± standard error) after treatment with 0.5 µ M 25- epi Ritterostatin G N 1 N for 48 and 72 h, for 3 independent measurements, according to Annexin V-FITC/propidium iodide staining analysis. (E and F) Giemsa staining for colony formation assessment in A375 and WM35 cells treated with varying concentrations of 25- epi Ritterostatin G N 1 N [or dimethyl sulfoxide (DMSO) only] for 14 days. The bar graphs represent the mean (± standard error of the mean) of three independent measurements. * p

    Article Snippet: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) and propidium iodide were purchased from Sigma-Aldrich (St. Louis, MO, USA), and an Annexin V-FITC kit was purchased from BD Pharmingen (San Jose, CA, USA).

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

    Normal melanocytes exhibit limited sensitivity to 25- epi Ritterostatin G N 1 N . (A) Percentage of viable normal melanocytes after treatment with 25- epi Ritterostatin G N 1 N , according to Annexin V-FITC/ propidium iodide staining and flow cytometry analysis. (B) Rhodamine 123 staining results showing the mitochondrial membrane potential change in normal melanocytes treated with 0.5 or 1 µ M 25- epi Ritterostatin G N 1 N for 48 h.

    Journal: International Journal of Oncology

    Article Title: Antitumor agent 25-epi Ritterostatin GN1N induces endoplasmic reticulum stress and autophagy mediated cell death in melanoma cells

    doi: 10.3892/ijo.2017.3944

    Figure Lengend Snippet: Normal melanocytes exhibit limited sensitivity to 25- epi Ritterostatin G N 1 N . (A) Percentage of viable normal melanocytes after treatment with 25- epi Ritterostatin G N 1 N , according to Annexin V-FITC/ propidium iodide staining and flow cytometry analysis. (B) Rhodamine 123 staining results showing the mitochondrial membrane potential change in normal melanocytes treated with 0.5 or 1 µ M 25- epi Ritterostatin G N 1 N for 48 h.

    Article Snippet: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) and propidium iodide were purchased from Sigma-Aldrich (St. Louis, MO, USA), and an Annexin V-FITC kit was purchased from BD Pharmingen (San Jose, CA, USA).

    Techniques: Staining, Flow Cytometry, Cytometry

    25- epi Ritterostatin G N 1 N triggers autophagic cell death in melanoma cells. (A and B) Rhodamine 123 staining results indicate the mitochondrial membrane potential changes in A375, WM35, A375SM, and UCSD-354L cells treated with 0.5 or 1 µ M 25- epi Ritterostatin G N 1 N for 48 h. The bar graphs represent the mean (± standard error of the mean) of 3 independent measurements. (C) Percentage of viable A375 cells at 24 h after treatment with 10 µ M chloroquine alone, 1 µ M 25- epi Ritterostatin G N 1 N alone, or 10 µ M chloroquine followed by 1 µ M 25- epi Ritterostatin G N 1 N , according to Annexin V-FITC/ propidium iodide staining and flow cytometry analysis. (D) The bar graphs represent the mean (± standard error of the mean) of three independent measurements for Annexin V-FITC/propidium iodide stained A375SM cells treated with 1 µ M 25- epi Ritterostatin G N 1 N ± 10 µ M chloroquine for 24 h. (E) Western blot results show expression of the autophagic proteins LC3B-I and LC3B-II in A375 cells treated with 0.5 µ M 25- epi Ritterostatin G N 1 N for the indicated time points. DMSO, dimethyl sulfoxide (control). * p

    Journal: International Journal of Oncology

    Article Title: Antitumor agent 25-epi Ritterostatin GN1N induces endoplasmic reticulum stress and autophagy mediated cell death in melanoma cells

    doi: 10.3892/ijo.2017.3944

    Figure Lengend Snippet: 25- epi Ritterostatin G N 1 N triggers autophagic cell death in melanoma cells. (A and B) Rhodamine 123 staining results indicate the mitochondrial membrane potential changes in A375, WM35, A375SM, and UCSD-354L cells treated with 0.5 or 1 µ M 25- epi Ritterostatin G N 1 N for 48 h. The bar graphs represent the mean (± standard error of the mean) of 3 independent measurements. (C) Percentage of viable A375 cells at 24 h after treatment with 10 µ M chloroquine alone, 1 µ M 25- epi Ritterostatin G N 1 N alone, or 10 µ M chloroquine followed by 1 µ M 25- epi Ritterostatin G N 1 N , according to Annexin V-FITC/ propidium iodide staining and flow cytometry analysis. (D) The bar graphs represent the mean (± standard error of the mean) of three independent measurements for Annexin V-FITC/propidium iodide stained A375SM cells treated with 1 µ M 25- epi Ritterostatin G N 1 N ± 10 µ M chloroquine for 24 h. (E) Western blot results show expression of the autophagic proteins LC3B-I and LC3B-II in A375 cells treated with 0.5 µ M 25- epi Ritterostatin G N 1 N for the indicated time points. DMSO, dimethyl sulfoxide (control). * p

    Article Snippet: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) and propidium iodide were purchased from Sigma-Aldrich (St. Louis, MO, USA), and an Annexin V-FITC kit was purchased from BD Pharmingen (San Jose, CA, USA).

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

    MLL -translocation cell lines are sensitive to inhibition of pan-Cullin-dependent and CKS1-dependent protein degradation. (A) Western blots for THP-1 ( MLL-AF9 ) and KOPN-8 ( MLL-ENL ) cells treated with vehicle control (DMSO), 0.1 μM MLN4924 or 1 μM C1 for 16 h and 24 h. Histone H3 was used as a loading control. Western blots are representative of 3 independent experiments. (B) Cell viability was assessed for THP-1, KOPN-8, healthy PBMC and CD34 + control cells after 3 days treatment with indicated concentrations of the pan-Cullin inhibitor (MLN4924) or the SKP2-CKS1 inhibitor (C1). Results represent the mean of 3 independent experiments with standard deviation bars. (C) Proportion of apoptotic CD34 + , THP-1 and KOPN-8 cells, in response to two different concentrations of C1 or MLN4924, was measured by PI and Annexin V-FITC staining using flow cytometry. Significance was tested using the Student's t -test versus DMSO treated cells (N = 3). (D) Cell cycle status was measured for KOPN-8 cells in response to DMSO vehicle control or MLN4924 (1 μM) using the Click-iT EdU incorporation kit. Graphs are representative of 3 independent experiments.

    Journal: Biochimica et Biophysica Acta

    Article Title: The Cks1/Cks2 axis fine-tunes Mll1 expression and is crucial for MLL-rearranged leukaemia cell viability

    doi: 10.1016/j.bbamcr.2017.09.009

    Figure Lengend Snippet: MLL -translocation cell lines are sensitive to inhibition of pan-Cullin-dependent and CKS1-dependent protein degradation. (A) Western blots for THP-1 ( MLL-AF9 ) and KOPN-8 ( MLL-ENL ) cells treated with vehicle control (DMSO), 0.1 μM MLN4924 or 1 μM C1 for 16 h and 24 h. Histone H3 was used as a loading control. Western blots are representative of 3 independent experiments. (B) Cell viability was assessed for THP-1, KOPN-8, healthy PBMC and CD34 + control cells after 3 days treatment with indicated concentrations of the pan-Cullin inhibitor (MLN4924) or the SKP2-CKS1 inhibitor (C1). Results represent the mean of 3 independent experiments with standard deviation bars. (C) Proportion of apoptotic CD34 + , THP-1 and KOPN-8 cells, in response to two different concentrations of C1 or MLN4924, was measured by PI and Annexin V-FITC staining using flow cytometry. Significance was tested using the Student's t -test versus DMSO treated cells (N = 3). (D) Cell cycle status was measured for KOPN-8 cells in response to DMSO vehicle control or MLN4924 (1 μM) using the Click-iT EdU incorporation kit. Graphs are representative of 3 independent experiments.

    Article Snippet: For the evaluation of apoptosis, cells were stained with Annexin V-FITC and PI (Biolegend, London, UK) following manufacturer's instructions.

    Techniques: Translocation Assay, Inhibition, Western Blot, Standard Deviation, Staining, Flow Cytometry, Cytometry

    GA-TAT induces apoptosis in EJ cells. ( A ) EJ cells were exposed to 1.0 μM TAT, GA, or GA-TAT for 24 h and then collected and mixed with 200 μL of dye mixture containing 100 μg/mL AO and EB. Cellular morphological changes were observed by using fluorescence microscopy (200× magnification). ( B ) After exposure to 1.0 μM TAT, GA, or GA-TAT for 24 h, EJ cells were harvested and stained with Annexin V-FITC and PI, followed by flow cytometric analysis of apoptosis. Arrows indicate apoptotic cells. * P

    Journal: Drug Design, Development and Therapy

    Article Title: Cell-penetrating peptide conjugates of gambogic acid enhance the antitumor effect on human bladder cancer EJ cells through ROS-mediated apoptosis

    doi: 10.2147/DDDT.S161821

    Figure Lengend Snippet: GA-TAT induces apoptosis in EJ cells. ( A ) EJ cells were exposed to 1.0 μM TAT, GA, or GA-TAT for 24 h and then collected and mixed with 200 μL of dye mixture containing 100 μg/mL AO and EB. Cellular morphological changes were observed by using fluorescence microscopy (200× magnification). ( B ) After exposure to 1.0 μM TAT, GA, or GA-TAT for 24 h, EJ cells were harvested and stained with Annexin V-FITC and PI, followed by flow cytometric analysis of apoptosis. Arrows indicate apoptotic cells. * P

    Article Snippet: Quantitation of cell apoptosis rates by flow cytometry The apoptosis rates of cells were determined by Annexin V-FITC and propidium iodide (PI) staining and flow cytometry, using the Annexin V-FITC/PI Apoptosis Detection Kit (KeyGen BioTECH, Nanjing, People’s Republic of China) according to the manufacturer’s instructions.

    Techniques: Fluorescence, Microscopy, Staining, Flow Cytometry

    Role of ROS in GA-TAT–induced apoptosis. EJ cells were pretreated with or without 5 mM NAC for 2 h and then were incubated with 1.0 μM TAT, GA, or GA-TAT for 24 h; ( A ) the qualitative ROS production analysis was undertaken using a fluorescence microplate reader; ( B ) levels of caspase-3 (processing), caspase-9 (processing), Bcl-2, and Bax protein were determined using Western blotting analysis, and GAPDH was used as a loading control; ( C ) caspase-3 and caspase-9 activity was determined using caspase-3 and caspase-9 assay kits, respectively; and ( D ) the percentage of apoptotic cells was detected by Annexin V-FITC and PI staining flow cytometry. * P

    Journal: Drug Design, Development and Therapy

    Article Title: Cell-penetrating peptide conjugates of gambogic acid enhance the antitumor effect on human bladder cancer EJ cells through ROS-mediated apoptosis

    doi: 10.2147/DDDT.S161821

    Figure Lengend Snippet: Role of ROS in GA-TAT–induced apoptosis. EJ cells were pretreated with or without 5 mM NAC for 2 h and then were incubated with 1.0 μM TAT, GA, or GA-TAT for 24 h; ( A ) the qualitative ROS production analysis was undertaken using a fluorescence microplate reader; ( B ) levels of caspase-3 (processing), caspase-9 (processing), Bcl-2, and Bax protein were determined using Western blotting analysis, and GAPDH was used as a loading control; ( C ) caspase-3 and caspase-9 activity was determined using caspase-3 and caspase-9 assay kits, respectively; and ( D ) the percentage of apoptotic cells was detected by Annexin V-FITC and PI staining flow cytometry. * P

    Article Snippet: Quantitation of cell apoptosis rates by flow cytometry The apoptosis rates of cells were determined by Annexin V-FITC and propidium iodide (PI) staining and flow cytometry, using the Annexin V-FITC/PI Apoptosis Detection Kit (KeyGen BioTECH, Nanjing, People’s Republic of China) according to the manufacturer’s instructions.

    Techniques: Incubation, Fluorescence, Western Blot, Activity Assay, Staining, Flow Cytometry, Cytometry

    Effects of GA-TAT on caspase-3 and caspase-9 activation in EJ cells. EJ cells were incubated with 1.0 μM TAT, GA, or GA-TAT for 24 h; ( A ) the activities of caspase-3 and caspase-9 were determined using caspase-3 and caspase-9 assay kits, respectively; and ( B ) Western blotting analysis of caspase-3 (processing), caspase-9 (processing), Bcl-2, and Bax was conducted. GAPDH was used as a loading control. ( C ) EJ cells were incubated with 1.0 μM TAT, GA, or GA-TAT for 24 h with or without 20 μM Ac-DEVD-CHO (caspase-3 inhibitor) or Z-LEHD-FMK (caspase-3 inhibitor); the percentage of apoptotic cells was detected by Annexin V-FITC and PI staining flow cytometry. * P

    Journal: Drug Design, Development and Therapy

    Article Title: Cell-penetrating peptide conjugates of gambogic acid enhance the antitumor effect on human bladder cancer EJ cells through ROS-mediated apoptosis

    doi: 10.2147/DDDT.S161821

    Figure Lengend Snippet: Effects of GA-TAT on caspase-3 and caspase-9 activation in EJ cells. EJ cells were incubated with 1.0 μM TAT, GA, or GA-TAT for 24 h; ( A ) the activities of caspase-3 and caspase-9 were determined using caspase-3 and caspase-9 assay kits, respectively; and ( B ) Western blotting analysis of caspase-3 (processing), caspase-9 (processing), Bcl-2, and Bax was conducted. GAPDH was used as a loading control. ( C ) EJ cells were incubated with 1.0 μM TAT, GA, or GA-TAT for 24 h with or without 20 μM Ac-DEVD-CHO (caspase-3 inhibitor) or Z-LEHD-FMK (caspase-3 inhibitor); the percentage of apoptotic cells was detected by Annexin V-FITC and PI staining flow cytometry. * P

    Article Snippet: Quantitation of cell apoptosis rates by flow cytometry The apoptosis rates of cells were determined by Annexin V-FITC and propidium iodide (PI) staining and flow cytometry, using the Annexin V-FITC/PI Apoptosis Detection Kit (KeyGen BioTECH, Nanjing, People’s Republic of China) according to the manufacturer’s instructions.

    Techniques: Activation Assay, Incubation, Western Blot, Staining, Flow Cytometry, Cytometry

    Apoptosis assay of mouse spleen cells before and after irreversible electroporation treatment using double-staining with annexin V-fluorescein isothiocyanate/propidium iodide. Apoptosis was quantified by flow cytometry. A: Control group; B: 1 d post-IRE; C: 3 d post-IRE; D: 7 d post-IRE; E: Percentages of apoptotic cells before and after the IRE intervention. Data are mean ± SD ( n = 8). IRE: irreversible electroporation.

    Journal: World Journal of Gastrointestinal Oncology

    Article Title: Histological analysis of human pancreatic carcinoma following irreversible electroporation in a nude mouse model

    doi: 10.4251/wjgo.v10.i12.476

    Figure Lengend Snippet: Apoptosis assay of mouse spleen cells before and after irreversible electroporation treatment using double-staining with annexin V-fluorescein isothiocyanate/propidium iodide. Apoptosis was quantified by flow cytometry. A: Control group; B: 1 d post-IRE; C: 3 d post-IRE; D: 7 d post-IRE; E: Percentages of apoptotic cells before and after the IRE intervention. Data are mean ± SD ( n = 8). IRE: irreversible electroporation.

    Article Snippet: Meanwhile, Annexin V-fluorescein isothiocyanate Apoptosis Detection Kit (BioVision) was employed for apoptosis quantitation, as directed by the manufacturer.

    Techniques: Apoptosis Assay, Electroporation, Double Staining, Flow Cytometry, Cytometry

    Holotoxin A 1 activations of acid SMase and neutral SMase induce apoptosis in human primary leukemia cells. ( A , B ) Holotoxin A 1 (6 h) induced apoptosis in ( A ) human primary leukemia cells, but not in ( B ) human hematopoietic progenitor CD34 + cells. Upper panels: Representative flow cytometry results show the percentage of apoptotic cells measured with annexin V-FITC/PI staining. Lower panels: Mean ± SD of three independent experiments. *** p

    Journal: Marine Drugs

    Article Title: Holotoxin A1 Induces Apoptosis by Activating Acid Sphingomyelinase and Neutral Sphingomyelinase in K562 and Human Primary Leukemia Cells

    doi: 10.3390/md16040123

    Figure Lengend Snippet: Holotoxin A 1 activations of acid SMase and neutral SMase induce apoptosis in human primary leukemia cells. ( A , B ) Holotoxin A 1 (6 h) induced apoptosis in ( A ) human primary leukemia cells, but not in ( B ) human hematopoietic progenitor CD34 + cells. Upper panels: Representative flow cytometry results show the percentage of apoptotic cells measured with annexin V-FITC/PI staining. Lower panels: Mean ± SD of three independent experiments. *** p

    Article Snippet: Briefly, cells were harvested, washed with PBS (pH 7.4), centrifuged, and stained with annexin V-FITC (Pharmingen) and 2 μg/mL PI in binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2 ) for 15 min at 37 °C in the dark.

    Techniques: Flow Cytometry, Cytometry, Staining

    Holotoxin A 1 induces apoptosis through activation of acid SMase and neutral SMase in human leukemic cells. ( A ) Immunohistochemistry images of K562 cells show that holotoxin A 1 stimulated the production of ceramide (green) by activating (left) acid SMase (red) and (right) neutral SMase (red); ( B ) SMase inhibitors block apoptosis. K562 cells (1 × 10 5 cells/well) were incubated for 6 h with holotoxin A 1 in the presence or absence of myriocin, fumonisin B 1 , desipramine, or GW4869, and the percentage of apoptotic cells was determined with annexin V-FITC/PI staining. Upper panel: Representative flow cytometry results of three experiments for each condition. Lower panel: Mean ± SD of three independent experiments. ** p

    Journal: Marine Drugs

    Article Title: Holotoxin A1 Induces Apoptosis by Activating Acid Sphingomyelinase and Neutral Sphingomyelinase in K562 and Human Primary Leukemia Cells

    doi: 10.3390/md16040123

    Figure Lengend Snippet: Holotoxin A 1 induces apoptosis through activation of acid SMase and neutral SMase in human leukemic cells. ( A ) Immunohistochemistry images of K562 cells show that holotoxin A 1 stimulated the production of ceramide (green) by activating (left) acid SMase (red) and (right) neutral SMase (red); ( B ) SMase inhibitors block apoptosis. K562 cells (1 × 10 5 cells/well) were incubated for 6 h with holotoxin A 1 in the presence or absence of myriocin, fumonisin B 1 , desipramine, or GW4869, and the percentage of apoptotic cells was determined with annexin V-FITC/PI staining. Upper panel: Representative flow cytometry results of three experiments for each condition. Lower panel: Mean ± SD of three independent experiments. ** p

    Article Snippet: Briefly, cells were harvested, washed with PBS (pH 7.4), centrifuged, and stained with annexin V-FITC (Pharmingen) and 2 μg/mL PI in binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2 ) for 15 min at 37 °C in the dark.

    Techniques: Activation Assay, Immunohistochemistry, Blocking Assay, Incubation, Staining, Flow Cytometry, Cytometry

    Acid SMase and neutral SMase knockdowns inhibit holotoxin A 1 -induced apoptosis in K562 cells. ( A – C ) K562 cells were transiently transfected by electroporation for 48 h with siRNAs against acid SMase (ASM) or neutral SMase (NSM). A nonspecific control (NC) siRNA and no siRNA (shock) served as controls. Then, cells were untreated (control) or treated with holotoxin A 1 (HA 1 ). ( A ) siRNA SMase silencing. Western blot analyses of cell lysates show pathway proteins stimulated by holotoxin A 1 treatment in (left) cells with normal or knocked down ASM expression and (right) cells with normal or knocked down NSM expression; ( B ) siRNA SMase silencing blocks ceramide production. K562 cells transfected with (left) ASM siRNAs or (right) NSM siRNAs were treated with holotoxin A 1 for 2 h, and then fixed and permeabilized. The samples were then stained with PE-conjugated (red) antibodies against acid SMase (three left columns), or neutral SMase (three right columns) and FITC-labeled ceramide antibody (green). Images are representative of three separate experiments; ( C ) siRNA SMase silencing inhibits apoptosis. Cells were untreated (control, shocked) or treated with NC, ASM, or NSM siRNAs and then incubated for 6 h with or without holotoxin A 1 . Upper panels: Representative flow cytometry results show the percentages of apoptotic cells determined with annexin V-FITC/PI staining. Lower panels: Mean ± SD of three independent experiments. ** p

    Journal: Marine Drugs

    Article Title: Holotoxin A1 Induces Apoptosis by Activating Acid Sphingomyelinase and Neutral Sphingomyelinase in K562 and Human Primary Leukemia Cells

    doi: 10.3390/md16040123

    Figure Lengend Snippet: Acid SMase and neutral SMase knockdowns inhibit holotoxin A 1 -induced apoptosis in K562 cells. ( A – C ) K562 cells were transiently transfected by electroporation for 48 h with siRNAs against acid SMase (ASM) or neutral SMase (NSM). A nonspecific control (NC) siRNA and no siRNA (shock) served as controls. Then, cells were untreated (control) or treated with holotoxin A 1 (HA 1 ). ( A ) siRNA SMase silencing. Western blot analyses of cell lysates show pathway proteins stimulated by holotoxin A 1 treatment in (left) cells with normal or knocked down ASM expression and (right) cells with normal or knocked down NSM expression; ( B ) siRNA SMase silencing blocks ceramide production. K562 cells transfected with (left) ASM siRNAs or (right) NSM siRNAs were treated with holotoxin A 1 for 2 h, and then fixed and permeabilized. The samples were then stained with PE-conjugated (red) antibodies against acid SMase (three left columns), or neutral SMase (three right columns) and FITC-labeled ceramide antibody (green). Images are representative of three separate experiments; ( C ) siRNA SMase silencing inhibits apoptosis. Cells were untreated (control, shocked) or treated with NC, ASM, or NSM siRNAs and then incubated for 6 h with or without holotoxin A 1 . Upper panels: Representative flow cytometry results show the percentages of apoptotic cells determined with annexin V-FITC/PI staining. Lower panels: Mean ± SD of three independent experiments. ** p

    Article Snippet: Briefly, cells were harvested, washed with PBS (pH 7.4), centrifuged, and stained with annexin V-FITC (Pharmingen) and 2 μg/mL PI in binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2 ) for 15 min at 37 °C in the dark.

    Techniques: Transfection, Electroporation, Western Blot, Expressing, Staining, Labeling, Incubation, Flow Cytometry, Cytometry

    Holotoxin A 1 induces apoptosis in human leukemic and colorectal cancer cells. ( A ) Structures of holotoxin A 1 and cladoloside C 2 ; ( B , C ) Cells were seeded, cultured for 4 h, and then treated with holotoxin A 1 , (left panels) for 6 h at various concentrations (0, 0.01, 0.03, 0.05, or 0.1 μM) and (right panels) for the indicated times (0.06 µM holotoxin A 1 ); The percentage of apoptotic cells was determined in ( B ) K562 cells and ( C ) HL-60 cells with annexin V-FITC/PI staining; ( D ) Cells were seeded, cultured for 24 h, and then treated for 24 h with various concentrations of holotoxin A 1 (0, 0.5, 1.0, or 2.0 μM). The percentage of apoptotic cells was measured in (left panel) SNU-C4 cells and (right panel) HT-29 cells with annexin V-FITC/PI staining; ( B – D ) Upper panels: Representative flow cytometry results indicate the extent of apoptosis. Lower panels: Mean ± SD of three independent experiments. * p

    Journal: Marine Drugs

    Article Title: Holotoxin A1 Induces Apoptosis by Activating Acid Sphingomyelinase and Neutral Sphingomyelinase in K562 and Human Primary Leukemia Cells

    doi: 10.3390/md16040123

    Figure Lengend Snippet: Holotoxin A 1 induces apoptosis in human leukemic and colorectal cancer cells. ( A ) Structures of holotoxin A 1 and cladoloside C 2 ; ( B , C ) Cells were seeded, cultured for 4 h, and then treated with holotoxin A 1 , (left panels) for 6 h at various concentrations (0, 0.01, 0.03, 0.05, or 0.1 μM) and (right panels) for the indicated times (0.06 µM holotoxin A 1 ); The percentage of apoptotic cells was determined in ( B ) K562 cells and ( C ) HL-60 cells with annexin V-FITC/PI staining; ( D ) Cells were seeded, cultured for 24 h, and then treated for 24 h with various concentrations of holotoxin A 1 (0, 0.5, 1.0, or 2.0 μM). The percentage of apoptotic cells was measured in (left panel) SNU-C4 cells and (right panel) HT-29 cells with annexin V-FITC/PI staining; ( B – D ) Upper panels: Representative flow cytometry results indicate the extent of apoptosis. Lower panels: Mean ± SD of three independent experiments. * p

    Article Snippet: Briefly, cells were harvested, washed with PBS (pH 7.4), centrifuged, and stained with annexin V-FITC (Pharmingen) and 2 μg/mL PI in binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2 ) for 15 min at 37 °C in the dark.

    Techniques: Cell Culture, Staining, Flow Cytometry, Cytometry