caspase reaction buffer Search Results


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  • 99
    Thermo Fisher reaction buffer
    Reaction Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5752 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore reaction buffer
    Reaction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore caspase reaction buffer
    TNF provokes a cholesterol-insensitive processing of SREBP-1 in ethanol-exposed cells that is promoted by <t>caspase-4</t> or -12 activation, resulting in the inappropriate induction of lipogenic enzymes and consequent accumulation of triglycerides. DISC , death inducing signaling complex.
    Caspase Reaction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega caspase 9 promega reaction buffer
    TNF provokes a cholesterol-insensitive processing of SREBP-1 in ethanol-exposed cells that is promoted by <t>caspase-4</t> or -12 activation, resulting in the inappropriate induction of lipogenic enzymes and consequent accumulation of triglycerides. DISC , death inducing signaling complex.
    Caspase 9 Promega Reaction Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioVision reaction buffer
    TNF provokes a cholesterol-insensitive processing of SREBP-1 in ethanol-exposed cells that is promoted by <t>caspase-4</t> or -12 activation, resulting in the inappropriate induction of lipogenic enzymes and consequent accumulation of triglycerides. DISC , death inducing signaling complex.
    Reaction Buffer, supplied by BioVision, used in various techniques. Bioz Stars score: 99/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioVision intratracheal active recombinant human caspase 6
    TNF provokes a cholesterol-insensitive processing of SREBP-1 in ethanol-exposed cells that is promoted by <t>caspase-4</t> or -12 activation, resulting in the inappropriate induction of lipogenic enzymes and consequent accumulation of triglycerides. DISC , death inducing signaling complex.
    Intratracheal Active Recombinant Human Caspase 6, supplied by BioVision, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega caspace assay system
    TNF provokes a cholesterol-insensitive processing of SREBP-1 in ethanol-exposed cells that is promoted by <t>caspase-4</t> or -12 activation, resulting in the inappropriate induction of lipogenic enzymes and consequent accumulation of triglycerides. DISC , death inducing signaling complex.
    Caspace Assay System, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 402 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc caspase 8
    Analysis of DZNep effects by Western-blot and RT-PCR. DZNep reduced EZH2 and ALOX5 levels, and induced caspase-dependent cell apoptosis. Cells were treated with 0.5 umol/L DZNep for 72 h, and then whole-cell lysates were analyzed by Western-blot analysis. β-Actin was used as an internal control. Treatment with DZNep reduced protein levels of EZH2 in MM cells. ALOX5 was over-expressed in H929 and was down-regulated greatly by DZNep treatment. In H929 and MM1.S, DZNep efficiently induced caspase-3 activation and the cleavage of PARP. Furthermore, DZNep treatment induced <t>caspase-8</t> activation and had little effect on full-length caspase-9 levels.
    Caspase 8, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam reaction buffer
    Analysis of DZNep effects by Western-blot and RT-PCR. DZNep reduced EZH2 and ALOX5 levels, and induced caspase-dependent cell apoptosis. Cells were treated with 0.5 umol/L DZNep for 72 h, and then whole-cell lysates were analyzed by Western-blot analysis. β-Actin was used as an internal control. Treatment with DZNep reduced protein levels of EZH2 in MM cells. ALOX5 was over-expressed in H929 and was down-regulated greatly by DZNep treatment. In H929 and MM1.S, DZNep efficiently induced caspase-3 activation and the cleavage of PARP. Furthermore, DZNep treatment induced <t>caspase-8</t> activation and had little effect on full-length caspase-9 levels.
    Reaction Buffer, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc cleaved caspase 8
    Bcl2-mediated SHP protein degradation is associated with activation of caspase 8 pathway. ( A ) Micrographs of DAPI staining (blue), MitoTracker staining (red), and GFP-SHP expression (green) in Huh7 cells transfected with GFP-SHP plasmid, alone or in combination with wide-type Bcl2 (Bcl2wt) or Bcl2 lacking the TM domain (Bcl2ΔTM). Magnification of 20X. ( B ) RT-PCR of Shp and Bcl2 mRNAs in various cells. ( C ) Western blot of SHP protein in Huh7 cells that were expressed with Bcl2 or Bcl2ΔTM in the absence or presence of protein synthesis inhibitor cycloheximide (CHX). ( D )  Left:  Western blot of SHP and BCL2 proteins in Huh7 cells. SHP protein was detected by anti-Flag or anti-SHP antibody.  Middle:  Western blot of SHP and BCL2 proteins in the liver. The mice were the same as in  Fig. 1 .  Left:  Western blot of SHP and BCL2 proteins in primary mouse hepatocytes. The corresponding mRNA level is in  Fig. S4A . Hepatocytes infected with ade-SHP served as a positive control. ( E )  Left and middle:  Western blot of SHP and BCL2 proteins in HepG2 cells treated with DMSO control or 5 μM MG132 for 6 hr. The endogenous SHP protein and exogenously expressed Flag-SHP protein were detected by anti-SHP or anti-Flag antibody, respectively.  Right : Western blot of BCL2 and cas-3 proteins in Bcl2 overexpressed liver. ( F ) Western blot of SHP and BCL2 proteins in HEK 293T cells.  Left:  Cells were pre-treated with caspase inhibitors (50 μM) for 1 hr followed by plasmid transfection for 24 hr.  Right:  Cells were pre-treated with various inhibitors for 1 hr followed by plasmid transfection for 24 hr. For TNFα+ CHX and staurosporine groups, cells were transfected with plasmids for 20 hr followed by a 4 hr treatment. Dose: SP600125 (SP) 50 μM, Rapamycin (Rapa) 10 μM, LY294002 (LY) 50 μM, U0126 10 μM, TNFα 50 ng/ml + CHX 50 μM, staurosporine (STS) 1 μM, and Z-VAD 50 μM.
    Cleaved Caspase 8, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1522 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences reaction buffer
    Bcl2-mediated SHP protein degradation is associated with activation of caspase 8 pathway. ( A ) Micrographs of DAPI staining (blue), MitoTracker staining (red), and GFP-SHP expression (green) in Huh7 cells transfected with GFP-SHP plasmid, alone or in combination with wide-type Bcl2 (Bcl2wt) or Bcl2 lacking the TM domain (Bcl2ΔTM). Magnification of 20X. ( B ) RT-PCR of Shp and Bcl2 mRNAs in various cells. ( C ) Western blot of SHP protein in Huh7 cells that were expressed with Bcl2 or Bcl2ΔTM in the absence or presence of protein synthesis inhibitor cycloheximide (CHX). ( D )  Left:  Western blot of SHP and BCL2 proteins in Huh7 cells. SHP protein was detected by anti-Flag or anti-SHP antibody.  Middle:  Western blot of SHP and BCL2 proteins in the liver. The mice were the same as in  Fig. 1 .  Left:  Western blot of SHP and BCL2 proteins in primary mouse hepatocytes. The corresponding mRNA level is in  Fig. S4A . Hepatocytes infected with ade-SHP served as a positive control. ( E )  Left and middle:  Western blot of SHP and BCL2 proteins in HepG2 cells treated with DMSO control or 5 μM MG132 for 6 hr. The endogenous SHP protein and exogenously expressed Flag-SHP protein were detected by anti-SHP or anti-Flag antibody, respectively.  Right : Western blot of BCL2 and cas-3 proteins in Bcl2 overexpressed liver. ( F ) Western blot of SHP and BCL2 proteins in HEK 293T cells.  Left:  Cells were pre-treated with caspase inhibitors (50 μM) for 1 hr followed by plasmid transfection for 24 hr.  Right:  Cells were pre-treated with various inhibitors for 1 hr followed by plasmid transfection for 24 hr. For TNFα+ CHX and staurosporine groups, cells were transfected with plasmids for 20 hr followed by a 4 hr treatment. Dose: SP600125 (SP) 50 μM, Rapamycin (Rapa) 10 μM, LY294002 (LY) 50 μM, U0126 10 μM, TNFα 50 ng/ml + CHX 50 μM, staurosporine (STS) 1 μM, and Z-VAD 50 μM.
    Reaction Buffer, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc caspase 1
    The effect of <t>caspase-1</t> inhibitor on ZIKV-induced inflammatory responses in mice. A129 mice (6 weeks old) were treated with DMSO or Ac-YVAD-cmk (8 mg/kg) by intraperitoneal injection 30 min and infected with ZIKV (5 × 10 5 PFU) or treated with PBS for the indicated times. a , b A129 mice were pretreated with DMSO ( n = 3; 2 males and 1 female) ( a ) or Ac-YVAD-cmk ( n = 3; 2 males and 1 female) ( b ) and then infected with ZIKV (5 × 10 5 PFU) for 0, 2, 4, and 6 days. ZIKV RNA in the mice blood was determined by RT-PCR. Data shown are whiskers: Min.–max.; * P
    Caspase 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 885 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem reaction buffer
    The effect of <t>caspase-1</t> inhibitor on ZIKV-induced inflammatory responses in mice. A129 mice (6 weeks old) were treated with DMSO or Ac-YVAD-cmk (8 mg/kg) by intraperitoneal injection 30 min and infected with ZIKV (5 × 10 5 PFU) or treated with PBS for the indicated times. a , b A129 mice were pretreated with DMSO ( n = 3; 2 males and 1 female) ( a ) or Ac-YVAD-cmk ( n = 3; 2 males and 1 female) ( b ) and then infected with ZIKV (5 × 10 5 PFU) for 0, 2, 4, and 6 days. ZIKV RNA in the mice blood was determined by RT-PCR. Data shown are whiskers: Min.–max.; * P
    Reaction Buffer, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 95/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc caspase 3
    CoQ10 reduced apoptosis in a rat SCI model. (a) The spine tissue sample protein expression of Bax, Bcl-2, and <t>Caspase-3</t> was measured by Western blot. (b) Immunohistochemical analysis determined the Caspase-3 protein expression. (c) TUNEL staining of the cell apoptosis rate. Scale bar = 50 μ m. Data are presented as mean ± SEM. ∗ P
    Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 21220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rpmi 1640 tissue culture media
    CoQ10 reduced apoptosis in a rat SCI model. (a) The spine tissue sample protein expression of Bax, Bcl-2, and <t>Caspase-3</t> was measured by Western blot. (b) Immunohistochemical analysis determined the Caspase-3 protein expression. (c) TUNEL staining of the cell apoptosis rate. Scale bar = 50 μ m. Data are presented as mean ± SEM. ∗ P
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    Cell Signaling Technology Inc 4 6 diamidino 2 phenylindole
    CoQ10 reduced apoptosis in a rat SCI model. (a) The spine tissue sample protein expression of Bax, Bcl-2, and <t>Caspase-3</t> was measured by Western blot. (b) Immunohistochemical analysis determined the Caspase-3 protein expression. (c) TUNEL staining of the cell apoptosis rate. Scale bar = 50 μ m. Data are presented as mean ± SEM. ∗ P
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    Millipore pp2a specific reaction buffer
    Untransformed hepatocytes showed lower I2PP2A and constitutive NF-κB activity than those in HCC cells and were resistant to isolie A. Basal levels of I2PP2A, <t>PP2A-C</t> and p65 phosphorylation at Ser536 in malignant and untransformed hepatocytes were compared. B. mRNA levels of NF-κB-responsive genes and I2PP2A in malignant and untransformed hepatocytes were compared. C, D. HL-7702 cells and PMHs were incubated with vehicle or 10 μg/mL isolie. After 24 h, transcription of NF-κB target genes and the binding between p65 and Bcl-2 promoter were detected by real-time PCR (C) and ChIP (D), respectively. E. PMH and HL-7702 cells were treated with vehicle or 10 μg/mL isolie. After 48 h, cell apoptosis was assessed using FACS (left) and caspase-3 activity assay (right).
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    Cell Signaling Technology Inc immunoblot analysis
    ( A , B ) Gene expression of CDK4, CD44, Vimentin, CD24, or E-cadherin was analyzed in 51 breast cancer cell line representing different breast cancer subtypes (basal-A, basal-B and luminal) using GOBO gene set analysis. ( C ) SUM159 cells were treated with or without 100 nM of palbociclib for 2 days and subjected to light microscopy (10× objective). ( D ) Representative histogram of MUC1 population. Percentage of MUC1+ subpopulation was analyzed by flow cytometry in SUM159 cells treated with palbociclib. ( E ) Gene expression of cytokeratin 8 (KRT8), mucin1 (MUC1), and alpha smooth muscle actin (ACTA2) in SUM159 cells were measured using RT-PCR. ( F ) SUM159 cells were infected with scr, cyclin D1, CDK4 or CDK6 shRNA overexpressing lentiviruses and imaged by light microscopy. ( G ) E-Cadherin expression was assessed in SUM159 treated or not with palbociclib, using both immunofluorescence and <t>immunoblot</t> analysis. For Western blots, 3 separate independent experiments were performed and quantified using densitometry. ( H ) SUM159 cells were infected with the scr, CDK4 or CDK6 shRNA lentiviruses. E-Cadherin expression was assessed using both immunofluorescence and immunoblot analysis. For Western blots, 3 separate independent experiments were performed and quantified using densitometry. ( I ) Caspase 3/7 activity of infected SUM159 cells was measured using the Caspase Glo 3/7 Assay.
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    Thermo Fisher trizol solution
    ( A , B ) Gene expression of CDK4, CD44, Vimentin, CD24, or E-cadherin was analyzed in 51 breast cancer cell line representing different breast cancer subtypes (basal-A, basal-B and luminal) using GOBO gene set analysis. ( C ) SUM159 cells were treated with or without 100 nM of palbociclib for 2 days and subjected to light microscopy (10× objective). ( D ) Representative histogram of MUC1 population. Percentage of MUC1+ subpopulation was analyzed by flow cytometry in SUM159 cells treated with palbociclib. ( E ) Gene expression of cytokeratin 8 (KRT8), mucin1 (MUC1), and alpha smooth muscle actin (ACTA2) in SUM159 cells were measured using RT-PCR. ( F ) SUM159 cells were infected with scr, cyclin D1, CDK4 or CDK6 shRNA overexpressing lentiviruses and imaged by light microscopy. ( G ) E-Cadherin expression was assessed in SUM159 treated or not with palbociclib, using both immunofluorescence and <t>immunoblot</t> analysis. For Western blots, 3 separate independent experiments were performed and quantified using densitometry. ( H ) SUM159 cells were infected with the scr, CDK4 or CDK6 shRNA lentiviruses. E-Cadherin expression was assessed using both immunofluorescence and immunoblot analysis. For Western blots, 3 separate independent experiments were performed and quantified using densitometry. ( I ) Caspase 3/7 activity of infected SUM159 cells was measured using the Caspase Glo 3/7 Assay.
    Trizol Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3584 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem ac devd afc reaction mix
    ( A , B ) Gene expression of CDK4, CD44, Vimentin, CD24, or E-cadherin was analyzed in 51 breast cancer cell line representing different breast cancer subtypes (basal-A, basal-B and luminal) using GOBO gene set analysis. ( C ) SUM159 cells were treated with or without 100 nM of palbociclib for 2 days and subjected to light microscopy (10× objective). ( D ) Representative histogram of MUC1 population. Percentage of MUC1+ subpopulation was analyzed by flow cytometry in SUM159 cells treated with palbociclib. ( E ) Gene expression of cytokeratin 8 (KRT8), mucin1 (MUC1), and alpha smooth muscle actin (ACTA2) in SUM159 cells were measured using RT-PCR. ( F ) SUM159 cells were infected with scr, cyclin D1, CDK4 or CDK6 shRNA overexpressing lentiviruses and imaged by light microscopy. ( G ) E-Cadherin expression was assessed in SUM159 treated or not with palbociclib, using both immunofluorescence and <t>immunoblot</t> analysis. For Western blots, 3 separate independent experiments were performed and quantified using densitometry. ( H ) SUM159 cells were infected with the scr, CDK4 or CDK6 shRNA lentiviruses. E-Cadherin expression was assessed using both immunofluorescence and immunoblot analysis. For Western blots, 3 separate independent experiments were performed and quantified using densitometry. ( I ) Caspase 3/7 activity of infected SUM159 cells was measured using the Caspase Glo 3/7 Assay.
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    Thermo Fisher propidium iodide solution pi
    PFKFB3 is important for cell survival during paclitaxel-induced mitotic arrest A . PFKFB3 mRNA and protein expression after siRNA-mediated depletion of PFKFB3 for 72 h was determined by qPCR and western blotting. B . The quantification of mitotically arrested cells by flow cytometry, C . caspase 3/7 activity using Caspase-Glo 3/7 assay D . <t>propidium</t> iodide incorporation by flow cytometry and E . ROS levels using CM-H2DCFDA staining and flow cytometry were assessed after siRNA-mediated depletion of PFKFB3 for 72 h followed by 50 nM paclitaxel for 16 h and a mitotic shake-off in SKOV3 cells. F . The levels of ROS after PFKFB3 depletion alone, without paclitaxel treatment or mitotic cell isolation, was also analysed using CM-H2DCFDA staining and flow cytometry. Data are expressed as +SEM of three experiments.
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    Image Search Results


    TNF provokes a cholesterol-insensitive processing of SREBP-1 in ethanol-exposed cells that is promoted by caspase-4 or -12 activation, resulting in the inappropriate induction of lipogenic enzymes and consequent accumulation of triglycerides. DISC , death inducing signaling complex.

    Journal: The Journal of Biological Chemistry

    Article Title: Tumor Necrosis Factor-? Can Provoke Cleavage and Activation of Sterol Regulatory Element-binding Protein in Ethanol-exposed Cells via a Caspase-dependent Pathway That Is Cholesterol Insensitive *

    doi: 10.1074/jbc.M800237200

    Figure Lengend Snippet: TNF provokes a cholesterol-insensitive processing of SREBP-1 in ethanol-exposed cells that is promoted by caspase-4 or -12 activation, resulting in the inappropriate induction of lipogenic enzymes and consequent accumulation of triglycerides. DISC , death inducing signaling complex.

    Article Snippet: The purified SREBP-1 was incubated with 5 units of recombinant caspase-3, -4, or -2 (EMD Biosciences) in caspase reaction buffer (50 m m HEPES, pH 7.2, 50 m m NaCl, 0.1% CHAPS, 10 m m EDTA, 5% glycerol, and 10 m m dithiothreitol).

    Techniques: Activation Assay

    The upper panel (A.) illustrates lung Caspase-3 activity (μM AFC/mg/min) in mice (n=5 mice/group) subjected to sham surgery compared to those subjected to hemorrhage followed 24 hrs later by cecal ligation and puncture (Hem/CLP) that were either

    Journal: Shock (Augusta, Ga.)

    Article Title: Local Tissue Expression of the Cell Death Ligand, FasL, Plays a Central Role in the Development of Extra-Pulmonary Acute Lung Injury

    doi: 10.1097/SHK.0b013e31821c236d

    Figure Lengend Snippet: The upper panel (A.) illustrates lung Caspase-3 activity (μM AFC/mg/min) in mice (n=5 mice/group) subjected to sham surgery compared to those subjected to hemorrhage followed 24 hrs later by cecal ligation and puncture (Hem/CLP) that were either

    Article Snippet: Fifty μg of tissue protein in fifty μl of buffer were added to 50 μl of Caspase-3 reaction buffer (containing 100mM HEPES, 2% Sucrose, 0.2% CHAPS, 10mM Dithiothreitol (all Sigma) and 100μM Ac-DEVD-AFC fluorogenic substrate (Biomol, Plymouth Meeting, PA)).

    Techniques: Activity Assay, Mouse Assay, Ligation

    Analysis of DZNep effects by Western-blot and RT-PCR. DZNep reduced EZH2 and ALOX5 levels, and induced caspase-dependent cell apoptosis. Cells were treated with 0.5 umol/L DZNep for 72 h, and then whole-cell lysates were analyzed by Western-blot analysis. β-Actin was used as an internal control. Treatment with DZNep reduced protein levels of EZH2 in MM cells. ALOX5 was over-expressed in H929 and was down-regulated greatly by DZNep treatment. In H929 and MM1.S, DZNep efficiently induced caspase-3 activation and the cleavage of PARP. Furthermore, DZNep treatment induced caspase-8 activation and had little effect on full-length caspase-9 levels.

    Journal: PLoS ONE

    Article Title: Determinants of Sensitivity to DZNep Induced Apoptosis in Multiple Myeloma Cells

    doi: 10.1371/journal.pone.0021583

    Figure Lengend Snippet: Analysis of DZNep effects by Western-blot and RT-PCR. DZNep reduced EZH2 and ALOX5 levels, and induced caspase-dependent cell apoptosis. Cells were treated with 0.5 umol/L DZNep for 72 h, and then whole-cell lysates were analyzed by Western-blot analysis. β-Actin was used as an internal control. Treatment with DZNep reduced protein levels of EZH2 in MM cells. ALOX5 was over-expressed in H929 and was down-regulated greatly by DZNep treatment. In H929 and MM1.S, DZNep efficiently induced caspase-3 activation and the cleavage of PARP. Furthermore, DZNep treatment induced caspase-8 activation and had little effect on full-length caspase-9 levels.

    Article Snippet: The blots were probed with antibodies against EZH2 (#3147), Cleaved Caspase-3 (Asp175) (#9661), Caspase-8 (#9746) and Caspase-9 (#9508) from Cell Signaling Technology.

    Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Activation Assay

    Bcl2-mediated SHP protein degradation is associated with activation of caspase 8 pathway. ( A ) Micrographs of DAPI staining (blue), MitoTracker staining (red), and GFP-SHP expression (green) in Huh7 cells transfected with GFP-SHP plasmid, alone or in combination with wide-type Bcl2 (Bcl2wt) or Bcl2 lacking the TM domain (Bcl2ΔTM). Magnification of 20X. ( B ) RT-PCR of Shp and Bcl2 mRNAs in various cells. ( C ) Western blot of SHP protein in Huh7 cells that were expressed with Bcl2 or Bcl2ΔTM in the absence or presence of protein synthesis inhibitor cycloheximide (CHX). ( D )  Left:  Western blot of SHP and BCL2 proteins in Huh7 cells. SHP protein was detected by anti-Flag or anti-SHP antibody.  Middle:  Western blot of SHP and BCL2 proteins in the liver. The mice were the same as in  Fig. 1 .  Left:  Western blot of SHP and BCL2 proteins in primary mouse hepatocytes. The corresponding mRNA level is in  Fig. S4A . Hepatocytes infected with ade-SHP served as a positive control. ( E )  Left and middle:  Western blot of SHP and BCL2 proteins in HepG2 cells treated with DMSO control or 5 μM MG132 for 6 hr. The endogenous SHP protein and exogenously expressed Flag-SHP protein were detected by anti-SHP or anti-Flag antibody, respectively.  Right : Western blot of BCL2 and cas-3 proteins in Bcl2 overexpressed liver. ( F ) Western blot of SHP and BCL2 proteins in HEK 293T cells.  Left:  Cells were pre-treated with caspase inhibitors (50 μM) for 1 hr followed by plasmid transfection for 24 hr.  Right:  Cells were pre-treated with various inhibitors for 1 hr followed by plasmid transfection for 24 hr. For TNFα+ CHX and staurosporine groups, cells were transfected with plasmids for 20 hr followed by a 4 hr treatment. Dose: SP600125 (SP) 50 μM, Rapamycin (Rapa) 10 μM, LY294002 (LY) 50 μM, U0126 10 μM, TNFα 50 ng/ml + CHX 50 μM, staurosporine (STS) 1 μM, and Z-VAD 50 μM.

    Journal: Scientific Reports

    Article Title: Bcl2 is a critical regulator of bile acid homeostasis by dictating Shp and lncRNA H19 function

    doi: 10.1038/srep20559

    Figure Lengend Snippet: Bcl2-mediated SHP protein degradation is associated with activation of caspase 8 pathway. ( A ) Micrographs of DAPI staining (blue), MitoTracker staining (red), and GFP-SHP expression (green) in Huh7 cells transfected with GFP-SHP plasmid, alone or in combination with wide-type Bcl2 (Bcl2wt) or Bcl2 lacking the TM domain (Bcl2ΔTM). Magnification of 20X. ( B ) RT-PCR of Shp and Bcl2 mRNAs in various cells. ( C ) Western blot of SHP protein in Huh7 cells that were expressed with Bcl2 or Bcl2ΔTM in the absence or presence of protein synthesis inhibitor cycloheximide (CHX). ( D ) Left: Western blot of SHP and BCL2 proteins in Huh7 cells. SHP protein was detected by anti-Flag or anti-SHP antibody. Middle: Western blot of SHP and BCL2 proteins in the liver. The mice were the same as in Fig. 1 . Left: Western blot of SHP and BCL2 proteins in primary mouse hepatocytes. The corresponding mRNA level is in Fig. S4A . Hepatocytes infected with ade-SHP served as a positive control. ( E ) Left and middle: Western blot of SHP and BCL2 proteins in HepG2 cells treated with DMSO control or 5 μM MG132 for 6 hr. The endogenous SHP protein and exogenously expressed Flag-SHP protein were detected by anti-SHP or anti-Flag antibody, respectively. Right : Western blot of BCL2 and cas-3 proteins in Bcl2 overexpressed liver. ( F ) Western blot of SHP and BCL2 proteins in HEK 293T cells. Left: Cells were pre-treated with caspase inhibitors (50 μM) for 1 hr followed by plasmid transfection for 24 hr. Right: Cells were pre-treated with various inhibitors for 1 hr followed by plasmid transfection for 24 hr. For TNFα+ CHX and staurosporine groups, cells were transfected with plasmids for 20 hr followed by a 4 hr treatment. Dose: SP600125 (SP) 50 μM, Rapamycin (Rapa) 10 μM, LY294002 (LY) 50 μM, U0126 10 μM, TNFα 50 ng/ml + CHX 50 μM, staurosporine (STS) 1 μM, and Z-VAD 50 μM.

    Article Snippet: The following antibodies were used for protein precipitation (IP) and Western blots (WB): antibodies against Flag (Sigma, F7425), GFP (Sigma, G1544), Bcl2 (Abcam, ab7973), β-actin (Sigma, A-1978), α-Tubulin (Sigma, T6074), PARP (Cell signaling, #9542), SHP (Santa Cruz, sc-30169), caspase-3 (Cell signaling, #9661), cleaved caspase-8 (Cell signaling, #9748).

    Techniques: Activation Assay, Staining, Expressing, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Mouse Assay, Infection, Positive Control

    The effect of caspase-1 inhibitor on ZIKV-induced inflammatory responses in mice. A129 mice (6 weeks old) were treated with DMSO or Ac-YVAD-cmk (8 mg/kg) by intraperitoneal injection 30 min and infected with ZIKV (5 × 10 5 PFU) or treated with PBS for the indicated times. a , b A129 mice were pretreated with DMSO ( n = 3; 2 males and 1 female) ( a ) or Ac-YVAD-cmk ( n = 3; 2 males and 1 female) ( b ) and then infected with ZIKV (5 × 10 5 PFU) for 0, 2, 4, and 6 days. ZIKV RNA in the mice blood was determined by RT-PCR. Data shown are whiskers: Min.–max.; * P

    Journal: Nature Communications

    Article Title: Zika virus infection induces host inflammatory responses by facilitating NLRP3 inflammasome assembly and interleukin-1β secretion

    doi: 10.1038/s41467-017-02645-3

    Figure Lengend Snippet: The effect of caspase-1 inhibitor on ZIKV-induced inflammatory responses in mice. A129 mice (6 weeks old) were treated with DMSO or Ac-YVAD-cmk (8 mg/kg) by intraperitoneal injection 30 min and infected with ZIKV (5 × 10 5 PFU) or treated with PBS for the indicated times. a , b A129 mice were pretreated with DMSO ( n = 3; 2 males and 1 female) ( a ) or Ac-YVAD-cmk ( n = 3; 2 males and 1 female) ( b ) and then infected with ZIKV (5 × 10 5 PFU) for 0, 2, 4, and 6 days. ZIKV RNA in the mice blood was determined by RT-PCR. Data shown are whiskers: Min.–max.; * P

    Article Snippet: The lyophilized product was dissolved in 100 μl phosphate-buffered saline (PBS) and mixed with sodium dodecyl sulfate (SDS) loading buffer for western blot analyses using antibodies against activated caspase-1 (D5782 1:500; Cell Signaling) or mature IL-1β (Asp116 1:500; Cell Signaling).

    Techniques: Mouse Assay, Injection, Infection, Reverse Transcription Polymerase Chain Reaction

    The role of ZIKV NS5 in the activation of the NLRP3 inflammasome. a , b HEK293T cells were co-transfected with plasmids encoding NLRP3, ASC, pro-Casp1, and pro-IL-1β. The cells were then transfected with pFlag-Env, pFlag-Capsid, pFlag-NS2A, pFlag-NS2B, pFlag-NS4B, or pFlag-NS5 ( a ) or transfected with pFlag-NS5 ( b ). IL-1β levels in the supernatants were determined by ELISA ( a , b , top). The mature IL-1β, cleaved pro-Casp-1, ZIKV NS5, and GAPDH levels in the cell lysates were determined by western blot ( b , bottom). c HEK293T cells were co-transfected with pcdna3.1(+)-ASC, pHA-NLRP3, or pFlag-NS5. Cell lysates were subjected to western blots (input) or IP using an IgG or anti-HA antibody and analyzed using immunoblotting with anti-HA, anti-ASC, or anti-Flag antibodies (IP). d HEK293T cells were co-transfected with pcdna3.1(+)-ASC, Flag-NLRP3 or HA-NS5 for 24 h. Cell lysates were prepared and the pellets were subjected to ASC oligomerization assays (top) or western blots (bottom). e – g THP-1 cells were infected with CT-lentivirus or NS5-lentivirus (NS5-encoding lentivirus), which were differentiated into macrophages by stimulating with TPA and then treated with 5 mM ATP for 2 h. IL-1β levels in the supernatants were determined by ELISA ( e ). p17 in the supernatants and pro-IL-1β, pro-Casp-1, and GAPDH in the cell lysates (Lys) were determined by western blot ( f ). Cell pellets were prepared and subjected to ASC oligomerization assays ( g , top). ASC in the cell lysates was determined by western blot ( g , bottom). h , i THP-1 cells were infected with CT-lentivirus or NS5-lentivirus, which were differentiated into macrophages by stimulating with TPA and then treated with caspase-1 inhibitor Ac-YVAD-cmk for 1 h. IL-1β levels in the supernatants were determined by ELISA ( h ). p17 in the supernatants and pro-IL-1β, pro-Casp-1, and GAPDH in the cell lysates (Lys) were determined by western blot ( i ). The number of replicates is two. Data shown are means ± s.e.m; * P

    Journal: Nature Communications

    Article Title: Zika virus infection induces host inflammatory responses by facilitating NLRP3 inflammasome assembly and interleukin-1β secretion

    doi: 10.1038/s41467-017-02645-3

    Figure Lengend Snippet: The role of ZIKV NS5 in the activation of the NLRP3 inflammasome. a , b HEK293T cells were co-transfected with plasmids encoding NLRP3, ASC, pro-Casp1, and pro-IL-1β. The cells were then transfected with pFlag-Env, pFlag-Capsid, pFlag-NS2A, pFlag-NS2B, pFlag-NS4B, or pFlag-NS5 ( a ) or transfected with pFlag-NS5 ( b ). IL-1β levels in the supernatants were determined by ELISA ( a , b , top). The mature IL-1β, cleaved pro-Casp-1, ZIKV NS5, and GAPDH levels in the cell lysates were determined by western blot ( b , bottom). c HEK293T cells were co-transfected with pcdna3.1(+)-ASC, pHA-NLRP3, or pFlag-NS5. Cell lysates were subjected to western blots (input) or IP using an IgG or anti-HA antibody and analyzed using immunoblotting with anti-HA, anti-ASC, or anti-Flag antibodies (IP). d HEK293T cells were co-transfected with pcdna3.1(+)-ASC, Flag-NLRP3 or HA-NS5 for 24 h. Cell lysates were prepared and the pellets were subjected to ASC oligomerization assays (top) or western blots (bottom). e – g THP-1 cells were infected with CT-lentivirus or NS5-lentivirus (NS5-encoding lentivirus), which were differentiated into macrophages by stimulating with TPA and then treated with 5 mM ATP for 2 h. IL-1β levels in the supernatants were determined by ELISA ( e ). p17 in the supernatants and pro-IL-1β, pro-Casp-1, and GAPDH in the cell lysates (Lys) were determined by western blot ( f ). Cell pellets were prepared and subjected to ASC oligomerization assays ( g , top). ASC in the cell lysates was determined by western blot ( g , bottom). h , i THP-1 cells were infected with CT-lentivirus or NS5-lentivirus, which were differentiated into macrophages by stimulating with TPA and then treated with caspase-1 inhibitor Ac-YVAD-cmk for 1 h. IL-1β levels in the supernatants were determined by ELISA ( h ). p17 in the supernatants and pro-IL-1β, pro-Casp-1, and GAPDH in the cell lysates (Lys) were determined by western blot ( i ). The number of replicates is two. Data shown are means ± s.e.m; * P

    Article Snippet: The lyophilized product was dissolved in 100 μl phosphate-buffered saline (PBS) and mixed with sodium dodecyl sulfate (SDS) loading buffer for western blot analyses using antibodies against activated caspase-1 (D5782 1:500; Cell Signaling) or mature IL-1β (Asp116 1:500; Cell Signaling).

    Techniques: Activation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot, Infection

    CoQ10 reduced apoptosis in a rat SCI model. (a) The spine tissue sample protein expression of Bax, Bcl-2, and Caspase-3 was measured by Western blot. (b) Immunohistochemical analysis determined the Caspase-3 protein expression. (c) TUNEL staining of the cell apoptosis rate. Scale bar = 50 μ m. Data are presented as mean ± SEM. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Coenzyme Q10 Regulation of Apoptosis and Oxidative Stress in H2O2 Induced BMSC Death by Modulating the Nrf-2/NQO-1 Signaling Pathway and Its Application in a Model of Spinal Cord Injury

    doi: 10.1155/2019/6493081

    Figure Lengend Snippet: CoQ10 reduced apoptosis in a rat SCI model. (a) The spine tissue sample protein expression of Bax, Bcl-2, and Caspase-3 was measured by Western blot. (b) Immunohistochemical analysis determined the Caspase-3 protein expression. (c) TUNEL staining of the cell apoptosis rate. Scale bar = 50 μ m. Data are presented as mean ± SEM. ∗ P

    Article Snippet: The sections were incubated overnight with Caspase-3 (1 : 50 in PBS, Cell Signaling Technology) at 4°C.

    Techniques: Expressing, Western Blot, Immunohistochemistry, TUNEL Assay, Staining

    CoQ10 alleviated H 2 O 2 -induced BMSC apoptosis. Cells were pretreated with CoQ10 (40 μ M) for 2 h followed with H 2 O 2 (300 μ M) for 24 h. (a) Annexin V-FITC/PI assay was used to evaluate the apoptosis (A) control group, (B) CoQ10 group, (C) H 2 O 2 group, (D) H 2 O 2 +CoQ10 group, and (E) H 2 O 2 +CoQ10+brusatol group. (b) TUNEL assays (with green fluorescence) measured the cell apoptosis. (c) The protein expression of Bax, Bcl-2, and Caspase-3 was measured by Western blot. (d) The gene expression of Bax, Bcl-2, and Caspase-3 was determined by real-time quantitative PCR. Data are presented as mean ± SEM. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Coenzyme Q10 Regulation of Apoptosis and Oxidative Stress in H2O2 Induced BMSC Death by Modulating the Nrf-2/NQO-1 Signaling Pathway and Its Application in a Model of Spinal Cord Injury

    doi: 10.1155/2019/6493081

    Figure Lengend Snippet: CoQ10 alleviated H 2 O 2 -induced BMSC apoptosis. Cells were pretreated with CoQ10 (40 μ M) for 2 h followed with H 2 O 2 (300 μ M) for 24 h. (a) Annexin V-FITC/PI assay was used to evaluate the apoptosis (A) control group, (B) CoQ10 group, (C) H 2 O 2 group, (D) H 2 O 2 +CoQ10 group, and (E) H 2 O 2 +CoQ10+brusatol group. (b) TUNEL assays (with green fluorescence) measured the cell apoptosis. (c) The protein expression of Bax, Bcl-2, and Caspase-3 was measured by Western blot. (d) The gene expression of Bax, Bcl-2, and Caspase-3 was determined by real-time quantitative PCR. Data are presented as mean ± SEM. ∗ P

    Article Snippet: The sections were incubated overnight with Caspase-3 (1 : 50 in PBS, Cell Signaling Technology) at 4°C.

    Techniques: TUNEL Assay, Fluorescence, Expressing, Western Blot, Real-time Polymerase Chain Reaction

    Untransformed hepatocytes showed lower I2PP2A and constitutive NF-κB activity than those in HCC cells and were resistant to isolie A. Basal levels of I2PP2A, PP2A-C and p65 phosphorylation at Ser536 in malignant and untransformed hepatocytes were compared. B. mRNA levels of NF-κB-responsive genes and I2PP2A in malignant and untransformed hepatocytes were compared. C, D. HL-7702 cells and PMHs were incubated with vehicle or 10 μg/mL isolie. After 24 h, transcription of NF-κB target genes and the binding between p65 and Bcl-2 promoter were detected by real-time PCR (C) and ChIP (D), respectively. E. PMH and HL-7702 cells were treated with vehicle or 10 μg/mL isolie. After 48 h, cell apoptosis was assessed using FACS (left) and caspase-3 activity assay (right).

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: Untransformed hepatocytes showed lower I2PP2A and constitutive NF-κB activity than those in HCC cells and were resistant to isolie A. Basal levels of I2PP2A, PP2A-C and p65 phosphorylation at Ser536 in malignant and untransformed hepatocytes were compared. B. mRNA levels of NF-κB-responsive genes and I2PP2A in malignant and untransformed hepatocytes were compared. C, D. HL-7702 cells and PMHs were incubated with vehicle or 10 μg/mL isolie. After 24 h, transcription of NF-κB target genes and the binding between p65 and Bcl-2 promoter were detected by real-time PCR (C) and ChIP (D), respectively. E. PMH and HL-7702 cells were treated with vehicle or 10 μg/mL isolie. After 48 h, cell apoptosis was assessed using FACS (left) and caspase-3 activity assay (right).

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: Activity Assay, Incubation, Binding Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, FACS, Caspase-3 Activity Assay

    Knockdown of PP2A-C abrogated isolie-induced p65 dephosphorylation and HCC cell apoptosis A. HCC cells were transfected with control or PP2A-C specific siRNA. After 48 h, protein levels of PP2A-C were detected. B, C, D, E. HepG2 and Huh-7 cells were transfected with control or PP2A-C siRNAs for 48 h. Then, cells were treated with 10 μg/mL isolie. After 24 h, NF-κB activities were detected by luciferase reporter assay (B). Levels of indicated proteins were examined by immunoblotting (C). mRNA levels of Bcl-2, Bcl-xL and MMP9 were detected by real-time PCR (D). Interactions between p65 and promoter regions of indicated genes were determined by ChIP (E). F. Indicated HCC cell transfectants were treated with 10 μg/mL isolie. After 48 h, cell apoptosis were quantified by FACS (left) and caspase-3 activity assay (right). ** p

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: Knockdown of PP2A-C abrogated isolie-induced p65 dephosphorylation and HCC cell apoptosis A. HCC cells were transfected with control or PP2A-C specific siRNA. After 48 h, protein levels of PP2A-C were detected. B, C, D, E. HepG2 and Huh-7 cells were transfected with control or PP2A-C siRNAs for 48 h. Then, cells were treated with 10 μg/mL isolie. After 24 h, NF-κB activities were detected by luciferase reporter assay (B). Levels of indicated proteins were examined by immunoblotting (C). mRNA levels of Bcl-2, Bcl-xL and MMP9 were detected by real-time PCR (D). Interactions between p65 and promoter regions of indicated genes were determined by ChIP (E). F. Indicated HCC cell transfectants were treated with 10 μg/mL isolie. After 48 h, cell apoptosis were quantified by FACS (left) and caspase-3 activity assay (right). ** p

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: De-Phosphorylation Assay, Transfection, Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, FACS, Caspase-3 Activity Assay

    Isolie suppressed PP2A/I2PP2A interaction A. HepG2 cells were incubated with indicated concentrations of isolie for 24 h. Then, PP2A/I2PP2A interaction was detected by immunoprecipitation (left). Phosphatase activity of immunoprecipitated PP2A was quantified by phosphatase activity assay (right). B. Indicated dosages of isolie or vehicle were injected intraperitoneally into nude mice bearing Huh-7 xenograft tumors. In the end, tumor lysates were prepared and subjected to the same experiments as described in A. C. Kunming mice bearing H22 transplanted tumors were treated with indicated dosages of isolie by gavage. In the end, tumor lysates were prepared and subjected to the same experiments as described in A. D. Purified 6 × his-tagged I2PP2A proteins already associated with Ni-NTA agarose were incubated with purified PP2A-C proteins and indicated concentrations of isolie. Amounts of PP2A-C proteins bound to I2PP2A proteins in vitro were determined by immunoblotting. * p

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: Isolie suppressed PP2A/I2PP2A interaction A. HepG2 cells were incubated with indicated concentrations of isolie for 24 h. Then, PP2A/I2PP2A interaction was detected by immunoprecipitation (left). Phosphatase activity of immunoprecipitated PP2A was quantified by phosphatase activity assay (right). B. Indicated dosages of isolie or vehicle were injected intraperitoneally into nude mice bearing Huh-7 xenograft tumors. In the end, tumor lysates were prepared and subjected to the same experiments as described in A. C. Kunming mice bearing H22 transplanted tumors were treated with indicated dosages of isolie by gavage. In the end, tumor lysates were prepared and subjected to the same experiments as described in A. D. Purified 6 × his-tagged I2PP2A proteins already associated with Ni-NTA agarose were incubated with purified PP2A-C proteins and indicated concentrations of isolie. Amounts of PP2A-C proteins bound to I2PP2A proteins in vitro were determined by immunoblotting. * p

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: Incubation, Immunoprecipitation, Activity Assay, Phosphatase Assay, Injection, Mouse Assay, Purification, In Vitro

    Isolie-provoked p65 dephosphorylation was initiated by decreased PP2A/I2PP2A interaction A, B, C. HCC cells were transfected with control or I2PP2A specific siRNA. After 48 h, levels of I2PP2A and effect of I2PP2A knockdown on p65/PP2A association as well as p65 phosphorylation at Ser536 were determined (A). NF-κB activities were measured by luciferase reporter assay (B). After 72 h, cell apoptosis were quantified by FACS (C, left) and caspase-3 activity assay (C, right). D. The encoding gene of human I2PP2A was stably transfected into indicated HCC cells, using empty vectors as the negative control. Then, I2PP2A proteins were detected. E, F. HCC cell transfectants as indicated were treated with 10 μg/mL isolie. After 24 h, levels of p65 phosphorylation at Ser536 (E) and NF-κB activity (F) were detected. G. Indicated HCC cell transfectants were incubated with 10 μg/mL isolie. After 48 h, cell apoptosis was detected by FACS (left) and caspase-3 activity assay (right). H, I. Indicated Huh-7 HCC cell transfectants were subcutaneously transplanted into nude mice. When volumes of xenograft tumors reached about 200 mm 3 , mice were treated with 10 mg/kg isolie or vehicle once daily for 20 d by gavage. Tumor growth was monitored every 5 d (H). Finally, xenograft tumor lysates were prepared and subjected to caspase-3 activity assay (I). ** p

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: Isolie-provoked p65 dephosphorylation was initiated by decreased PP2A/I2PP2A interaction A, B, C. HCC cells were transfected with control or I2PP2A specific siRNA. After 48 h, levels of I2PP2A and effect of I2PP2A knockdown on p65/PP2A association as well as p65 phosphorylation at Ser536 were determined (A). NF-κB activities were measured by luciferase reporter assay (B). After 72 h, cell apoptosis were quantified by FACS (C, left) and caspase-3 activity assay (C, right). D. The encoding gene of human I2PP2A was stably transfected into indicated HCC cells, using empty vectors as the negative control. Then, I2PP2A proteins were detected. E, F. HCC cell transfectants as indicated were treated with 10 μg/mL isolie. After 24 h, levels of p65 phosphorylation at Ser536 (E) and NF-κB activity (F) were detected. G. Indicated HCC cell transfectants were incubated with 10 μg/mL isolie. After 48 h, cell apoptosis was detected by FACS (left) and caspase-3 activity assay (right). H, I. Indicated Huh-7 HCC cell transfectants were subcutaneously transplanted into nude mice. When volumes of xenograft tumors reached about 200 mm 3 , mice were treated with 10 mg/kg isolie or vehicle once daily for 20 d by gavage. Tumor growth was monitored every 5 d (H). Finally, xenograft tumor lysates were prepared and subjected to caspase-3 activity assay (I). ** p

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: De-Phosphorylation Assay, Transfection, Luciferase, Reporter Assay, FACS, Caspase-3 Activity Assay, Stable Transfection, Negative Control, Activity Assay, Incubation, Mouse Assay

    Summary by a schematic panel In untreated HCC cells, I2PP2A binds to PP2A. PP2A/I2PP2A association reduces the phosphatase activity of PP2A and prevents PP2A from binding to NF-κB p65 subunit. These events benefit p65 hyperphosphorylation at Ser536 and hyperactivation of NF-κB, which is important for the survival of HCC cells. Isolie disrupts PP2A/I2PP2A interaction, increases p65/PP2A association and upregulates the phosphatase activity of PP2A. In this way, p65 is dephosphorylated at Ser536, giving rise to the inhibition of NF-κB and subsequent apoptosis of HCC cells.

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: Summary by a schematic panel In untreated HCC cells, I2PP2A binds to PP2A. PP2A/I2PP2A association reduces the phosphatase activity of PP2A and prevents PP2A from binding to NF-κB p65 subunit. These events benefit p65 hyperphosphorylation at Ser536 and hyperactivation of NF-κB, which is important for the survival of HCC cells. Isolie disrupts PP2A/I2PP2A interaction, increases p65/PP2A association and upregulates the phosphatase activity of PP2A. In this way, p65 is dephosphorylated at Ser536, giving rise to the inhibition of NF-κB and subsequent apoptosis of HCC cells.

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: Activity Assay, Binding Assay, Inhibition

    PP2A participated in isolie-induced p65 dephosphorylation A. Indicated HCC cells were treated with different concentrations of isolie. After 24 h, protein levels of PP2A-C (left) and p65/PP2A interaction (right) were determined. B. Indicated dosages of isolie or vehicle were intraperitoneally injected into tumor-bearing nude mice over the course of 3 weeks. Finally, effects of isolie on p65/PP2A interaction in Huh-7 tumor tissues were determined. C. Kunming mice bearing H22 tumors were treated with indicated dosages of isolie by gavage for 10 d. Then, effects of isolie on p65/PP2A interaction in transplanted tumor tissues were detected. D, E. HCC cells were treated with 10 μg/mL isolie and indicated concentrations of OA. After 24 h, phosphorylation of p65 at Ser536 was determined (D), and NF-κB activity was measured by NF-κB-dependent luciferase reporter assay (E). F. Cells were subjected to the same treatment as described in D and E. Cell apoptosis was quantified by FACS (left) and caspase-3 activity assay (right) 48 h later. * p

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: PP2A participated in isolie-induced p65 dephosphorylation A. Indicated HCC cells were treated with different concentrations of isolie. After 24 h, protein levels of PP2A-C (left) and p65/PP2A interaction (right) were determined. B. Indicated dosages of isolie or vehicle were intraperitoneally injected into tumor-bearing nude mice over the course of 3 weeks. Finally, effects of isolie on p65/PP2A interaction in Huh-7 tumor tissues were determined. C. Kunming mice bearing H22 tumors were treated with indicated dosages of isolie by gavage for 10 d. Then, effects of isolie on p65/PP2A interaction in transplanted tumor tissues were detected. D, E. HCC cells were treated with 10 μg/mL isolie and indicated concentrations of OA. After 24 h, phosphorylation of p65 at Ser536 was determined (D), and NF-κB activity was measured by NF-κB-dependent luciferase reporter assay (E). F. Cells were subjected to the same treatment as described in D and E. Cell apoptosis was quantified by FACS (left) and caspase-3 activity assay (right) 48 h later. * p

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: De-Phosphorylation Assay, Injection, Mouse Assay, Activity Assay, Luciferase, Reporter Assay, FACS, Caspase-3 Activity Assay

    ( A , B ) Gene expression of CDK4, CD44, Vimentin, CD24, or E-cadherin was analyzed in 51 breast cancer cell line representing different breast cancer subtypes (basal-A, basal-B and luminal) using GOBO gene set analysis. ( C ) SUM159 cells were treated with or without 100 nM of palbociclib for 2 days and subjected to light microscopy (10× objective). ( D ) Representative histogram of MUC1 population. Percentage of MUC1+ subpopulation was analyzed by flow cytometry in SUM159 cells treated with palbociclib. ( E ) Gene expression of cytokeratin 8 (KRT8), mucin1 (MUC1), and alpha smooth muscle actin (ACTA2) in SUM159 cells were measured using RT-PCR. ( F ) SUM159 cells were infected with scr, cyclin D1, CDK4 or CDK6 shRNA overexpressing lentiviruses and imaged by light microscopy. ( G ) E-Cadherin expression was assessed in SUM159 treated or not with palbociclib, using both immunofluorescence and immunoblot analysis. For Western blots, 3 separate independent experiments were performed and quantified using densitometry. ( H ) SUM159 cells were infected with the scr, CDK4 or CDK6 shRNA lentiviruses. E-Cadherin expression was assessed using both immunofluorescence and immunoblot analysis. For Western blots, 3 separate independent experiments were performed and quantified using densitometry. ( I ) Caspase 3/7 activity of infected SUM159 cells was measured using the Caspase Glo 3/7 Assay.

    Journal: Scientific Reports

    Article Title: CDK4 regulates cancer stemness and is a novel therapeutic target for triple-negative breast cancer

    doi: 10.1038/srep35383

    Figure Lengend Snippet: ( A , B ) Gene expression of CDK4, CD44, Vimentin, CD24, or E-cadherin was analyzed in 51 breast cancer cell line representing different breast cancer subtypes (basal-A, basal-B and luminal) using GOBO gene set analysis. ( C ) SUM159 cells were treated with or without 100 nM of palbociclib for 2 days and subjected to light microscopy (10× objective). ( D ) Representative histogram of MUC1 population. Percentage of MUC1+ subpopulation was analyzed by flow cytometry in SUM159 cells treated with palbociclib. ( E ) Gene expression of cytokeratin 8 (KRT8), mucin1 (MUC1), and alpha smooth muscle actin (ACTA2) in SUM159 cells were measured using RT-PCR. ( F ) SUM159 cells were infected with scr, cyclin D1, CDK4 or CDK6 shRNA overexpressing lentiviruses and imaged by light microscopy. ( G ) E-Cadherin expression was assessed in SUM159 treated or not with palbociclib, using both immunofluorescence and immunoblot analysis. For Western blots, 3 separate independent experiments were performed and quantified using densitometry. ( H ) SUM159 cells were infected with the scr, CDK4 or CDK6 shRNA lentiviruses. E-Cadherin expression was assessed using both immunofluorescence and immunoblot analysis. For Western blots, 3 separate independent experiments were performed and quantified using densitometry. ( I ) Caspase 3/7 activity of infected SUM159 cells was measured using the Caspase Glo 3/7 Assay.

    Article Snippet: Cell lysates were boiled with 6× sodium dodecyl sulfate (SDS) Laemmli sample buffer for five minutes and subjected to immunoblot analysis using antibodies against cyclin D1 (Neomarker), phospho-retinoblastoma Ser780 (Cell Signaling Technology), and anti-CDK4 (Santa Cruz Biotechnology).

    Techniques: Expressing, Light Microscopy, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Infection, shRNA, Immunofluorescence, Western Blot, Activity Assay, Caspase-Glo Assay

    PFKFB3 is important for cell survival during paclitaxel-induced mitotic arrest A . PFKFB3 mRNA and protein expression after siRNA-mediated depletion of PFKFB3 for 72 h was determined by qPCR and western blotting. B . The quantification of mitotically arrested cells by flow cytometry, C . caspase 3/7 activity using Caspase-Glo 3/7 assay D . propidium iodide incorporation by flow cytometry and E . ROS levels using CM-H2DCFDA staining and flow cytometry were assessed after siRNA-mediated depletion of PFKFB3 for 72 h followed by 50 nM paclitaxel for 16 h and a mitotic shake-off in SKOV3 cells. F . The levels of ROS after PFKFB3 depletion alone, without paclitaxel treatment or mitotic cell isolation, was also analysed using CM-H2DCFDA staining and flow cytometry. Data are expressed as +SEM of three experiments.

    Journal: Oncotarget

    Article Title: Loss of PFKFB4 induces cell death in mitotically arrested ovarian cancer cells

    doi: 10.18632/oncotarget.14910

    Figure Lengend Snippet: PFKFB3 is important for cell survival during paclitaxel-induced mitotic arrest A . PFKFB3 mRNA and protein expression after siRNA-mediated depletion of PFKFB3 for 72 h was determined by qPCR and western blotting. B . The quantification of mitotically arrested cells by flow cytometry, C . caspase 3/7 activity using Caspase-Glo 3/7 assay D . propidium iodide incorporation by flow cytometry and E . ROS levels using CM-H2DCFDA staining and flow cytometry were assessed after siRNA-mediated depletion of PFKFB3 for 72 h followed by 50 nM paclitaxel for 16 h and a mitotic shake-off in SKOV3 cells. F . The levels of ROS after PFKFB3 depletion alone, without paclitaxel treatment or mitotic cell isolation, was also analysed using CM-H2DCFDA staining and flow cytometry. Data are expressed as +SEM of three experiments.

    Article Snippet: Cells were finally resuspended in 0.5 ml 3 μM Propidium Iodide solution (PI) (Thermo Fisher Scientific) diluted in PBS to exclude dead cells as PI is membrane impermeant and analysed on the CyAn ADP Analyser (Beckman Coulter).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry, Cytometry, Activity Assay, Caspase-Glo Assay, Staining, Cell Isolation

    PFKFB4 depletion increases death of mitotically arrested SKOV3 cells A . Cell cycle analysis after siRNA-mediated depletion of PFKFB4 for 72 h was assessed by propidium iodide staining and flow cytometry. B . Mitotic cell death was assessed using time-lapse microscopy for 16 h and following the fate of over 200 mitotically arrested cells in each condition. C . After PFKFB4 depletion and mitotic cell isolation, the incorporation of propidium iodide was assessed by flow cytrometry and D . the activity of caspases 3 and 7 was measured using the Caspase-Glo 3/7 assay. Data are expressed as the mean +SEM of three experiments.

    Journal: Oncotarget

    Article Title: Loss of PFKFB4 induces cell death in mitotically arrested ovarian cancer cells

    doi: 10.18632/oncotarget.14910

    Figure Lengend Snippet: PFKFB4 depletion increases death of mitotically arrested SKOV3 cells A . Cell cycle analysis after siRNA-mediated depletion of PFKFB4 for 72 h was assessed by propidium iodide staining and flow cytometry. B . Mitotic cell death was assessed using time-lapse microscopy for 16 h and following the fate of over 200 mitotically arrested cells in each condition. C . After PFKFB4 depletion and mitotic cell isolation, the incorporation of propidium iodide was assessed by flow cytrometry and D . the activity of caspases 3 and 7 was measured using the Caspase-Glo 3/7 assay. Data are expressed as the mean +SEM of three experiments.

    Article Snippet: Cells were finally resuspended in 0.5 ml 3 μM Propidium Iodide solution (PI) (Thermo Fisher Scientific) diluted in PBS to exclude dead cells as PI is membrane impermeant and analysed on the CyAn ADP Analyser (Beckman Coulter).

    Techniques: Cell Cycle Assay, Staining, Flow Cytometry, Cytometry, Time-lapse Microscopy, Cell Isolation, Activity Assay, Caspase-Glo Assay