phosphatase inhibitors  (Thermo Fisher)

 
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    Name:
    Pierce Protease and Phosphatase Inhibitor Mini Tablets EDTA free
    Description:
    Thermo Scientific Pierce Protease and Phosphatase Inhibitor Mini Tablets EDTA free contain both protease inhibitors and phosphatase inhibitors to provide broad spectrum complete protection from dephosphorylation and proteolytic degradation Features of Protease and Phosphatase Inhibitor Mini Tablets EDTA free • Improved formulation tablets dissolve quickly to form a clear solution • Economical more cost effective than other commercially available formulations for equivalent volumes • Convenient ready to use fully disclosed broad spectrum formulations • Compatible use directly with Pierce BCA assays • EDTA free formulated without EDTA a metalloproteinase inhibitor only for EDTA free formulations The Pierce Protease and Phosphatase Inhibitor Mini Tablets contain aprotinin bestatin E 64 and leupeptin to provide protection from degradation by serine proteases cysteine proteases and aspartic acid proteases To prevent the dephosphorylation activity of serine threonine and tyrosine phosphatases each tablet also contains sodium fluoride sodium orthovanadate sodium pyrophosphate and beta glycerophosphate Pierce Protease and Phosphatase Inhibitor Tablets are stable at 4°C for up to 12 months and each tablet is sufficient for 10 mL of solution Pierce Protease and Phosphatase Inhibitor Mini Tablets are the first complete inhibitor tablet Adding one tablet to 10 mL of lysis buffer provides complete protection from protease and phosphatase activity during protein extraction and protein purification There are two formulations of this inhibitor available one containing EDTA and one without While both formulations have metalloprotease inhibitors an EDTA free formulation is available for applications that require divalent cations for protein activity Similarly EDTA and certain phosphatase inhibitors interfere with immobilized metal chelate affinity chromatography IMAC and 2D electrophoresis Dialyze or desalt the sample before performing these procedures The Protease and Phosphatase Inhibitor formulation is mass spectrometry compatible because it does not contain AEBSF Related products Pierce Phosphatase Inhibitor Mini Tablets Pierce Protease Inhibitor Mini Tablets EDTA free
    Catalog Number:
    a32961
    Price:
    None
    Applications:
    Cell Lysis & Fractionation|Protease and Phosphatase Inhibition|Protein Biology|Protein Purification & Isolation
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher phosphatase inhibitors
    Thermo Scientific Pierce Protease and Phosphatase Inhibitor Mini Tablets EDTA free contain both protease inhibitors and phosphatase inhibitors to provide broad spectrum complete protection from dephosphorylation and proteolytic degradation Features of Protease and Phosphatase Inhibitor Mini Tablets EDTA free • Improved formulation tablets dissolve quickly to form a clear solution • Economical more cost effective than other commercially available formulations for equivalent volumes • Convenient ready to use fully disclosed broad spectrum formulations • Compatible use directly with Pierce BCA assays • EDTA free formulated without EDTA a metalloproteinase inhibitor only for EDTA free formulations The Pierce Protease and Phosphatase Inhibitor Mini Tablets contain aprotinin bestatin E 64 and leupeptin to provide protection from degradation by serine proteases cysteine proteases and aspartic acid proteases To prevent the dephosphorylation activity of serine threonine and tyrosine phosphatases each tablet also contains sodium fluoride sodium orthovanadate sodium pyrophosphate and beta glycerophosphate Pierce Protease and Phosphatase Inhibitor Tablets are stable at 4°C for up to 12 months and each tablet is sufficient for 10 mL of solution Pierce Protease and Phosphatase Inhibitor Mini Tablets are the first complete inhibitor tablet Adding one tablet to 10 mL of lysis buffer provides complete protection from protease and phosphatase activity during protein extraction and protein purification There are two formulations of this inhibitor available one containing EDTA and one without While both formulations have metalloprotease inhibitors an EDTA free formulation is available for applications that require divalent cations for protein activity Similarly EDTA and certain phosphatase inhibitors interfere with immobilized metal chelate affinity chromatography IMAC and 2D electrophoresis Dialyze or desalt the sample before performing these procedures The Protease and Phosphatase Inhibitor formulation is mass spectrometry compatible because it does not contain AEBSF Related products Pierce Phosphatase Inhibitor Mini Tablets Pierce Protease Inhibitor Mini Tablets EDTA free
    https://www.bioz.com/result/phosphatase inhibitors/product/Thermo Fisher
    Average 99 stars, based on 1380 article reviews
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    99/100 stars

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    Related Articles

    Lysis:

    Article Title: Inhibition of β2-adrenergic receptor reduces triple-negative breast cancer brain metastases: The potential benefit of perioperative β-blockade
    Article Snippet: .. Collected cells were homogenized by lysis buffer [glycerol, 3 M KCl and 10% NP-40 (all from Sigma-Aldrich), 1 M Tris (Bio-Rad) and 1 M DTT (Sigma-Aldrich)] in the presence of phosphatase inhibitor, EDTA, and protease inhibitor (all from Thermo Scientific) for 15 min. .. Forty micrograms of protein boiled for 5 min with loading buffer [95% laemmli, and 5% β-mercaptoethanol (both from Bio-Rad)] were loaded onto Mini-PROTEAN TGX gels (Bio-Rad).

    Article Title: Kinase Inhibitors with Antiepileptic Properties Identified with a Novel in Vitro Screening Platform
    Article Snippet: .. After 24 h, cultures were collected from the 6-well plates and lysed in lysis buffer: RIPA buffer, phosphatase and protease inhibitors (Thermo Scientific, Waltham, MA, USA). .. Protein concentrations were assessed by micro BCA protein assay kit from Thermo Scientific.

    Protease Inhibitor:

    Article Title: Inhibition of β2-adrenergic receptor reduces triple-negative breast cancer brain metastases: The potential benefit of perioperative β-blockade
    Article Snippet: .. Collected cells were homogenized by lysis buffer [glycerol, 3 M KCl and 10% NP-40 (all from Sigma-Aldrich), 1 M Tris (Bio-Rad) and 1 M DTT (Sigma-Aldrich)] in the presence of phosphatase inhibitor, EDTA, and protease inhibitor (all from Thermo Scientific) for 15 min. .. Forty micrograms of protein boiled for 5 min with loading buffer [95% laemmli, and 5% β-mercaptoethanol (both from Bio-Rad)] were loaded onto Mini-PROTEAN TGX gels (Bio-Rad).

    Radio Immunoprecipitation:

    Article Title: Expression of tumor suppressor programmed cell death 4 in endometrioid endometrial carcinomas and clinicopathological significance
    Article Snippet: .. Frozen, fresh EEC and control endometrium specimens were ground mechanically, and total protein was extracted using radioimmunoprecipitation assay buffer containing 1% phenylmethanesulfonyl fluoride and 0.5% phosphatase inhibitor (Thermo Fisher Scientific, Inc., Waltham, MA, USA). .. Following centrifugation at 12,000 × g for 30 min at 4°C, the protein concentration of supernatant was measured with a bicinchoninic acid kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

    Western Blot:

    Article Title: An optochemical tool for light-induced dissociation of adherens junctions to control mechanical coupling between cells
    Article Snippet: .. Western blotting Cells were lysed in RIPA buffer supplemented with protease/phosphatase inhibitors and 5 mM EDTA (all Thermo Fisher Scientific). .. SDS-PAGE and Western Blot was done according to the NuPAGE standard protocol using 4–12% Bis-Tris gels and MOPS SDS running buffer.

    Article Title: The high-fat high-fructose hamster as an animal model for niacin's biological activities in humans.
    Article Snippet: .. Niacin has been used for more than 50 years to treat dyslipidemia, yet the mechanisms underlying its lipid-modifying effects remain unknown, a situation stemming at least in part from a lack of validated animal models. ..

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    Thermo Fisher gene exp ppp1r1b hs00259967 m1
    Neuroimaging analyses (structure and function) of common haplotype in <t>PPP1R1B</t> in control sample of white subjects. Top row shows haplotype effects on volume ( A ) or activation ( C and E ) in striatum; bottom row shows haplotype effects on structural ( B ) and functional ( D and F ) connectivity of striatum with prefrontal cortex. Structural MRI analyses (voxel-based morphometry): ( A ) significantly reduced volume in striatum ( P
    Gene Exp Ppp1r1b Hs00259967 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 87 stars, based on 1 article reviews
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    97
    Thermo Fisher phosphatase inhibitor cocktail without edta
    N-Cadherin from POMGNT1-deficient cells shows enhanced homotypic interactions in vitro . (A) Semi-quantitative analysis of the mean size of dynabead aggregates coated with the recombinant extracellular domain of recombinant N-Cdh purified from WT (gray line) and ΔPOMGnT1 cells (black line). Protein-coated dynabeads were imaged following incubation in the presence of either CaCl 2 (solid lines) or <t>EDTA</t> (broken lines) at the indicated time intervals. Mean aggregate size is plotted against time. Assays were performed at quadruplicate from two independent experiments. Data are represented as means ± SD. Asterisks denote statistical significance in comparison to WT cells: * p ≤ 0.05, ** p ≤ 0.01. (B) Western Blot analysis evaluating levels of protein from WT and ΔPOMGnT1 (Δ) bound to dynabeads. Dynabead-bound recombinant N-Cdh was eluted in <t>SDS</t> sample buffer (beads), resolved on a 10% SDS PAA gel. For detection on Western blot an anti-5xHis antibody was used. In addition, protein before binding to beads (input) and unbound fractions (void) are shown.
    Phosphatase Inhibitor Cocktail Without Edta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher gene exp ppp1r15a hs00169585 m1
    Fluphenazine induces ATF4 overactivation in hypoxic spheroids. ( a ) Analysis of all protein-encoding genes by deep sequencing of HCT116 cells treated for 24 h with either 1 mM DFO or 1 mM DFO+5 μ M Fluphenazine (see also Supplementary Figure S6 ) showed upregulation of ATF4/CHOP-dependent stress-response pathway upon Fluphenazine and DFO co-treatment. Bars show mean with S.D. ( n =1, median of 4 replicates). ( b ) Gene expression analysis (RT-qPCR) of ATF4 pro-apoptotic target genes in HCT116 spheroids treated for 24 h in normoxia or hypoxia with DMSO control or 5 μ M Fluphenazine. Ct values of each sample were normalized with the internal control RPL32 and normalized to the normoxia DMSO control. Bars show mean with S.D. ( n =3). ( c ) HCT116 cells were incubated with ATF4 siRNA or lipid only control and grown as spheroids for 3 days under normoxic conditions. Later, spheroids were incubated for 24 h under hypoxia with DMSO control or 5 μ M Fluphenazine. Gene expression level for ATF4 target genes <t>PPP1R15A</t> and DDIT3 were determined using RT-qPCR. Ct values of each sample were normalized with the internal control RPL32 and normalized to the hypoxia lipid only control sample. Bars show mean with S.D. ( n =3). ( d ) siRNA-treated cells grown as spheroids (for ATF4) or spheroids from HCT116 cells stably transfected with HIF shRNA (see Materials and Methods) were treated with either DMSO control or Fluphenazine (3 μ M) (or 100 μ M N -Palmitoyl- D -Sphingomyelin (SM d18:1/16)) for 3 days under hypoxia. Spheroid nuclei were stained with Hoechst (red) and dead cells were stained with SytoxGreen (green). ATF4 ameliorates Fluphenazine- or SM-induced cell death under hypoxia. n =3. Scale bar 100 μ m. ** = P -value between 0.001 and 0.01, *** = P -value between 0.0001 and 0.001
    Gene Exp Ppp1r15a Hs00169585 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Neuroimaging analyses (structure and function) of common haplotype in PPP1R1B in control sample of white subjects. Top row shows haplotype effects on volume ( A ) or activation ( C and E ) in striatum; bottom row shows haplotype effects on structural ( B ) and functional ( D and F ) connectivity of striatum with prefrontal cortex. Structural MRI analyses (voxel-based morphometry): ( A ) significantly reduced volume in striatum ( P

    Journal: Journal of Clinical Investigation

    Article Title: Genetic evidence implicating DARPP-32 in human frontostriatal structure, function, and cognition

    doi: 10.1172/JCI30413

    Figure Lengend Snippet: Neuroimaging analyses (structure and function) of common haplotype in PPP1R1B in control sample of white subjects. Top row shows haplotype effects on volume ( A ) or activation ( C and E ) in striatum; bottom row shows haplotype effects on structural ( B ) and functional ( D and F ) connectivity of striatum with prefrontal cortex. Structural MRI analyses (voxel-based morphometry): ( A ) significantly reduced volume in striatum ( P

    Article Snippet: Three probes were based on the common exon structure as found in full-length cDNA: exons 4 and 5 (type 9967; Hs00259967), 1 and 2 (type 10; Hs00938410_m1) and 5 and 6 (type 14; Hs00938414_m1); splice variant 5183, exons 2 and 5; and t-DARPP (type 16; Hs00938416_g1).

    Techniques: Activation Assay, Functional Assay, Magnetic Resonance Imaging

    Genetic variation in PPP1R1B.

    Journal: Journal of Clinical Investigation

    Article Title: Genetic evidence implicating DARPP-32 in human frontostriatal structure, function, and cognition

    doi: 10.1172/JCI30413

    Figure Lengend Snippet: Genetic variation in PPP1R1B.

    Article Snippet: Three probes were based on the common exon structure as found in full-length cDNA: exons 4 and 5 (type 9967; Hs00259967), 1 and 2 (type 10; Hs00938410_m1) and 5 and 6 (type 14; Hs00938414_m1); splice variant 5183, exons 2 and 5; and t-DARPP (type 16; Hs00938416_g1).

    Techniques:

    Effect of PPP1R1B haplotype on mRNA expression in postmortem human brain. ( A ) Exon position of primers used for quantitative PCR for isoform amplification, showing which isoform was amplified by which probe. ( B ) Effect of PPP1R1B haplotypes on mRNA expression for the common isoform. The PPP1R1B haplotype has an impact on expression of all probes assaying the expression of the full-length mRNA (type 14, P

    Journal: Journal of Clinical Investigation

    Article Title: Genetic evidence implicating DARPP-32 in human frontostriatal structure, function, and cognition

    doi: 10.1172/JCI30413

    Figure Lengend Snippet: Effect of PPP1R1B haplotype on mRNA expression in postmortem human brain. ( A ) Exon position of primers used for quantitative PCR for isoform amplification, showing which isoform was amplified by which probe. ( B ) Effect of PPP1R1B haplotypes on mRNA expression for the common isoform. The PPP1R1B haplotype has an impact on expression of all probes assaying the expression of the full-length mRNA (type 14, P

    Article Snippet: Three probes were based on the common exon structure as found in full-length cDNA: exons 4 and 5 (type 9967; Hs00259967), 1 and 2 (type 10; Hs00938410_m1) and 5 and 6 (type 14; Hs00938414_m1); splice variant 5183, exons 2 and 5; and t-DARPP (type 16; Hs00938416_g1).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Amplification

    Genetic variation in PPP1R1B.

    Journal: Journal of Clinical Investigation

    Article Title: Genetic evidence implicating DARPP-32 in human frontostriatal structure, function, and cognition

    doi: 10.1172/JCI30413

    Figure Lengend Snippet: Genetic variation in PPP1R1B.

    Article Snippet: Three probes were based on the common exon structure as found in full-length cDNA: exons 4 and 5 (type 9967; Hs00259967), 1 and 2 (type 10; Hs00938410_m1) and 5 and 6 (type 14; Hs00938414_m1); splice variant 5183, exons 2 and 5; and t-DARPP (type 16; Hs00938416_g1).

    Techniques:

    N-Cadherin from POMGNT1-deficient cells shows enhanced homotypic interactions in vitro . (A) Semi-quantitative analysis of the mean size of dynabead aggregates coated with the recombinant extracellular domain of recombinant N-Cdh purified from WT (gray line) and ΔPOMGnT1 cells (black line). Protein-coated dynabeads were imaged following incubation in the presence of either CaCl 2 (solid lines) or EDTA (broken lines) at the indicated time intervals. Mean aggregate size is plotted against time. Assays were performed at quadruplicate from two independent experiments. Data are represented as means ± SD. Asterisks denote statistical significance in comparison to WT cells: * p ≤ 0.05, ** p ≤ 0.01. (B) Western Blot analysis evaluating levels of protein from WT and ΔPOMGnT1 (Δ) bound to dynabeads. Dynabead-bound recombinant N-Cdh was eluted in SDS sample buffer (beads), resolved on a 10% SDS PAA gel. For detection on Western blot an anti-5xHis antibody was used. In addition, protein before binding to beads (input) and unbound fractions (void) are shown.

    Journal: bioRxiv

    Article Title: Glycosyltransferase POMGNT1 deficiency affects N-cadherin-mediated cell-cell adhesion

    doi: 10.1101/2020.09.09.289306

    Figure Lengend Snippet: N-Cadherin from POMGNT1-deficient cells shows enhanced homotypic interactions in vitro . (A) Semi-quantitative analysis of the mean size of dynabead aggregates coated with the recombinant extracellular domain of recombinant N-Cdh purified from WT (gray line) and ΔPOMGnT1 cells (black line). Protein-coated dynabeads were imaged following incubation in the presence of either CaCl 2 (solid lines) or EDTA (broken lines) at the indicated time intervals. Mean aggregate size is plotted against time. Assays were performed at quadruplicate from two independent experiments. Data are represented as means ± SD. Asterisks denote statistical significance in comparison to WT cells: * p ≤ 0.05, ** p ≤ 0.01. (B) Western Blot analysis evaluating levels of protein from WT and ΔPOMGnT1 (Δ) bound to dynabeads. Dynabead-bound recombinant N-Cdh was eluted in SDS sample buffer (beads), resolved on a 10% SDS PAA gel. For detection on Western blot an anti-5xHis antibody was used. In addition, protein before binding to beads (input) and unbound fractions (void) are shown.

    Article Snippet: Whole cell lysate and crude membrane preparation Cells at 90% confluency (~5.0 x 106 cells) were washed with ice-cold PBS and lysed with 500 μl of lysis buffer (10 mM Tris-HCl pH 7.6, 1% (w/v) SDS, 1 mM EDTA, supplemented with Halt™ protease and phosphatase inhibitor cocktail without EDTA (ThermoFisher Scientific)).

    Techniques: In Vitro, Recombinant, Purification, Incubation, Western Blot, Binding Assay

    Lentiviral vector is released from LDLR3-ELP after internalization by cells A549 were starved for 24 hours then were treated in serum free media with the indicated treatments. Cells were treated for 24 hours with the lentiviral vector containing nanoparticles that consisted of 1 µM of biotinylated LDLR3-ELP and 1 µM KGF-ELP. At the indicated time points, cells were put on ice for five minutes followed by three ice cold washed in PBS. The cells were lysed with radio immune protection assay (RIPA) buffer containing 2X halt protease and phosphatase inhibitors cocktail with 1X ethylene diamine triacetic acid (EDTA). The lysates were immunoprecipitated using streptavidin conjugated magnetic beads (dynabeads). Then a magnetic bar was used to separate the beads from the supernatant. Following a few washes, the immunoprecipitated complexes (dynabeads) and the supernatant were analyzed for the presence of virus using western blots. Anti VSV-G antibody was used to detect the lentiviral vector.

    Journal: Gene therapy

    Article Title: Recombinant elastin based nanoparticles for targeted gene therapy

    doi: 10.1038/gt.2017.54

    Figure Lengend Snippet: Lentiviral vector is released from LDLR3-ELP after internalization by cells A549 were starved for 24 hours then were treated in serum free media with the indicated treatments. Cells were treated for 24 hours with the lentiviral vector containing nanoparticles that consisted of 1 µM of biotinylated LDLR3-ELP and 1 µM KGF-ELP. At the indicated time points, cells were put on ice for five minutes followed by three ice cold washed in PBS. The cells were lysed with radio immune protection assay (RIPA) buffer containing 2X halt protease and phosphatase inhibitors cocktail with 1X ethylene diamine triacetic acid (EDTA). The lysates were immunoprecipitated using streptavidin conjugated magnetic beads (dynabeads). Then a magnetic bar was used to separate the beads from the supernatant. Following a few washes, the immunoprecipitated complexes (dynabeads) and the supernatant were analyzed for the presence of virus using western blots. Anti VSV-G antibody was used to detect the lentiviral vector.

    Article Snippet: The cells were then lysed with radio immune protection assay (RIPA) buffer containing 2X halt protease and phosphatase inhibitors cocktail with 1X ethylene diamine triacetic acid (EDTA) (Thermo scientific cat #78440).

    Techniques: Plasmid Preparation, Immunoprecipitation, Magnetic Beads, Western Blot

    Fluphenazine induces ATF4 overactivation in hypoxic spheroids. ( a ) Analysis of all protein-encoding genes by deep sequencing of HCT116 cells treated for 24 h with either 1 mM DFO or 1 mM DFO+5 μ M Fluphenazine (see also Supplementary Figure S6 ) showed upregulation of ATF4/CHOP-dependent stress-response pathway upon Fluphenazine and DFO co-treatment. Bars show mean with S.D. ( n =1, median of 4 replicates). ( b ) Gene expression analysis (RT-qPCR) of ATF4 pro-apoptotic target genes in HCT116 spheroids treated for 24 h in normoxia or hypoxia with DMSO control or 5 μ M Fluphenazine. Ct values of each sample were normalized with the internal control RPL32 and normalized to the normoxia DMSO control. Bars show mean with S.D. ( n =3). ( c ) HCT116 cells were incubated with ATF4 siRNA or lipid only control and grown as spheroids for 3 days under normoxic conditions. Later, spheroids were incubated for 24 h under hypoxia with DMSO control or 5 μ M Fluphenazine. Gene expression level for ATF4 target genes PPP1R15A and DDIT3 were determined using RT-qPCR. Ct values of each sample were normalized with the internal control RPL32 and normalized to the hypoxia lipid only control sample. Bars show mean with S.D. ( n =3). ( d ) siRNA-treated cells grown as spheroids (for ATF4) or spheroids from HCT116 cells stably transfected with HIF shRNA (see Materials and Methods) were treated with either DMSO control or Fluphenazine (3 μ M) (or 100 μ M N -Palmitoyl- D -Sphingomyelin (SM d18:1/16)) for 3 days under hypoxia. Spheroid nuclei were stained with Hoechst (red) and dead cells were stained with SytoxGreen (green). ATF4 ameliorates Fluphenazine- or SM-induced cell death under hypoxia. n =3. Scale bar 100 μ m. ** = P -value between 0.001 and 0.01, *** = P -value between 0.0001 and 0.001

    Journal: Cell Death & Disease

    Article Title: Functional inhibition of acid sphingomyelinase by Fluphenazine triggers hypoxia-specific tumor cell death

    doi: 10.1038/cddis.2017.130

    Figure Lengend Snippet: Fluphenazine induces ATF4 overactivation in hypoxic spheroids. ( a ) Analysis of all protein-encoding genes by deep sequencing of HCT116 cells treated for 24 h with either 1 mM DFO or 1 mM DFO+5 μ M Fluphenazine (see also Supplementary Figure S6 ) showed upregulation of ATF4/CHOP-dependent stress-response pathway upon Fluphenazine and DFO co-treatment. Bars show mean with S.D. ( n =1, median of 4 replicates). ( b ) Gene expression analysis (RT-qPCR) of ATF4 pro-apoptotic target genes in HCT116 spheroids treated for 24 h in normoxia or hypoxia with DMSO control or 5 μ M Fluphenazine. Ct values of each sample were normalized with the internal control RPL32 and normalized to the normoxia DMSO control. Bars show mean with S.D. ( n =3). ( c ) HCT116 cells were incubated with ATF4 siRNA or lipid only control and grown as spheroids for 3 days under normoxic conditions. Later, spheroids were incubated for 24 h under hypoxia with DMSO control or 5 μ M Fluphenazine. Gene expression level for ATF4 target genes PPP1R15A and DDIT3 were determined using RT-qPCR. Ct values of each sample were normalized with the internal control RPL32 and normalized to the hypoxia lipid only control sample. Bars show mean with S.D. ( n =3). ( d ) siRNA-treated cells grown as spheroids (for ATF4) or spheroids from HCT116 cells stably transfected with HIF shRNA (see Materials and Methods) were treated with either DMSO control or Fluphenazine (3 μ M) (or 100 μ M N -Palmitoyl- D -Sphingomyelin (SM d18:1/16)) for 3 days under hypoxia. Spheroid nuclei were stained with Hoechst (red) and dead cells were stained with SytoxGreen (green). ATF4 ameliorates Fluphenazine- or SM-induced cell death under hypoxia. n =3. Scale bar 100 μ m. ** = P -value between 0.001 and 0.01, *** = P -value between 0.0001 and 0.001

    Article Snippet: Hs00851655_g1), SLC2a3 (solute carrier family 2 (facilitated glucose transporter), member 3, encoding Glut3 protein, Hs00359840_m1), VEGFA (vascular endothelial growth factor A, Hs00900055_m1), BNIP3 (BCL2/ adenovirus E1B 19kDa interacting protein 3, Hs00969291_m1), PPP1R15A (Protein phosphatase 1 regulatory subunit 15A, Hs00169585_m1), DDIT3 (DNA-damage-inducible transcript 3, Hs99999172_m1), ATF4 (Activating transcription factor 4, Hs00909569_g1), HIF-1-α (Hypoxia inducible factor 1 alpha, Hs00153153_m1), and EPAS1 (Endothelial PAS domain-containing protein 1, Hs01026149_m1).

    Techniques: Sequencing, Expressing, Quantitative RT-PCR, Incubation, Stable Transfection, Transfection, shRNA, Staining