protease inhibitor mix  (Millipore)


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    Name:
    Protease Inhibitor Cocktail
    Description:

    Catalog Number:
    p8340
    Price:
    None
    Applications:
    Protease Inhibitor Cocktail has been used-. as a buffer component during sonication of GFP (green fluorescent protein)-huntingtin-transfected HEK 293 cells in GST (glutathione S-transferase) pull down assay. as a component of lysis buffer. as a component of radioimmunoprecipitation assay buffer (RIPA)
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    Structured Review

    Millipore protease inhibitor mix

    https://www.bioz.com/result/protease inhibitor mix/product/Millipore
    Average 99 stars, based on 278 article reviews
    Price from $9.99 to $1999.99
    protease inhibitor mix - by Bioz Stars, 2020-11
    99/100 stars

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    Related Articles

    Sonication:

    Article Title: G-patch domain and KOW motifs-containing protein, GPKOW; a nuclear RNA-binding protein regulated by protein kinase A
    Article Snippet: .. Two wells with 293T cells were washed in PBS and lysed by sonication in immunoprecipitation buffer (150 mM NaCl, 50 mM Tris pH 7.5, 0.5% Triton X-100, 1 × protease inhibitor cocktail (Sigma P8340), 1 mM PMSF (Sigma P7626) and 1 mM Na3 VO4 (Sigma S6508)). .. The lysates were cleared by centrifugation and adjusted to equal protein concentration (between 2.5 - 3.0 μg protein/μl) by the Bradford method (BioRad 500-0006).

    Protease Inhibitor:

    Article Title: G-patch domain and KOW motifs-containing protein, GPKOW; a nuclear RNA-binding protein regulated by protein kinase A
    Article Snippet: .. Two wells with 293T cells were washed in PBS and lysed by sonication in immunoprecipitation buffer (150 mM NaCl, 50 mM Tris pH 7.5, 0.5% Triton X-100, 1 × protease inhibitor cocktail (Sigma P8340), 1 mM PMSF (Sigma P7626) and 1 mM Na3 VO4 (Sigma S6508)). .. The lysates were cleared by centrifugation and adjusted to equal protein concentration (between 2.5 - 3.0 μg protein/μl) by the Bradford method (BioRad 500-0006).

    Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin
    Article Snippet: .. After 24 h, the medium was replaced with Optimem supplemented with 0.1% protease inhibitor cocktail (PIC) (P8340, Sigma-Aldrich, Saint Louis, MO), 0.1% DMSO, 0.1 mM AEBSF, 0.08 μM aprotinin, 1.4 μM E64, 4 μM bestatin, 2 μM leupeptin and 1.5 μM pepstatin A (Sigma-Aldrich). .. Immunofluorescence analysis was performed after 24 hr of treatment.

    Article Title: Chemically monoubiquitinated PEX5 binds to the components of the peroxisomal docking and export machinery
    Article Snippet: .. For cytosol extraction four to seven 175 cm2 culture flasks of 90% confluent ΔPEX5 fibroblasts were harvested and resuspended in cytosol extraction buffer (20 mM HEPES, 150 mM NaCl, 5 mM imidazole, 20% glycerol, pH 7.4, protease inhibitor cocktail (PI) (Sigma; 1:200)). ..

    Article Title: Chemotherapy Enhances Cross-Presentation of Nuclear Tumor Antigens
    Article Snippet: .. Preparation of whole cell lysates and secretory fractions for EGFP ELISA assay Whole cell lysates were extracted from each cell line by resuspending 5×106 tumor cells in 500 µL of radio immuno-precipitation assay (RIPA) lysis buffer (150 mM sodium chloride; 1.0% Triton X-100; 0.1% sodium dodecyl sulphate; 50 mM Tris, pH 8.0; 1/100 dilution Protease Inhibitor Cocktail (P8340, Sigma Aldrich) and 0.5% sodium deoxycholate) and incubating for 30 minutes at 4°C. .. Cell debris was removed by centrifugation (500×g for 10 mins) and supernatants containing whole cell extracts gently transferred to a fresh tube.

    Article Title: Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins
    Article Snippet: .. Recombinant insect cell pellets were either solubilized directly into non-reducing SDS Laemmli buffer (BioRad), or for use in western blot competition studies, we employed a non-reducing insect cell lysis buffer (1% Triton-X-100, 10mM Tris pH 8.0, 140 mM NaCl, 1x Sigma protease inhibitor cocktail). .. Production of recombinant schistosome membrane proteins in E . coli DNA encoding the predicted extracellular domains of SmLy6B, C, F, SmTsp-2, and Sm29 was amplified by PCR and ligated into a pET32 E. coli expression plasmid in frame with the E . coli thioredoxin (Trx) coding DNA and containing hexahistidine and epitope tags.

    Article Title: Amyloid-?1–42 Slows Clearance of Synaptically Released Glutamate by Mislocalizing Astrocytic GLT-1
    Article Snippet: .. Protein lysates from slices and cultured astrocytes were prepared using 1% Triton X-100 buffer [in m m : 20 Tris, 1 EDTA, 0.5 EGTA, 250 sucrose, Protease Inhibitor Cocktail (EMD Millipore)]. .. Lysate protein concentrations were determined by bicinchoninic acid assay (Thermo Scientific).

    Article Title: The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion
    Article Snippet: .. Cells were resuspended in ice-cold lysis buffer (50 mM Tris-HCl at pH 7.5, 1 mM EDTA, 400 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1× protease inhibitor cocktail III [Calbiochem]) and incubated for 10 min on ice prior to addition of an equal amount of ice-cold water. .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce).

    Cell Culture:

    Article Title: Amyloid-?1–42 Slows Clearance of Synaptically Released Glutamate by Mislocalizing Astrocytic GLT-1
    Article Snippet: .. Protein lysates from slices and cultured astrocytes were prepared using 1% Triton X-100 buffer [in m m : 20 Tris, 1 EDTA, 0.5 EGTA, 250 sucrose, Protease Inhibitor Cocktail (EMD Millipore)]. .. Lysate protein concentrations were determined by bicinchoninic acid assay (Thermo Scientific).

    Radio IP Assay:

    Article Title: Chemotherapy Enhances Cross-Presentation of Nuclear Tumor Antigens
    Article Snippet: .. Preparation of whole cell lysates and secretory fractions for EGFP ELISA assay Whole cell lysates were extracted from each cell line by resuspending 5×106 tumor cells in 500 µL of radio immuno-precipitation assay (RIPA) lysis buffer (150 mM sodium chloride; 1.0% Triton X-100; 0.1% sodium dodecyl sulphate; 50 mM Tris, pH 8.0; 1/100 dilution Protease Inhibitor Cocktail (P8340, Sigma Aldrich) and 0.5% sodium deoxycholate) and incubating for 30 minutes at 4°C. .. Cell debris was removed by centrifugation (500×g for 10 mins) and supernatants containing whole cell extracts gently transferred to a fresh tube.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Chemotherapy Enhances Cross-Presentation of Nuclear Tumor Antigens
    Article Snippet: .. Preparation of whole cell lysates and secretory fractions for EGFP ELISA assay Whole cell lysates were extracted from each cell line by resuspending 5×106 tumor cells in 500 µL of radio immuno-precipitation assay (RIPA) lysis buffer (150 mM sodium chloride; 1.0% Triton X-100; 0.1% sodium dodecyl sulphate; 50 mM Tris, pH 8.0; 1/100 dilution Protease Inhibitor Cocktail (P8340, Sigma Aldrich) and 0.5% sodium deoxycholate) and incubating for 30 minutes at 4°C. .. Cell debris was removed by centrifugation (500×g for 10 mins) and supernatants containing whole cell extracts gently transferred to a fresh tube.

    Immunoprecipitation:

    Article Title: G-patch domain and KOW motifs-containing protein, GPKOW; a nuclear RNA-binding protein regulated by protein kinase A
    Article Snippet: .. Two wells with 293T cells were washed in PBS and lysed by sonication in immunoprecipitation buffer (150 mM NaCl, 50 mM Tris pH 7.5, 0.5% Triton X-100, 1 × protease inhibitor cocktail (Sigma P8340), 1 mM PMSF (Sigma P7626) and 1 mM Na3 VO4 (Sigma S6508)). .. The lysates were cleared by centrifugation and adjusted to equal protein concentration (between 2.5 - 3.0 μg protein/μl) by the Bradford method (BioRad 500-0006).

    Incubation:

    Article Title: The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion
    Article Snippet: .. Cells were resuspended in ice-cold lysis buffer (50 mM Tris-HCl at pH 7.5, 1 mM EDTA, 400 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1× protease inhibitor cocktail III [Calbiochem]) and incubated for 10 min on ice prior to addition of an equal amount of ice-cold water. .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce).

    Lysis:

    Article Title: Chemotherapy Enhances Cross-Presentation of Nuclear Tumor Antigens
    Article Snippet: .. Preparation of whole cell lysates and secretory fractions for EGFP ELISA assay Whole cell lysates were extracted from each cell line by resuspending 5×106 tumor cells in 500 µL of radio immuno-precipitation assay (RIPA) lysis buffer (150 mM sodium chloride; 1.0% Triton X-100; 0.1% sodium dodecyl sulphate; 50 mM Tris, pH 8.0; 1/100 dilution Protease Inhibitor Cocktail (P8340, Sigma Aldrich) and 0.5% sodium deoxycholate) and incubating for 30 minutes at 4°C. .. Cell debris was removed by centrifugation (500×g for 10 mins) and supernatants containing whole cell extracts gently transferred to a fresh tube.

    Article Title: Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins
    Article Snippet: .. Recombinant insect cell pellets were either solubilized directly into non-reducing SDS Laemmli buffer (BioRad), or for use in western blot competition studies, we employed a non-reducing insect cell lysis buffer (1% Triton-X-100, 10mM Tris pH 8.0, 140 mM NaCl, 1x Sigma protease inhibitor cocktail). .. Production of recombinant schistosome membrane proteins in E . coli DNA encoding the predicted extracellular domains of SmLy6B, C, F, SmTsp-2, and Sm29 was amplified by PCR and ligated into a pET32 E. coli expression plasmid in frame with the E . coli thioredoxin (Trx) coding DNA and containing hexahistidine and epitope tags.

    Article Title: The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion
    Article Snippet: .. Cells were resuspended in ice-cold lysis buffer (50 mM Tris-HCl at pH 7.5, 1 mM EDTA, 400 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1× protease inhibitor cocktail III [Calbiochem]) and incubated for 10 min on ice prior to addition of an equal amount of ice-cold water. .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce).

    Western Blot:

    Article Title: Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins
    Article Snippet: .. Recombinant insect cell pellets were either solubilized directly into non-reducing SDS Laemmli buffer (BioRad), or for use in western blot competition studies, we employed a non-reducing insect cell lysis buffer (1% Triton-X-100, 10mM Tris pH 8.0, 140 mM NaCl, 1x Sigma protease inhibitor cocktail). .. Production of recombinant schistosome membrane proteins in E . coli DNA encoding the predicted extracellular domains of SmLy6B, C, F, SmTsp-2, and Sm29 was amplified by PCR and ligated into a pET32 E. coli expression plasmid in frame with the E . coli thioredoxin (Trx) coding DNA and containing hexahistidine and epitope tags.

    Recombinant:

    Article Title: Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins
    Article Snippet: .. Recombinant insect cell pellets were either solubilized directly into non-reducing SDS Laemmli buffer (BioRad), or for use in western blot competition studies, we employed a non-reducing insect cell lysis buffer (1% Triton-X-100, 10mM Tris pH 8.0, 140 mM NaCl, 1x Sigma protease inhibitor cocktail). .. Production of recombinant schistosome membrane proteins in E . coli DNA encoding the predicted extracellular domains of SmLy6B, C, F, SmTsp-2, and Sm29 was amplified by PCR and ligated into a pET32 E. coli expression plasmid in frame with the E . coli thioredoxin (Trx) coding DNA and containing hexahistidine and epitope tags.

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  • 99
    Millipore z vad fmk
    ORM-induced autophagy is upstream of apoptosis and pro-death in nature. ( A ) PA-1 cells were transfected for siRNA against either LC3 or Beclin 1, two critical autophagy proteins and cellular levels of LC3-II and cPARP were observed as markers of autophagic and apoptotic activity. ( B) PA-1 cells were treated with ORM alone (IC 50 dose), or pre-treated with lysosomal fusion inhibitor chloroquine (CQ; 10 μM, 2 h) or chemical chaperone and UPR inhibitor 4-PBA (5 mM; 2 h) before ORM treatment, stained with Annexin V-FITC/PI and analyzed by flow cytometry to assess apoptotic cell death (lower right quadrant = %early apoptotic population). ( C ) Viability (percentage of control) of PA-1 and OVCAR-3 cells pre-treated with CQ or 4-PBA and treated with ORM for 48 h measured through SRB assay; ( D , E ) PA-1 cells were pre-treated with 2.5 mM 3-MA or with 20 μM <t>z-VAD-fmk</t> or with 20 μM z-DEVD-fmk followed by 24 h ORM treatment (3-MA, z-DEVD) or 48 h ORM treatment (zVAD). PARP and LC3-II levels were analysed by immunoblotting.
    Z Vad Fmk, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    99
    Millipore pepstatin a
    Effects of enzymes and inhibitors on cleavage of the RLQLKL ACPP  in vitro . A , cleavage of 5 μ m  Cy5-labeled RLQLKL ACPP for 2 h with 2% cystic fluid obtained from PyMT tumors, in the absence of inhibitor or with 5 m m  Ca-DTPA, 50 μ m  TPEN, 18 μ m  SB3CT (  43 ), 50 n m  GM6001 (also tested at 1 μ m  and no inhibition; data not shown), 0.26 μ m  prinomastat, 150 n m  aprotinin, 10 μ m  E-64, 100 μ m  leupeptin, 1 μ m  pepstatin A, Calbiochem mixture III (diluted 1:1000), mixture III + DTPA. All inhibitors were used at the manufacturers' recommended concentrations, which should inhibit ≥95% of the target enzymes' activity.  B , cleavage of RLQLKL ACPP for 2 h with 50 n m  MMP-1, MMP-2, MMP-7, MMP-9, MMP-14, kallikrein 5, thrombin, plasmin, urokinase plasminogen activator, tissue plasminogen activator, factor VIIa, factor IXa, factor Xa, factor XIa, factor XIIa, trypsin (cathepsins B, G, L, and H), and neutrophil elastase. The percentage of cleavage measured with UVP software was 93.0 ± 2.9% for trypsin, 79.0 ± 2.8% for neutrophil elastase, 77.9 ± 2.1% for plasmin, 60% ± 2.3% for cathepsin G, 23.4 ± 2.5% for MMP7, 9.7 ± 1.1% for MMP1, and less than 3% for all other enzymes tested, including hepsin, enterokinase, and prostate-specific antigen ( supplemental Fig. 4 ). MMP-8, MMP-13, matriptase, urokinase, and legumain also showed no cleavage (data not shown). Trypsin and trypsin-2 cleavage of various ACPP are also shown in  supplemental Fig. 4 .
    Pepstatin A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 396 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pepstatin a/product/Millipore
    Average 99 stars, based on 396 article reviews
    Price from $9.99 to $1999.99
    pepstatin a - by Bioz Stars, 2020-11
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    99
    Millipore complete protease inhibitor
    DAG treatment causes S/G 2 phase cell cycle arrest and induces replication-dependent DNA damage. a , b Serum-starved (ST for 24 h) M059K and PC-3 cells were treated with or without 5 μM DAG in <t>complete</t> medium for the indicated periods of time. Then the cells were collected for cell cycle analysis using PI staining as described in “Materials and Methods”. The representative flow cytometric histograms from two individual experiments are shown with the corresponding percentages of cell populations at G 0 /G 1 , S, and G 2 /M phases. c PC-3 cells were synchronized at G 0 /G 1 phase by 24 h serum starvation before treatment with 50 μM DAG for 1 h in complete medium. After the treatment, DAG was removed and the cells were continued to incubate in complete medium for another 24 h (DAG 1 h + WO 24 h). WO stands for washout. After that, cells were fixed, permeabilized, and immunostained with anti-cyclin A2 (green) and anti-ɣH2AX (red) antibodies. Representative IF images are shown from two independent experiments. The scale bar stands for 10 μm. d From each experimental condition in c , we examined 100–120 cells and calculated the percentages of cyclin A2-/ɣH2AX- (double negative) and cyclin A2 + /ɣH2AX + (double positive) populations of cells. The corresponding statistical analysis shows significance (** p ≤ 0.01; *** p ≤ 0.001). e PC-3 and M059K cells were treated with 20 μM DAG for 48 h or 50 μM DAG for 1 h (in quiescent cells by 24 h serum starvation) followed by 24 h incubation without the drug in complete medium (DAG 1 h + WO 24 h). DSBs were analyzed by neutral comet assay and representative images are shown. The scale bar stands for 50 μm. f Tail moment from 50–100 cells at each experimental condition in e was analyzed by ImageJ (OpenComet) software and shows significance compare to untreated cells (** p ≤ 0.01; **** p ≤ 0.0001). g , h PC-3 or M059K cells were seeded in 96-well plates and cultured for 24 h in either serum-deprived medium or complete medium. Then the cells were treated with different concentrations of DAG for 72 h followed by crystal violet assay. Cell survival rates compared to untreated condition were shown as mean ± standard deviation.
    Complete Protease Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complete protease inhibitor/product/Millipore
    Average 99 stars, based on 86 article reviews
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    Image Search Results


    ORM-induced autophagy is upstream of apoptosis and pro-death in nature. ( A ) PA-1 cells were transfected for siRNA against either LC3 or Beclin 1, two critical autophagy proteins and cellular levels of LC3-II and cPARP were observed as markers of autophagic and apoptotic activity. ( B) PA-1 cells were treated with ORM alone (IC 50 dose), or pre-treated with lysosomal fusion inhibitor chloroquine (CQ; 10 μM, 2 h) or chemical chaperone and UPR inhibitor 4-PBA (5 mM; 2 h) before ORM treatment, stained with Annexin V-FITC/PI and analyzed by flow cytometry to assess apoptotic cell death (lower right quadrant = %early apoptotic population). ( C ) Viability (percentage of control) of PA-1 and OVCAR-3 cells pre-treated with CQ or 4-PBA and treated with ORM for 48 h measured through SRB assay; ( D , E ) PA-1 cells were pre-treated with 2.5 mM 3-MA or with 20 μM z-VAD-fmk or with 20 μM z-DEVD-fmk followed by 24 h ORM treatment (3-MA, z-DEVD) or 48 h ORM treatment (zVAD). PARP and LC3-II levels were analysed by immunoblotting.

    Journal: Scientific Reports

    Article Title: Ormeloxifene-induced unfolded protein response contributes to autophagy-associated apoptosis via disruption of Akt/mTOR and activation of JNK

    doi: 10.1038/s41598-018-20541-8

    Figure Lengend Snippet: ORM-induced autophagy is upstream of apoptosis and pro-death in nature. ( A ) PA-1 cells were transfected for siRNA against either LC3 or Beclin 1, two critical autophagy proteins and cellular levels of LC3-II and cPARP were observed as markers of autophagic and apoptotic activity. ( B) PA-1 cells were treated with ORM alone (IC 50 dose), or pre-treated with lysosomal fusion inhibitor chloroquine (CQ; 10 μM, 2 h) or chemical chaperone and UPR inhibitor 4-PBA (5 mM; 2 h) before ORM treatment, stained with Annexin V-FITC/PI and analyzed by flow cytometry to assess apoptotic cell death (lower right quadrant = %early apoptotic population). ( C ) Viability (percentage of control) of PA-1 and OVCAR-3 cells pre-treated with CQ or 4-PBA and treated with ORM for 48 h measured through SRB assay; ( D , E ) PA-1 cells were pre-treated with 2.5 mM 3-MA or with 20 μM z-VAD-fmk or with 20 μM z-DEVD-fmk followed by 24 h ORM treatment (3-MA, z-DEVD) or 48 h ORM treatment (zVAD). PARP and LC3-II levels were analysed by immunoblotting.

    Article Snippet: JC-1, antibiotic, AlexaFluor 488 and 568 conjugated antibodies, Lipofectamine 2000 were purchased from Invitrogen; z-VAD-fmk, protease inhibitor cocktail and GAPDH antibody were from Millipore.

    Techniques: Transfection, Activity Assay, Staining, Flow Cytometry, Cytometry, Sulforhodamine B Assay

    Effects of enzymes and inhibitors on cleavage of the RLQLKL ACPP  in vitro . A , cleavage of 5 μ m  Cy5-labeled RLQLKL ACPP for 2 h with 2% cystic fluid obtained from PyMT tumors, in the absence of inhibitor or with 5 m m  Ca-DTPA, 50 μ m  TPEN, 18 μ m  SB3CT (  43 ), 50 n m  GM6001 (also tested at 1 μ m  and no inhibition; data not shown), 0.26 μ m  prinomastat, 150 n m  aprotinin, 10 μ m  E-64, 100 μ m  leupeptin, 1 μ m  pepstatin A, Calbiochem mixture III (diluted 1:1000), mixture III + DTPA. All inhibitors were used at the manufacturers' recommended concentrations, which should inhibit ≥95% of the target enzymes' activity.  B , cleavage of RLQLKL ACPP for 2 h with 50 n m  MMP-1, MMP-2, MMP-7, MMP-9, MMP-14, kallikrein 5, thrombin, plasmin, urokinase plasminogen activator, tissue plasminogen activator, factor VIIa, factor IXa, factor Xa, factor XIa, factor XIIa, trypsin (cathepsins B, G, L, and H), and neutrophil elastase. The percentage of cleavage measured with UVP software was 93.0 ± 2.9% for trypsin, 79.0 ± 2.8% for neutrophil elastase, 77.9 ± 2.1% for plasmin, 60% ± 2.3% for cathepsin G, 23.4 ± 2.5% for MMP7, 9.7 ± 1.1% for MMP1, and less than 3% for all other enzymes tested, including hepsin, enterokinase, and prostate-specific antigen ( supplemental Fig. 4 ). MMP-8, MMP-13, matriptase, urokinase, and legumain also showed no cleavage (data not shown). Trypsin and trypsin-2 cleavage of various ACPP are also shown in  supplemental Fig. 4 .

    Journal: The Journal of Biological Chemistry

    Article Title: Parallel in Vivo and in Vitro Selection Using Phage Display Identifies Protease-dependent Tumor-targeting Peptides *

    doi: 10.1074/jbc.M110.138297

    Figure Lengend Snippet: Effects of enzymes and inhibitors on cleavage of the RLQLKL ACPP in vitro . A , cleavage of 5 μ m Cy5-labeled RLQLKL ACPP for 2 h with 2% cystic fluid obtained from PyMT tumors, in the absence of inhibitor or with 5 m m Ca-DTPA, 50 μ m TPEN, 18 μ m SB3CT ( 43 ), 50 n m GM6001 (also tested at 1 μ m and no inhibition; data not shown), 0.26 μ m prinomastat, 150 n m aprotinin, 10 μ m E-64, 100 μ m leupeptin, 1 μ m pepstatin A, Calbiochem mixture III (diluted 1:1000), mixture III + DTPA. All inhibitors were used at the manufacturers' recommended concentrations, which should inhibit ≥95% of the target enzymes' activity. B , cleavage of RLQLKL ACPP for 2 h with 50 n m MMP-1, MMP-2, MMP-7, MMP-9, MMP-14, kallikrein 5, thrombin, plasmin, urokinase plasminogen activator, tissue plasminogen activator, factor VIIa, factor IXa, factor Xa, factor XIa, factor XIIa, trypsin (cathepsins B, G, L, and H), and neutrophil elastase. The percentage of cleavage measured with UVP software was 93.0 ± 2.9% for trypsin, 79.0 ± 2.8% for neutrophil elastase, 77.9 ± 2.1% for plasmin, 60% ± 2.3% for cathepsin G, 23.4 ± 2.5% for MMP7, 9.7 ± 1.1% for MMP1, and less than 3% for all other enzymes tested, including hepsin, enterokinase, and prostate-specific antigen ( supplemental Fig. 4 ). MMP-8, MMP-13, matriptase, urokinase, and legumain also showed no cleavage (data not shown). Trypsin and trypsin-2 cleavage of various ACPP are also shown in supplemental Fig. 4 .

    Article Snippet: Tumors were excised and homogenized in PBS in the presence of protease inhibitor containing 100 μm 4-(2-aminoethyl)-benzenesulfonyl fluoride-HCl, 80 nm aprotinin, 5 μm bestatin, 1.5 μm E-64, 2 μm leupeptin hemisulfate, and 1 μm pepstatin A (EMD Biosciences mixture III).

    Techniques: In Vitro, Labeling, Inhibition, Activity Assay, Software

    DAG treatment causes S/G 2 phase cell cycle arrest and induces replication-dependent DNA damage. a , b Serum-starved (ST for 24 h) M059K and PC-3 cells were treated with or without 5 μM DAG in complete medium for the indicated periods of time. Then the cells were collected for cell cycle analysis using PI staining as described in “Materials and Methods”. The representative flow cytometric histograms from two individual experiments are shown with the corresponding percentages of cell populations at G 0 /G 1 , S, and G 2 /M phases. c PC-3 cells were synchronized at G 0 /G 1 phase by 24 h serum starvation before treatment with 50 μM DAG for 1 h in complete medium. After the treatment, DAG was removed and the cells were continued to incubate in complete medium for another 24 h (DAG 1 h + WO 24 h). WO stands for washout. After that, cells were fixed, permeabilized, and immunostained with anti-cyclin A2 (green) and anti-ɣH2AX (red) antibodies. Representative IF images are shown from two independent experiments. The scale bar stands for 10 μm. d From each experimental condition in c , we examined 100–120 cells and calculated the percentages of cyclin A2-/ɣH2AX- (double negative) and cyclin A2 + /ɣH2AX + (double positive) populations of cells. The corresponding statistical analysis shows significance (** p ≤ 0.01; *** p ≤ 0.001). e PC-3 and M059K cells were treated with 20 μM DAG for 48 h or 50 μM DAG for 1 h (in quiescent cells by 24 h serum starvation) followed by 24 h incubation without the drug in complete medium (DAG 1 h + WO 24 h). DSBs were analyzed by neutral comet assay and representative images are shown. The scale bar stands for 50 μm. f Tail moment from 50–100 cells at each experimental condition in e was analyzed by ImageJ (OpenComet) software and shows significance compare to untreated cells (** p ≤ 0.01; **** p ≤ 0.0001). g , h PC-3 or M059K cells were seeded in 96-well plates and cultured for 24 h in either serum-deprived medium or complete medium. Then the cells were treated with different concentrations of DAG for 72 h followed by crystal violet assay. Cell survival rates compared to untreated condition were shown as mean ± standard deviation.

    Journal: Cell Death & Disease

    Article Title: Dianhydrogalactitol synergizes with topoisomerase poisons to overcome DNA repair activity in tumor cells

    doi: 10.1038/s41419-020-02780-8

    Figure Lengend Snippet: DAG treatment causes S/G 2 phase cell cycle arrest and induces replication-dependent DNA damage. a , b Serum-starved (ST for 24 h) M059K and PC-3 cells were treated with or without 5 μM DAG in complete medium for the indicated periods of time. Then the cells were collected for cell cycle analysis using PI staining as described in “Materials and Methods”. The representative flow cytometric histograms from two individual experiments are shown with the corresponding percentages of cell populations at G 0 /G 1 , S, and G 2 /M phases. c PC-3 cells were synchronized at G 0 /G 1 phase by 24 h serum starvation before treatment with 50 μM DAG for 1 h in complete medium. After the treatment, DAG was removed and the cells were continued to incubate in complete medium for another 24 h (DAG 1 h + WO 24 h). WO stands for washout. After that, cells were fixed, permeabilized, and immunostained with anti-cyclin A2 (green) and anti-ɣH2AX (red) antibodies. Representative IF images are shown from two independent experiments. The scale bar stands for 10 μm. d From each experimental condition in c , we examined 100–120 cells and calculated the percentages of cyclin A2-/ɣH2AX- (double negative) and cyclin A2 + /ɣH2AX + (double positive) populations of cells. The corresponding statistical analysis shows significance (** p ≤ 0.01; *** p ≤ 0.001). e PC-3 and M059K cells were treated with 20 μM DAG for 48 h or 50 μM DAG for 1 h (in quiescent cells by 24 h serum starvation) followed by 24 h incubation without the drug in complete medium (DAG 1 h + WO 24 h). DSBs were analyzed by neutral comet assay and representative images are shown. The scale bar stands for 50 μm. f Tail moment from 50–100 cells at each experimental condition in e was analyzed by ImageJ (OpenComet) software and shows significance compare to untreated cells (** p ≤ 0.01; **** p ≤ 0.0001). g , h PC-3 or M059K cells were seeded in 96-well plates and cultured for 24 h in either serum-deprived medium or complete medium. Then the cells were treated with different concentrations of DAG for 72 h followed by crystal violet assay. Cell survival rates compared to untreated condition were shown as mean ± standard deviation.

    Article Snippet: Western blot analysisCells were lysed with EBC buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 1% NP-40, and 1 mM ethylenediaminetetraacetic acid; EDTA) supplemented with PhosSTOP phosphatase inhibitor and cOmplete protease inhibitor (MilliporeSigma, Oakville, Canada).

    Techniques: Cell Cycle Assay, Staining, Incubation, Neutral Comet Assay, Software, Cell Culture, Crystal Violet Assay, Standard Deviation

    DAG-induced DNA damage activates HR pathway. a PC-3 and M059K cells were synchronized at G 0 /G 1 phase by serum starvation for 24 h. The quiescent cells were treated with 50 μM DAG for 1 h followed by washout and additional incubation of 0, 20, 24, or 48 h in complete medium. Cell lysates were then analyzed for HR mediators by western blot from three independent experiments. b Quiescent PC-3 cells were treated with 50 μM DAG for 1 h followed by incubation without the drug for 24 h. Cells were pre-extracted by cytoskeletal buffer, fixed, permeabilized, and probed with corresponding antibodies for indicated proteins. Representative confocal images are shown from three independent experiments. The scale bar represents 5 μm. c PC-3 (80–150 cells) from each condition in b were quantified for foci-positive cells with statistical analysis (** p ≤ 0.01; *** p ≤ 0.001). d , e PC-3 or M059K cells were knocked down of BRCA1 by transient transfection using non-overlapping BRCA1-targeting siRNAs (B1, siBRCA1–2; B2, siBRCA1–14; B3, siBRCA1–25; B4, siBRCA1–15; or B5, siBRCA1–17). The negative control siRNA (C) was also included. After 24 h transfection, the cells were treated with different concentrations of DAG (0, 100 nM, 500 nM, 1 μM, 1.5 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM) for 5 days followed by crystal violet assay. The IC 50 values of DAG in control or BRCA1 knockdown cells were calculated using GraphPad Prism 6. The data are presented as mean ± standard deviation from three independent experiments with statistical significance (* p ≤ 0.05; ** p ≤ 0.01). In parallel western blot analysis confirmed BRCA1 knockdown. f Cell lysates were extracted from two pairs of isogenic colorectal cancer cell lines. Western blot analysis showed the expression of MLH1 and MSH2 in HCT116-PS-MLH1 and LoVo-PS-MSH2 cells, respectively, from three independent experiments. g The two pairs of isogenic colorectal cancer cells were treated with different concentrations of DAG for 3 days followed by crystal violet assay. The data in the curve are presented as mean ± standard deviation from three independent experiments.

    Journal: Cell Death & Disease

    Article Title: Dianhydrogalactitol synergizes with topoisomerase poisons to overcome DNA repair activity in tumor cells

    doi: 10.1038/s41419-020-02780-8

    Figure Lengend Snippet: DAG-induced DNA damage activates HR pathway. a PC-3 and M059K cells were synchronized at G 0 /G 1 phase by serum starvation for 24 h. The quiescent cells were treated with 50 μM DAG for 1 h followed by washout and additional incubation of 0, 20, 24, or 48 h in complete medium. Cell lysates were then analyzed for HR mediators by western blot from three independent experiments. b Quiescent PC-3 cells were treated with 50 μM DAG for 1 h followed by incubation without the drug for 24 h. Cells were pre-extracted by cytoskeletal buffer, fixed, permeabilized, and probed with corresponding antibodies for indicated proteins. Representative confocal images are shown from three independent experiments. The scale bar represents 5 μm. c PC-3 (80–150 cells) from each condition in b were quantified for foci-positive cells with statistical analysis (** p ≤ 0.01; *** p ≤ 0.001). d , e PC-3 or M059K cells were knocked down of BRCA1 by transient transfection using non-overlapping BRCA1-targeting siRNAs (B1, siBRCA1–2; B2, siBRCA1–14; B3, siBRCA1–25; B4, siBRCA1–15; or B5, siBRCA1–17). The negative control siRNA (C) was also included. After 24 h transfection, the cells were treated with different concentrations of DAG (0, 100 nM, 500 nM, 1 μM, 1.5 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM) for 5 days followed by crystal violet assay. The IC 50 values of DAG in control or BRCA1 knockdown cells were calculated using GraphPad Prism 6. The data are presented as mean ± standard deviation from three independent experiments with statistical significance (* p ≤ 0.05; ** p ≤ 0.01). In parallel western blot analysis confirmed BRCA1 knockdown. f Cell lysates were extracted from two pairs of isogenic colorectal cancer cell lines. Western blot analysis showed the expression of MLH1 and MSH2 in HCT116-PS-MLH1 and LoVo-PS-MSH2 cells, respectively, from three independent experiments. g The two pairs of isogenic colorectal cancer cells were treated with different concentrations of DAG for 3 days followed by crystal violet assay. The data in the curve are presented as mean ± standard deviation from three independent experiments.

    Article Snippet: Western blot analysisCells were lysed with EBC buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 1% NP-40, and 1 mM ethylenediaminetetraacetic acid; EDTA) supplemented with PhosSTOP phosphatase inhibitor and cOmplete protease inhibitor (MilliporeSigma, Oakville, Canada).

    Techniques: Incubation, Western Blot, Transfection, Negative Control, Crystal Violet Assay, Standard Deviation, Expressing

    Cytotoxic effect of DAG in GBM and PCa cell lines. a M059K and PC-3 cells were treated with or without 10 μM DAG in complete medium for 72 h. The representative bright-field images were shown and the scale bar represents 100 μm. b , c Two GBM (M059K and M059J) and four PCa (PC-3, LNCaP, DU-145, and PC-3-DR) cell lines were treated with different concentrations of DAG (0, 50 nM, 100 nM, 500 nM, 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM) for 72 h in 96-well culture plates. Following the treatment, crystal violet assay was performed and the IC 50 values of DAG for each cell line were calculated by fitting a sigmoidal dose-response curve to the data using GraphPad Prism 6. The data on the curve are presented as mean ± standard deviation. Three individual experiments were performed for each cell line.

    Journal: Cell Death & Disease

    Article Title: Dianhydrogalactitol synergizes with topoisomerase poisons to overcome DNA repair activity in tumor cells

    doi: 10.1038/s41419-020-02780-8

    Figure Lengend Snippet: Cytotoxic effect of DAG in GBM and PCa cell lines. a M059K and PC-3 cells were treated with or without 10 μM DAG in complete medium for 72 h. The representative bright-field images were shown and the scale bar represents 100 μm. b , c Two GBM (M059K and M059J) and four PCa (PC-3, LNCaP, DU-145, and PC-3-DR) cell lines were treated with different concentrations of DAG (0, 50 nM, 100 nM, 500 nM, 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM) for 72 h in 96-well culture plates. Following the treatment, crystal violet assay was performed and the IC 50 values of DAG for each cell line were calculated by fitting a sigmoidal dose-response curve to the data using GraphPad Prism 6. The data on the curve are presented as mean ± standard deviation. Three individual experiments were performed for each cell line.

    Article Snippet: Western blot analysisCells were lysed with EBC buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 1% NP-40, and 1 mM ethylenediaminetetraacetic acid; EDTA) supplemented with PhosSTOP phosphatase inhibitor and cOmplete protease inhibitor (MilliporeSigma, Oakville, Canada).

    Techniques: Crystal Violet Assay, Standard Deviation