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  • 99
    ATCC 3 sulfonyl nor β lapachones 7a g staphylococcus aureus 2065 ma
    3 Sulfonyl Nor β Lapachones 7a G Staphylococcus Aureus 2065 Ma, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore β lapachone
    ROS reagents inhibit the activity of the SMN complex in vitro and in cells (A) Effect of H 2 O 2 on the activity of the SMN complex in vitro . Magnetic beads snRNP assembly assay was carried out in the presence of increasing amounts of H 2 O 2 . IC 50 was calculated from the dose-response curve. Error bars represent SDs from triplicate measurements. (B) Effect of menadione on SMN complex activity in vitro . The same experimental procedure was carried out as in (A), except that menadione was used instead. (C) Dose-dependent effect of menadione on SMN complex activity in cells. HeLa cells were treated with menadione at the indicated concentrations or with DMSO control for 1 hour. SMN complex activity in extracts from various treated cells was measured in comparison to DMSO control cell extract (100% activity) by magnetic beads snRNP assembly assay. Error bars represent SDs from 3 independent measurements. (D) Extracts from (C) mixed with sample buffer without or with DTT were resolved by SDS-PAGE and analyzed by Western blot of the entire membrane with anti-SMN antibody 62E7. The molecular mass markers in kDa are indicated on the left. “redSMN” indicates monomer SMN migrating at normal molecular mass and “oxSMN” indicates disulfide-crosslinked SMN upon oxidation. (E) <t>β-lapachone</t> and menadione generate ROS in live cells. HeLa cells were incubated 30 minutes with ROS indicator dye H 2 DCFDA (10 μM) or without dye as a control, then treated with compounds at indicated concentrations or DMSO as control. Fluorescence images were acquired 30 minutes after treatment.
    β Lapachone, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam β lapachone
    ROS reagents inhibit the activity of the SMN complex in vitro and in cells (A) Effect of H 2 O 2 on the activity of the SMN complex in vitro . Magnetic beads snRNP assembly assay was carried out in the presence of increasing amounts of H 2 O 2 . IC 50 was calculated from the dose-response curve. Error bars represent SDs from triplicate measurements. (B) Effect of menadione on SMN complex activity in vitro . The same experimental procedure was carried out as in (A), except that menadione was used instead. (C) Dose-dependent effect of menadione on SMN complex activity in cells. HeLa cells were treated with menadione at the indicated concentrations or with DMSO control for 1 hour. SMN complex activity in extracts from various treated cells was measured in comparison to DMSO control cell extract (100% activity) by magnetic beads snRNP assembly assay. Error bars represent SDs from 3 independent measurements. (D) Extracts from (C) mixed with sample buffer without or with DTT were resolved by SDS-PAGE and analyzed by Western blot of the entire membrane with anti-SMN antibody 62E7. The molecular mass markers in kDa are indicated on the left. “redSMN” indicates monomer SMN migrating at normal molecular mass and “oxSMN” indicates disulfide-crosslinked SMN upon oxidation. (E) <t>β-lapachone</t> and menadione generate ROS in live cells. HeLa cells were incubated 30 minutes with ROS indicator dye H 2 DCFDA (10 μM) or without dye as a control, then treated with compounds at indicated concentrations or DMSO as control. Fluorescence images were acquired 30 minutes after treatment.
    β Lapachone, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Tocris β lapachone
    ULK1 kinase activity promotes PARP1 activation and sensitizes cells to oxidative stress-induced death. ( a ) Ulk1 KO/ Ulk2 stably expressing vector control, WT ULK1, or KI ULK1 were cotransfected with a vector containing PARP1-GFP and incubated in complete media (CM) with or without H 2 O 2 for 20 min. PARP1 was depleted from the nucleoli of cells expressing WT ULK1 but not from those of cells expressing the KI mutant. Scale bars=10 μ M. Representative immunoblots ( b ) and a graph ( c ) show the levels of PARylated proteins in the indicated MEF lines incubated in the presence or the absence of 500 μ M H 2 O 2 for 10 min. ( d and e ) Trypan blue-exclusion assays demonstrate that the KI-ULK1 mutant fails to promote cell death to the same extent as WT ULK1 does when stably expressed in Ulk1 -deficient MEFs. ( f ) The 293T cells cotransfected with PARP1-GFP and either WT ULK1 or the R266A-ULK1 mutant were treated for 10 min with 100 μ M H 2 O 2 . Immunoblot analyses demonstrate a decrease in PARylated proteins in cells transfected with the R266A mutant. ( g ) The 293T cells transfected with the indicated expression constructs were treated with 100 μ M H 2 O 2 . ATP levels were analyzed in cells collected 10, 30, or 60 min after treatment with 100 μ M H 2 O 2 and normalized to levels in untreated cells. ( h ) Trypan blue-exclusion assays performed 6 h after treatment with the indicated dose of H 2 O 2 demonstrate increased viability of 293T transfected with the R266A-ULK1 mutant expression construct compared with that of those transfected with the WT ULK1 construct. ( i ) The 293T cells were transfected with empty vector (−), WT ULK1 (WT), or the following mutant forms of ULK1: K46A (kinase-defective mutant), 4SA (AMPK-resistant, autophagy-defective mutant), or R266A (NLS mutant). Transfected cells were treated with 4.5 μ M <t>β</t> -lapachone for 72 h and then cell viability was assessed using CellTiter-Glo assays. Signals were normalized to those in DMSO-treated controls ( n =4 biologic replicates per condition), P
    β Lapachone, supplied by Tocris, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ArQule β lapachone
    <t>β-Lapachone</t> lethality is dependent on NQO1 status, but not oncogenic driver or passenger mutations NSCLC cell lines were screened in a double-blind manner for lethality (LD 50 values), NQO1 ) and oncogene mutational statuses. NQO1 polymorphic statuses were: WT (wild-type); hets (WT + polymorphic alleles: ^, *2 (C609T) or #, *3 (C465T), HCC2935); or pm, homozygous polymorphic (*2 or *3) alleles. Relative β-lap sensitivities were expressed as LD 50 values (µM, 2 hr). Values with ‘greater than ( > )’ signs indicate highest concentrations (µM) tested. DNA from NSCLC cells were sequenced by the UTSW-MDA lung cancer SPORE (red: mutant, blue: null (deletion), black: wild-type, white: no sequence available). .
    β Lapachone, supplied by ArQule, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Biomol GmbH β lapachone
    Camptothecin induces degradation of DNA topoisomerase I in fibroblasts . (A) Presence of DNA toposiomerase I in the immunocomplexes described in 1B was confirmed by Western blotting analyses. (B) Fibroblasts described in 1B were treated with 25 μM camptothecin or 25 μM <t>β-lapachone,</t> and DNA topoisomerase I present in the lysates was detected by Western blotting. The same blots were stripped and reprobed with anti-β-tubulin antibodies as a loading control. Relative ratios of DNA topoisomerase I to tubulin levels are indicated. All the data shown here are representative of at least two independent experiments. CPT = camptothecin, Topo I = DNA topoisomerase I, IP = immunoprecipitation, IB = immunoblotting, and β-LP = β-lapachone.
    β Lapachone, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 89/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ciba Geigy β lapachone
    Dow-response and temporal kinetics of apoptosis induction in HL-60 cells by β -lapachone. In A and B, log-phase human HL-60 cells were exposed to 5.0 μ M <t>β</t> -lapachone or camptothecin and then assayed for apoptotic responses in the first 4 h. A , apoptotic DNA laddering by 1.8% agarose gel electrophoresis; B , cell cycle and apoptotic responses by flow cytometry. Treatment conditions were: a , control; b–e , β -lapachone; or f , camptothecin. In C and D , log-phase HL-60 cells were treated with increasing concentrations of β -lapachone (0.05–5.0 μ M) or camptothecin (5.0 μ M) for 4 h, and apoptotic responses were noted by 1.8% agarose gel electrophoresis ( C ) in D and flow cytometry ( D ) as described in “Materials and Methods.” β -Lapachone induced a G 0 /G 1 block and caused apoptosis in a concentration-dependent fashion. Treatment conditions (4 h at 37°C) in D were: a , 0.25% DMSO-treated control; b , 0.5 μ M β -lapachone; c , 1.0 μ M β -lapachone; d , 5.0 μ M β -lapachone; and e , 5.0 μ M camptothecin. In B and D , flow cytometric analyses were generated by computer as described in “Materials and Methods.” Shaded areas , G 0 /G 1 and G 2 /M cell populations; parallel lines , S-phase cells. Single line tracings are the actual flow cytometric data.
    β Lapachone, supplied by Ciba Geigy, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cayman Chemical beta lapachone
    Dow-response and temporal kinetics of apoptosis induction in HL-60 cells by β -lapachone. In A and B, log-phase human HL-60 cells were exposed to 5.0 μ M <t>β</t> -lapachone or camptothecin and then assayed for apoptotic responses in the first 4 h. A , apoptotic DNA laddering by 1.8% agarose gel electrophoresis; B , cell cycle and apoptotic responses by flow cytometry. Treatment conditions were: a , control; b–e , β -lapachone; or f , camptothecin. In C and D , log-phase HL-60 cells were treated with increasing concentrations of β -lapachone (0.05–5.0 μ M) or camptothecin (5.0 μ M) for 4 h, and apoptotic responses were noted by 1.8% agarose gel electrophoresis ( C ) in D and flow cytometry ( D ) as described in “Materials and Methods.” β -Lapachone induced a G 0 /G 1 block and caused apoptosis in a concentration-dependent fashion. Treatment conditions (4 h at 37°C) in D were: a , 0.25% DMSO-treated control; b , 0.5 μ M β -lapachone; c , 1.0 μ M β -lapachone; d , 5.0 μ M β -lapachone; and e , 5.0 μ M camptothecin. In B and D , flow cytometric analyses were generated by computer as described in “Materials and Methods.” Shaded areas , G 0 /G 1 and G 2 /M cell populations; parallel lines , S-phase cells. Single line tracings are the actual flow cytometric data.
    Beta Lapachone, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nanotherapeutics β lapachone
    ( A ) Colloidal stability of LDNLC in PBS (pH 7.4) at 37°C for up to 48 h. ( B ) Hemolysis of LDNLC at various concentrations. Data were shown as mean ± SD (n = 3). Abbreviations: LDNLC, NLC co-delivering Lapa and DOX; DOX, doxorubicin; NLC, nanostructured lipid carrier; Lapa, <t>β-lapachone.</t>
    β Lapachone, supplied by Nanotherapeutics, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem β lapachone
    <t>β-Lapachone-induced</t> necrosis and release of proteins from cardiac myocytes. A : Sytox Green staining in neonatal rat ventricular cardiac myocytes treated for increasing times with 20 μM β-lapachone (βLap). B : silver staining
    β Lapachone, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    University of Pittsburgh - Drug Discovery Institute β lapachone
    <t>β-Lapachone-induced</t> necrosis and release of proteins from cardiac myocytes. A : Sytox Green staining in neonatal rat ventricular cardiac myocytes treated for increasing times with 20 μM β-lapachone (βLap). B : silver staining
    β Lapachone, supplied by University of Pittsburgh - Drug Discovery Institute, used in various techniques. Bioz Stars score: 89/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nanotherapeutics beta lapachone micellar nanotherapeutics
    <t>β-Lapachone-induced</t> necrosis and release of proteins from cardiac myocytes. A : Sytox Green staining in neonatal rat ventricular cardiac myocytes treated for increasing times with 20 μM β-lapachone (βLap). B : silver staining
    Beta Lapachone Micellar Nanotherapeutics, supplied by Nanotherapeutics, used in various techniques. Bioz Stars score: 88/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biomol GmbH antibodies β lapachone
    Effect of <t>β-lapachone</t> on the levels of phosphorylation of PI3K and Akt in AGS cells. The cells were incubated with the indicated concentrations of β-lapachone for 24 h. The cells were lysed, and the cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were probed with specific antibodies to p-Akt (Ser 473), total Akt, p-PI3K (p85 Tyr 458/p55 Tyr 199), and total PI3K. The proteins were visualized using an ECL detection system. Actin was used as the internal control.
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    Nanotherapeutics β lapachone micellar nanotherapeutics
    Effect of <t>β-lapachone</t> on the levels of phosphorylation of PI3K and Akt in AGS cells. The cells were incubated with the indicated concentrations of β-lapachone for 24 h. The cells were lysed, and the cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were probed with specific antibodies to p-Akt (Ser 473), total Akt, p-PI3K (p85 Tyr 458/p55 Tyr 199), and total PI3K. The proteins were visualized using an ECL detection system. Actin was used as the internal control.
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    Nanotherapeutics gao j beta lapachone micellar nanotherapeutics
    Effect of <t>β-lapachone</t> on the levels of phosphorylation of PI3K and Akt in AGS cells. The cells were incubated with the indicated concentrations of β-lapachone for 24 h. The cells were lysed, and the cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were probed with specific antibodies to p-Akt (Ser 473), total Akt, p-PI3K (p85 Tyr 458/p55 Tyr 199), and total PI3K. The proteins were visualized using an ECL detection system. Actin was used as the internal control.
    Gao J Beta Lapachone Micellar Nanotherapeutics, supplied by Nanotherapeutics, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nanotherapeutics gao j β lapachone micellar nanotherapeutics
    Effect of <t>β-lapachone</t> on the levels of phosphorylation of PI3K and Akt in AGS cells. The cells were incubated with the indicated concentrations of β-lapachone for 24 h. The cells were lysed, and the cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were probed with specific antibodies to p-Akt (Ser 473), total Akt, p-PI3K (p85 Tyr 458/p55 Tyr 199), and total PI3K. The proteins were visualized using an ECL detection system. Actin was used as the internal control.
    Gao J β Lapachone Micellar Nanotherapeutics, supplied by Nanotherapeutics, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore materials β lapachone
    Chemical structure of <t>β-lapachone.</t>
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    Nanotherapeutics beta lapachone containing peg pla polymer micelles
    Chemical structure of <t>β-lapachone.</t>
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    ArQule arq 501
    Chemical structure of <t>β-lapachone.</t>
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    Nanotherapeutics against nqo1 overexpressing tumor cells
    Chemical structure of <t>β-lapachone.</t>
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    Image Search Results


    ROS reagents inhibit the activity of the SMN complex in vitro and in cells (A) Effect of H 2 O 2 on the activity of the SMN complex in vitro . Magnetic beads snRNP assembly assay was carried out in the presence of increasing amounts of H 2 O 2 . IC 50 was calculated from the dose-response curve. Error bars represent SDs from triplicate measurements. (B) Effect of menadione on SMN complex activity in vitro . The same experimental procedure was carried out as in (A), except that menadione was used instead. (C) Dose-dependent effect of menadione on SMN complex activity in cells. HeLa cells were treated with menadione at the indicated concentrations or with DMSO control for 1 hour. SMN complex activity in extracts from various treated cells was measured in comparison to DMSO control cell extract (100% activity) by magnetic beads snRNP assembly assay. Error bars represent SDs from 3 independent measurements. (D) Extracts from (C) mixed with sample buffer without or with DTT were resolved by SDS-PAGE and analyzed by Western blot of the entire membrane with anti-SMN antibody 62E7. The molecular mass markers in kDa are indicated on the left. “redSMN” indicates monomer SMN migrating at normal molecular mass and “oxSMN” indicates disulfide-crosslinked SMN upon oxidation. (E) β-lapachone and menadione generate ROS in live cells. HeLa cells were incubated 30 minutes with ROS indicator dye H 2 DCFDA (10 μM) or without dye as a control, then treated with compounds at indicated concentrations or DMSO as control. Fluorescence images were acquired 30 minutes after treatment.

    Journal: Molecular cell

    Article Title: Inactivation of the SMN complex by Oxidative Stress

    doi: 10.1016/j.molcel.2008.06.004

    Figure Lengend Snippet: ROS reagents inhibit the activity of the SMN complex in vitro and in cells (A) Effect of H 2 O 2 on the activity of the SMN complex in vitro . Magnetic beads snRNP assembly assay was carried out in the presence of increasing amounts of H 2 O 2 . IC 50 was calculated from the dose-response curve. Error bars represent SDs from triplicate measurements. (B) Effect of menadione on SMN complex activity in vitro . The same experimental procedure was carried out as in (A), except that menadione was used instead. (C) Dose-dependent effect of menadione on SMN complex activity in cells. HeLa cells were treated with menadione at the indicated concentrations or with DMSO control for 1 hour. SMN complex activity in extracts from various treated cells was measured in comparison to DMSO control cell extract (100% activity) by magnetic beads snRNP assembly assay. Error bars represent SDs from 3 independent measurements. (D) Extracts from (C) mixed with sample buffer without or with DTT were resolved by SDS-PAGE and analyzed by Western blot of the entire membrane with anti-SMN antibody 62E7. The molecular mass markers in kDa are indicated on the left. “redSMN” indicates monomer SMN migrating at normal molecular mass and “oxSMN” indicates disulfide-crosslinked SMN upon oxidation. (E) β-lapachone and menadione generate ROS in live cells. HeLa cells were incubated 30 minutes with ROS indicator dye H 2 DCFDA (10 μM) or without dye as a control, then treated with compounds at indicated concentrations or DMSO as control. Fluorescence images were acquired 30 minutes after treatment.

    Article Snippet: For confirmation studies and treatment on cells, β-lapachone, H2 O2 , cumene hydroperoxide, and menadione were purchased from Sigma-Aldrich Chemical Co.

    Techniques: Activity Assay, In Vitro, Magnetic Beads, SDS Page, Western Blot, Incubation, Fluorescence

    ROS mapping of disulfide-crosslinked cysteines in SMN (A) Sequence alignment of human ( Homo sapien s), mouse ( Mus musculus ), and frog ( Xenopus laevies ) SMN protein sequences. Conserved residues are shaded in gray. Cysteine residues are highlighted. Exons and their boundaries are indicated with opposite-directed arrows. The schematic diagram shows SMN protein domain organization and positions of cysteines. Two cysteines (C60 and C250) that form disulfide bridges are marked (-S-S-). (B) Human SMN protein (WT, wild type; ΔEx7, exon7 deletion mutant; no Cys, mutation of all 8 cysteines to alanines; C60, C98, C123, and C250, mutation of 7 cysteines to alanines except for cysteine at positions 60, 98, 123, and 250, respectively) were produced by in vitro transcription and translation in the presence of 35 S-Met and then treated with 40 μM β-lapachone or DMSO for 1 hour. Samples were mixed with sample buffer without DTT, and then analyzed by SDS-PAGE and autoradiography. Molecular mass markers in kDa are indicated on the left. Protein bands corresponding to monomer SMN (redSMN), disulfide-crosslinked SMN (oxSMN) and SMN dimer (oxSMN dimer) are indicated on the right. (C) SMN amino terminus deletion (Ex3-7) and carboxyl terminus deletion (Ex1-4) mutants were tested for crosslinking, as described in panel (B). Note that these mutants were constructed in pcDNA3-myc-pyruvate kinase (PK, ~60kD) vector to facilitate detection of otherwise small fragments.

    Journal: Molecular cell

    Article Title: Inactivation of the SMN complex by Oxidative Stress

    doi: 10.1016/j.molcel.2008.06.004

    Figure Lengend Snippet: ROS mapping of disulfide-crosslinked cysteines in SMN (A) Sequence alignment of human ( Homo sapien s), mouse ( Mus musculus ), and frog ( Xenopus laevies ) SMN protein sequences. Conserved residues are shaded in gray. Cysteine residues are highlighted. Exons and their boundaries are indicated with opposite-directed arrows. The schematic diagram shows SMN protein domain organization and positions of cysteines. Two cysteines (C60 and C250) that form disulfide bridges are marked (-S-S-). (B) Human SMN protein (WT, wild type; ΔEx7, exon7 deletion mutant; no Cys, mutation of all 8 cysteines to alanines; C60, C98, C123, and C250, mutation of 7 cysteines to alanines except for cysteine at positions 60, 98, 123, and 250, respectively) were produced by in vitro transcription and translation in the presence of 35 S-Met and then treated with 40 μM β-lapachone or DMSO for 1 hour. Samples were mixed with sample buffer without DTT, and then analyzed by SDS-PAGE and autoradiography. Molecular mass markers in kDa are indicated on the left. Protein bands corresponding to monomer SMN (redSMN), disulfide-crosslinked SMN (oxSMN) and SMN dimer (oxSMN dimer) are indicated on the right. (C) SMN amino terminus deletion (Ex3-7) and carboxyl terminus deletion (Ex1-4) mutants were tested for crosslinking, as described in panel (B). Note that these mutants were constructed in pcDNA3-myc-pyruvate kinase (PK, ~60kD) vector to facilitate detection of otherwise small fragments.

    Article Snippet: For confirmation studies and treatment on cells, β-lapachone, H2 O2 , cumene hydroperoxide, and menadione were purchased from Sigma-Aldrich Chemical Co.

    Techniques: Sequencing, Mutagenesis, Produced, In Vitro, SDS Page, Autoradiography, Construct, Plasmid Preparation

    β-lapachone potently and selectively inhibits the SMN complex-mediated snRNP assembly in vitro and in cells (A) Chemical structure of β-lapachone. (B) Concentration-dependent inhibition of SMN complex activity by β-lapachone in cells. HeLa cells were treated with various concentrations of β-lapachone or with DMSO (control) for 1 hour. SMN complex activity in extracts from treated cells was measured using magnetic beads snRNP assembly assay and compared to DMSO-treated control cells (100% activity). IC 50 was calculated from the dose-reponse graph. Error bars represent SDs from 3 independent measurements. (C) β-lapachone selectively inhibits SMN complex-mediated Sm core assembly. Assembly reactions were performed using either cell extracts or purified native snRNP proteins lacking the SMN complex (Sm proteins). Both samples were adjusted to contain a similar amount of Sm proteins. Magnetic beads snRNP assembly assay was carried out with U4 or control U4ΔSm snRNA in the presence of either 20 or 100 μM β-lapachone or DMSO control. Sm core assembly on U4 snRNA in the presence of DMSO was considered 100% activity. The error bars represent SDs from 3 independent measurements.

    Journal: Molecular cell

    Article Title: Inactivation of the SMN complex by Oxidative Stress

    doi: 10.1016/j.molcel.2008.06.004

    Figure Lengend Snippet: β-lapachone potently and selectively inhibits the SMN complex-mediated snRNP assembly in vitro and in cells (A) Chemical structure of β-lapachone. (B) Concentration-dependent inhibition of SMN complex activity by β-lapachone in cells. HeLa cells were treated with various concentrations of β-lapachone or with DMSO (control) for 1 hour. SMN complex activity in extracts from treated cells was measured using magnetic beads snRNP assembly assay and compared to DMSO-treated control cells (100% activity). IC 50 was calculated from the dose-reponse graph. Error bars represent SDs from 3 independent measurements. (C) β-lapachone selectively inhibits SMN complex-mediated Sm core assembly. Assembly reactions were performed using either cell extracts or purified native snRNP proteins lacking the SMN complex (Sm proteins). Both samples were adjusted to contain a similar amount of Sm proteins. Magnetic beads snRNP assembly assay was carried out with U4 or control U4ΔSm snRNA in the presence of either 20 or 100 μM β-lapachone or DMSO control. Sm core assembly on U4 snRNA in the presence of DMSO was considered 100% activity. The error bars represent SDs from 3 independent measurements.

    Article Snippet: For confirmation studies and treatment on cells, β-lapachone, H2 O2 , cumene hydroperoxide, and menadione were purchased from Sigma-Aldrich Chemical Co.

    Techniques: In Vitro, Concentration Assay, Inhibition, Activity Assay, Magnetic Beads, Purification

    SMN protein is oxidized to form intermolecular disulfide bonds upon β-lapachone treatment (A) Indirect immunofluorescence staining of SMN (2B1; green) and snRNPs (Y12; red) in HeLa PV cells treated 3 hours with 5 μM β-lapachone or DMSO control. (B) HeLa total cell extracts prepared from cells treated 3 hours with 5 μM β-lapachone or DMSO control were resolved by SDS-PAGE and analyzed by quantitative Western blotting, using JBP1 and Magoh as loading controls. Extracts were prepared and mixed with sample buffer without DTT. The membrane was cut into strips for probing of each protein at the corresponding molecular mass. ) were used for in vitro assembly reactions in the presence of either 100 μM β-lapachone or DMSO control. The SMN complex was isolated by anti-Flag immunoprecipitation, mixed with sample buffer without or with DTT and resolved by SDS-PAGE. Western blot analysis was performed on the entire membrane with anti-SMN antibody 62E7. Molecular mass markers in kDa are indicated on the left. “redSMN” indicates monomer SMN migrating at normal molecular mass and “oxSMN” indicates disulfide-crosslinked SMN upon oxidation.

    Journal: Molecular cell

    Article Title: Inactivation of the SMN complex by Oxidative Stress

    doi: 10.1016/j.molcel.2008.06.004

    Figure Lengend Snippet: SMN protein is oxidized to form intermolecular disulfide bonds upon β-lapachone treatment (A) Indirect immunofluorescence staining of SMN (2B1; green) and snRNPs (Y12; red) in HeLa PV cells treated 3 hours with 5 μM β-lapachone or DMSO control. (B) HeLa total cell extracts prepared from cells treated 3 hours with 5 μM β-lapachone or DMSO control were resolved by SDS-PAGE and analyzed by quantitative Western blotting, using JBP1 and Magoh as loading controls. Extracts were prepared and mixed with sample buffer without DTT. The membrane was cut into strips for probing of each protein at the corresponding molecular mass. ) were used for in vitro assembly reactions in the presence of either 100 μM β-lapachone or DMSO control. The SMN complex was isolated by anti-Flag immunoprecipitation, mixed with sample buffer without or with DTT and resolved by SDS-PAGE. Western blot analysis was performed on the entire membrane with anti-SMN antibody 62E7. Molecular mass markers in kDa are indicated on the left. “redSMN” indicates monomer SMN migrating at normal molecular mass and “oxSMN” indicates disulfide-crosslinked SMN upon oxidation.

    Article Snippet: For confirmation studies and treatment on cells, β-lapachone, H2 O2 , cumene hydroperoxide, and menadione were purchased from Sigma-Aldrich Chemical Co.

    Techniques: Immunofluorescence, Staining, SDS Page, Western Blot, In Vitro, Isolation, Immunoprecipitation

    DTT prevents the inhibition of the activity of the SMN complex by β-lapachone Cell extracts treated with 20 μM β-lapachone, or 20 μM β-lapachone together with 20 mM DTT, or DMSO control were analyzed by non-reducing Western blot. The relative levels of monomer SMN (“redSMN”) were calculated as the percentage of that in DMSO control and shown by the blue bar. Assembly activities of the SMN complex were measured by magnetic beads snRNP assembly assay using the same set of treated extracts and shown by the red bar. Error bars represent SDs from 3 independent experiments.

    Journal: Molecular cell

    Article Title: Inactivation of the SMN complex by Oxidative Stress

    doi: 10.1016/j.molcel.2008.06.004

    Figure Lengend Snippet: DTT prevents the inhibition of the activity of the SMN complex by β-lapachone Cell extracts treated with 20 μM β-lapachone, or 20 μM β-lapachone together with 20 mM DTT, or DMSO control were analyzed by non-reducing Western blot. The relative levels of monomer SMN (“redSMN”) were calculated as the percentage of that in DMSO control and shown by the blue bar. Assembly activities of the SMN complex were measured by magnetic beads snRNP assembly assay using the same set of treated extracts and shown by the red bar. Error bars represent SDs from 3 independent experiments.

    Article Snippet: For confirmation studies and treatment on cells, β-lapachone, H2 O2 , cumene hydroperoxide, and menadione were purchased from Sigma-Aldrich Chemical Co.

    Techniques: Inhibition, Activity Assay, Western Blot, Magnetic Beads

    Defects in heart-looping, valve formation, and contractile performance were detected in β-lapachone-treated embryos . A: Embryos at 24 hours post-fertilization (hpf) were treated with DMSO or β-lapachone for 4 h and fixed at 48 and 72 hpf for nppa and cmlc2 hybridization (a-h). A Q-RT-PCR analysis indicated nppa expression levels in β-lapachone-treated 48- and 72-hpf embryos (i). B: Images of respective hearts with ventricles at either end-diastolic volume of DMSO (a), β-lapachone-treated embryo containing few erythrocytes (b), and β-lapachone-treated embryo containing no erythrocytes (c), or at end-systolic volume of DMSO (a'), β-lapachone-treated embryo containing few erythrocytes (b'), and β-lapachone-treated embryo containing no erythrocytes (c') are shown. Fractional shortening (FS) of the atrial and ventricular chamber of DMSO or β-lapachone-treated 52-hpf embryos was measured and calculated according to the formula, FS = (ED - ES)/ED × 100%, where ED is the end-diastolic diameter and ES is the end-systolic diameter of either the atrial or ventricular chambers (d). In β-lapachone-treated embryos, embryos containing few or no erythrocytes were recorded. Error bars indicate the standard error. Student's t -test was used to compare DMSO- and β-lapachone-treated embryos. * p

    Journal: Journal of Biomedical Science

    Article Title: ?-Lapachone induces heart morphogenetic and functional defects by promoting the death of erythrocytes and the endocardium in zebrafish embryos

    doi: 10.1186/1423-0127-18-70

    Figure Lengend Snippet: Defects in heart-looping, valve formation, and contractile performance were detected in β-lapachone-treated embryos . A: Embryos at 24 hours post-fertilization (hpf) were treated with DMSO or β-lapachone for 4 h and fixed at 48 and 72 hpf for nppa and cmlc2 hybridization (a-h). A Q-RT-PCR analysis indicated nppa expression levels in β-lapachone-treated 48- and 72-hpf embryos (i). B: Images of respective hearts with ventricles at either end-diastolic volume of DMSO (a), β-lapachone-treated embryo containing few erythrocytes (b), and β-lapachone-treated embryo containing no erythrocytes (c), or at end-systolic volume of DMSO (a'), β-lapachone-treated embryo containing few erythrocytes (b'), and β-lapachone-treated embryo containing no erythrocytes (c') are shown. Fractional shortening (FS) of the atrial and ventricular chamber of DMSO or β-lapachone-treated 52-hpf embryos was measured and calculated according to the formula, FS = (ED - ES)/ED × 100%, where ED is the end-diastolic diameter and ES is the end-systolic diameter of either the atrial or ventricular chambers (d). In β-lapachone-treated embryos, embryos containing few or no erythrocytes were recorded. Error bars indicate the standard error. Student's t -test was used to compare DMSO- and β-lapachone-treated embryos. * p

    Article Snippet: β-Lapachone treatment Embryos were treated with β-lapachone (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 2 μM diluted with egg water at 24 or 48 hpf for 4 h at 28°C.

    Techniques: Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing

    Both dicoumarol and BAPTA-AM rescued the erythrocyte-deficiency in circulation and heart-looping defect phenotypes in β-lapachone-treated embryos . Embryos at 24 hours post-fertilization (hpf) were treated with either 0.2% DMSO (A, B), 5 μM dicoumarol (C, D), 2 μM β-lapachone (E, F), or 2 μM β-lapachone and 5 μM dicoumarol (G, H) for 4 h and fixed for nppa hybridization and o -dianisidine (ODA) staining at 48 hpf. Higher-magnification ODA-stained images of the ventral tail region are shown (B, D, F, H). (I, J) Evaluation of the rescue effects by dicoumarol or BAPTA-AM. Error bars indicate the standard error. Scale bars represent 100 μm.

    Journal: Journal of Biomedical Science

    Article Title: ?-Lapachone induces heart morphogenetic and functional defects by promoting the death of erythrocytes and the endocardium in zebrafish embryos

    doi: 10.1186/1423-0127-18-70

    Figure Lengend Snippet: Both dicoumarol and BAPTA-AM rescued the erythrocyte-deficiency in circulation and heart-looping defect phenotypes in β-lapachone-treated embryos . Embryos at 24 hours post-fertilization (hpf) were treated with either 0.2% DMSO (A, B), 5 μM dicoumarol (C, D), 2 μM β-lapachone (E, F), or 2 μM β-lapachone and 5 μM dicoumarol (G, H) for 4 h and fixed for nppa hybridization and o -dianisidine (ODA) staining at 48 hpf. Higher-magnification ODA-stained images of the ventral tail region are shown (B, D, F, H). (I, J) Evaluation of the rescue effects by dicoumarol or BAPTA-AM. Error bars indicate the standard error. Scale bars represent 100 μm.

    Article Snippet: β-Lapachone treatment Embryos were treated with β-lapachone (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 2 μM diluted with egg water at 24 or 48 hpf for 4 h at 28°C.

    Techniques: Hybridization, Staining

    Phenotype and circulation defect of β-lapachone-treated embryos . Embryos at 24 hours post-fertilization (hpf) were treated with DMSO or β-lapachone for 4 h and examined at 30 (A, B), 52 (C-F), 72 (G-J), and 96 hpf (K, L). Dextran rhodamine dye-injected and DMSO-treated 48-hpf embryos 2 min after dye injection (M, N), and dextran rhodamine dye-injected β-lapachone-treated 48-hpf embryos 2 min (O) and 16 min after dye injection (P) are shown. Arrowheads indicate erythrocytes. The star indicates yolk edema. Black arrows point to the linear arrangement of the heart chambers and pericardial edema, while white arrows in panels N-P indicate dye injection sites. Scale bars represent 100 μm.

    Journal: Journal of Biomedical Science

    Article Title: ?-Lapachone induces heart morphogenetic and functional defects by promoting the death of erythrocytes and the endocardium in zebrafish embryos

    doi: 10.1186/1423-0127-18-70

    Figure Lengend Snippet: Phenotype and circulation defect of β-lapachone-treated embryos . Embryos at 24 hours post-fertilization (hpf) were treated with DMSO or β-lapachone for 4 h and examined at 30 (A, B), 52 (C-F), 72 (G-J), and 96 hpf (K, L). Dextran rhodamine dye-injected and DMSO-treated 48-hpf embryos 2 min after dye injection (M, N), and dextran rhodamine dye-injected β-lapachone-treated 48-hpf embryos 2 min (O) and 16 min after dye injection (P) are shown. Arrowheads indicate erythrocytes. The star indicates yolk edema. Black arrows point to the linear arrangement of the heart chambers and pericardial edema, while white arrows in panels N-P indicate dye injection sites. Scale bars represent 100 μm.

    Article Snippet: β-Lapachone treatment Embryos were treated with β-lapachone (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 2 μM diluted with egg water at 24 or 48 hpf for 4 h at 28°C.

    Techniques: Injection

    Induction of ROS and DNA fragmentation in erythrocytes and the endocardium by β-lapachone treatment . A: Embryos at 24 hours post-fertilization (hpf) were treated with DMSO or β-lapachone for 4 h and fixed at 30 and 48 hpf for either hbae1 hybridization or o -dianisidine (ODA) staining. B: DMSO- and β-lapachone-treated embryos were incubated with CM-H 2 DCFDA for 1 h at 29 hpf, and both bright-field (a, c) and fluorescent (b, d-f) images under a green fluorescent protein (GFP) filter were recorded. Atrial boundary is depicted by white dotted lines. Arrows indicate flowing erythrocytes with green fluorescence from the common cardinal vein to the atrium of the heart (d-f) of β-lapachone-treated embryos. C: Embryos were treated with DMSO or β-lapachone at 48 hpf for 4 h, fixed at 52 hpf, and stained with ODA. After paraffin sectioning, TUNEL reactions were conducted, and fluorescein-dUTP-labeled erythrocytes were detected in β-lapachone-treated embryos (f, h). DIC images are shown (a, e), and the inset figure in panel a shows the position of sectioning. Tail border is illustrated by white dotted lines. Arrows in panels a and e indicate ODA-stained erythrocytes. D: Embryos were treated with DMSO or β-lapachone at 48 hpf for 4 h and fixed at 52 hpf. After paraffin sectioning, TUNEL reactions were conducted, and fluorescein-dUTP-labeled cells located in the endocardium were detected in β-lapachone-treated embryos (f, h, j, l). In addition, fluorescein-dUTP-labeled erythrocytes were detected in the yolk near the heart (f, j). Red dotted lines indicate borders of head and yolk while white dotted lines illustrate ventricle boundaries. * indicates erythrocytes. V, ventricle. Scale bars represent 100 μm.

    Journal: Journal of Biomedical Science

    Article Title: ?-Lapachone induces heart morphogenetic and functional defects by promoting the death of erythrocytes and the endocardium in zebrafish embryos

    doi: 10.1186/1423-0127-18-70

    Figure Lengend Snippet: Induction of ROS and DNA fragmentation in erythrocytes and the endocardium by β-lapachone treatment . A: Embryos at 24 hours post-fertilization (hpf) were treated with DMSO or β-lapachone for 4 h and fixed at 30 and 48 hpf for either hbae1 hybridization or o -dianisidine (ODA) staining. B: DMSO- and β-lapachone-treated embryos were incubated with CM-H 2 DCFDA for 1 h at 29 hpf, and both bright-field (a, c) and fluorescent (b, d-f) images under a green fluorescent protein (GFP) filter were recorded. Atrial boundary is depicted by white dotted lines. Arrows indicate flowing erythrocytes with green fluorescence from the common cardinal vein to the atrium of the heart (d-f) of β-lapachone-treated embryos. C: Embryos were treated with DMSO or β-lapachone at 48 hpf for 4 h, fixed at 52 hpf, and stained with ODA. After paraffin sectioning, TUNEL reactions were conducted, and fluorescein-dUTP-labeled erythrocytes were detected in β-lapachone-treated embryos (f, h). DIC images are shown (a, e), and the inset figure in panel a shows the position of sectioning. Tail border is illustrated by white dotted lines. Arrows in panels a and e indicate ODA-stained erythrocytes. D: Embryos were treated with DMSO or β-lapachone at 48 hpf for 4 h and fixed at 52 hpf. After paraffin sectioning, TUNEL reactions were conducted, and fluorescein-dUTP-labeled cells located in the endocardium were detected in β-lapachone-treated embryos (f, h, j, l). In addition, fluorescein-dUTP-labeled erythrocytes were detected in the yolk near the heart (f, j). Red dotted lines indicate borders of head and yolk while white dotted lines illustrate ventricle boundaries. * indicates erythrocytes. V, ventricle. Scale bars represent 100 μm.

    Article Snippet: β-Lapachone treatment Embryos were treated with β-lapachone (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 2 μM diluted with egg water at 24 or 48 hpf for 4 h at 28°C.

    Techniques: Hybridization, Staining, Incubation, Fluorescence, TUNEL Assay, Labeling

    Decreased wall shear stress was detected in β-lapachone-treated embryos . Tg( gata1 : DsRed ) was treated with DMSO or β-lapachone for 4 h at 24 h post-fertilization (hpf), and blood cell circulation was recorded at 30 hpf. A: A DIC image of 30-hpf embryos is shown. The inset figure in panel A indicates the recorded region, and DsRed-labeled erythrocytes were recorded under the TRITC mode (a-f). Images corresponding to an individual DsRed-labeled erythrocyte (arrow) at time 0 and after traveling for some distance at time t are shown for respective DMSO-treated (a, b) and 2 representative β-lapachone-treated (c, d; e, f) embryos. B: The relative wall shear stress was calculated based on τ = 4 μQ/πR 3 where μ is viscosity, Q is the flow rate, and R is the blood vessel diameter. Student's t -test was used to compare DMSO- and β-lapachone-treated embryos. * p

    Journal: Journal of Biomedical Science

    Article Title: ?-Lapachone induces heart morphogenetic and functional defects by promoting the death of erythrocytes and the endocardium in zebrafish embryos

    doi: 10.1186/1423-0127-18-70

    Figure Lengend Snippet: Decreased wall shear stress was detected in β-lapachone-treated embryos . Tg( gata1 : DsRed ) was treated with DMSO or β-lapachone for 4 h at 24 h post-fertilization (hpf), and blood cell circulation was recorded at 30 hpf. A: A DIC image of 30-hpf embryos is shown. The inset figure in panel A indicates the recorded region, and DsRed-labeled erythrocytes were recorded under the TRITC mode (a-f). Images corresponding to an individual DsRed-labeled erythrocyte (arrow) at time 0 and after traveling for some distance at time t are shown for respective DMSO-treated (a, b) and 2 representative β-lapachone-treated (c, d; e, f) embryos. B: The relative wall shear stress was calculated based on τ = 4 μQ/πR 3 where μ is viscosity, Q is the flow rate, and R is the blood vessel diameter. Student's t -test was used to compare DMSO- and β-lapachone-treated embryos. * p

    Article Snippet: β-Lapachone treatment Embryos were treated with β-lapachone (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 2 μM diluted with egg water at 24 or 48 hpf for 4 h at 28°C.

    Techniques: Labeling, Flow Cytometry

    ULK1 kinase activity promotes PARP1 activation and sensitizes cells to oxidative stress-induced death. ( a ) Ulk1 KO/ Ulk2 stably expressing vector control, WT ULK1, or KI ULK1 were cotransfected with a vector containing PARP1-GFP and incubated in complete media (CM) with or without H 2 O 2 for 20 min. PARP1 was depleted from the nucleoli of cells expressing WT ULK1 but not from those of cells expressing the KI mutant. Scale bars=10 μ M. Representative immunoblots ( b ) and a graph ( c ) show the levels of PARylated proteins in the indicated MEF lines incubated in the presence or the absence of 500 μ M H 2 O 2 for 10 min. ( d and e ) Trypan blue-exclusion assays demonstrate that the KI-ULK1 mutant fails to promote cell death to the same extent as WT ULK1 does when stably expressed in Ulk1 -deficient MEFs. ( f ) The 293T cells cotransfected with PARP1-GFP and either WT ULK1 or the R266A-ULK1 mutant were treated for 10 min with 100 μ M H 2 O 2 . Immunoblot analyses demonstrate a decrease in PARylated proteins in cells transfected with the R266A mutant. ( g ) The 293T cells transfected with the indicated expression constructs were treated with 100 μ M H 2 O 2 . ATP levels were analyzed in cells collected 10, 30, or 60 min after treatment with 100 μ M H 2 O 2 and normalized to levels in untreated cells. ( h ) Trypan blue-exclusion assays performed 6 h after treatment with the indicated dose of H 2 O 2 demonstrate increased viability of 293T transfected with the R266A-ULK1 mutant expression construct compared with that of those transfected with the WT ULK1 construct. ( i ) The 293T cells were transfected with empty vector (−), WT ULK1 (WT), or the following mutant forms of ULK1: K46A (kinase-defective mutant), 4SA (AMPK-resistant, autophagy-defective mutant), or R266A (NLS mutant). Transfected cells were treated with 4.5 μ M β -lapachone for 72 h and then cell viability was assessed using CellTiter-Glo assays. Signals were normalized to those in DMSO-treated controls ( n =4 biologic replicates per condition), P

    Journal: Cell Death and Differentiation

    Article Title: Nuclear ULK1 promotes cell death in response to oxidative stress through PARP1

    doi: 10.1038/cdd.2015.88

    Figure Lengend Snippet: ULK1 kinase activity promotes PARP1 activation and sensitizes cells to oxidative stress-induced death. ( a ) Ulk1 KO/ Ulk2 stably expressing vector control, WT ULK1, or KI ULK1 were cotransfected with a vector containing PARP1-GFP and incubated in complete media (CM) with or without H 2 O 2 for 20 min. PARP1 was depleted from the nucleoli of cells expressing WT ULK1 but not from those of cells expressing the KI mutant. Scale bars=10 μ M. Representative immunoblots ( b ) and a graph ( c ) show the levels of PARylated proteins in the indicated MEF lines incubated in the presence or the absence of 500 μ M H 2 O 2 for 10 min. ( d and e ) Trypan blue-exclusion assays demonstrate that the KI-ULK1 mutant fails to promote cell death to the same extent as WT ULK1 does when stably expressed in Ulk1 -deficient MEFs. ( f ) The 293T cells cotransfected with PARP1-GFP and either WT ULK1 or the R266A-ULK1 mutant were treated for 10 min with 100 μ M H 2 O 2 . Immunoblot analyses demonstrate a decrease in PARylated proteins in cells transfected with the R266A mutant. ( g ) The 293T cells transfected with the indicated expression constructs were treated with 100 μ M H 2 O 2 . ATP levels were analyzed in cells collected 10, 30, or 60 min after treatment with 100 μ M H 2 O 2 and normalized to levels in untreated cells. ( h ) Trypan blue-exclusion assays performed 6 h after treatment with the indicated dose of H 2 O 2 demonstrate increased viability of 293T transfected with the R266A-ULK1 mutant expression construct compared with that of those transfected with the WT ULK1 construct. ( i ) The 293T cells were transfected with empty vector (−), WT ULK1 (WT), or the following mutant forms of ULK1: K46A (kinase-defective mutant), 4SA (AMPK-resistant, autophagy-defective mutant), or R266A (NLS mutant). Transfected cells were treated with 4.5 μ M β -lapachone for 72 h and then cell viability was assessed using CellTiter-Glo assays. Signals were normalized to those in DMSO-treated controls ( n =4 biologic replicates per condition), P

    Article Snippet: The β -lapachone (CAS 4707-32-8, Tocris 1293, Bristol, UK) was dissolved in DMSO and used at a final concentration of 4.5 μ M. For camptothecin treatment, cells were plated in 96-well plates (Corning catalogue number 3917, Corning, NY, USA) at the following densities: untransfected WT and Ulk1 -deficient MEFs (2.0 × 104 cells/well) and transfected 293T cells (2.5 × 104 cells/well).

    Techniques: Activity Assay, Activation Assay, Stable Transfection, Expressing, Plasmid Preparation, Incubation, Mutagenesis, Western Blot, Transfection, Construct

    β-Lapachone lethality is dependent on NQO1 status, but not oncogenic driver or passenger mutations NSCLC cell lines were screened in a double-blind manner for lethality (LD 50 values), NQO1 ) and oncogene mutational statuses. NQO1 polymorphic statuses were: WT (wild-type); hets (WT + polymorphic alleles: ^, *2 (C609T) or #, *3 (C465T), HCC2935); or pm, homozygous polymorphic (*2 or *3) alleles. Relative β-lap sensitivities were expressed as LD 50 values (µM, 2 hr). Values with ‘greater than ( > )’ signs indicate highest concentrations (µM) tested. DNA from NSCLC cells were sequenced by the UTSW-MDA lung cancer SPORE (red: mutant, blue: null (deletion), black: wild-type, white: no sequence available). .

    Journal: Cancer cell

    Article Title: Leveraging an NQO1 Bioactivatable Drug for Tumor-Selective Use of Poly(ADP-ribose) Polymerase (PARP) Inhibitors

    doi: 10.1016/j.ccell.2016.11.006

    Figure Lengend Snippet: β-Lapachone lethality is dependent on NQO1 status, but not oncogenic driver or passenger mutations NSCLC cell lines were screened in a double-blind manner for lethality (LD 50 values), NQO1 ) and oncogene mutational statuses. NQO1 polymorphic statuses were: WT (wild-type); hets (WT + polymorphic alleles: ^, *2 (C609T) or #, *3 (C465T), HCC2935); or pm, homozygous polymorphic (*2 or *3) alleles. Relative β-lap sensitivities were expressed as LD 50 values (µM, 2 hr). Values with ‘greater than ( > )’ signs indicate highest concentrations (µM) tested. DNA from NSCLC cells were sequenced by the UTSW-MDA lung cancer SPORE (red: mutant, blue: null (deletion), black: wild-type, white: no sequence available). .

    Article Snippet: We show that the NAD(P)H:quinone oxidoreductase 1 (NQO1) bioactivatable drug, β-lapachone (ARQ761, ArQule, Woburn, MA, in clinical form), capitalizes on elevated NQO1:CAT ratios in recalcitrant pancreatic, non-small cell lung (NSCLC) and breast cancers to elicit tumor-selective programmed necrosis.

    Techniques: Multiple Displacement Amplification, Mutagenesis, Sequencing

    Synergy between nontoxic doses of PARP inhibitors and sublethal β-lapachone doses (A–D) A549 NSCLC cells were pretreated for 2 hr with: Rucaparib (A) , Olaparib (B) , Veliparib (C) , each at 15 µM, or Talazoparib at 1.25 µM (D) ). Synergy values for Rucaparib (η=0.452, p value=0.003), (Olaparib (η=0.494, p value=0.0013) and Talazoparib (η=0.548, p value=0.036) were reported based on multiple dose-responses, or on comparative p values indicated. (E, H) PAR and γH2AX formation alterations for DMSO or β-lap (3 µM)-exposed A549 cells treated with various doses of Rucaparib (E) or Olaparib (H) . (F, I, G, J) Relative PARP activity inhibition for doses of Rucaparib (F) or Olaparib (I) and dose enhancement ratio (DER) correlations for Rucaparib (G) or Olaparib (J) dose-responses when combined with β-lap (3 µM). .

    Journal: Cancer cell

    Article Title: Leveraging an NQO1 Bioactivatable Drug for Tumor-Selective Use of Poly(ADP-ribose) Polymerase (PARP) Inhibitors

    doi: 10.1016/j.ccell.2016.11.006

    Figure Lengend Snippet: Synergy between nontoxic doses of PARP inhibitors and sublethal β-lapachone doses (A–D) A549 NSCLC cells were pretreated for 2 hr with: Rucaparib (A) , Olaparib (B) , Veliparib (C) , each at 15 µM, or Talazoparib at 1.25 µM (D) ). Synergy values for Rucaparib (η=0.452, p value=0.003), (Olaparib (η=0.494, p value=0.0013) and Talazoparib (η=0.548, p value=0.036) were reported based on multiple dose-responses, or on comparative p values indicated. (E, H) PAR and γH2AX formation alterations for DMSO or β-lap (3 µM)-exposed A549 cells treated with various doses of Rucaparib (E) or Olaparib (H) . (F, I, G, J) Relative PARP activity inhibition for doses of Rucaparib (F) or Olaparib (I) and dose enhancement ratio (DER) correlations for Rucaparib (G) or Olaparib (J) dose-responses when combined with β-lap (3 µM). .

    Article Snippet: We show that the NAD(P)H:quinone oxidoreductase 1 (NQO1) bioactivatable drug, β-lapachone (ARQ761, ArQule, Woburn, MA, in clinical form), capitalizes on elevated NQO1:CAT ratios in recalcitrant pancreatic, non-small cell lung (NSCLC) and breast cancers to elicit tumor-selective programmed necrosis.

    Techniques: Activity Assay, Inhibition

    PARP inhibition and β-lapachone synergy is NQO1-dependent and broadly applied to various types of NQO1 over-expressing cancers (A,B) Polymorphic *2 H596 NSCLC cells corrected for NQO1 expression (A) were pretreated with Rucaparib (15 µM, 2 hr) and then exposed or not to Rucaparib (15 µM) + β-lap (0–4 µM) for 2 hr. Cells were also exposed to β-lap, ± dicoumarol (DIC, 50 µM) for 2 hr and survival assessed. Genetically matched NQO1-deficient H596 NSCLC cells (B) were treated as in (A) , and survival assessed. (C,D) NQO1 + MiaPaCa2 PDA cells (C) were pretreated with Rucaparib (15 µM, 2 hr), then exposed or not to Rucaparib (15 µM) + β-lap (0–3 µM or 0–6 µM for D , respectively) for 2 hr, ± dicoumarol (DIC, 50 µM). Drugs were removed and survival assessed. Stable shRNA- NQO1 down MiaPaCa2 (clone 17–7) vs shSCR MiaPaCa2 cells (D) were treated as in (C) , but without dicoumarol and assessed for survival. (E,F) NQO1 + Suit2 (S2-013) PDA cells (E) harboring a CMV-NQO1 over-expression vector (see Western, inset) were pretreated with Rucaparib (15 µM) and then exposed or not to β-lap (0–4 µM), ± dicoumarol (DIC, 50 µM) for 2 hr. Genetically matched, NQO1 − *2 polymorphic S2-013 chemo- and radio-resistant PDA cells (F) expressing shSCR were treated as in (E) and survival assessed. See inset (E) for NQO1 expression. (G,H) MDA-MB-231 *2 polymorphic TNBC cells corrected for NQO1 expression (G) were pretreated with Rucaparib (15 µM, 2 hr), + β-lap (0–2.5 µM), ± dicoumarol (DIC, 50 µM) for 2 hr. Drugs were removed and survival assessed. shSCR NQO1 − MDA-MB-231 TNBC cells (H) lacking NQO1 expression were treated as in (G) and assessed for survival. ( A–H ). Synergy values (η=0.452, p value=0.0003) were reported based on multiple dose-responses, or on comparative p values indicated. All error bars are means of six replicates from three independent experiments; means ± SEM. ***, p

    Journal: Cancer cell

    Article Title: Leveraging an NQO1 Bioactivatable Drug for Tumor-Selective Use of Poly(ADP-ribose) Polymerase (PARP) Inhibitors

    doi: 10.1016/j.ccell.2016.11.006

    Figure Lengend Snippet: PARP inhibition and β-lapachone synergy is NQO1-dependent and broadly applied to various types of NQO1 over-expressing cancers (A,B) Polymorphic *2 H596 NSCLC cells corrected for NQO1 expression (A) were pretreated with Rucaparib (15 µM, 2 hr) and then exposed or not to Rucaparib (15 µM) + β-lap (0–4 µM) for 2 hr. Cells were also exposed to β-lap, ± dicoumarol (DIC, 50 µM) for 2 hr and survival assessed. Genetically matched NQO1-deficient H596 NSCLC cells (B) were treated as in (A) , and survival assessed. (C,D) NQO1 + MiaPaCa2 PDA cells (C) were pretreated with Rucaparib (15 µM, 2 hr), then exposed or not to Rucaparib (15 µM) + β-lap (0–3 µM or 0–6 µM for D , respectively) for 2 hr, ± dicoumarol (DIC, 50 µM). Drugs were removed and survival assessed. Stable shRNA- NQO1 down MiaPaCa2 (clone 17–7) vs shSCR MiaPaCa2 cells (D) were treated as in (C) , but without dicoumarol and assessed for survival. (E,F) NQO1 + Suit2 (S2-013) PDA cells (E) harboring a CMV-NQO1 over-expression vector (see Western, inset) were pretreated with Rucaparib (15 µM) and then exposed or not to β-lap (0–4 µM), ± dicoumarol (DIC, 50 µM) for 2 hr. Genetically matched, NQO1 − *2 polymorphic S2-013 chemo- and radio-resistant PDA cells (F) expressing shSCR were treated as in (E) and survival assessed. See inset (E) for NQO1 expression. (G,H) MDA-MB-231 *2 polymorphic TNBC cells corrected for NQO1 expression (G) were pretreated with Rucaparib (15 µM, 2 hr), + β-lap (0–2.5 µM), ± dicoumarol (DIC, 50 µM) for 2 hr. Drugs were removed and survival assessed. shSCR NQO1 − MDA-MB-231 TNBC cells (H) lacking NQO1 expression were treated as in (G) and assessed for survival. ( A–H ). Synergy values (η=0.452, p value=0.0003) were reported based on multiple dose-responses, or on comparative p values indicated. All error bars are means of six replicates from three independent experiments; means ± SEM. ***, p

    Article Snippet: We show that the NAD(P)H:quinone oxidoreductase 1 (NQO1) bioactivatable drug, β-lapachone (ARQ761, ArQule, Woburn, MA, in clinical form), capitalizes on elevated NQO1:CAT ratios in recalcitrant pancreatic, non-small cell lung (NSCLC) and breast cancers to elicit tumor-selective programmed necrosis.

    Techniques: Inhibition, Expressing, shRNA, Over Expression, Plasmid Preparation, Western Blot, Multiple Displacement Amplification

    Inhibition of β-lapachone-stimulated, NQO1-dependent PARP1 hyperactivation by Rucaparib spares catastrophic energy loss (A–D) NQO1 + A549 NSCLC cells were pretreated with Rucaparib (15 µM, 2 hr), then exposed or not to Rucaparib (15 µM) + β-lap (3 or 8 µM), ± dicoumarol (DIC, 50 µM) for 2 hr. Cells were also treated with β-lap (3 or 8 µM, 2 hr) alone. Cells were then monitored for: Long-term ROS formation (i.e., relative H 2 O 2 levels) at 2 hr (A) ; Real-time oxygen consumption rates (OCRs) after various drug treatments (added at t=20 min, arrow) by Seahorse XF analyses: Ruc, Rucaparib; Oligo, oligomycin (B) ; Total NAD + and NADH levels (C) ; and Relative ATP levels after 2 hr treatments ( D ). Results were separately repeated at least three times in triplicate each. Results (means ± SEM) from three independent experiments. ***, p

    Journal: Cancer cell

    Article Title: Leveraging an NQO1 Bioactivatable Drug for Tumor-Selective Use of Poly(ADP-ribose) Polymerase (PARP) Inhibitors

    doi: 10.1016/j.ccell.2016.11.006

    Figure Lengend Snippet: Inhibition of β-lapachone-stimulated, NQO1-dependent PARP1 hyperactivation by Rucaparib spares catastrophic energy loss (A–D) NQO1 + A549 NSCLC cells were pretreated with Rucaparib (15 µM, 2 hr), then exposed or not to Rucaparib (15 µM) + β-lap (3 or 8 µM), ± dicoumarol (DIC, 50 µM) for 2 hr. Cells were also treated with β-lap (3 or 8 µM, 2 hr) alone. Cells were then monitored for: Long-term ROS formation (i.e., relative H 2 O 2 levels) at 2 hr (A) ; Real-time oxygen consumption rates (OCRs) after various drug treatments (added at t=20 min, arrow) by Seahorse XF analyses: Ruc, Rucaparib; Oligo, oligomycin (B) ; Total NAD + and NADH levels (C) ; and Relative ATP levels after 2 hr treatments ( D ). Results were separately repeated at least three times in triplicate each. Results (means ± SEM) from three independent experiments. ***, p

    Article Snippet: We show that the NAD(P)H:quinone oxidoreductase 1 (NQO1) bioactivatable drug, β-lapachone (ARQ761, ArQule, Woburn, MA, in clinical form), capitalizes on elevated NQO1:CAT ratios in recalcitrant pancreatic, non-small cell lung (NSCLC) and breast cancers to elicit tumor-selective programmed necrosis.

    Techniques: Inhibition

    Camptothecin induces degradation of DNA topoisomerase I in fibroblasts . (A) Presence of DNA toposiomerase I in the immunocomplexes described in 1B was confirmed by Western blotting analyses. (B) Fibroblasts described in 1B were treated with 25 μM camptothecin or 25 μM β-lapachone, and DNA topoisomerase I present in the lysates was detected by Western blotting. The same blots were stripped and reprobed with anti-β-tubulin antibodies as a loading control. Relative ratios of DNA topoisomerase I to tubulin levels are indicated. All the data shown here are representative of at least two independent experiments. CPT = camptothecin, Topo I = DNA topoisomerase I, IP = immunoprecipitation, IB = immunoblotting, and β-LP = β-lapachone.

    Journal: BMC Cell Biology

    Article Title: Increased susceptibility of spinal muscular atrophy fibroblasts to camptothecin is p53-independent

    doi: 10.1186/1471-2121-10-40

    Figure Lengend Snippet: Camptothecin induces degradation of DNA topoisomerase I in fibroblasts . (A) Presence of DNA toposiomerase I in the immunocomplexes described in 1B was confirmed by Western blotting analyses. (B) Fibroblasts described in 1B were treated with 25 μM camptothecin or 25 μM β-lapachone, and DNA topoisomerase I present in the lysates was detected by Western blotting. The same blots were stripped and reprobed with anti-β-tubulin antibodies as a loading control. Relative ratios of DNA topoisomerase I to tubulin levels are indicated. All the data shown here are representative of at least two independent experiments. CPT = camptothecin, Topo I = DNA topoisomerase I, IP = immunoprecipitation, IB = immunoblotting, and β-LP = β-lapachone.

    Article Snippet: Eighteen hours after seeding, cells were left untreated or treated with camptothecin (Sigma, St. Louis, MO), β-lapachone (Biomol, Plymouth Meeting, PA), or menadione (Sigma) at the indicated time points (Fig. ).

    Techniques: Western Blot, Cycling Probe Technology, Immunoprecipitation

    Activation of p53 and cell death induced by the DNA topoisomerase I inhibitors camptothecin and β-lapachone . (A) A type I SMA fibroblasts as described [ 29 ] were treated with 25 μM camptothecin and levels of p53 were analyzed by Western blotting. The same blots were stripped and reprobed with anti-β-tubulin antibodies as a loading control. (B) A type II/III SMA fibroblasts as described [ 29 ] were treated with 10 μM menadione and levels of p53 were analyzed as described for (A). (C) Control and SMA fibroblasts were treated with 25 μM camptothecin or 25 μM β-lapachone and levels of p53 were analyzed as described for (A). Relative ratios of p53 to tubulin levels are indicated. (D) Three control and three SMA fibroblasts (one type I, one type II, and one type III) were treated with camptothecin for 72 h or β-lapachone for 24 h at the indicated concentrations, respectively. Cell survival of treated cells was measured by the CellTiter-Blue assay, and the relative cell viability was calculated and presented as percentage of the untreated cells. Each condition was set up as replicates of four and repeated three times. The data presented here are combined mean values ± sem for three control and three SMA fibroblasts. Statistical analyses (unpaired t test) indicate that SMA fibroblasts are significantly sensitive to camptothecin at each tested concentration than control fibroblasts (*** p

    Journal: BMC Cell Biology

    Article Title: Increased susceptibility of spinal muscular atrophy fibroblasts to camptothecin is p53-independent

    doi: 10.1186/1471-2121-10-40

    Figure Lengend Snippet: Activation of p53 and cell death induced by the DNA topoisomerase I inhibitors camptothecin and β-lapachone . (A) A type I SMA fibroblasts as described [ 29 ] were treated with 25 μM camptothecin and levels of p53 were analyzed by Western blotting. The same blots were stripped and reprobed with anti-β-tubulin antibodies as a loading control. (B) A type II/III SMA fibroblasts as described [ 29 ] were treated with 10 μM menadione and levels of p53 were analyzed as described for (A). (C) Control and SMA fibroblasts were treated with 25 μM camptothecin or 25 μM β-lapachone and levels of p53 were analyzed as described for (A). Relative ratios of p53 to tubulin levels are indicated. (D) Three control and three SMA fibroblasts (one type I, one type II, and one type III) were treated with camptothecin for 72 h or β-lapachone for 24 h at the indicated concentrations, respectively. Cell survival of treated cells was measured by the CellTiter-Blue assay, and the relative cell viability was calculated and presented as percentage of the untreated cells. Each condition was set up as replicates of four and repeated three times. The data presented here are combined mean values ± sem for three control and three SMA fibroblasts. Statistical analyses (unpaired t test) indicate that SMA fibroblasts are significantly sensitive to camptothecin at each tested concentration than control fibroblasts (*** p

    Article Snippet: Eighteen hours after seeding, cells were left untreated or treated with camptothecin (Sigma, St. Louis, MO), β-lapachone (Biomol, Plymouth Meeting, PA), or menadione (Sigma) at the indicated time points (Fig. ).

    Techniques: Activation Assay, Western Blot, CtB Assay, Concentration Assay

    Dow-response and temporal kinetics of apoptosis induction in HL-60 cells by β -lapachone. In A and B, log-phase human HL-60 cells were exposed to 5.0 μ M β -lapachone or camptothecin and then assayed for apoptotic responses in the first 4 h. A , apoptotic DNA laddering by 1.8% agarose gel electrophoresis; B , cell cycle and apoptotic responses by flow cytometry. Treatment conditions were: a , control; b–e , β -lapachone; or f , camptothecin. In C and D , log-phase HL-60 cells were treated with increasing concentrations of β -lapachone (0.05–5.0 μ M) or camptothecin (5.0 μ M) for 4 h, and apoptotic responses were noted by 1.8% agarose gel electrophoresis ( C ) in D and flow cytometry ( D ) as described in “Materials and Methods.” β -Lapachone induced a G 0 /G 1 block and caused apoptosis in a concentration-dependent fashion. Treatment conditions (4 h at 37°C) in D were: a , 0.25% DMSO-treated control; b , 0.5 μ M β -lapachone; c , 1.0 μ M β -lapachone; d , 5.0 μ M β -lapachone; and e , 5.0 μ M camptothecin. In B and D , flow cytometric analyses were generated by computer as described in “Materials and Methods.” Shaded areas , G 0 /G 1 and G 2 /M cell populations; parallel lines , S-phase cells. Single line tracings are the actual flow cytometric data.

    Journal: Cancer research

    Article Title: β-Lapachone-mediated Apoptosis in Human Promyelocytic Leukemia (HL-60) and Human Prostate Cancer Cells: A p53-independent Response

    doi:

    Figure Lengend Snippet: Dow-response and temporal kinetics of apoptosis induction in HL-60 cells by β -lapachone. In A and B, log-phase human HL-60 cells were exposed to 5.0 μ M β -lapachone or camptothecin and then assayed for apoptotic responses in the first 4 h. A , apoptotic DNA laddering by 1.8% agarose gel electrophoresis; B , cell cycle and apoptotic responses by flow cytometry. Treatment conditions were: a , control; b–e , β -lapachone; or f , camptothecin. In C and D , log-phase HL-60 cells were treated with increasing concentrations of β -lapachone (0.05–5.0 μ M) or camptothecin (5.0 μ M) for 4 h, and apoptotic responses were noted by 1.8% agarose gel electrophoresis ( C ) in D and flow cytometry ( D ) as described in “Materials and Methods.” β -Lapachone induced a G 0 /G 1 block and caused apoptosis in a concentration-dependent fashion. Treatment conditions (4 h at 37°C) in D were: a , 0.25% DMSO-treated control; b , 0.5 μ M β -lapachone; c , 1.0 μ M β -lapachone; d , 5.0 μ M β -lapachone; and e , 5.0 μ M camptothecin. In B and D , flow cytometric analyses were generated by computer as described in “Materials and Methods.” Shaded areas , G 0 /G 1 and G 2 /M cell populations; parallel lines , S-phase cells. Single line tracings are the actual flow cytometric data.

    Article Snippet: We discovered that the β -lapachone available from Ciba-Geigy is roughly two-thirds pure and contains a high molecular weight (mw: 377) degradation byproduct, which has no apparent biological activity.

    Techniques: DNA Laddering, Agarose Gel Electrophoresis, Flow Cytometry, Cytometry, Blocking Assay, Concentration Assay, Generated

    ( A ) Colloidal stability of LDNLC in PBS (pH 7.4) at 37°C for up to 48 h. ( B ) Hemolysis of LDNLC at various concentrations. Data were shown as mean ± SD (n = 3). Abbreviations: LDNLC, NLC co-delivering Lapa and DOX; DOX, doxorubicin; NLC, nanostructured lipid carrier; Lapa, β-lapachone.

    Journal: International Journal of Nanomedicine

    Article Title: Nanostructured lipid carriers co-delivering lapachone and doxorubicin for overcoming multidrug resistance in breast cancer therapy

    doi: 10.2147/IJN.S163929

    Figure Lengend Snippet: ( A ) Colloidal stability of LDNLC in PBS (pH 7.4) at 37°C for up to 48 h. ( B ) Hemolysis of LDNLC at various concentrations. Data were shown as mean ± SD (n = 3). Abbreviations: LDNLC, NLC co-delivering Lapa and DOX; DOX, doxorubicin; NLC, nanostructured lipid carrier; Lapa, β-lapachone.

    Article Snippet: Zhang L, Chen Z, Yang K, et al. β-Lapachone and paclitaxel combination micelles with improved drug encapsulation and therapeutic synergy as novel nanotherapeutics for NQO1-targeted cancer therapy.

    Techniques:

    Drug release profile of free Lapa, free DOX, and Lapa/DOX from LDNLC for up to 48 h. Note: Data were shown as mean ± SD (n = 3). Abbreviations: LDNLC, NLC co-delivering Lapa and DOX; DOX, doxorubicin; NLC, nanostructured lipid carrier; Lapa, β-lapachone.

    Journal: International Journal of Nanomedicine

    Article Title: Nanostructured lipid carriers co-delivering lapachone and doxorubicin for overcoming multidrug resistance in breast cancer therapy

    doi: 10.2147/IJN.S163929

    Figure Lengend Snippet: Drug release profile of free Lapa, free DOX, and Lapa/DOX from LDNLC for up to 48 h. Note: Data were shown as mean ± SD (n = 3). Abbreviations: LDNLC, NLC co-delivering Lapa and DOX; DOX, doxorubicin; NLC, nanostructured lipid carrier; Lapa, β-lapachone.

    Article Snippet: Zhang L, Chen Z, Yang K, et al. β-Lapachone and paclitaxel combination micelles with improved drug encapsulation and therapeutic synergy as novel nanotherapeutics for NQO1-targeted cancer therapy.

    Techniques:

    ( A ) In vivo imaging of biodistribution of MCF-7 ADR tumor xenograft mice after injection of free DiR or DiR-loaded LDNLC. ( B ) DOX concentration in blood as a function of time after a single intravenous administration of free DOX or LDNLC. ( C ) Distribution profiles of total DOX in tissues at 24 h after a single intravenous administration of free DOX or LDNLC. Data were shown as mean ± SD (n = 3). Abbreviations: LDNLC, NLC co-delivering Lapa and DOX; DOX, doxorubicin; NLC, nanostructured lipid carrier; Lapa, β-lapachone; DiR,.

    Journal: International Journal of Nanomedicine

    Article Title: Nanostructured lipid carriers co-delivering lapachone and doxorubicin for overcoming multidrug resistance in breast cancer therapy

    doi: 10.2147/IJN.S163929

    Figure Lengend Snippet: ( A ) In vivo imaging of biodistribution of MCF-7 ADR tumor xenograft mice after injection of free DiR or DiR-loaded LDNLC. ( B ) DOX concentration in blood as a function of time after a single intravenous administration of free DOX or LDNLC. ( C ) Distribution profiles of total DOX in tissues at 24 h after a single intravenous administration of free DOX or LDNLC. Data were shown as mean ± SD (n = 3). Abbreviations: LDNLC, NLC co-delivering Lapa and DOX; DOX, doxorubicin; NLC, nanostructured lipid carrier; Lapa, β-lapachone; DiR,.

    Article Snippet: Zhang L, Chen Z, Yang K, et al. β-Lapachone and paclitaxel combination micelles with improved drug encapsulation and therapeutic synergy as novel nanotherapeutics for NQO1-targeted cancer therapy.

    Techniques: In Vivo Imaging, Mouse Assay, Injection, Concentration Assay

    Intracellular uptake at 12 h post-incubation of DNLC and LDNLC (pretreated with/without 60 μM of dicoumarol [Di]). Note: Scale bar: 20 μM. Abbreviations: DNLC, DOX mono-delivery NLC; LDNLC, NLC co-delivering Lapa and DOX; DOX, doxorubicin; NLC, nanostructured lipid carrier; Lapa, β-lapachone.

    Journal: International Journal of Nanomedicine

    Article Title: Nanostructured lipid carriers co-delivering lapachone and doxorubicin for overcoming multidrug resistance in breast cancer therapy

    doi: 10.2147/IJN.S163929

    Figure Lengend Snippet: Intracellular uptake at 12 h post-incubation of DNLC and LDNLC (pretreated with/without 60 μM of dicoumarol [Di]). Note: Scale bar: 20 μM. Abbreviations: DNLC, DOX mono-delivery NLC; LDNLC, NLC co-delivering Lapa and DOX; DOX, doxorubicin; NLC, nanostructured lipid carrier; Lapa, β-lapachone.

    Article Snippet: Zhang L, Chen Z, Yang K, et al. β-Lapachone and paclitaxel combination micelles with improved drug encapsulation and therapeutic synergy as novel nanotherapeutics for NQO1-targeted cancer therapy.

    Techniques: Incubation

    β-Lapachone-induced necrosis and release of proteins from cardiac myocytes. A : Sytox Green staining in neonatal rat ventricular cardiac myocytes treated for increasing times with 20 μM β-lapachone (βLap). B : silver staining

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Proteomic mapping of proteins released during necrosis and apoptosis from cultured neonatal cardiac myocytes

    doi: 10.1152/ajpcell.00167.2013

    Figure Lengend Snippet: β-Lapachone-induced necrosis and release of proteins from cardiac myocytes. A : Sytox Green staining in neonatal rat ventricular cardiac myocytes treated for increasing times with 20 μM β-lapachone (βLap). B : silver staining

    Article Snippet: Sytox Green was purchased from Invitrogen; M199 medium, Hanks' buffered saline solution (HBSS), penicillin/streptomycin, and bovine growth serum (BGS) were purchased from Hyclone; the neonatal rat myocyte isolation kit was purchased from Worthington; the silver staining kit was purchased from Thermo Pierce; zVAD-FMK was purchased from Promega; β-lapachone was from Enzo Life Sciences; the TUNEL kit was purchased from Roche; and all other chemicals/reagents were from Fisher and Sigma-Aldrich.

    Techniques: Staining, Silver Staining

    Effect of β-lapachone on the levels of phosphorylation of PI3K and Akt in AGS cells. The cells were incubated with the indicated concentrations of β-lapachone for 24 h. The cells were lysed, and the cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were probed with specific antibodies to p-Akt (Ser 473), total Akt, p-PI3K (p85 Tyr 458/p55 Tyr 199), and total PI3K. The proteins were visualized using an ECL detection system. Actin was used as the internal control.

    Journal: Biomolecules & Therapeutics

    Article Title: ?-lapachone-Induced Apoptosis of Human Gastric Carcinoma AGS Cells Is Caspase-Dependent and Regulated by the PI3K/Akt Pathway

    doi: 10.4062/biomolther.2014.026

    Figure Lengend Snippet: Effect of β-lapachone on the levels of phosphorylation of PI3K and Akt in AGS cells. The cells were incubated with the indicated concentrations of β-lapachone for 24 h. The cells were lysed, and the cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were probed with specific antibodies to p-Akt (Ser 473), total Akt, p-PI3K (p85 Tyr 458/p55 Tyr 199), and total PI3K. The proteins were visualized using an ECL detection system. Actin was used as the internal control.

    Article Snippet: Reagents and antibodies β-lapachone was purchased from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Incubation, Polyacrylamide Gel Electrophoresis

    β-lapachone inhibits cell viability in AGS cells. Cells were seeded at 2×10 5 cell per well onto 96-well culture plates overnight and then treated with the indicated concentrations of β-lapachone for 24 h. Cell viability was determined by the MTT assay. Data are mean ± SD of three independent experiments ( * p

    Journal: Biomolecules & Therapeutics

    Article Title: ?-lapachone-Induced Apoptosis of Human Gastric Carcinoma AGS Cells Is Caspase-Dependent and Regulated by the PI3K/Akt Pathway

    doi: 10.4062/biomolther.2014.026

    Figure Lengend Snippet: β-lapachone inhibits cell viability in AGS cells. Cells were seeded at 2×10 5 cell per well onto 96-well culture plates overnight and then treated with the indicated concentrations of β-lapachone for 24 h. Cell viability was determined by the MTT assay. Data are mean ± SD of three independent experiments ( * p

    Article Snippet: Reagents and antibodies β-lapachone was purchased from Biomol (Plymouth Meeting, PA, USA).

    Techniques: MTT Assay

    β-lapachone induces apoptosis in AGS cells. (A) The cells were treated with the indicated concentrations of β-lapachone for 24 h, sampled, fixed, and stained with DAPI solution. The stained nuclei were observed under a fluorescent microscope (original magnification, 400×). (B) To quantify the degree of apoptosis induced by β-lapachone, cells grown under the same conditions as (A) were evaluated by a flow cytometry for sub-G1 DNA content, which represents the cells undergoing apoptotic DNA degradation. Data are mean ± SD of three independent experiments ( * p

    Journal: Biomolecules & Therapeutics

    Article Title: ?-lapachone-Induced Apoptosis of Human Gastric Carcinoma AGS Cells Is Caspase-Dependent and Regulated by the PI3K/Akt Pathway

    doi: 10.4062/biomolther.2014.026

    Figure Lengend Snippet: β-lapachone induces apoptosis in AGS cells. (A) The cells were treated with the indicated concentrations of β-lapachone for 24 h, sampled, fixed, and stained with DAPI solution. The stained nuclei were observed under a fluorescent microscope (original magnification, 400×). (B) To quantify the degree of apoptosis induced by β-lapachone, cells grown under the same conditions as (A) were evaluated by a flow cytometry for sub-G1 DNA content, which represents the cells undergoing apoptotic DNA degradation. Data are mean ± SD of three independent experiments ( * p

    Article Snippet: Reagents and antibodies β-lapachone was purchased from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Staining, Microscopy, Flow Cytometry, Cytometry

    Caspase-mediated apoptosis induced by β-lapachone in AGS cells. (A) Cells were incubated with 4 μM β-lapachone for 24 h after 1 h pretreatment with the pan-caspase inhibitor, z-VAD-fmk (50 μM). Equal amounts of cell lysates isolated from cells were subjected to SDS-polyacrylamide gel electrophoresis and analyzed by Western blot for caspase-3 and PARP. (B) Cells grown under the same conditions as (A) were fixed and stained with DAPI solution. Stained nuclei were observed under a fluorescent microscope. (C) Cells were evaluated by flow cytometry for sub-G1 DNA content, suggestive of apoptotic cell death. (D) Cell viability was determined by the MTT assay. Results are mean ± SD of three independent experiments ( * p

    Journal: Biomolecules & Therapeutics

    Article Title: ?-lapachone-Induced Apoptosis of Human Gastric Carcinoma AGS Cells Is Caspase-Dependent and Regulated by the PI3K/Akt Pathway

    doi: 10.4062/biomolther.2014.026

    Figure Lengend Snippet: Caspase-mediated apoptosis induced by β-lapachone in AGS cells. (A) Cells were incubated with 4 μM β-lapachone for 24 h after 1 h pretreatment with the pan-caspase inhibitor, z-VAD-fmk (50 μM). Equal amounts of cell lysates isolated from cells were subjected to SDS-polyacrylamide gel electrophoresis and analyzed by Western blot for caspase-3 and PARP. (B) Cells grown under the same conditions as (A) were fixed and stained with DAPI solution. Stained nuclei were observed under a fluorescent microscope. (C) Cells were evaluated by flow cytometry for sub-G1 DNA content, suggestive of apoptotic cell death. (D) Cell viability was determined by the MTT assay. Results are mean ± SD of three independent experiments ( * p

    Article Snippet: Reagents and antibodies β-lapachone was purchased from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Incubation, Isolation, Polyacrylamide Gel Electrophoresis, Western Blot, Staining, Microscopy, Flow Cytometry, Cytometry, MTT Assay

    The role of PI3K/Akt signaling in β-lapachone-induced apoptosis in AGS cells. (A) Cells were stimulated with 3 μM β-lapachone for 24 h after pretreatment with 20 μM LY290042 for 1 h. Equal amounts of cell lysates were resolved by SDS-polyacrylamide gel electrophoresis, transferred to membranes, and probed with the indicated antibodies. Anti-actin antibody was a protein loading control. (B and D) The percentage of the sub-G1 population (B) and cell viability (D) were assessed by flow cytometry and the MTT assay, respectively. Results are mean ± SD of three independent experiments ( * p

    Journal: Biomolecules & Therapeutics

    Article Title: ?-lapachone-Induced Apoptosis of Human Gastric Carcinoma AGS Cells Is Caspase-Dependent and Regulated by the PI3K/Akt Pathway

    doi: 10.4062/biomolther.2014.026

    Figure Lengend Snippet: The role of PI3K/Akt signaling in β-lapachone-induced apoptosis in AGS cells. (A) Cells were stimulated with 3 μM β-lapachone for 24 h after pretreatment with 20 μM LY290042 for 1 h. Equal amounts of cell lysates were resolved by SDS-polyacrylamide gel electrophoresis, transferred to membranes, and probed with the indicated antibodies. Anti-actin antibody was a protein loading control. (B and D) The percentage of the sub-G1 population (B) and cell viability (D) were assessed by flow cytometry and the MTT assay, respectively. Results are mean ± SD of three independent experiments ( * p

    Article Snippet: Reagents and antibodies β-lapachone was purchased from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Polyacrylamide Gel Electrophoresis, Flow Cytometry, Cytometry, MTT Assay

    Effect of β-lapachone on levels of MMP, Bcl-2 family proteins, and cytochrome c in AGS cells. (A) Cells were treated with indicated concentrations of β-lapachone for 24 h. They were collected and incubated with 10 μM JC-1 for 15 min at 37°C in the dark. The cells were washed once with PBS and analyzed by a DNA flow cytometer. Results are presented as the mean of two independent experiments. (B) Equal amounts of cell lysates was resolved by SDS-polyacrylamide gel electrophoresis, transferred to membranes, and probed with specific antibodies. The anti-actin antibody was a protein loading control. (C) Cytosolic and mitochondrial proteins were extracted from cells grown under the same conditions and analyzed by Western blotting using anti-cytochrome c antibody. Actin and cytochrome oxidase subunit IV (COX IV) were used as internal controls for the cytosolic and mitochondrial fractions, respectively.

    Journal: Biomolecules & Therapeutics

    Article Title: ?-lapachone-Induced Apoptosis of Human Gastric Carcinoma AGS Cells Is Caspase-Dependent and Regulated by the PI3K/Akt Pathway

    doi: 10.4062/biomolther.2014.026

    Figure Lengend Snippet: Effect of β-lapachone on levels of MMP, Bcl-2 family proteins, and cytochrome c in AGS cells. (A) Cells were treated with indicated concentrations of β-lapachone for 24 h. They were collected and incubated with 10 μM JC-1 for 15 min at 37°C in the dark. The cells were washed once with PBS and analyzed by a DNA flow cytometer. Results are presented as the mean of two independent experiments. (B) Equal amounts of cell lysates was resolved by SDS-polyacrylamide gel electrophoresis, transferred to membranes, and probed with specific antibodies. The anti-actin antibody was a protein loading control. (C) Cytosolic and mitochondrial proteins were extracted from cells grown under the same conditions and analyzed by Western blotting using anti-cytochrome c antibody. Actin and cytochrome oxidase subunit IV (COX IV) were used as internal controls for the cytosolic and mitochondrial fractions, respectively.

    Article Snippet: Reagents and antibodies β-lapachone was purchased from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Incubation, Flow Cytometry, Cytometry, Polyacrylamide Gel Electrophoresis, Western Blot

    Activation of caspases and degradation of PARP by β-lapachone in AGS cells. (A) Cells were treated with the indicated concentrations of β-lapachone for 24 h. The cells were lysed, and cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The proteins were visualized using the indicated antibodies and an ECL detection system. Proteolytic cleavage of PARP is indicated by the arrow. Actin was used as the internal control. (B) After a 24 h incubation with the indicated concentrations of β-lapachone, the cells were lysed, and aliquots were assayed for in vitro caspase-3, -8, and -9 activity using Ac-DEVD-pNA, Ac-IETD-pNA, and Ac-LEHD-pNA as substrates, respectively, at 37°C. After incubation of 1 h, the amount of pNA released was measured at 405 nm using an ELISA microplate reader. Each point represents the mean ± SD of three independent experiments ( * p

    Journal: Biomolecules & Therapeutics

    Article Title: ?-lapachone-Induced Apoptosis of Human Gastric Carcinoma AGS Cells Is Caspase-Dependent and Regulated by the PI3K/Akt Pathway

    doi: 10.4062/biomolther.2014.026

    Figure Lengend Snippet: Activation of caspases and degradation of PARP by β-lapachone in AGS cells. (A) Cells were treated with the indicated concentrations of β-lapachone for 24 h. The cells were lysed, and cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The proteins were visualized using the indicated antibodies and an ECL detection system. Proteolytic cleavage of PARP is indicated by the arrow. Actin was used as the internal control. (B) After a 24 h incubation with the indicated concentrations of β-lapachone, the cells were lysed, and aliquots were assayed for in vitro caspase-3, -8, and -9 activity using Ac-DEVD-pNA, Ac-IETD-pNA, and Ac-LEHD-pNA as substrates, respectively, at 37°C. After incubation of 1 h, the amount of pNA released was measured at 405 nm using an ELISA microplate reader. Each point represents the mean ± SD of three independent experiments ( * p

    Article Snippet: Reagents and antibodies β-lapachone was purchased from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Activation Assay, Polyacrylamide Gel Electrophoresis, Incubation, In Vitro, Activity Assay, Enzyme-linked Immunosorbent Assay

    Chemical structure of β-lapachone.

    Journal: Biomolecules & Therapeutics

    Article Title: Preclinical Pharmacokinetic Evaluation of β-Lapachone: Characteristics of Oral Bioavailability and First-Pass Metabolism in Rats

    doi: 10.4062/biomolther.2015.029

    Figure Lengend Snippet: Chemical structure of β-lapachone.

    Article Snippet: Materials β-Lapachone ( > 98% determined by thin-layer chromatography) and nifedipine were obtained from Sigma-Aldrich Company (St. Louis, MO, USA).

    Techniques:

    The profiles of levels of β-lapachone in plasma as a function of time after oral and intravenous administration of β-lapachone to rats at dose of 40 mg/kg and 1.5 mg/kg. Each data point indicates mean ± SD (n=5).

    Journal: Biomolecules & Therapeutics

    Article Title: Preclinical Pharmacokinetic Evaluation of β-Lapachone: Characteristics of Oral Bioavailability and First-Pass Metabolism in Rats

    doi: 10.4062/biomolther.2015.029

    Figure Lengend Snippet: The profiles of levels of β-lapachone in plasma as a function of time after oral and intravenous administration of β-lapachone to rats at dose of 40 mg/kg and 1.5 mg/kg. Each data point indicates mean ± SD (n=5).

    Article Snippet: Materials β-Lapachone ( > 98% determined by thin-layer chromatography) and nifedipine were obtained from Sigma-Aldrich Company (St. Louis, MO, USA).

    Techniques:

    Metabolic degradation of β-lapachone in liver, small and large intestine homogenates. Each data point indicates mean ± SD (n=3).

    Journal: Biomolecules & Therapeutics

    Article Title: Preclinical Pharmacokinetic Evaluation of β-Lapachone: Characteristics of Oral Bioavailability and First-Pass Metabolism in Rats

    doi: 10.4062/biomolther.2015.029

    Figure Lengend Snippet: Metabolic degradation of β-lapachone in liver, small and large intestine homogenates. Each data point indicates mean ± SD (n=3).

    Article Snippet: Materials β-Lapachone ( > 98% determined by thin-layer chromatography) and nifedipine were obtained from Sigma-Aldrich Company (St. Louis, MO, USA).

    Techniques:

    Stability profiles of β-lapachone in rat plasma and dimethyl sulfoxide (DMSO) used to solubilize the drug. Each data point indicates mean ± SD (n=3).

    Journal: Biomolecules & Therapeutics

    Article Title: Preclinical Pharmacokinetic Evaluation of β-Lapachone: Characteristics of Oral Bioavailability and First-Pass Metabolism in Rats

    doi: 10.4062/biomolther.2015.029

    Figure Lengend Snippet: Stability profiles of β-lapachone in rat plasma and dimethyl sulfoxide (DMSO) used to solubilize the drug. Each data point indicates mean ± SD (n=3).

    Article Snippet: Materials β-Lapachone ( > 98% determined by thin-layer chromatography) and nifedipine were obtained from Sigma-Aldrich Company (St. Louis, MO, USA).

    Techniques: