ethidium bromide  (Millipore)


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    Structured Review

    Millipore ethidium bromide
    DNA (1000 μ M) and <t>ethidium</t> bromide (50 μ M). ( a ) Absorption spectra of ethidium bromide ( dashed line ) and ethidium bromide-ct-DNA ( solid line ). ( Inset ) Enlarged region of ethidium bromide band. ( b ) Fluorescence excitation spectra with all emitted photons detected, ( c ) LD spectra of ct-DNA ( dashed line ) and ethidium bromide-ct-DNA ( solid line ), and ( d ) FDFLD spectra. All solutions prepared using a sodium cacodylate buffer (10 mM, pH 7).
    Ethidium Bromide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium bromide/product/Millipore
    Average 99 stars, based on 60 article reviews
    Price from $9.99 to $1999.99
    ethidium bromide - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) Product Images from "Micro-Volume Couette Flow Sample Orientation for Absorbance and Fluorescence Linear Dichroism"

    Article Title: Micro-Volume Couette Flow Sample Orientation for Absorbance and Fluorescence Linear Dichroism

    Journal: Biophysical Journal

    doi: 10.1529/biophysj.103.035022

    DNA (1000 μ M) and ethidium bromide (50 μ M). ( a ) Absorption spectra of ethidium bromide ( dashed line ) and ethidium bromide-ct-DNA ( solid line ). ( Inset ) Enlarged region of ethidium bromide band. ( b ) Fluorescence excitation spectra with all emitted photons detected, ( c ) LD spectra of ct-DNA ( dashed line ) and ethidium bromide-ct-DNA ( solid line ), and ( d ) FDFLD spectra. All solutions prepared using a sodium cacodylate buffer (10 mM, pH 7).
    Figure Legend Snippet: DNA (1000 μ M) and ethidium bromide (50 μ M). ( a ) Absorption spectra of ethidium bromide ( dashed line ) and ethidium bromide-ct-DNA ( solid line ). ( Inset ) Enlarged region of ethidium bromide band. ( b ) Fluorescence excitation spectra with all emitted photons detected, ( c ) LD spectra of ct-DNA ( dashed line ) and ethidium bromide-ct-DNA ( solid line ), and ( d ) FDFLD spectra. All solutions prepared using a sodium cacodylate buffer (10 mM, pH 7).

    Techniques Used: Fluorescence

    LD spectra of DNA (200 μ M) and different concentrations of ethidium bromide (0–50 μ M) using a sodium cacodylate buffer (10 mM, pH 7) and NaCl (10 mM).
    Figure Legend Snippet: LD spectra of DNA (200 μ M) and different concentrations of ethidium bromide (0–50 μ M) using a sodium cacodylate buffer (10 mM, pH 7) and NaCl (10 mM).

    Techniques Used:

    2) Product Images from "Adipose-Derived Mesenchymal Stem Cells Migrate and Rescue RPE in the Setting of Oxidative Stress"

    Article Title: Adipose-Derived Mesenchymal Stem Cells Migrate and Rescue RPE in the Setting of Oxidative Stress

    Journal: Stem Cells International

    doi: 10.1155/2018/9682856

    ASCs rescue RPE cell death under oxidative stress. RPE cells were incubated with P3-CM or with controls comprising P5-CM or non-CM for 48 hours, followed by exposure to H 2 O 2 (1 mM, 7 h). Cells were harvested and cell death was analyzed. (a, b) Necrotic cell death was determined using PI staining followed by flow cytometry analysis. (c) Cell death visualized by acridine orange and ethidium bromide staining. Live cells appear green stained by acridine orange only (×20 magnification). CM: conditioned medium. Each experiment was performed a minimum of 3 samples from 3 different patients. Each experiment was performed a minimum of 3 times.
    Figure Legend Snippet: ASCs rescue RPE cell death under oxidative stress. RPE cells were incubated with P3-CM or with controls comprising P5-CM or non-CM for 48 hours, followed by exposure to H 2 O 2 (1 mM, 7 h). Cells were harvested and cell death was analyzed. (a, b) Necrotic cell death was determined using PI staining followed by flow cytometry analysis. (c) Cell death visualized by acridine orange and ethidium bromide staining. Live cells appear green stained by acridine orange only (×20 magnification). CM: conditioned medium. Each experiment was performed a minimum of 3 samples from 3 different patients. Each experiment was performed a minimum of 3 times.

    Techniques Used: Incubation, Staining, Flow Cytometry, Cytometry

    3) Product Images from "Adipose-Derived Mesenchymal Stem Cells Migrate and Rescue RPE in the Setting of Oxidative Stress"

    Article Title: Adipose-Derived Mesenchymal Stem Cells Migrate and Rescue RPE in the Setting of Oxidative Stress

    Journal: Stem Cells International

    doi: 10.1155/2018/9682856

    ASCs rescue RPE cell death under oxidative stress. RPE cells were incubated with P3-CM or with controls comprising P5-CM or non-CM for 48 hours, followed by exposure to H 2 O 2 (1 mM, 7 h). Cells were harvested and cell death was analyzed. (a, b) Necrotic cell death was determined using PI staining followed by flow cytometry analysis. (c) Cell death visualized by acridine orange and ethidium bromide staining. Live cells appear green stained by acridine orange only (×20 magnification). CM: conditioned medium. Each experiment was performed a minimum of 3 samples from 3 different patients. Each experiment was performed a minimum of 3 times.
    Figure Legend Snippet: ASCs rescue RPE cell death under oxidative stress. RPE cells were incubated with P3-CM or with controls comprising P5-CM or non-CM for 48 hours, followed by exposure to H 2 O 2 (1 mM, 7 h). Cells were harvested and cell death was analyzed. (a, b) Necrotic cell death was determined using PI staining followed by flow cytometry analysis. (c) Cell death visualized by acridine orange and ethidium bromide staining. Live cells appear green stained by acridine orange only (×20 magnification). CM: conditioned medium. Each experiment was performed a minimum of 3 samples from 3 different patients. Each experiment was performed a minimum of 3 times.

    Techniques Used: Incubation, Staining, Flow Cytometry, Cytometry

    Related Articles

    Amplification:

    Article Title: Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]
    Article Snippet: .. The amplified products were determined on a 1.0% agarose gel (Sigma, St. Louis, USA) stained with 0.1% ethidium bromide (Sigma, St. Louis, USA) and photographed using the Gel Documentation system (UVITEC, Cambridge, UK). qRT-PCR analysis was performed in LightCycler® 480 Real-time PCR system (Roche, Germany). .. The expression analysis of the RT-PCR positive (T1 ) and NT plants were conducted using Prime Script™ RT Reagent Kit (Takara Bio Inc, Japan) and p68 gene-specific primer set (FP: 5′-CCTCGCATTCTCTTCCTCGTA-3′ and RP: 5′-CGACGAGAACCATTGGCTAGA-3′) (Banu et al. ).

    Article Title: Molecular screening of pathogenic Escherichia coli strains isolated from dairy neonatal calves in Cordoba province, Argentina.
    Article Snippet: The amplified PCR products were separated by electrophoresis on a 2 % agarose gel with Tris---acetate---EDTA (TAE) buffer (Sigma---Aldrich®). .. The gel was stained with ethidium bromide (Sigma---Aldrich ® ) and visualized under a UV-transilluminator at 302 nm.

    Article Title: Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]
    Article Snippet: .. The amplified products were analyzed by electrophoresis on a 1.0% agarose (Sigma, St. Louis, USA) gel stained with 0.1% ethidium bromide (Sigma, St. Louis, USA) and photographed using the Gel Documentation system (UVITEC, Cambridge, UK). .. Five µg of genomic DNA extracted from the transformed plants (T1 ), non-transformed (NT) plant and 5 µg of binary vector pCAMBIA 1300- p68 were dotted into 1 cm square grids on a nylon membrane (Hybond N+ , GE Health care Limted, Buckinghamshire, UK).

    Article Title: Agro-morphological description, genetic diversity and population structure of sugarcane varieties from sub-tropical India
    Article Snippet: .. The amplification products were resolved on 12% polyacrylamide gel with 1 × Tris-borate EDTA buffer, stained with 0.5 µg/ml ethidium bromide (Sigma-Aldrich, USA) and visualized using a gel documentation system (G:Box, Syngene, UK). .. The clear and unambiguous amplified products were scored as dominant markers with 1 (present) or 0 (absent) since it is difficult to identify and differentiate between amplicons from homeologous chromosomes.

    Quantitative RT-PCR:

    Article Title: Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]
    Article Snippet: .. The amplified products were determined on a 1.0% agarose gel (Sigma, St. Louis, USA) stained with 0.1% ethidium bromide (Sigma, St. Louis, USA) and photographed using the Gel Documentation system (UVITEC, Cambridge, UK). qRT-PCR analysis was performed in LightCycler® 480 Real-time PCR system (Roche, Germany). .. The expression analysis of the RT-PCR positive (T1 ) and NT plants were conducted using Prime Script™ RT Reagent Kit (Takara Bio Inc, Japan) and p68 gene-specific primer set (FP: 5′-CCTCGCATTCTCTTCCTCGTA-3′ and RP: 5′-CGACGAGAACCATTGGCTAGA-3′) (Banu et al. ).

    Real-time Polymerase Chain Reaction:

    Article Title: Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]
    Article Snippet: .. The amplified products were determined on a 1.0% agarose gel (Sigma, St. Louis, USA) stained with 0.1% ethidium bromide (Sigma, St. Louis, USA) and photographed using the Gel Documentation system (UVITEC, Cambridge, UK). qRT-PCR analysis was performed in LightCycler® 480 Real-time PCR system (Roche, Germany). .. The expression analysis of the RT-PCR positive (T1 ) and NT plants were conducted using Prime Script™ RT Reagent Kit (Takara Bio Inc, Japan) and p68 gene-specific primer set (FP: 5′-CCTCGCATTCTCTTCCTCGTA-3′ and RP: 5′-CGACGAGAACCATTGGCTAGA-3′) (Banu et al. ).

    Proliferation Assay:

    Article Title: Acquisition of Apoptotic Resistance in Cadmium-Transformed Human Prostate Epithelial Cells: Bcl-2 Overexpression Blocks the Activation of JNK Signal Transduction Pathway
    Article Snippet: Chemicals and reagents Cadmium chloride (CdCl2 ), etoposide, cisplatin, chloroform, isopropanol, formaldehyde, and ethidium bromide were purchased from Sigma Chemical Co. (St. Louis, MO). .. A nonradioactive cell proliferation assay kit was obtained from Promega (Madison, WI).

    Expressing:

    Article Title: Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]
    Article Snippet: The amplified products were determined on a 1.0% agarose gel (Sigma, St. Louis, USA) stained with 0.1% ethidium bromide (Sigma, St. Louis, USA) and photographed using the Gel Documentation system (UVITEC, Cambridge, UK). qRT-PCR analysis was performed in LightCycler® 480 Real-time PCR system (Roche, Germany). .. The expression analysis of the RT-PCR positive (T1 ) and NT plants were conducted using Prime Script™ RT Reagent Kit (Takara Bio Inc, Japan) and p68 gene-specific primer set (FP: 5′-CCTCGCATTCTCTTCCTCGTA-3′ and RP: 5′-CGACGAGAACCATTGGCTAGA-3′) (Banu et al. ).

    Modification:

    Article Title: Impact of different cell penetrating peptides on the efficacy of antisense therapeutics for targeting intracellular pathogens
    Article Snippet: .. Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s Phosphate Buffered Saline (PBS), nisin, chloroform, isopropanol, agarose, ethidium bromide, Tris-Borate-EDTA buffer, free water and primers were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). .. Gentamicin, TRIzol Max Bacterial RNA Isolation Kit, SYBR Green PCR Master Mix, SuperScript II Reverse Transcriptase, 1 kb plus DNA ladder were purchased from Invitrogen (Carlsbad, CA, USA).

    Article Title: Cytotoxicity and genotoxicity of coronaridine from Tabernaemontana catharinensis A.DC in a human laryngeal epithelial carcinoma cell line (Hep-2)
    Article Snippet: .. Penicillin, streptomycin, doxorubicin, Dulbecco’s modified Eagle’s and HAM-F10 culture media, DMSO, isopropanol, MTT, actinomycin D, acridine orange, ethidium bromide and trypsin were purchased from Sigma Chemicals (St. Louis, MO, USA). .. Plant material Root barks of T. catharinensis were collected in Assis, São Paulo, Brazil, and a voucher specimen was deposited at the herbarium (SPFR) of FFCLRP, University of São Paulo, Ribeirão Preto, Brazil (Registration No. 02940).

    Transformation Assay:

    Article Title: Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]
    Article Snippet: Genomic DNA was isolated from the young leaf tissues collected from seventeen transformed lines (T1 ) and non-transformed (NT) plant using the CTAB method as earlier reported by Dellaporta et al. ( ). .. The amplified products were analyzed by electrophoresis on a 1.0% agarose (Sigma, St. Louis, USA) gel stained with 0.1% ethidium bromide (Sigma, St. Louis, USA) and photographed using the Gel Documentation system (UVITEC, Cambridge, UK).

    Hybridization:

    Article Title: Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]
    Article Snippet: Total RNA was extracted from Southern hybridization positive (T1 ) and NT plants using a RNAqueous kit (Ambion Inc., Austin, USA) and the DNA contamination were eliminated by treatment with DNase I. RT-PCR was performed by a one-step RT-PCR kit (Qiagen, USA) as instructed by the manufacturer. .. The amplified products were determined on a 1.0% agarose gel (Sigma, St. Louis, USA) stained with 0.1% ethidium bromide (Sigma, St. Louis, USA) and photographed using the Gel Documentation system (UVITEC, Cambridge, UK). qRT-PCR analysis was performed in LightCycler® 480 Real-time PCR system (Roche, Germany).

    Transfection:

    Article Title: X protein variants of the autochthonous Latin American hepatitis B virus F genotype promotes human hepatocyte death by the induction of apoptosis and autophagy.
    Article Snippet: .. Acridine orange and ethidium bromide staining After transfections, cells were harvested, washed with PBS and resuspended in 1 ml PBS containing 4 μg/ml acridine orange (Sigma, USA) and 4 μg/ml ethidium bromide (Sigma, USA). .. The cell suspension was immediately dispensed onto slides, viewed under fluorescent microscopy (Leitz Dialux 20, Germany) and photographed.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]
    Article Snippet: Paragraph title: Reverse transcriptase-PCR (RT-PCR) and Quantitative real-time PCR (qRT-PCR) ... The amplified products were determined on a 1.0% agarose gel (Sigma, St. Louis, USA) stained with 0.1% ethidium bromide (Sigma, St. Louis, USA) and photographed using the Gel Documentation system (UVITEC, Cambridge, UK). qRT-PCR analysis was performed in LightCycler® 480 Real-time PCR system (Roche, Germany).

    Molecular Weight:

    Article Title: Molecular screening of pathogenic Escherichia coli strains isolated from dairy neonatal calves in Cordoba province, Argentina.
    Article Snippet: A 100-bp DNA ladder (CienMarker, Biodynamics) was used as molecular weight marker. .. The gel was stained with ethidium bromide (Sigma---Aldrich ® ) and visualized under a UV-transilluminator at 302 nm.

    MTT Assay:

    Article Title: Cytotoxicity and genotoxicity of coronaridine from Tabernaemontana catharinensis A.DC in a human laryngeal epithelial carcinoma cell line (Hep-2)
    Article Snippet: .. Penicillin, streptomycin, doxorubicin, Dulbecco’s modified Eagle’s and HAM-F10 culture media, DMSO, isopropanol, MTT, actinomycin D, acridine orange, ethidium bromide and trypsin were purchased from Sigma Chemicals (St. Louis, MO, USA). .. Plant material Root barks of T. catharinensis were collected in Assis, São Paulo, Brazil, and a voucher specimen was deposited at the herbarium (SPFR) of FFCLRP, University of São Paulo, Ribeirão Preto, Brazil (Registration No. 02940).

    Fluorescence:

    Article Title: Micro-Volume Couette Flow Sample Orientation for Absorbance and Fluorescence Linear Dichroism
    Article Snippet: Fluorescence data were collected using a Perkin Elmer LS50B fluorimeter (Buckinghamshire, UK) not adapted for the use of capillaries, comparing a masked (mimicking the location of the rod) capillary and a 5-mm pathlength quartz cuvette with four polished sides. .. Aqueous solutions of ct-DNA (317 μ M, Sigma), ethidium bromide (30 μ M, Sigma) in sodium cacodylate buffer (10 mM, pH 7, Sigma), and NaCl (10 mM, Sigma) were used.

    Article Title: X protein variants of the autochthonous Latin American hepatitis B virus F genotype promotes human hepatocyte death by the induction of apoptosis and autophagy.
    Article Snippet: Acridine orange and ethidium bromide staining After transfections, cells were harvested, washed with PBS and resuspended in 1 ml PBS containing 4 μg/ml acridine orange (Sigma, USA) and 4 μg/ml ethidium bromide (Sigma, USA). .. Distinctive fluorescence patterns were observed according to the state of the cells: viable, bright green fluorescent nuclei with an organized structure; early apoptotic, nuclei containing green chromatin were highly condensed or fragmented; late apoptotic, bright orange chromatin with nuclei highly condensed and fragmented; necrotic, diffuse orange nuclei.

    Isolation:

    Article Title: Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]
    Article Snippet: Genomic DNA was isolated from the young leaf tissues collected from seventeen transformed lines (T1 ) and non-transformed (NT) plant using the CTAB method as earlier reported by Dellaporta et al. ( ). .. The amplified products were analyzed by electrophoresis on a 1.0% agarose (Sigma, St. Louis, USA) gel stained with 0.1% ethidium bromide (Sigma, St. Louis, USA) and photographed using the Gel Documentation system (UVITEC, Cambridge, UK).

    Article Title: Impact of different cell penetrating peptides on the efficacy of antisense therapeutics for targeting intracellular pathogens
    Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s Phosphate Buffered Saline (PBS), nisin, chloroform, isopropanol, agarose, ethidium bromide, Tris-Borate-EDTA buffer, free water and primers were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). .. Gentamicin, TRIzol Max Bacterial RNA Isolation Kit, SYBR Green PCR Master Mix, SuperScript II Reverse Transcriptase, 1 kb plus DNA ladder were purchased from Invitrogen (Carlsbad, CA, USA).

    Article Title: Agro-morphological description, genetic diversity and population structure of sugarcane varieties from sub-tropical India
    Article Snippet: Genomic DNA was isolated from the young leaves of each variety using Cetyl trimethyl ammonium bromide (CTAB) method as described by Doyle and Doyle ( ). .. The amplification products were resolved on 12% polyacrylamide gel with 1 × Tris-borate EDTA buffer, stained with 0.5 µg/ml ethidium bromide (Sigma-Aldrich, USA) and visualized using a gel documentation system (G:Box, Syngene, UK).

    Microscopy:

    Article Title: X protein variants of the autochthonous Latin American hepatitis B virus F genotype promotes human hepatocyte death by the induction of apoptosis and autophagy.
    Article Snippet: Acridine orange and ethidium bromide staining After transfections, cells were harvested, washed with PBS and resuspended in 1 ml PBS containing 4 μg/ml acridine orange (Sigma, USA) and 4 μg/ml ethidium bromide (Sigma, USA). .. The cell suspension was immediately dispensed onto slides, viewed under fluorescent microscopy (Leitz Dialux 20, Germany) and photographed.

    Polymerase Chain Reaction:

    Article Title: Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]
    Article Snippet: The PCR amplification profile consisted of an initial denaturation of cDNA at 95 °C for 5 min and followed by 28 cycles for 40 s at 94 °C, 40 s at 58 °C and 20 s at 72 °C, followed by a final extension at 72 °C for 5 min. .. The amplified products were determined on a 1.0% agarose gel (Sigma, St. Louis, USA) stained with 0.1% ethidium bromide (Sigma, St. Louis, USA) and photographed using the Gel Documentation system (UVITEC, Cambridge, UK). qRT-PCR analysis was performed in LightCycler® 480 Real-time PCR system (Roche, Germany).

    Article Title: Molecular screening of pathogenic Escherichia coli strains isolated from dairy neonatal calves in Cordoba province, Argentina.
    Article Snippet: Paragraph title: Molecular typing by PCR ... The gel was stained with ethidium bromide (Sigma---Aldrich ® ) and visualized under a UV-transilluminator at 302 nm.

    Article Title: Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]
    Article Snippet: Paragraph title: Polymerase chain reaction (PCR) ... The amplified products were analyzed by electrophoresis on a 1.0% agarose (Sigma, St. Louis, USA) gel stained with 0.1% ethidium bromide (Sigma, St. Louis, USA) and photographed using the Gel Documentation system (UVITEC, Cambridge, UK).

    Article Title: Impact of different cell penetrating peptides on the efficacy of antisense therapeutics for targeting intracellular pathogens
    Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s Phosphate Buffered Saline (PBS), nisin, chloroform, isopropanol, agarose, ethidium bromide, Tris-Borate-EDTA buffer, free water and primers were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). .. Gentamicin, TRIzol Max Bacterial RNA Isolation Kit, SYBR Green PCR Master Mix, SuperScript II Reverse Transcriptase, 1 kb plus DNA ladder were purchased from Invitrogen (Carlsbad, CA, USA).

    Article Title: Agro-morphological description, genetic diversity and population structure of sugarcane varieties from sub-tropical India
    Article Snippet: The DNA amplification was carried out in 15 µl reaction volume consisting of 1 × PCR assay buffer, 200 mM of each dNTPs (Fermentas, USA), 12 ng (1.8 pmol) each of forward and reverse primers (Operon Biotechnologies, GmbH, Germany), 0.5 unit of Taq DNA polymerase (Fermentas, USA) and 25 ng genomic DNA using a thermal cycler (MyCycler, Biorad, USA). .. The amplification products were resolved on 12% polyacrylamide gel with 1 × Tris-borate EDTA buffer, stained with 0.5 µg/ml ethidium bromide (Sigma-Aldrich, USA) and visualized using a gel documentation system (G:Box, Syngene, UK).

    Gel Extraction:

    Article Title: Impact of different cell penetrating peptides on the efficacy of antisense therapeutics for targeting intracellular pathogens
    Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s Phosphate Buffered Saline (PBS), nisin, chloroform, isopropanol, agarose, ethidium bromide, Tris-Borate-EDTA buffer, free water and primers were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). .. Random hexamers (Applied Biosystems, Carlsbad, CA, USA), QIAquick Gel Extraction Kit (Germantown, MD, USA) and In-Cell ELISA Kit (MitoSciences Inc., Eugene, OR, USA) were also used in this study.

    In-Cell ELISA:

    Article Title: Impact of different cell penetrating peptides on the efficacy of antisense therapeutics for targeting intracellular pathogens
    Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s Phosphate Buffered Saline (PBS), nisin, chloroform, isopropanol, agarose, ethidium bromide, Tris-Borate-EDTA buffer, free water and primers were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). .. Random hexamers (Applied Biosystems, Carlsbad, CA, USA), QIAquick Gel Extraction Kit (Germantown, MD, USA) and In-Cell ELISA Kit (MitoSciences Inc., Eugene, OR, USA) were also used in this study.

    Plasmid Preparation:

    Article Title: Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]
    Article Snippet: The NT plant genomic DNA and binary vector pCAMBIA 1300- p68 were used as negative and positive controls, respectively. .. The amplified products were analyzed by electrophoresis on a 1.0% agarose (Sigma, St. Louis, USA) gel stained with 0.1% ethidium bromide (Sigma, St. Louis, USA) and photographed using the Gel Documentation system (UVITEC, Cambridge, UK).

    SYBR Green Assay:

    Article Title: Impact of different cell penetrating peptides on the efficacy of antisense therapeutics for targeting intracellular pathogens
    Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s Phosphate Buffered Saline (PBS), nisin, chloroform, isopropanol, agarose, ethidium bromide, Tris-Borate-EDTA buffer, free water and primers were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). .. Gentamicin, TRIzol Max Bacterial RNA Isolation Kit, SYBR Green PCR Master Mix, SuperScript II Reverse Transcriptase, 1 kb plus DNA ladder were purchased from Invitrogen (Carlsbad, CA, USA).

    Agarose Gel Electrophoresis:

    Article Title: Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]
    Article Snippet: .. The amplified products were determined on a 1.0% agarose gel (Sigma, St. Louis, USA) stained with 0.1% ethidium bromide (Sigma, St. Louis, USA) and photographed using the Gel Documentation system (UVITEC, Cambridge, UK). qRT-PCR analysis was performed in LightCycler® 480 Real-time PCR system (Roche, Germany). .. The expression analysis of the RT-PCR positive (T1 ) and NT plants were conducted using Prime Script™ RT Reagent Kit (Takara Bio Inc, Japan) and p68 gene-specific primer set (FP: 5′-CCTCGCATTCTCTTCCTCGTA-3′ and RP: 5′-CGACGAGAACCATTGGCTAGA-3′) (Banu et al. ).

    Article Title: Molecular screening of pathogenic Escherichia coli strains isolated from dairy neonatal calves in Cordoba province, Argentina.
    Article Snippet: The amplified PCR products were separated by electrophoresis on a 2 % agarose gel with Tris---acetate---EDTA (TAE) buffer (Sigma---Aldrich®). .. The gel was stained with ethidium bromide (Sigma---Aldrich ® ) and visualized under a UV-transilluminator at 302 nm.

    Electrophoresis:

    Article Title: Molecular screening of pathogenic Escherichia coli strains isolated from dairy neonatal calves in Cordoba province, Argentina.
    Article Snippet: The amplified PCR products were separated by electrophoresis on a 2 % agarose gel with Tris---acetate---EDTA (TAE) buffer (Sigma---Aldrich®). .. The gel was stained with ethidium bromide (Sigma---Aldrich ® ) and visualized under a UV-transilluminator at 302 nm.

    Article Title: Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]
    Article Snippet: .. The amplified products were analyzed by electrophoresis on a 1.0% agarose (Sigma, St. Louis, USA) gel stained with 0.1% ethidium bromide (Sigma, St. Louis, USA) and photographed using the Gel Documentation system (UVITEC, Cambridge, UK). .. Five µg of genomic DNA extracted from the transformed plants (T1 ), non-transformed (NT) plant and 5 µg of binary vector pCAMBIA 1300- p68 were dotted into 1 cm square grids on a nylon membrane (Hybond N+ , GE Health care Limted, Buckinghamshire, UK).

    Concentration Assay:

    Article Title: Agro-morphological description, genetic diversity and population structure of sugarcane varieties from sub-tropical India
    Article Snippet: The quality and quantity of genomic DNA was checked, and DNA was diluted to a concentration of 25 ng/µl. .. The amplification products were resolved on 12% polyacrylamide gel with 1 × Tris-borate EDTA buffer, stained with 0.5 µg/ml ethidium bromide (Sigma-Aldrich, USA) and visualized using a gel documentation system (G:Box, Syngene, UK).

    Marker:

    Article Title: Molecular screening of pathogenic Escherichia coli strains isolated from dairy neonatal calves in Cordoba province, Argentina.
    Article Snippet: A 100-bp DNA ladder (CienMarker, Biodynamics) was used as molecular weight marker. .. The gel was stained with ethidium bromide (Sigma---Aldrich ® ) and visualized under a UV-transilluminator at 302 nm.

    Staining:

    Article Title: Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]
    Article Snippet: .. The amplified products were determined on a 1.0% agarose gel (Sigma, St. Louis, USA) stained with 0.1% ethidium bromide (Sigma, St. Louis, USA) and photographed using the Gel Documentation system (UVITEC, Cambridge, UK). qRT-PCR analysis was performed in LightCycler® 480 Real-time PCR system (Roche, Germany). .. The expression analysis of the RT-PCR positive (T1 ) and NT plants were conducted using Prime Script™ RT Reagent Kit (Takara Bio Inc, Japan) and p68 gene-specific primer set (FP: 5′-CCTCGCATTCTCTTCCTCGTA-3′ and RP: 5′-CGACGAGAACCATTGGCTAGA-3′) (Banu et al. ).

    Article Title: Molecular screening of pathogenic Escherichia coli strains isolated from dairy neonatal calves in Cordoba province, Argentina.
    Article Snippet: .. The gel was stained with ethidium bromide (Sigma---Aldrich ® ) and visualized under a UV-transilluminator at 302 nm. .. Molecular typing by PCR The E. coli strains were classified into different pathotypes, based on their specific virulence genes 26 .

    Article Title: X protein variants of the autochthonous Latin American hepatitis B virus F genotype promotes human hepatocyte death by the induction of apoptosis and autophagy.
    Article Snippet: .. Acridine orange and ethidium bromide staining After transfections, cells were harvested, washed with PBS and resuspended in 1 ml PBS containing 4 μg/ml acridine orange (Sigma, USA) and 4 μg/ml ethidium bromide (Sigma, USA). .. The cell suspension was immediately dispensed onto slides, viewed under fluorescent microscopy (Leitz Dialux 20, Germany) and photographed.

    Article Title: Adipose-Derived Mesenchymal Stem Cells Migrate and Rescue RPE in the Setting of Oxidative Stress
    Article Snippet: .. Ethidium Bromide and Acridine Orange Fluorescent Staining Following rescue studies as described above, RPE cells were collected by trypsinization; the apoptosis and necrosis rates of RPE were assessed using ethidium bromide and acridine orange fluorescent staining as follows: fluorescent staining solution (0.5 μ l) containing an equal volume of 100 μ g/ml acridine orange and 100 μ g/ml ethidium bromide (Sigma) was added to each cell suspension sample and then covered with a coverslip. ..

    Article Title: Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]
    Article Snippet: .. The amplified products were analyzed by electrophoresis on a 1.0% agarose (Sigma, St. Louis, USA) gel stained with 0.1% ethidium bromide (Sigma, St. Louis, USA) and photographed using the Gel Documentation system (UVITEC, Cambridge, UK). .. Five µg of genomic DNA extracted from the transformed plants (T1 ), non-transformed (NT) plant and 5 µg of binary vector pCAMBIA 1300- p68 were dotted into 1 cm square grids on a nylon membrane (Hybond N+ , GE Health care Limted, Buckinghamshire, UK).

    Article Title: Adipose-Derived Mesenchymal Stem Cells Migrate and Rescue RPE in the Setting of Oxidative Stress
    Article Snippet: .. Following rescue studies as described above, RPE cells were collected by trypsinization; the apoptosis and necrosis rates of RPE were assessed using ethidium bromide and acridine orange fluorescent staining as follows: fluorescent staining solution (0.5 μ l) containing an equal volume of 100 μ g/ml acridine orange and 100 μ g/ml ethidium bromide (Sigma) was added to each cell suspension sample and then covered with a coverslip. ..

    Article Title: Agro-morphological description, genetic diversity and population structure of sugarcane varieties from sub-tropical India
    Article Snippet: .. The amplification products were resolved on 12% polyacrylamide gel with 1 × Tris-borate EDTA buffer, stained with 0.5 µg/ml ethidium bromide (Sigma-Aldrich, USA) and visualized using a gel documentation system (G:Box, Syngene, UK). .. The clear and unambiguous amplified products were scored as dominant markers with 1 (present) or 0 (absent) since it is difficult to identify and differentiate between amplicons from homeologous chromosomes.

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    Millipore α factor
    Sir2p affects both the firing efficiency and distribution of replication origins in the rDNA. Wild-type and sir2Δ hmlΔ cells (E1385) were synchronized in G 1 with <t>α-factor</t> and released into S phase in the presence of HU. Ribosomal DNA fibers were analyzed by DMC as described previously. ( A ) Representative rDNA fibers. (Red) FISH; (green) BrdU. Bar, 50 kb. ( B ) Size distribution of BrdU tracks ( b ) and gaps ( a ) in sir2Δ rDNA fibers. ( C ) Model of the distribution of active origins in the rDNA of wild-type and sir2Δ cells (see text).
    α Factor, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore e cadherin
    184- hTERT -L9 cells have a normal karyotype and form acini in three dimensions. (A) 184- hTERT -L9 cells were arrested in metaphase using colcemid prior to multicolour fluorescence in situ hybridization analysis. Metaphase spreads had a normal diploid chromosome complement devoid of numerical or structural changes ( n = 7). (B) Cells cultured in three-dimensional Matrigel were fixed after 21 days and probed with antibodies targeting GM130 (apical polarity), CD49f (basal polarity) and MUC1 (luminal marker). Either Oregon Green–labelled phalloidin or an antibody targeting <t>E-cadherin</t> was used as a counterstain, with DRAQ5 nuclear stain. For viability assessment, unfixed structures were incubated for 20 minutes with calcein AM, ethidium homodimer 1 (EthD-1) and Hoechst 33452 immediately prior to being imaged on a Nikon confocal laser scanning microscope.
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    Millipore smad3 specific phosphorylation inhibitor sis3
    Misregulated Smad2/3 signaling in Lmna −/− muscle cells. ( A ) Increased Smad2/3 expression in Lmna −/− cells. WT and Lmna −/− muscle cells were collected at different timepoints: proliferating MBs (MB) and days 1–4 of differentiation (d1-d4). Cell pellets were separated into nuclear (n) and cytoplasmic (c) fractions and subjected to immunoblotting using antibodies to Smad2/3. Vinculin was used as a loading control. ( B ) Quantitation of Smad2/3 in nuclear and cytoplasmic fractions. Values greater than 0.5 indicate a predominantly nucleoplasmic localization ( n = 3 experiments per time point ± SEM). ( C ) WT and Lmna −/− satellite cells were incubated with differentiation medium containing 10 ng/ml of TGFβ1 for 54 h, then immunostained with antibodies to Smad2/3 (green) and desmin (red) and co-stained with DAPI (blue) to visualize nuclei. In untreated WT cells, some Smad2/3 expression can be seen in the nuclei of MBs whereas nuclear localization is decreased in myotubes. In response to treatment with TGFβ1, Smad2/3 localize to the nuclei of both MBs and myotubes (green). In Lmna −/− cells, Smad2/3 are generally upregulated but localization is equally distributed in nuclear and cytoplasmic compartments. In response to TGFβ1 treatment in Lmna −/− MBs, localization in Lmna −/− myotubes remains unclear with more cytoplasmic staining. Scale, 50 μm. Arrow indicates location of inset. Scale, 20 μm. ( D ) LIVE-DEAD assay showing live calcein+ve (green) and dead EthD-1 (red) staining. WT and Lmna −/− H-2K cells were differentiated for 5 days, then analyzed using the calcein and EthD-1 permeability assay. When <t>Smad3</t> blocker <t>SIS3</t> was included in the differentiation media, cell death in Lmna −/− cells was decreased. Scale bar, 100 μm. ( E ) Lmna −/− myotubes show increased cell death, which is significantly decreased by SIS3 treatment (n = 3 experiments; *, P
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    Millipore western blot analysis whole cell lysates
    C/EBPβ-knockdown inhibits <t>cell</t> cycle progression. ( A ) A549 cells were transfectected with siNC or siC/EBPβ and incubated for 48 h. As a positive control for cell death, cells were treated with 20 M cisplatin for 48 h. Live cells were stained with green calcein-AM, while dead cells were stained with red ethidium homodimer-1 (EthD-1). Cell images were taken at a magnification of 100X using an Operetta High Content Screening (HCS) System. Scale bar: 200 μm. ( B ) A549 cells were transfected with control siRNA or C/EBPβ siRNA for 48 h. The cell cycle was analyzed by fluorescence-activated cell sorting (FACS) after DNA staining with propidium iodide (PI). M1: subG 0 /G 1 , M2: G 0 /G 1 , M3: S, and M4: G 2 /M phase. Percentage of cells in each cell cycle phase is shown as a bar graph. ( C ) <t>Whole</t> cell <t>lysates</t> were prepared 48 h after transfection and the levels of the G 2 /M cell cycle-related proteins in control or C/EBPβ-knockdown cells were analyzed by <t>Western</t> blotting. β-actin was used as a loading control. Data are presented as mean ± SD. Statistical significance was determined using the t -test, * p
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    Image Search Results


    Sir2p affects both the firing efficiency and distribution of replication origins in the rDNA. Wild-type and sir2Δ hmlΔ cells (E1385) were synchronized in G 1 with α-factor and released into S phase in the presence of HU. Ribosomal DNA fibers were analyzed by DMC as described previously. ( A ) Representative rDNA fibers. (Red) FISH; (green) BrdU. Bar, 50 kb. ( B ) Size distribution of BrdU tracks ( b ) and gaps ( a ) in sir2Δ rDNA fibers. ( C ) Model of the distribution of active origins in the rDNA of wild-type and sir2Δ cells (see text).

    Journal: Genes & Development

    Article Title: Single-molecule analysis reveals clustering and epigenetic regulation of replication origins at the yeast rDNA locus

    doi: 10.1101/gad.232902

    Figure Lengend Snippet: Sir2p affects both the firing efficiency and distribution of replication origins in the rDNA. Wild-type and sir2Δ hmlΔ cells (E1385) were synchronized in G 1 with α-factor and released into S phase in the presence of HU. Ribosomal DNA fibers were analyzed by DMC as described previously. ( A ) Representative rDNA fibers. (Red) FISH; (green) BrdU. Bar, 50 kb. ( B ) Size distribution of BrdU tracks ( b ) and gaps ( a ) in sir2Δ rDNA fibers. ( C ) Model of the distribution of active origins in the rDNA of wild-type and sir2Δ cells (see text).

    Article Snippet: Cells were synchronized in G1 with 2 μg/mL α-factor (E1000 and E1385) or by centrifugal elutriation (E1244) and were released into S phase in the presence of 0.4 mg/mL BrdU by the addition of 50 μg/mL pronase (Calbiochem).

    Techniques: Fluorescence In Situ Hybridization

    184- hTERT -L9 cells have a normal karyotype and form acini in three dimensions. (A) 184- hTERT -L9 cells were arrested in metaphase using colcemid prior to multicolour fluorescence in situ hybridization analysis. Metaphase spreads had a normal diploid chromosome complement devoid of numerical or structural changes ( n = 7). (B) Cells cultured in three-dimensional Matrigel were fixed after 21 days and probed with antibodies targeting GM130 (apical polarity), CD49f (basal polarity) and MUC1 (luminal marker). Either Oregon Green–labelled phalloidin or an antibody targeting E-cadherin was used as a counterstain, with DRAQ5 nuclear stain. For viability assessment, unfixed structures were incubated for 20 minutes with calcein AM, ethidium homodimer 1 (EthD-1) and Hoechst 33452 immediately prior to being imaged on a Nikon confocal laser scanning microscope.

    Journal: Breast Cancer Research : BCR

    Article Title: A co-culture genome-wide RNAi screen with mammary epithelial cells reveals transmembrane signals required for growth and differentiation

    doi: 10.1186/s13058-014-0510-y

    Figure Lengend Snippet: 184- hTERT -L9 cells have a normal karyotype and form acini in three dimensions. (A) 184- hTERT -L9 cells were arrested in metaphase using colcemid prior to multicolour fluorescence in situ hybridization analysis. Metaphase spreads had a normal diploid chromosome complement devoid of numerical or structural changes ( n = 7). (B) Cells cultured in three-dimensional Matrigel were fixed after 21 days and probed with antibodies targeting GM130 (apical polarity), CD49f (basal polarity) and MUC1 (luminal marker). Either Oregon Green–labelled phalloidin or an antibody targeting E-cadherin was used as a counterstain, with DRAQ5 nuclear stain. For viability assessment, unfixed structures were incubated for 20 minutes with calcein AM, ethidium homodimer 1 (EthD-1) and Hoechst 33452 immediately prior to being imaged on a Nikon confocal laser scanning microscope.

    Article Snippet: E-cadherin

    Techniques: Fluorescence, In Situ Hybridization, Cell Culture, Marker, Staining, Incubation, Ethidium Homodimer Assay, Laser-Scanning Microscopy

    Misregulated Smad2/3 signaling in Lmna −/− muscle cells. ( A ) Increased Smad2/3 expression in Lmna −/− cells. WT and Lmna −/− muscle cells were collected at different timepoints: proliferating MBs (MB) and days 1–4 of differentiation (d1-d4). Cell pellets were separated into nuclear (n) and cytoplasmic (c) fractions and subjected to immunoblotting using antibodies to Smad2/3. Vinculin was used as a loading control. ( B ) Quantitation of Smad2/3 in nuclear and cytoplasmic fractions. Values greater than 0.5 indicate a predominantly nucleoplasmic localization ( n = 3 experiments per time point ± SEM). ( C ) WT and Lmna −/− satellite cells were incubated with differentiation medium containing 10 ng/ml of TGFβ1 for 54 h, then immunostained with antibodies to Smad2/3 (green) and desmin (red) and co-stained with DAPI (blue) to visualize nuclei. In untreated WT cells, some Smad2/3 expression can be seen in the nuclei of MBs whereas nuclear localization is decreased in myotubes. In response to treatment with TGFβ1, Smad2/3 localize to the nuclei of both MBs and myotubes (green). In Lmna −/− cells, Smad2/3 are generally upregulated but localization is equally distributed in nuclear and cytoplasmic compartments. In response to TGFβ1 treatment in Lmna −/− MBs, localization in Lmna −/− myotubes remains unclear with more cytoplasmic staining. Scale, 50 μm. Arrow indicates location of inset. Scale, 20 μm. ( D ) LIVE-DEAD assay showing live calcein+ve (green) and dead EthD-1 (red) staining. WT and Lmna −/− H-2K cells were differentiated for 5 days, then analyzed using the calcein and EthD-1 permeability assay. When Smad3 blocker SIS3 was included in the differentiation media, cell death in Lmna −/− cells was decreased. Scale bar, 100 μm. ( E ) Lmna −/− myotubes show increased cell death, which is significantly decreased by SIS3 treatment (n = 3 experiments; *, P

    Journal: Human Molecular Genetics

    Article Title: Defective skeletal muscle growth in lamin A/C-deficient mice is rescued by loss of Lap2α

    doi: 10.1093/hmg/ddt135

    Figure Lengend Snippet: Misregulated Smad2/3 signaling in Lmna −/− muscle cells. ( A ) Increased Smad2/3 expression in Lmna −/− cells. WT and Lmna −/− muscle cells were collected at different timepoints: proliferating MBs (MB) and days 1–4 of differentiation (d1-d4). Cell pellets were separated into nuclear (n) and cytoplasmic (c) fractions and subjected to immunoblotting using antibodies to Smad2/3. Vinculin was used as a loading control. ( B ) Quantitation of Smad2/3 in nuclear and cytoplasmic fractions. Values greater than 0.5 indicate a predominantly nucleoplasmic localization ( n = 3 experiments per time point ± SEM). ( C ) WT and Lmna −/− satellite cells were incubated with differentiation medium containing 10 ng/ml of TGFβ1 for 54 h, then immunostained with antibodies to Smad2/3 (green) and desmin (red) and co-stained with DAPI (blue) to visualize nuclei. In untreated WT cells, some Smad2/3 expression can be seen in the nuclei of MBs whereas nuclear localization is decreased in myotubes. In response to treatment with TGFβ1, Smad2/3 localize to the nuclei of both MBs and myotubes (green). In Lmna −/− cells, Smad2/3 are generally upregulated but localization is equally distributed in nuclear and cytoplasmic compartments. In response to TGFβ1 treatment in Lmna −/− MBs, localization in Lmna −/− myotubes remains unclear with more cytoplasmic staining. Scale, 50 μm. Arrow indicates location of inset. Scale, 20 μm. ( D ) LIVE-DEAD assay showing live calcein+ve (green) and dead EthD-1 (red) staining. WT and Lmna −/− H-2K cells were differentiated for 5 days, then analyzed using the calcein and EthD-1 permeability assay. When Smad3 blocker SIS3 was included in the differentiation media, cell death in Lmna −/− cells was decreased. Scale bar, 100 μm. ( E ) Lmna −/− myotubes show increased cell death, which is significantly decreased by SIS3 treatment (n = 3 experiments; *, P

    Article Snippet: For Smad3 blocking assay, MBs were treated with indicated concentrations of the Smad3-specific phosphorylation inhibitor SIS3 (EMD Biosciences, Billerica, MA, USA) for 5 days, then incubated with LIVE-DEAD ® cytotoxicity assay reagent or fixed and immunostained using an anti-myosin heavy chain antibody.

    Techniques: Expressing, Quantitation Assay, Incubation, Staining, Live Dead Assay, Ethidium Homodimer Assay, Permeability

    Blockade of Smad3 increases myotubes size in Lmna −/− Lap2α −/− . ( A ) MHC immunostaining showing decreased myotubes in Lmna −/− cultures, compared to both WT and Lmna −/− Lap2α −/− . Treatment with SIS3 (bottom panels) increases myotube size. Scale, 50 μm. ( B ) Quantitation of nuclei per myotubes in SIS3-treated and control (CTRL) cultures ( n = 6 non-overlapping fields from 2 independent experiments. *, P

    Journal: Human Molecular Genetics

    Article Title: Defective skeletal muscle growth in lamin A/C-deficient mice is rescued by loss of Lap2α

    doi: 10.1093/hmg/ddt135

    Figure Lengend Snippet: Blockade of Smad3 increases myotubes size in Lmna −/− Lap2α −/− . ( A ) MHC immunostaining showing decreased myotubes in Lmna −/− cultures, compared to both WT and Lmna −/− Lap2α −/− . Treatment with SIS3 (bottom panels) increases myotube size. Scale, 50 μm. ( B ) Quantitation of nuclei per myotubes in SIS3-treated and control (CTRL) cultures ( n = 6 non-overlapping fields from 2 independent experiments. *, P

    Article Snippet: For Smad3 blocking assay, MBs were treated with indicated concentrations of the Smad3-specific phosphorylation inhibitor SIS3 (EMD Biosciences, Billerica, MA, USA) for 5 days, then incubated with LIVE-DEAD ® cytotoxicity assay reagent or fixed and immunostained using an anti-myosin heavy chain antibody.

    Techniques: Immunostaining, Quantitation Assay

    C/EBPβ-knockdown inhibits cell cycle progression. ( A ) A549 cells were transfectected with siNC or siC/EBPβ and incubated for 48 h. As a positive control for cell death, cells were treated with 20 M cisplatin for 48 h. Live cells were stained with green calcein-AM, while dead cells were stained with red ethidium homodimer-1 (EthD-1). Cell images were taken at a magnification of 100X using an Operetta High Content Screening (HCS) System. Scale bar: 200 μm. ( B ) A549 cells were transfected with control siRNA or C/EBPβ siRNA for 48 h. The cell cycle was analyzed by fluorescence-activated cell sorting (FACS) after DNA staining with propidium iodide (PI). M1: subG 0 /G 1 , M2: G 0 /G 1 , M3: S, and M4: G 2 /M phase. Percentage of cells in each cell cycle phase is shown as a bar graph. ( C ) Whole cell lysates were prepared 48 h after transfection and the levels of the G 2 /M cell cycle-related proteins in control or C/EBPβ-knockdown cells were analyzed by Western blotting. β-actin was used as a loading control. Data are presented as mean ± SD. Statistical significance was determined using the t -test, * p

    Journal: Cells

    Article Title: C/EBPβ Is a Transcriptional Regulator of Wee1 at the G2/M Phase of the Cell Cycle

    doi: 10.3390/cells8020145

    Figure Lengend Snippet: C/EBPβ-knockdown inhibits cell cycle progression. ( A ) A549 cells were transfectected with siNC or siC/EBPβ and incubated for 48 h. As a positive control for cell death, cells were treated with 20 M cisplatin for 48 h. Live cells were stained with green calcein-AM, while dead cells were stained with red ethidium homodimer-1 (EthD-1). Cell images were taken at a magnification of 100X using an Operetta High Content Screening (HCS) System. Scale bar: 200 μm. ( B ) A549 cells were transfected with control siRNA or C/EBPβ siRNA for 48 h. The cell cycle was analyzed by fluorescence-activated cell sorting (FACS) after DNA staining with propidium iodide (PI). M1: subG 0 /G 1 , M2: G 0 /G 1 , M3: S, and M4: G 2 /M phase. Percentage of cells in each cell cycle phase is shown as a bar graph. ( C ) Whole cell lysates were prepared 48 h after transfection and the levels of the G 2 /M cell cycle-related proteins in control or C/EBPβ-knockdown cells were analyzed by Western blotting. β-actin was used as a loading control. Data are presented as mean ± SD. Statistical significance was determined using the t -test, * p

    Article Snippet: Western Blot Analysis Whole-cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer, supplemented with protease inhibitor cocktail, phosphatase inhibitor (Calbiochem, San Diego, CA, USA), phenylmethylsulfonyl fluoride (PMSF), and dithiothreitol (DTT).

    Techniques: Incubation, Positive Control, Staining, Ethidium Homodimer Assay, High Content Screening, Transfection, Fluorescence, FACS, Western Blot

    C/EBPβ regulates Wee1 expression at the transcription levels and interacts with HDAC2. ( A ) Quantitative real-time RT-PCR (qRT-PCR) was used to determine Wee1 and Cdc25B mRNA levels relative to the control gene GAPDH in C/EBPβ-knockdown A549 cells. Data are presented as mean ± SD. ( B ) The position of the four predicted C/EBPβ binding sites in the WEE1 promoter are represented. C/EBPβ–ChIP on chip data and TFSEARCH based binding sites are indicated as a rectangle and an oval, respectively. The prediction of WEE1 promoter regions was based on NCBI accession number (NC_000011.10, GRCh37.p11) ( C ) The C/EBPβ-ChIP assay followed by qRT-PCR on putative C/EBPβ binding regions on the WEE1 promoter was performed to determine endogenous C/EBPβ occupancy at the specified region. The fold enrichment of C/EBPβ occupancy over GAPDH exon (negative control) is shown. Data are presented as mean ± SE. ( D ) A549 cell lysates were immunoprecipitated using anti-C/EBPβ antibodies. Immunocomplexes were analyzed by Western blot with either anti-HDAC1 or -HDAC2 antibodies. IgG was used as a negative control. ( E ) HDAC2-ChIP assay followed by qRT-PCR on putative C/EBPβ binding regions at the WEE1 promoter was performed. The fold enrichment of HDAC2 occupancy over GAPDH exon (negative control) is shown. Data are presented as mean ± SE. ( F ) The C/EBPβ-ChIP or HDAC2-ChIP assay followed by PCR on putative C/EBPβ binding regions at the WEE1 promoter was performed. The PCR products resolved on 2% agarose gel were visualized. ( G ) A549 cells were co-transfected with a WEE1 promoter-luciferase construct containing R2, or R3 along with C/EBPβ and/or HDAC2, as indicated, for 48 h, and then luciferase activities were measured. Data are expressed as relative luciferase activity/ug protein standardized by a control pGL3-promoter vector. Data are presented as mean ± SD. Statistical significance was determined using the t -test, * p

    Journal: Cells

    Article Title: C/EBPβ Is a Transcriptional Regulator of Wee1 at the G2/M Phase of the Cell Cycle

    doi: 10.3390/cells8020145

    Figure Lengend Snippet: C/EBPβ regulates Wee1 expression at the transcription levels and interacts with HDAC2. ( A ) Quantitative real-time RT-PCR (qRT-PCR) was used to determine Wee1 and Cdc25B mRNA levels relative to the control gene GAPDH in C/EBPβ-knockdown A549 cells. Data are presented as mean ± SD. ( B ) The position of the four predicted C/EBPβ binding sites in the WEE1 promoter are represented. C/EBPβ–ChIP on chip data and TFSEARCH based binding sites are indicated as a rectangle and an oval, respectively. The prediction of WEE1 promoter regions was based on NCBI accession number (NC_000011.10, GRCh37.p11) ( C ) The C/EBPβ-ChIP assay followed by qRT-PCR on putative C/EBPβ binding regions on the WEE1 promoter was performed to determine endogenous C/EBPβ occupancy at the specified region. The fold enrichment of C/EBPβ occupancy over GAPDH exon (negative control) is shown. Data are presented as mean ± SE. ( D ) A549 cell lysates were immunoprecipitated using anti-C/EBPβ antibodies. Immunocomplexes were analyzed by Western blot with either anti-HDAC1 or -HDAC2 antibodies. IgG was used as a negative control. ( E ) HDAC2-ChIP assay followed by qRT-PCR on putative C/EBPβ binding regions at the WEE1 promoter was performed. The fold enrichment of HDAC2 occupancy over GAPDH exon (negative control) is shown. Data are presented as mean ± SE. ( F ) The C/EBPβ-ChIP or HDAC2-ChIP assay followed by PCR on putative C/EBPβ binding regions at the WEE1 promoter was performed. The PCR products resolved on 2% agarose gel were visualized. ( G ) A549 cells were co-transfected with a WEE1 promoter-luciferase construct containing R2, or R3 along with C/EBPβ and/or HDAC2, as indicated, for 48 h, and then luciferase activities were measured. Data are expressed as relative luciferase activity/ug protein standardized by a control pGL3-promoter vector. Data are presented as mean ± SD. Statistical significance was determined using the t -test, * p

    Article Snippet: Western Blot Analysis Whole-cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer, supplemented with protease inhibitor cocktail, phosphatase inhibitor (Calbiochem, San Diego, CA, USA), phenylmethylsulfonyl fluoride (PMSF), and dithiothreitol (DTT).

    Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Chromatin Immunoprecipitation, Negative Control, Immunoprecipitation, Western Blot, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transfection, Luciferase, Construct, Activity Assay, Plasmid Preparation

    C/EBPβ promotes cell proliferation in various subtypes of non-small cell lung cancer (NSCLC) cell lines. ( A ) C/EBPβ protein levels in normal human bronchial epithelial cells (NHBE), immortalized human bronchial epithelial cells, BEAS-2B and various NSCLC cell lines were determined by Western blot analysis. Aenocarcinoma, A549, NCI-H1975, NCI-H23, NCI-H1703, NCI-H522, A427, Calu-3, NCI-H358; adeno-squamous cell carcinoma, HCC2279; squamous cell carcinoma, HCC95, HCC1588; large cell carcinoma, H460, NCI-H1299; anaplastic carcinoma, Calu-6. ( B ) Live cells of A549 transfected with si-Negative Control (siNC), siC/EBPβ #1, or siC/EBPβ #2 were counted with trypan blue staining at indicated times after transfection. Data are presented as fold increase. ( C ) Doxycycline-inducible shC/EBPβ cells using A549 were generated and treated with or without doxycycline (100 ng/mL). Using the IncuCyte live cell imaging system, proliferation cells was monitored and quantified by the percentage of cell confluence. ( D ) The protein levels of C/EBPβ were detected by Western blotting to check C/EBPβ-knockdown in each cell line. ( E ) Cell number of lung cancer cell lines transfected with siNC or siC/EBPβ (#1 + #2) was counted using a Coulter counter at intervals of 24 h up to 120 h after siRNA transfection. K-RAS : mutant K-RAS /wild-type EGFR , EGFR : wild-type K-RAS /mutant EGFR , WT: wild-type K-RAS /wild-type EGFR . Data are presented as mean ± standard deviation (SD). The statistical significance was determined using t -tests, * p

    Journal: Cells

    Article Title: C/EBPβ Is a Transcriptional Regulator of Wee1 at the G2/M Phase of the Cell Cycle

    doi: 10.3390/cells8020145

    Figure Lengend Snippet: C/EBPβ promotes cell proliferation in various subtypes of non-small cell lung cancer (NSCLC) cell lines. ( A ) C/EBPβ protein levels in normal human bronchial epithelial cells (NHBE), immortalized human bronchial epithelial cells, BEAS-2B and various NSCLC cell lines were determined by Western blot analysis. Aenocarcinoma, A549, NCI-H1975, NCI-H23, NCI-H1703, NCI-H522, A427, Calu-3, NCI-H358; adeno-squamous cell carcinoma, HCC2279; squamous cell carcinoma, HCC95, HCC1588; large cell carcinoma, H460, NCI-H1299; anaplastic carcinoma, Calu-6. ( B ) Live cells of A549 transfected with si-Negative Control (siNC), siC/EBPβ #1, or siC/EBPβ #2 were counted with trypan blue staining at indicated times after transfection. Data are presented as fold increase. ( C ) Doxycycline-inducible shC/EBPβ cells using A549 were generated and treated with or without doxycycline (100 ng/mL). Using the IncuCyte live cell imaging system, proliferation cells was monitored and quantified by the percentage of cell confluence. ( D ) The protein levels of C/EBPβ were detected by Western blotting to check C/EBPβ-knockdown in each cell line. ( E ) Cell number of lung cancer cell lines transfected with siNC or siC/EBPβ (#1 + #2) was counted using a Coulter counter at intervals of 24 h up to 120 h after siRNA transfection. K-RAS : mutant K-RAS /wild-type EGFR , EGFR : wild-type K-RAS /mutant EGFR , WT: wild-type K-RAS /wild-type EGFR . Data are presented as mean ± standard deviation (SD). The statistical significance was determined using t -tests, * p

    Article Snippet: Western Blot Analysis Whole-cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer, supplemented with protease inhibitor cocktail, phosphatase inhibitor (Calbiochem, San Diego, CA, USA), phenylmethylsulfonyl fluoride (PMSF), and dithiothreitol (DTT).

    Techniques: Western Blot, Transfection, Negative Control, Staining, Generated, Live Cell Imaging, Mutagenesis, Standard Deviation

    C/EBPβ knockdown delays G 2 /M-cell cycle transition. ( A ) A time course study of the cell cycle analysis was performed in control or C/EBPβ-knockdown A549 cells. Cells were released from thymidine double block-induced G 1 /S synchronization; and 0, 2, 4, 6, 8, 10, 12, 14, 16, and 26 h after releasing, cells were collected and stained with PI to measure DNA content using FACS. ( B ) The data are expressed as the percentage of cells in the subG 0 /G 1 , G 0 /G 1 , S, and G 2 /M phase at the indicated time points. ( C ) Whole cell lysates were prepared at the indicated times and Western blot analysis was performed for expression of proteins associated with the G 2 /M transition (Y15-pCDK1, Wee1, Cdc25B and Cyclin B1). SE; short exposure, LE; long exposure. ( D ) Mitotic duration of control or C/EBPβ-knockdown A549 cells stained with NucBlue ® was monitored by an Operetta High Content Screening (HCS) System. Representative images of siNC and siC/EBPβ-transfected cells from time lapse series were shown. Images were acquired every 10 min. Data are presented as mean ± SD of mitotic duration in 30 cells in each group. Mitotic duration was measured f rom nuclear envelope breakdown (prometaphase) to anaphase onsets [ 52 ]. Statistical significance was determined using the t -test; n.s., not significant. Cell images were taken at a magnification of 200X using an Operetta High Content Screening (HCS) System. Scale bar: 20 μm.

    Journal: Cells

    Article Title: C/EBPβ Is a Transcriptional Regulator of Wee1 at the G2/M Phase of the Cell Cycle

    doi: 10.3390/cells8020145

    Figure Lengend Snippet: C/EBPβ knockdown delays G 2 /M-cell cycle transition. ( A ) A time course study of the cell cycle analysis was performed in control or C/EBPβ-knockdown A549 cells. Cells were released from thymidine double block-induced G 1 /S synchronization; and 0, 2, 4, 6, 8, 10, 12, 14, 16, and 26 h after releasing, cells were collected and stained with PI to measure DNA content using FACS. ( B ) The data are expressed as the percentage of cells in the subG 0 /G 1 , G 0 /G 1 , S, and G 2 /M phase at the indicated time points. ( C ) Whole cell lysates were prepared at the indicated times and Western blot analysis was performed for expression of proteins associated with the G 2 /M transition (Y15-pCDK1, Wee1, Cdc25B and Cyclin B1). SE; short exposure, LE; long exposure. ( D ) Mitotic duration of control or C/EBPβ-knockdown A549 cells stained with NucBlue ® was monitored by an Operetta High Content Screening (HCS) System. Representative images of siNC and siC/EBPβ-transfected cells from time lapse series were shown. Images were acquired every 10 min. Data are presented as mean ± SD of mitotic duration in 30 cells in each group. Mitotic duration was measured f rom nuclear envelope breakdown (prometaphase) to anaphase onsets [ 52 ]. Statistical significance was determined using the t -test; n.s., not significant. Cell images were taken at a magnification of 200X using an Operetta High Content Screening (HCS) System. Scale bar: 20 μm.

    Article Snippet: Western Blot Analysis Whole-cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer, supplemented with protease inhibitor cocktail, phosphatase inhibitor (Calbiochem, San Diego, CA, USA), phenylmethylsulfonyl fluoride (PMSF), and dithiothreitol (DTT).

    Techniques: Cell Cycle Assay, Blocking Assay, Staining, FACS, Western Blot, Expressing, High Content Screening, Transfection