vitro transcribed full length hcv 1a  (Qiagen)

 
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    QIAquick Gel Extraction Kit
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    For gel extraction cleanup of up to 10 μg DNA 70 bp to 10 kb from enzymatic reactions Kit contents Qiagen QIAquick Gel Extraction Kit 50 rxns 30 to 50L Elution Volume 10g Binding Capacity DNA Sample Tube Format Silica Technology Manual Processing 70 bp to 10 kb Fragment Fast and Convenient Procedure For Gel Extraction Cleanup of up to 10μg DNA 70 bp to 10 kb from Enzymatic Reactions Includes 50 QIAquick Spin Columns Buffers Collection Tubes 2mL Benefits Up to 95 recovery of ready to use DNA Fast and convenient procedure Cleanup of DNA up to 10 kb in three easy steps Gel loading dye for convenient sample analysis
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    QIAquick Gel Extraction Kit
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    Qiagen vitro transcribed full length hcv 1a
    QIAquick Gel Extraction Kit
    For gel extraction cleanup of up to 10 μg DNA 70 bp to 10 kb from enzymatic reactions Kit contents Qiagen QIAquick Gel Extraction Kit 50 rxns 30 to 50L Elution Volume 10g Binding Capacity DNA Sample Tube Format Silica Technology Manual Processing 70 bp to 10 kb Fragment Fast and Convenient Procedure For Gel Extraction Cleanup of up to 10μg DNA 70 bp to 10 kb from Enzymatic Reactions Includes 50 QIAquick Spin Columns Buffers Collection Tubes 2mL Benefits Up to 95 recovery of ready to use DNA Fast and convenient procedure Cleanup of DNA up to 10 kb in three easy steps Gel loading dye for convenient sample analysis
    https://www.bioz.com/result/vitro transcribed full length hcv 1a/product/Qiagen
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    vitro transcribed full length hcv 1a - by Bioz Stars, 2021-01
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    1) Product Images from "Hepatitis C Virus (HCV) Proteins Induce NADPH Oxidase 4 Expression in a Transforming Growth Factor ?-Dependent Manner: a New Contributor to HCV-Induced Oxidative Stress ▿"

    Article Title: Hepatitis C Virus (HCV) Proteins Induce NADPH Oxidase 4 Expression in a Transforming Growth Factor ?-Dependent Manner: a New Contributor to HCV-Induced Oxidative Stress ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01059-09

    Effect of HCV on the expression of Nox4. (A) Nox-specific PCR amplification of reverse-transcribed total RNA (1 μg) from HepG2 cells transfected 48 h previously with either pcDNA3.1 empty vector plasmid (1 μg) or full-length HCV-1a cDNA plasmid. Results shown are representative of four independent experiments. (B) Quantitative real-time RT-PCR (qPCR) analysis of Nox4 from total RNA extracted from HepG2 cells. RNAs were collected from cells transfected for 48 h with either empty vector plasmid (1 μg) or HCV plasmid (1 μg), followed by reverse transcription. Equal amounts of cDNA were subjected to quantitative RT-PCR using human Nox4-specific primers. GAPDH-specific primers were used as internal control ( n = 2, in quadruplicate). (C) HepG2 cells were transfected as described for panel A with increasing amounts of HCV plasmid (0.25, 0.5, and 1.0 μg). p22 phox -specific primers were used to determine mRNA expression. Primers specific for the HCV NS5b gene were used to confirm the presence of HCV RNA. Results shown are representative of three independent experiments.
    Figure Legend Snippet: Effect of HCV on the expression of Nox4. (A) Nox-specific PCR amplification of reverse-transcribed total RNA (1 μg) from HepG2 cells transfected 48 h previously with either pcDNA3.1 empty vector plasmid (1 μg) or full-length HCV-1a cDNA plasmid. Results shown are representative of four independent experiments. (B) Quantitative real-time RT-PCR (qPCR) analysis of Nox4 from total RNA extracted from HepG2 cells. RNAs were collected from cells transfected for 48 h with either empty vector plasmid (1 μg) or HCV plasmid (1 μg), followed by reverse transcription. Equal amounts of cDNA were subjected to quantitative RT-PCR using human Nox4-specific primers. GAPDH-specific primers were used as internal control ( n = 2, in quadruplicate). (C) HepG2 cells were transfected as described for panel A with increasing amounts of HCV plasmid (0.25, 0.5, and 1.0 μg). p22 phox -specific primers were used to determine mRNA expression. Primers specific for the HCV NS5b gene were used to confirm the presence of HCV RNA. Results shown are representative of three independent experiments.

    Techniques Used: Expressing, Polymerase Chain Reaction, Amplification, Transfection, Plasmid Preparation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Effect of HCV RNA or infectious virus on superoxide generation and Nox4 mRNA. (A) Left, HepG2 cells were transfected with in vitro-transcribed full-length HCV-1a or in vitro-transcribed HCVΔNS5b (ΔHCV) RNA as mentioned in Materials and Methods. After 48 h, cells were assayed for superoxide generation for 1 h in the presence or absence of DPI (10 μM) by Diogenes chemiluminescence ( n = 3, in triplicate). Right, quantitative real-time RT-PCR (qPCR) analysis of Nox4 from total RNA extracted from cells used for experiments whose results are shown at the left using human Nox4-specific primers. Actin-specific primers were used as internal control ( n = 3, in triplicate). RLU, relative light units. (B) Huh7.5 cells were infected with cell culture of replicating JFH-AM2 HCV (viral titer 1 × 10 5 viral copies per milliliter) or were mock infected. Cells were collected at days 2 and 5 postinfection and assayed for superoxide generation. Fold Δ, fold change; w/o, without.
    Figure Legend Snippet: Effect of HCV RNA or infectious virus on superoxide generation and Nox4 mRNA. (A) Left, HepG2 cells were transfected with in vitro-transcribed full-length HCV-1a or in vitro-transcribed HCVΔNS5b (ΔHCV) RNA as mentioned in Materials and Methods. After 48 h, cells were assayed for superoxide generation for 1 h in the presence or absence of DPI (10 μM) by Diogenes chemiluminescence ( n = 3, in triplicate). Right, quantitative real-time RT-PCR (qPCR) analysis of Nox4 from total RNA extracted from cells used for experiments whose results are shown at the left using human Nox4-specific primers. Actin-specific primers were used as internal control ( n = 3, in triplicate). RLU, relative light units. (B) Huh7.5 cells were infected with cell culture of replicating JFH-AM2 HCV (viral titer 1 × 10 5 viral copies per milliliter) or were mock infected. Cells were collected at days 2 and 5 postinfection and assayed for superoxide generation. Fold Δ, fold change; w/o, without.

    Techniques Used: Transfection, In Vitro, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Infection, Cell Culture

    2) Product Images from "Hepatitis C Virus (HCV) Proteins Induce NADPH Oxidase 4 Expression in a Transforming Growth Factor ?-Dependent Manner: a New Contributor to HCV-Induced Oxidative Stress ▿"

    Article Title: Hepatitis C Virus (HCV) Proteins Induce NADPH Oxidase 4 Expression in a Transforming Growth Factor ?-Dependent Manner: a New Contributor to HCV-Induced Oxidative Stress ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01059-09

    HCV structural and nonstructural genes regulate Nox4 expression and Nox4-derived ROS generation. (A) Schematic map of the full-length (FL) HCV genome displaying the structural and nonstructural genomic regions. The structural (Str) genes include core, E1, E2, and p7 viral genes. The nonstructural (NStr) genes include NS2, NS3, NS4a, NS4b, NS5a, and NS5b. HCV deletion constructs (Str and NStr) were generated by PCR and cloned into pcDNA3.3 as described in Materials and Methods. (B) HepG2 cells were transfected with empty-vector (EV), HCV FL, HCV Str, or HCV NStr plasmid (1 μg). After 48 h, whole-cell lysates were collected and 30 μg amounts of protein were analyzed by Western blotting, being probed sequentially with anticore, anti-E2, anti-NS5a, anti-Nox4, and anti-GAPDH. Lower panel displays densitometry values of Nox4 protein relative to GAPDH protein expression. Results are indicated as the factor of change compared to the level in cells transfected with vector alone. IB, immunoblot. (C) HepG2 cells were cotransfected with vector (V) and HCV FL, Str, or NStr constructs or cotransfected with Nox4-ΔCT and HCV FL, Str, or NStr constructs. Cells were assayed by Diogenes luminescence for superoxide generation for 45 min in the presence or absence of DPI (10 μM) ( n = 3, in triplicate). (D) HepG2 cells stably expressing Nox4 shRNA (clone #89) were transfected as described for panel B. Cells were assayed with Diogenes reagent for superoxide generation for 45 min in the presence or absence of DPI (10 μM) ( n = 3, in triplicate; *, P
    Figure Legend Snippet: HCV structural and nonstructural genes regulate Nox4 expression and Nox4-derived ROS generation. (A) Schematic map of the full-length (FL) HCV genome displaying the structural and nonstructural genomic regions. The structural (Str) genes include core, E1, E2, and p7 viral genes. The nonstructural (NStr) genes include NS2, NS3, NS4a, NS4b, NS5a, and NS5b. HCV deletion constructs (Str and NStr) were generated by PCR and cloned into pcDNA3.3 as described in Materials and Methods. (B) HepG2 cells were transfected with empty-vector (EV), HCV FL, HCV Str, or HCV NStr plasmid (1 μg). After 48 h, whole-cell lysates were collected and 30 μg amounts of protein were analyzed by Western blotting, being probed sequentially with anticore, anti-E2, anti-NS5a, anti-Nox4, and anti-GAPDH. Lower panel displays densitometry values of Nox4 protein relative to GAPDH protein expression. Results are indicated as the factor of change compared to the level in cells transfected with vector alone. IB, immunoblot. (C) HepG2 cells were cotransfected with vector (V) and HCV FL, Str, or NStr constructs or cotransfected with Nox4-ΔCT and HCV FL, Str, or NStr constructs. Cells were assayed by Diogenes luminescence for superoxide generation for 45 min in the presence or absence of DPI (10 μM) ( n = 3, in triplicate). (D) HepG2 cells stably expressing Nox4 shRNA (clone #89) were transfected as described for panel B. Cells were assayed with Diogenes reagent for superoxide generation for 45 min in the presence or absence of DPI (10 μM) ( n = 3, in triplicate; *, P

    Techniques Used: Expressing, Derivative Assay, Construct, Generated, Polymerase Chain Reaction, Clone Assay, Transfection, Plasmid Preparation, Western Blot, Stable Transfection, shRNA

    HCV induces both human and murine Nox4 promoters. (A) 5′-End deletion constructs of the human (top) and murine (bottom) Nox4 promoters were cloned into pGL3 luciferase reporter plasmids. Positive regulatory regions of the Nox4 promoter are indicated in dark gray. ChipMapper, a transcription factor binding site-screening algorithm, was used to analyze 2 kb of promoter sequence upstream of the Nox4 transcription start sites. (B) HepG2 cells were cotransfected with a reporter plasmid (1 μg) containing the luciferase gene under the regulation of the Nox4 promoter (pGL3 −1848) and GFP plasmid (1 μg) or increasing amounts of HCV plasmid (0.25, 0.5, and 1.0 μg). Whole-cell lysates were collected, and luciferase activity was determined by luminescence 48 h later ( n = 3, in triplicate). +, present; −, absent. (C) HepG2 cell were cotransfected as described for panel A with serially deleted Nox4 promoter constructs (1 μg) tied to luciferase. Results shown are representative of two independent experiments performed in triplicate. (D) Human Huh-7 or CHL hepatoma cells were cotransfected with Nox4 promoter/reporter plasmid (pGL3 −1848) and GFP or HCV plasmid (1 μg). Whole-cell lysates were collected and analyzed for luciferase activity as described for panel A ( n = 4, in triplicate). (E) Hepa 1-6 murine hepatocyte cells were cotransfected with a reporter plasmid containing the luciferase gene under the regulation of the Nox4 murine promoter (pGL3 −1291 or pGL3 −1707) and GFP or HCV plasmid. Luciferase activity was determined 48 h posttransfection ( n = 3, in triplicate). (F) Hepa 1-6 cells were transfected with empty vector (1 μg), full-length HCV (1 μg), or murine Nox4 (1 μg) for 48 h. Cells were assayed with Diogenes luminescence for superoxide generation (45 min) in the presence (+) or absence (−) of Nox inhibitor DPI (10 μM) ( n = 3, in triplicate; *, P
    Figure Legend Snippet: HCV induces both human and murine Nox4 promoters. (A) 5′-End deletion constructs of the human (top) and murine (bottom) Nox4 promoters were cloned into pGL3 luciferase reporter plasmids. Positive regulatory regions of the Nox4 promoter are indicated in dark gray. ChipMapper, a transcription factor binding site-screening algorithm, was used to analyze 2 kb of promoter sequence upstream of the Nox4 transcription start sites. (B) HepG2 cells were cotransfected with a reporter plasmid (1 μg) containing the luciferase gene under the regulation of the Nox4 promoter (pGL3 −1848) and GFP plasmid (1 μg) or increasing amounts of HCV plasmid (0.25, 0.5, and 1.0 μg). Whole-cell lysates were collected, and luciferase activity was determined by luminescence 48 h later ( n = 3, in triplicate). +, present; −, absent. (C) HepG2 cell were cotransfected as described for panel A with serially deleted Nox4 promoter constructs (1 μg) tied to luciferase. Results shown are representative of two independent experiments performed in triplicate. (D) Human Huh-7 or CHL hepatoma cells were cotransfected with Nox4 promoter/reporter plasmid (pGL3 −1848) and GFP or HCV plasmid (1 μg). Whole-cell lysates were collected and analyzed for luciferase activity as described for panel A ( n = 4, in triplicate). (E) Hepa 1-6 murine hepatocyte cells were cotransfected with a reporter plasmid containing the luciferase gene under the regulation of the Nox4 murine promoter (pGL3 −1291 or pGL3 −1707) and GFP or HCV plasmid. Luciferase activity was determined 48 h posttransfection ( n = 3, in triplicate). (F) Hepa 1-6 cells were transfected with empty vector (1 μg), full-length HCV (1 μg), or murine Nox4 (1 μg) for 48 h. Cells were assayed with Diogenes luminescence for superoxide generation (45 min) in the presence (+) or absence (−) of Nox inhibitor DPI (10 μM) ( n = 3, in triplicate; *, P

    Techniques Used: Construct, Clone Assay, Luciferase, Binding Assay, Sequencing, Plasmid Preparation, Activity Assay, Transfection

    Effect of HCV on the expression of Nox4. (A) Nox-specific PCR amplification of reverse-transcribed total RNA (1 μg) from HepG2 cells transfected 48 h previously with either pcDNA3.1 empty vector plasmid (1 μg) or full-length HCV-1a cDNA plasmid. Results shown are representative of four independent experiments. (B) Quantitative real-time RT-PCR (qPCR) analysis of Nox4 from total RNA extracted from HepG2 cells. RNAs were collected from cells transfected for 48 h with either empty vector plasmid (1 μg) or HCV plasmid (1 μg), followed by reverse transcription. Equal amounts of cDNA were subjected to quantitative RT-PCR using human Nox4-specific primers. GAPDH-specific primers were used as internal control ( n = 2, in quadruplicate). (C) HepG2 cells were transfected as described for panel A with increasing amounts of HCV plasmid (0.25, 0.5, and 1.0 μg). p22 phox -specific primers were used to determine mRNA expression. Primers specific for the HCV NS5b gene were used to confirm the presence of HCV RNA. Results shown are representative of three independent experiments.
    Figure Legend Snippet: Effect of HCV on the expression of Nox4. (A) Nox-specific PCR amplification of reverse-transcribed total RNA (1 μg) from HepG2 cells transfected 48 h previously with either pcDNA3.1 empty vector plasmid (1 μg) or full-length HCV-1a cDNA plasmid. Results shown are representative of four independent experiments. (B) Quantitative real-time RT-PCR (qPCR) analysis of Nox4 from total RNA extracted from HepG2 cells. RNAs were collected from cells transfected for 48 h with either empty vector plasmid (1 μg) or HCV plasmid (1 μg), followed by reverse transcription. Equal amounts of cDNA were subjected to quantitative RT-PCR using human Nox4-specific primers. GAPDH-specific primers were used as internal control ( n = 2, in quadruplicate). (C) HepG2 cells were transfected as described for panel A with increasing amounts of HCV plasmid (0.25, 0.5, and 1.0 μg). p22 phox -specific primers were used to determine mRNA expression. Primers specific for the HCV NS5b gene were used to confirm the presence of HCV RNA. Results shown are representative of three independent experiments.

    Techniques Used: Expressing, Polymerase Chain Reaction, Amplification, Transfection, Plasmid Preparation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Regulation of Nox4 by HCV core protein involves TGF-β1 autocrine signals. (A) Exogenous TGF-β1 induces the Nox4 promoter. HepG2 cells were transfected with the Nox4 promoter reporter plasmid (pGL3 −1848) with or without GFP plasmid (1.0 μg). Twenty-four hours later, transfected cells were treated with or without TGF-β1 (2 ng/ml) for 24 h. Whole-cell lysates were collected, and luciferase activity was determined by luminescence ( n = 4, in triplicate). (B) Nox4 mRNA induction by TGF-β1-treated HepG2 cells. Cells were treated with or without (UT, untreated) increasing concentrations of TGF-β1 (0.5 to 2.0 ng/ml) in full-serum culture medium for 48 h. Total RNA was collected and reverse transcribed. Nox4 and GAPDH expression levels were determined by PCR amplification of total cDNA with gene-specific primers. (C) HepG2 cells were treated with TGF-β1 for 48 h as described for panel B. Cells were assayed with Diogenes reagent for superoxide generation for 45 min in the presence (+) or absence (−) of DPI (10 μM) ( n = 3, in triplicate). (D) Left, HepG2 cells were transfected with either empty-vector plasmid or HCV core plasmid (1 μg). Twenty-four hours later, core-transfected cells were treated or not treated (−) with TGF-β monoclonal antibody (MAb; 2 μg/ml) for 24 h. Nox4-, core-, TGF-β1-, and GAPDH-specific PCR products were amplified from reverse-transcribed total RNA (1 μg) from cells subjected to different treatments. Right, HepG2 cells were transfected and treated as described for the left panel. Thirty-microgram amounts of total cell lysate were analyzed for protein expression by Western blotting. The immunoblot (IB) was sequentially probed with antibodies to Nox4, core, TGF-β1, and actin. Densitometry values given below blots indicate Nox4 or TGF-β protein levels relative to GAPDH protein expression. (E) HepG2 cells were transfected with empty-vector plasmid (1 μg), core plasmid (1 μg), or dominant negative TGF-βRII (ΔTβRII; 1 μg). Twenty-four hours later, cells were treated without or with TGF-β monoclonal antibody (TβMAb; 2 μg/ml) for 24 h. Cells were then assayed for superoxide production as described for panel C ( n = 3). w/o DPI, without DPI. (F) HepG2 cells were transfected with empty-vector or core plasmid or cotransfected with core plasmid and dominant negative TGF-βRII (ΔTβRII) (1.0 μg each). Forty-eight hours following transfection, total RNA was collected and reverse transcribed to determine Nox4 mRNA expression by quantitative PCR (qPCR) ( n = 3, in triplicate; *, P
    Figure Legend Snippet: Regulation of Nox4 by HCV core protein involves TGF-β1 autocrine signals. (A) Exogenous TGF-β1 induces the Nox4 promoter. HepG2 cells were transfected with the Nox4 promoter reporter plasmid (pGL3 −1848) with or without GFP plasmid (1.0 μg). Twenty-four hours later, transfected cells were treated with or without TGF-β1 (2 ng/ml) for 24 h. Whole-cell lysates were collected, and luciferase activity was determined by luminescence ( n = 4, in triplicate). (B) Nox4 mRNA induction by TGF-β1-treated HepG2 cells. Cells were treated with or without (UT, untreated) increasing concentrations of TGF-β1 (0.5 to 2.0 ng/ml) in full-serum culture medium for 48 h. Total RNA was collected and reverse transcribed. Nox4 and GAPDH expression levels were determined by PCR amplification of total cDNA with gene-specific primers. (C) HepG2 cells were treated with TGF-β1 for 48 h as described for panel B. Cells were assayed with Diogenes reagent for superoxide generation for 45 min in the presence (+) or absence (−) of DPI (10 μM) ( n = 3, in triplicate). (D) Left, HepG2 cells were transfected with either empty-vector plasmid or HCV core plasmid (1 μg). Twenty-four hours later, core-transfected cells were treated or not treated (−) with TGF-β monoclonal antibody (MAb; 2 μg/ml) for 24 h. Nox4-, core-, TGF-β1-, and GAPDH-specific PCR products were amplified from reverse-transcribed total RNA (1 μg) from cells subjected to different treatments. Right, HepG2 cells were transfected and treated as described for the left panel. Thirty-microgram amounts of total cell lysate were analyzed for protein expression by Western blotting. The immunoblot (IB) was sequentially probed with antibodies to Nox4, core, TGF-β1, and actin. Densitometry values given below blots indicate Nox4 or TGF-β protein levels relative to GAPDH protein expression. (E) HepG2 cells were transfected with empty-vector plasmid (1 μg), core plasmid (1 μg), or dominant negative TGF-βRII (ΔTβRII; 1 μg). Twenty-four hours later, cells were treated without or with TGF-β monoclonal antibody (TβMAb; 2 μg/ml) for 24 h. Cells were then assayed for superoxide production as described for panel C ( n = 3). w/o DPI, without DPI. (F) HepG2 cells were transfected with empty-vector or core plasmid or cotransfected with core plasmid and dominant negative TGF-βRII (ΔTβRII) (1.0 μg each). Forty-eight hours following transfection, total RNA was collected and reverse transcribed to determine Nox4 mRNA expression by quantitative PCR (qPCR) ( n = 3, in triplicate; *, P

    Techniques Used: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Polymerase Chain Reaction, Amplification, Western Blot, Dominant Negative Mutation, Real-time Polymerase Chain Reaction

    ROS generation by HCV-transfected human hepatocytes. (A) Schematic of the HCV genome. HCV is a positive-strand RNA [(+) RNA] virus whose genes are translated by host cell translational machinery upon infection. Both host and viral proteases cleave the viral polyprotein into 10 individual viral proteins with specific functions. UTR, untranslated region; IRES, internal ribosome entry site. (B) Western blot detection of HCV core and E2 proteins in HepG2 cells transfected with pcDNA3.3 plasmid containing the full-length HCV genome (genotype 1a) (1 μg). Whole-cell lysates were collected 48 h posttransfection, and 30-μg amounts of protein were probed with anti-core, anti-E2, and anti-actin. (C) Human hepatoma cell lines (Huh-7, HepG2, and CHL) were transfected with empty vector or plasmid containing the full-length HCV genome (genotype 1a) (1 μg) for 48 h. Cell lines were assayed for superoxide generation with Diogenes luminescence (45 min) in the presence (+) or absence (−) of Nox inhibitor DPI (10 μM) ( n = 5, in triplicate). RLU, relative light units. (D) HepG2 cells were transfected as described for panel C for 48 h. Single-cell intracellular H 2 O 2 was measured by flow cytometry after exposure to CM-H 2 DCF-DA (DCF; 2 μM) for 15 min and with (+) or without (−) DPI for 10 min ( n = 3, in triplicate; *, P
    Figure Legend Snippet: ROS generation by HCV-transfected human hepatocytes. (A) Schematic of the HCV genome. HCV is a positive-strand RNA [(+) RNA] virus whose genes are translated by host cell translational machinery upon infection. Both host and viral proteases cleave the viral polyprotein into 10 individual viral proteins with specific functions. UTR, untranslated region; IRES, internal ribosome entry site. (B) Western blot detection of HCV core and E2 proteins in HepG2 cells transfected with pcDNA3.3 plasmid containing the full-length HCV genome (genotype 1a) (1 μg). Whole-cell lysates were collected 48 h posttransfection, and 30-μg amounts of protein were probed with anti-core, anti-E2, and anti-actin. (C) Human hepatoma cell lines (Huh-7, HepG2, and CHL) were transfected with empty vector or plasmid containing the full-length HCV genome (genotype 1a) (1 μg) for 48 h. Cell lines were assayed for superoxide generation with Diogenes luminescence (45 min) in the presence (+) or absence (−) of Nox inhibitor DPI (10 μM) ( n = 5, in triplicate). RLU, relative light units. (D) HepG2 cells were transfected as described for panel C for 48 h. Single-cell intracellular H 2 O 2 was measured by flow cytometry after exposure to CM-H 2 DCF-DA (DCF; 2 μM) for 15 min and with (+) or without (−) DPI for 10 min ( n = 3, in triplicate; *, P

    Techniques Used: Transfection, Infection, Western Blot, Plasmid Preparation, Flow Cytometry, Cytometry

    Effect of HCV RNA or infectious virus on superoxide generation and Nox4 mRNA. (A) Left, HepG2 cells were transfected with in vitro-transcribed full-length HCV-1a or in vitro-transcribed HCVΔNS5b (ΔHCV) RNA as mentioned in Materials and Methods. After 48 h, cells were assayed for superoxide generation for 1 h in the presence or absence of DPI (10 μM) by Diogenes chemiluminescence ( n = 3, in triplicate). Right, quantitative real-time RT-PCR (qPCR) analysis of Nox4 from total RNA extracted from cells used for experiments whose results are shown at the left using human Nox4-specific primers. Actin-specific primers were used as internal control ( n = 3, in triplicate). RLU, relative light units. (B) Huh7.5 cells were infected with cell culture of replicating JFH-AM2 HCV (viral titer 1 × 10 5 viral copies per milliliter) or were mock infected. Cells were collected at days 2 and 5 postinfection and assayed for superoxide generation. Fold Δ, fold change; w/o, without.
    Figure Legend Snippet: Effect of HCV RNA or infectious virus on superoxide generation and Nox4 mRNA. (A) Left, HepG2 cells were transfected with in vitro-transcribed full-length HCV-1a or in vitro-transcribed HCVΔNS5b (ΔHCV) RNA as mentioned in Materials and Methods. After 48 h, cells were assayed for superoxide generation for 1 h in the presence or absence of DPI (10 μM) by Diogenes chemiluminescence ( n = 3, in triplicate). Right, quantitative real-time RT-PCR (qPCR) analysis of Nox4 from total RNA extracted from cells used for experiments whose results are shown at the left using human Nox4-specific primers. Actin-specific primers were used as internal control ( n = 3, in triplicate). RLU, relative light units. (B) Huh7.5 cells were infected with cell culture of replicating JFH-AM2 HCV (viral titer 1 × 10 5 viral copies per milliliter) or were mock infected. Cells were collected at days 2 and 5 postinfection and assayed for superoxide generation. Fold Δ, fold change; w/o, without.

    Techniques Used: Transfection, In Vitro, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Infection, Cell Culture

    Dominant negative Nox4 (Nox4-ΔCT) or knockdown of Nox4 decreases HCV-induced superoxide production. (A) HepG2 cells were stably transfected with plasmids encoding shRNAs specific to Nox4 (shRNA Nox4 #88 or shRNA Nox4 #89) or control nontargeting shRNA plasmid and analyzed by Western blotting. The same blot was sequentially probed with anti-Nox4 antibody and anti-GAPDH antibody. Densitometry values given below blots indicate the level of Nox4 protein expression relative to the level of GAPDH protein. (B) Nontargeting control or Nox4 (clone #89) shRNA stable cell lines described for panel A were transfected with HCV and assayed for superoxide generation for 45 min in the presence (+) or absence (−) of DPI (10 μM) ( n = 4, in triplicate). (C) Deletion of the FAD and NADPH domains of Nox4 results in a truncated inactive enzyme that has dominant negative effects. (D) HepG2 cells were transfected with vector alone, HCV cDNA, or V5-tagged Nox4-ΔCT or with HCV and V5-tagged Nox4-ΔCT. Forty-eight hours posttransfection, cells were collected and assayed for superoxide generation in the presence or absence (w/o, without) of DPI ( n = 3; *, P
    Figure Legend Snippet: Dominant negative Nox4 (Nox4-ΔCT) or knockdown of Nox4 decreases HCV-induced superoxide production. (A) HepG2 cells were stably transfected with plasmids encoding shRNAs specific to Nox4 (shRNA Nox4 #88 or shRNA Nox4 #89) or control nontargeting shRNA plasmid and analyzed by Western blotting. The same blot was sequentially probed with anti-Nox4 antibody and anti-GAPDH antibody. Densitometry values given below blots indicate the level of Nox4 protein expression relative to the level of GAPDH protein. (B) Nontargeting control or Nox4 (clone #89) shRNA stable cell lines described for panel A were transfected with HCV and assayed for superoxide generation for 45 min in the presence (+) or absence (−) of DPI (10 μM) ( n = 4, in triplicate). (C) Deletion of the FAD and NADPH domains of Nox4 results in a truncated inactive enzyme that has dominant negative effects. (D) HepG2 cells were transfected with vector alone, HCV cDNA, or V5-tagged Nox4-ΔCT or with HCV and V5-tagged Nox4-ΔCT. Forty-eight hours posttransfection, cells were collected and assayed for superoxide generation in the presence or absence (w/o, without) of DPI ( n = 3; *, P

    Techniques Used: Dominant Negative Mutation, Stable Transfection, Transfection, shRNA, Plasmid Preparation, Western Blot, Expressing

    HCV core protein induces Nox4 expression and ROS release in human hepatocytes. (A) Left, HepG2 cells were transfected for 48 h with pcDNA3.3 expression plasmid encoding the HCV core protein or NS3 protein (1 μg). Nox4- and actin-specific primers were used to determine mRNA expression by RT-PCR. Right, HepG2 cells were transfected with either empty-vector plasmid or HCV core plasmid (1 μg) for 48 h. Whole-cell lysates were then collected, and 30-μg amounts of protein were analyzed by Western blotting. The blot was sequentially probed with anti-Nox4 and anti-core. IB, immunoblot. (B) HepG2 cells stably expressing Nox4 shRNA (clone #89) were transfected as described for panel A, right. Cells were assayed for superoxide generation with Diogenes reagent for 45 min in the presence or absence of DPI (10 μM) ( n = 3, in triplicate). (C) HepG2 cells were transfected for 48 h with empty vector or increasing amounts of pcDNA3.3 core plasmid (0.25, 0.5, 1.0, or 2.0 μg). Total RNA was collected and reverse transcribed to determine the level of Nox4 mRNA expression by quantitative PCR (qPCR) ( n = 3, in triplicate). (D) HepG2 cells were transfected for 48 h with empty vector or increasing amounts of pcDNA3.3 core as described for panel D. After 48 h, the cells were collected and analyzed for extracellular superoxide production in the presence or absence of DPI (10 μM). (E) HepG2 cells were cotransfected with the Nox4 promoter-luciferase reporter plasmid pGL3 −1848 and GFP, core, or NS3 plasmids (1.0 μg). Whole-cell lysates were collected, and luciferase activity was determined by luminescence 48 h later ( n = 3, in triplicate; *, P
    Figure Legend Snippet: HCV core protein induces Nox4 expression and ROS release in human hepatocytes. (A) Left, HepG2 cells were transfected for 48 h with pcDNA3.3 expression plasmid encoding the HCV core protein or NS3 protein (1 μg). Nox4- and actin-specific primers were used to determine mRNA expression by RT-PCR. Right, HepG2 cells were transfected with either empty-vector plasmid or HCV core plasmid (1 μg) for 48 h. Whole-cell lysates were then collected, and 30-μg amounts of protein were analyzed by Western blotting. The blot was sequentially probed with anti-Nox4 and anti-core. IB, immunoblot. (B) HepG2 cells stably expressing Nox4 shRNA (clone #89) were transfected as described for panel A, right. Cells were assayed for superoxide generation with Diogenes reagent for 45 min in the presence or absence of DPI (10 μM) ( n = 3, in triplicate). (C) HepG2 cells were transfected for 48 h with empty vector or increasing amounts of pcDNA3.3 core plasmid (0.25, 0.5, 1.0, or 2.0 μg). Total RNA was collected and reverse transcribed to determine the level of Nox4 mRNA expression by quantitative PCR (qPCR) ( n = 3, in triplicate). (D) HepG2 cells were transfected for 48 h with empty vector or increasing amounts of pcDNA3.3 core as described for panel D. After 48 h, the cells were collected and analyzed for extracellular superoxide production in the presence or absence of DPI (10 μM). (E) HepG2 cells were cotransfected with the Nox4 promoter-luciferase reporter plasmid pGL3 −1848 and GFP, core, or NS3 plasmids (1.0 μg). Whole-cell lysates were collected, and luciferase activity was determined by luminescence 48 h later ( n = 3, in triplicate; *, P

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Stable Transfection, shRNA, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay

    3) Product Images from "Nanolipoparticles-mediated MDR1 siRNA delivery reduces doxorubicin resistance in breast cancer cells and silences MDR1 expression in xenograft model of human breast cancer"

    Article Title: Nanolipoparticles-mediated MDR1 siRNA delivery reduces doxorubicin resistance in breast cancer cells and silences MDR1 expression in xenograft model of human breast cancer

    Journal: Iranian Journal of Basic Medical Sciences

    doi:

    Relative mRNA levels for MDR1 in MCF-7/ADR human multidrug-resistant breast cancer cells. MCF-7/ADR Cells transfected with different formulations and incubated for 24 or 48 hr. MDR1 mRNA downregulation efficiency was calculated by comparing the MDR1 mRNA expression value in each transfected group to the untreated group. The MDR1 mRNA expression value was normalized to β-actin . Data are means±SEM. (n=3; * P
    Figure Legend Snippet: Relative mRNA levels for MDR1 in MCF-7/ADR human multidrug-resistant breast cancer cells. MCF-7/ADR Cells transfected with different formulations and incubated for 24 or 48 hr. MDR1 mRNA downregulation efficiency was calculated by comparing the MDR1 mRNA expression value in each transfected group to the untreated group. The MDR1 mRNA expression value was normalized to β-actin . Data are means±SEM. (n=3; * P

    Techniques Used: Transfection, Incubation, Expressing

    Relative mRNA levels for MDR1 in MCF-7/ADR human multidrug-resistant breast cancer cells and their parental cells MCF-7. The MDR1 mRNA expression value was normalized to β-actin . Data are means±SEM. (n = 3; *** P
    Figure Legend Snippet: Relative mRNA levels for MDR1 in MCF-7/ADR human multidrug-resistant breast cancer cells and their parental cells MCF-7. The MDR1 mRNA expression value was normalized to β-actin . Data are means±SEM. (n = 3; *** P

    Techniques Used: Expressing

    Western blot assay to determine the effect of MDR1 siRNA loaded nanoparticles on the P-gp expression. MCF-7/ADR Cells transfected with different formulations at the 100 nM and incubated for 48, 72 or 96 hr. P-gp downregulation efficiency was calculated by comparing the level of P-gp expression in each transfected group to the untreated group. (A) Top: P-glycoprotein was detected with C219 antibody. Bottom: The same membrane was reprobed with anti-actin antibody. (B) The quantitation of Western blot images carried out by using UVtec software and the protein levels were normalized against actin intensity. Data are means±SEM (n= 3; ** P
    Figure Legend Snippet: Western blot assay to determine the effect of MDR1 siRNA loaded nanoparticles on the P-gp expression. MCF-7/ADR Cells transfected with different formulations at the 100 nM and incubated for 48, 72 or 96 hr. P-gp downregulation efficiency was calculated by comparing the level of P-gp expression in each transfected group to the untreated group. (A) Top: P-glycoprotein was detected with C219 antibody. Bottom: The same membrane was reprobed with anti-actin antibody. (B) The quantitation of Western blot images carried out by using UVtec software and the protein levels were normalized against actin intensity. Data are means±SEM (n= 3; ** P

    Techniques Used: Western Blot, Expressing, Transfection, Incubation, Quantitation Assay, Software

    Enhancement of chemosensitivity to doxorubicin. Cytotoxicity of doxorubicin in MCF-7/ADR cells was assessed by MTT. Untransfected cells (control), cells transfected with liposomes containing MDR1 siRNA 100 nM (NLP siM, OFA siM) or cells transfected with empty NLP (NLP) or NLP containing control siRNA 100 nM (NLP siC) (as negative controls). After 48 hr, cells were incubated with 10 µM doxorubicin for 48 hr. Relative cell viability compared with control group has been reported. Data are means±SEM (n=3; ** P
    Figure Legend Snippet: Enhancement of chemosensitivity to doxorubicin. Cytotoxicity of doxorubicin in MCF-7/ADR cells was assessed by MTT. Untransfected cells (control), cells transfected with liposomes containing MDR1 siRNA 100 nM (NLP siM, OFA siM) or cells transfected with empty NLP (NLP) or NLP containing control siRNA 100 nM (NLP siC) (as negative controls). After 48 hr, cells were incubated with 10 µM doxorubicin for 48 hr. Relative cell viability compared with control group has been reported. Data are means±SEM (n=3; ** P

    Techniques Used: MTT Assay, Transfection, Incubation

    4) Product Images from "Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR"

    Article Title: Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011337

    Determination of lysis speed and burst sizes for bacteriophages ϕA1122, L-413C, and P2 vir1 on Y. pestis CO92 pgm − . Phage burst sizes (an average phage progeny produced by one bacterial cell) correspond to plateaus on the curves.
    Figure Legend Snippet: Determination of lysis speed and burst sizes for bacteriophages ϕA1122, L-413C, and P2 vir1 on Y. pestis CO92 pgm − . Phage burst sizes (an average phage progeny produced by one bacterial cell) correspond to plateaus on the curves.

    Techniques Used: Lysis, Produced

    Lytic properties of bacteriophages ϕA1122, L-413C, and P2 vir1 towards Y. pestis CO92 pgm − . The dynamics of lysis was determined in BHI broth at multiplicity of infection of 0.1. Optical density was normalized to the start of infection (1 on the Y axis corresponds to the initial OD 600 = 0.2).
    Figure Legend Snippet: Lytic properties of bacteriophages ϕA1122, L-413C, and P2 vir1 towards Y. pestis CO92 pgm − . The dynamics of lysis was determined in BHI broth at multiplicity of infection of 0.1. Optical density was normalized to the start of infection (1 on the Y axis corresponds to the initial OD 600 = 0.2).

    Techniques Used: Lysis, Infection

    5) Product Images from "Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR"

    Article Title: Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011337

    qPCR tests on simulated clinical human blood samples. A. Linear regression of ϕA1122 DNA concentration in blood diluted 1∶20 in comparison with SM buffer data. B. ϕA1122-based detection of Y. pestis in artificially contaminated blood diluted 10-fold with BHI broth. To calculate the actual bacterial loads in the undiluted blood samples, the CFU numbers shown should be multiplied by 10. The starting points of phage infection correspond to 100 PFU per 1 µl sample and are normalized to 10 0 = 1.
    Figure Legend Snippet: qPCR tests on simulated clinical human blood samples. A. Linear regression of ϕA1122 DNA concentration in blood diluted 1∶20 in comparison with SM buffer data. B. ϕA1122-based detection of Y. pestis in artificially contaminated blood diluted 10-fold with BHI broth. To calculate the actual bacterial loads in the undiluted blood samples, the CFU numbers shown should be multiplied by 10. The starting points of phage infection correspond to 100 PFU per 1 µl sample and are normalized to 10 0 = 1.

    Techniques Used: Real-time Polymerase Chain Reaction, Concentration Assay, Infection

    Dynamics of growth of phages ϕA1122 and L-413C on different concentrations of Y. pestis cells detected by qPCR. The starting points of phage infection correspond to 100 PFU per 1 µl sample and are normalized to 1. A. The titer rise of ϕA1122. B. L-413C amplification.
    Figure Legend Snippet: Dynamics of growth of phages ϕA1122 and L-413C on different concentrations of Y. pestis cells detected by qPCR. The starting points of phage infection correspond to 100 PFU per 1 µl sample and are normalized to 1. A. The titer rise of ϕA1122. B. L-413C amplification.

    Techniques Used: Real-time Polymerase Chain Reaction, Infection, Amplification

    Parameters of ϕA1122- and L-413C-based qPCR tests for phage DNA and live phage particles determined by linear regression method. A and B, standard curves plotted for DNA concentrations of ϕA1122 and L-413C, respectively. C and D, standard curves plotted for live phage particles of ϕA1122 and L-413C, respectively.
    Figure Legend Snippet: Parameters of ϕA1122- and L-413C-based qPCR tests for phage DNA and live phage particles determined by linear regression method. A and B, standard curves plotted for DNA concentrations of ϕA1122 and L-413C, respectively. C and D, standard curves plotted for live phage particles of ϕA1122 and L-413C, respectively.

    Techniques Used: Real-time Polymerase Chain Reaction

    6) Product Images from "A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies"

    Article Title: A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005679

    Chromosomal microdeletions contribute to mitochondrial respiratory chain complex deficiencies. ( A ) The 17p12 deletion was detected by high-density oligonucleotide arrays. This heterozygous deletion of 17p12 involved the last 2 exons of COX10 . ( B and C ) Family pedigrees of Pt657 and Pt369. In Pt369, the 17p12 deletion was paternally inherited ( C ). Eu means uninformative DNA test. ( D ) Analysis of endogenous COX10 mRNA expression relative to that in normal cells (fHDF and NHDF; Normal neonatal human dermal fibroblast); Pt223 harbored compound heterozygous non-synonymous mutations in COX10 . The COX10 expression in Pt369 and Pt657 was decreased by 50%. ( E – G ) Wild type COX10 cDNA rescue of complex IV assembly and activity in Pt657 fibroblasts. Mitochondria were isolated from control or Pt657 fibroblasts, and mitochondrial respiratory complex assembly was analyzed by BN-PAGE and Western blotting. Wild-type COX10-V5 cDNA expression rescued complex IV assembly in Pt657 fibroblasts ( E ). Mitochondrial TurboRFP-V5 (RFP) and COX10-V5 (COX10) were detected in control and Pt657 fibroblasts ( F ). Mitochondrial respiratory chain complex activities were measured twice. Compared with TurboRFP-V5 expressing Pt657 fibroblasts, expression of wild-type COX10-V5 in Pt657 fibroblasts resulted in a significant increase in complex IV activity ( G ).
    Figure Legend Snippet: Chromosomal microdeletions contribute to mitochondrial respiratory chain complex deficiencies. ( A ) The 17p12 deletion was detected by high-density oligonucleotide arrays. This heterozygous deletion of 17p12 involved the last 2 exons of COX10 . ( B and C ) Family pedigrees of Pt657 and Pt369. In Pt369, the 17p12 deletion was paternally inherited ( C ). Eu means uninformative DNA test. ( D ) Analysis of endogenous COX10 mRNA expression relative to that in normal cells (fHDF and NHDF; Normal neonatal human dermal fibroblast); Pt223 harbored compound heterozygous non-synonymous mutations in COX10 . The COX10 expression in Pt369 and Pt657 was decreased by 50%. ( E – G ) Wild type COX10 cDNA rescue of complex IV assembly and activity in Pt657 fibroblasts. Mitochondria were isolated from control or Pt657 fibroblasts, and mitochondrial respiratory complex assembly was analyzed by BN-PAGE and Western blotting. Wild-type COX10-V5 cDNA expression rescued complex IV assembly in Pt657 fibroblasts ( E ). Mitochondrial TurboRFP-V5 (RFP) and COX10-V5 (COX10) were detected in control and Pt657 fibroblasts ( F ). Mitochondrial respiratory chain complex activities were measured twice. Compared with TurboRFP-V5 expressing Pt657 fibroblasts, expression of wild-type COX10-V5 in Pt657 fibroblasts resulted in a significant increase in complex IV activity ( G ).

    Techniques Used: Expressing, Activity Assay, Isolation, Polyacrylamide Gel Electrophoresis, Western Blot

    7) Product Images from "Occurrence of Horizontal Gene Transfer of PIB-type ATPase Genes among Bacteria Isolated from the Uranium Rich Deposit of Domiasiat in North East India"

    Article Title: Occurrence of Horizontal Gene Transfer of PIB-type ATPase Genes among Bacteria Isolated from the Uranium Rich Deposit of Domiasiat in North East India

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0048199

    Molecular evidence for horizontal gene transfer among Domiasiat isolates. The genes encoding (A) 16S rRNA and (B) zntA/cadA/pbrA -like transporters.of uranium and heavy metal tolerant isolates obtained from subsurface soils of U-rich deposits of the Domiasiat site were subjected to neighbor-joining analysis. Respective accession numbers of gene nucleotide sequences are indicated in brackets. P IB -type ATPase positive isolates predicted to have undergone HGT are connected by dotted lines. The scale bars indicate 0.05 change per nucleotide position for the 16S rRNA gene and 0.1 change per amino acid position for P IB -type ATPase phylogeny.
    Figure Legend Snippet: Molecular evidence for horizontal gene transfer among Domiasiat isolates. The genes encoding (A) 16S rRNA and (B) zntA/cadA/pbrA -like transporters.of uranium and heavy metal tolerant isolates obtained from subsurface soils of U-rich deposits of the Domiasiat site were subjected to neighbor-joining analysis. Respective accession numbers of gene nucleotide sequences are indicated in brackets. P IB -type ATPase positive isolates predicted to have undergone HGT are connected by dotted lines. The scale bars indicate 0.05 change per nucleotide position for the 16S rRNA gene and 0.1 change per amino acid position for P IB -type ATPase phylogeny.

    Techniques Used:

    8) Product Images from "Occurrence of Horizontal Gene Transfer of PIB-type ATPase Genes among Bacteria Isolated from the Uranium Rich Deposit of Domiasiat in North East India"

    Article Title: Occurrence of Horizontal Gene Transfer of PIB-type ATPase Genes among Bacteria Isolated from the Uranium Rich Deposit of Domiasiat in North East India

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0048199

    Molecular evidence for horizontal gene transfer among Domiasiat isolates. The genes encoding (A) 16S rRNA and (B) zntA/cadA/pbrA -like transporters.of uranium and heavy metal tolerant isolates obtained from subsurface soils of U-rich deposits of the Domiasiat site were subjected to neighbor-joining analysis. Respective accession numbers of gene nucleotide sequences are indicated in brackets. P IB -type ATPase positive isolates predicted to have undergone HGT are connected by dotted lines. The scale bars indicate 0.05 change per nucleotide position for the 16S rRNA gene and 0.1 change per amino acid position for P IB -type ATPase phylogeny.
    Figure Legend Snippet: Molecular evidence for horizontal gene transfer among Domiasiat isolates. The genes encoding (A) 16S rRNA and (B) zntA/cadA/pbrA -like transporters.of uranium and heavy metal tolerant isolates obtained from subsurface soils of U-rich deposits of the Domiasiat site were subjected to neighbor-joining analysis. Respective accession numbers of gene nucleotide sequences are indicated in brackets. P IB -type ATPase positive isolates predicted to have undergone HGT are connected by dotted lines. The scale bars indicate 0.05 change per nucleotide position for the 16S rRNA gene and 0.1 change per amino acid position for P IB -type ATPase phylogeny.

    Techniques Used:

    9) Product Images from "Live Cell Monitoring of hiPSC Generation and Differentiation Using Differential Expression of Endogenous microRNAs"

    Article Title: Live Cell Monitoring of hiPSC Generation and Differentiation Using Differential Expression of Endogenous microRNAs

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011834

    hiPSCs transduced with the reporter vector containing miRNA targets show differentiation-specific reporter expression in EBs. hESC (H1) and 4 different hiPSC clones (E/m#1, E/m#5, E/m#101, and hiPSC#19) were differentiated into EBs and maintained 25 days in IMDM containing 10% FBS. EBs were then dissociated with 0.25% trypsin/EDTA and the reporter expression was analyzed by flow cytometry. Histograms filled with black are undifferentiated controls. Histograms filled with blue (EGFP) and pink (mCherry) are differentiated cells, respectively. The numbers indicated in histogram show percentage of positive cells (EGFP) and negative cells (mCherry). MFI: mean fluorescence intensity. U: MFI of undifferentiated cells. D: MFI of differentiated cells.
    Figure Legend Snippet: hiPSCs transduced with the reporter vector containing miRNA targets show differentiation-specific reporter expression in EBs. hESC (H1) and 4 different hiPSC clones (E/m#1, E/m#5, E/m#101, and hiPSC#19) were differentiated into EBs and maintained 25 days in IMDM containing 10% FBS. EBs were then dissociated with 0.25% trypsin/EDTA and the reporter expression was analyzed by flow cytometry. Histograms filled with black are undifferentiated controls. Histograms filled with blue (EGFP) and pink (mCherry) are differentiated cells, respectively. The numbers indicated in histogram show percentage of positive cells (EGFP) and negative cells (mCherry). MFI: mean fluorescence intensity. U: MFI of undifferentiated cells. D: MFI of differentiated cells.

    Techniques Used: Transduction, Plasmid Preparation, Expressing, Clone Assay, Flow Cytometry, Cytometry, Fluorescence

    Transduction of the reporter vector containing miRNA targets does not grossly affect expression of hESC-specific markers. (A and B) Single-cell suspensions of hESC (H1), hiPSCs transduced with a lentiviral vector encoding EGFP miR-T/mCherry miR-T (E/m#1, E/m#5, and E/m#101) or untransduced (hiPSC#19) were analyzed for the expression of EGFP and mCherry (A) and that of hESC-specific markers (SSEA1, SSEA3, TRA1-60, and TRA-1-81) (B) by flow cytometry. The number (%) in each quadrant is listed on each plot. (C) hESCs (H1), hiPSCs transduced with a lentiviral vector encoding EGFP miR-T/mCherry miR-T (E/m#1, E/m#5, and E/m#101) or untransduced (hiPSC#19) were plated on poly-L-lysine and Matrigel coated glass coverslips and expanded for a week. Cells were then fixed with 1% formaldehyde, permeabilized with 0.2% Triton X-100 for 5 min on ice, and stained with anti-Nanog antibody and DyLight488 conjugated anti-rabbit IgGs. 7-amino-actinomycin D (7-AAD) was used for nuclear staining.
    Figure Legend Snippet: Transduction of the reporter vector containing miRNA targets does not grossly affect expression of hESC-specific markers. (A and B) Single-cell suspensions of hESC (H1), hiPSCs transduced with a lentiviral vector encoding EGFP miR-T/mCherry miR-T (E/m#1, E/m#5, and E/m#101) or untransduced (hiPSC#19) were analyzed for the expression of EGFP and mCherry (A) and that of hESC-specific markers (SSEA1, SSEA3, TRA1-60, and TRA-1-81) (B) by flow cytometry. The number (%) in each quadrant is listed on each plot. (C) hESCs (H1), hiPSCs transduced with a lentiviral vector encoding EGFP miR-T/mCherry miR-T (E/m#1, E/m#5, and E/m#101) or untransduced (hiPSC#19) were plated on poly-L-lysine and Matrigel coated glass coverslips and expanded for a week. Cells were then fixed with 1% formaldehyde, permeabilized with 0.2% Triton X-100 for 5 min on ice, and stained with anti-Nanog antibody and DyLight488 conjugated anti-rabbit IgGs. 7-amino-actinomycin D (7-AAD) was used for nuclear staining.

    Techniques Used: Transduction, Plasmid Preparation, Expressing, Flow Cytometry, Cytometry, Staining

    Molecular characterization of hiPSCs transduced with the reporter vector containing miRNA targets. Total RNA was isolated using QIAGEN's RNeasy Mini kit from HFFs transduced with (4Fs/HFF) or without 4 reprogramming factors (HFF), hESCs (H1), and 4 different hiPSC clones transduced with (E/m#1, E/m#5, and E/m#101) or without (hiPSC#19) the reporter vector encoding EGFP miR-T/mCherry miR-T. Total RNA (250 ng) was reverse-transcribed using QIAGEN's Omniscript reverse transcription kit and used as a template in subsequent PCR with 5-PRIME's HotMaster Taq DNA polymerase. PCR products were analyzed on a 2% agarose gel. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.
    Figure Legend Snippet: Molecular characterization of hiPSCs transduced with the reporter vector containing miRNA targets. Total RNA was isolated using QIAGEN's RNeasy Mini kit from HFFs transduced with (4Fs/HFF) or without 4 reprogramming factors (HFF), hESCs (H1), and 4 different hiPSC clones transduced with (E/m#1, E/m#5, and E/m#101) or without (hiPSC#19) the reporter vector encoding EGFP miR-T/mCherry miR-T. Total RNA (250 ng) was reverse-transcribed using QIAGEN's Omniscript reverse transcription kit and used as a template in subsequent PCR with 5-PRIME's HotMaster Taq DNA polymerase. PCR products were analyzed on a 2% agarose gel. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

    Techniques Used: Transduction, Plasmid Preparation, Isolation, Clone Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    10) Product Images from "A putative genomic island, PGI-1, in Ralstonia solanacearum biovar 2 revealed by subtractive hybridization"

    Article Title: A putative genomic island, PGI-1, in Ralstonia solanacearum biovar 2 revealed by subtractive hybridization

    Journal: Antonie Van Leeuwenhoek

    doi: 10.1007/s10482-010-9450-4

    Primer systems used to characterize the identified region ( A ) and the composition of the IS blocks ( B ). A Upper line : inferred localization of IS elements and the relA / spoT region and PCR systems based on genomic information of strain 1609. IS: unassigned nucleotides in the 1609 genome which represent insertion sequences. Superscript 1 Numbers: primer systems, corresponding to primers shown in Table 3 (ps1 through ps15; ps15 not indicated here). PCR systems consisted of two PCR primers (e.g. 1: ps1-F/ps1-R, 2: ps2-F/ps2-R, etc.). *: ps10 amplifies ISRso3 . **: ps13 amplifies ISRso2s . Second line : Presence and absence of sequences in strain KZR-5 on the basis of PCR and hybridization. : The absence in strain KZR-5 was confirmed by Southern blot analysis using the corresponding PCR products of strain 1609 as DIG labeled DNA probes. B Insertion sequence (IS) regions determined by sequence analysis of PCR products of strains 715 and 1609 that were obtained with primer combinations ps9-F/ps11-R or ps6-F/ps14-R. The position of the sequencing primers (ps9-F, ps6-F, ps11-R, ps14-R and IS2/3) is indicated. Superscript 2 : IS regions correspond to the two IS regions, which are similar, shown in A (IS)
    Figure Legend Snippet: Primer systems used to characterize the identified region ( A ) and the composition of the IS blocks ( B ). A Upper line : inferred localization of IS elements and the relA / spoT region and PCR systems based on genomic information of strain 1609. IS: unassigned nucleotides in the 1609 genome which represent insertion sequences. Superscript 1 Numbers: primer systems, corresponding to primers shown in Table 3 (ps1 through ps15; ps15 not indicated here). PCR systems consisted of two PCR primers (e.g. 1: ps1-F/ps1-R, 2: ps2-F/ps2-R, etc.). *: ps10 amplifies ISRso3 . **: ps13 amplifies ISRso2s . Second line : Presence and absence of sequences in strain KZR-5 on the basis of PCR and hybridization. : The absence in strain KZR-5 was confirmed by Southern blot analysis using the corresponding PCR products of strain 1609 as DIG labeled DNA probes. B Insertion sequence (IS) regions determined by sequence analysis of PCR products of strains 715 and 1609 that were obtained with primer combinations ps9-F/ps11-R or ps6-F/ps14-R. The position of the sequencing primers (ps9-F, ps6-F, ps11-R, ps14-R and IS2/3) is indicated. Superscript 2 : IS regions correspond to the two IS regions, which are similar, shown in A (IS)

    Techniques Used: Polymerase Chain Reaction, Hybridization, Southern Blot, Labeling, Sequencing

    Detection of a deletion incurred in R. solanacearum biovar 2 strain KZR-5. A Localization of five sequences of strain 715 in the strain 1609 genome. The numbers 52–56 correspond to sequences of clones shown in Table 2 . The spoT-F and spoT-R: indicate primers used for PCR amplification of the relA/spoT 1.6 Kb fragment. Positions 1 and 1,461 of relA/spoT (RSIPO_04909) correspond to positions 238,741 and 237,281 in the genome of strain 1609. B Southern blot analysis of genomic DNA of different R. solanacearum strains after restriction with Pst I using a 1.6 Kb relA/spoT fragment as DNA probe. The R. solanacearum strains used for hybridization are indicated in the figure. Lane P is unlabeled DNA probe
    Figure Legend Snippet: Detection of a deletion incurred in R. solanacearum biovar 2 strain KZR-5. A Localization of five sequences of strain 715 in the strain 1609 genome. The numbers 52–56 correspond to sequences of clones shown in Table 2 . The spoT-F and spoT-R: indicate primers used for PCR amplification of the relA/spoT 1.6 Kb fragment. Positions 1 and 1,461 of relA/spoT (RSIPO_04909) correspond to positions 238,741 and 237,281 in the genome of strain 1609. B Southern blot analysis of genomic DNA of different R. solanacearum strains after restriction with Pst I using a 1.6 Kb relA/spoT fragment as DNA probe. The R. solanacearum strains used for hybridization are indicated in the figure. Lane P is unlabeled DNA probe

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Amplification, Southern Blot, Hybridization

    11) Product Images from "PCR-Based Molecular Diagnosis of Hepatitis Virus (HBV and HDV) in HCV Infected Patients and Their Biochemical Study"

    Article Title: PCR-Based Molecular Diagnosis of Hepatitis Virus (HBV and HDV) in HCV Infected Patients and Their Biochemical Study

    Journal: Journal of Pathogens

    doi: 10.1155/2016/3219793

    A representative 1.5% agarose gel of PCR products for the detection of HBV in HCV positive patients. Lane M: DNA marker, lane 1: positive control (242 bp), lane 2: negative control, and lanes 3 to 7: patients positive for HBV DNA.
    Figure Legend Snippet: A representative 1.5% agarose gel of PCR products for the detection of HBV in HCV positive patients. Lane M: DNA marker, lane 1: positive control (242 bp), lane 2: negative control, and lanes 3 to 7: patients positive for HBV DNA.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker, Positive Control, Negative Control

    12) Product Images from "Metabolic engineering of CHO cells for the development of a robust protein production platform"

    Article Title: Metabolic engineering of CHO cells for the development of a robust protein production platform

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0181455

    Comparative fed batch study of mAb expression in CHO-PYC2 clone 12 and CHO-S control cells. (A) Cell density, ( B) Cell viability, ( C) Lactate profile ( D) Specific productivity and (E) Titer profile.
    Figure Legend Snippet: Comparative fed batch study of mAb expression in CHO-PYC2 clone 12 and CHO-S control cells. (A) Cell density, ( B) Cell viability, ( C) Lactate profile ( D) Specific productivity and (E) Titer profile.

    Techniques Used: Expressing

    Comparative glycoform analysis of mAb secreted from the clone expressing PYC2 gene and a set of control without PYC2 over-expression. Relative abundance of glycan composition of a mAb.
    Figure Legend Snippet: Comparative glycoform analysis of mAb secreted from the clone expressing PYC2 gene and a set of control without PYC2 over-expression. Relative abundance of glycan composition of a mAb.

    Techniques Used: Expressing, Over Expression

    Culture performance of the clone 12 expressing PYC2 and parental CHO cell in culture medium MamPF77, CDM4perMAB and Dynamis in batch mode. Graph representing culture profile: (A and D) Cell density, (B and E) cell viability, (C and F) lactate profile.
    Figure Legend Snippet: Culture performance of the clone 12 expressing PYC2 and parental CHO cell in culture medium MamPF77, CDM4perMAB and Dynamis in batch mode. Graph representing culture profile: (A and D) Cell density, (B and E) cell viability, (C and F) lactate profile.

    Techniques Used: Expressing

    A comparative shake flask fed-batch study of the screened clones expressing PYC2 gene. Graph representing culture profile: (A) Cell density ( B) cell viability ( C) lactate profile ( D) glucose consumption profile for PYC2-expressing cells in shake-flask culture.
    Figure Legend Snippet: A comparative shake flask fed-batch study of the screened clones expressing PYC2 gene. Graph representing culture profile: (A) Cell density ( B) cell viability ( C) lactate profile ( D) glucose consumption profile for PYC2-expressing cells in shake-flask culture.

    Techniques Used: Clone Assay, Expressing

    Culture performance of the clone#12 expressing PYC2 and parental CHO cell in shake flask fed-batch culture. Graph representing culture profile: (A) Cell density,( B) cell viability, ( C) Glutamine ( D) lactate and ( E) glucose consumption profiles.
    Figure Legend Snippet: Culture performance of the clone#12 expressing PYC2 and parental CHO cell in shake flask fed-batch culture. Graph representing culture profile: (A) Cell density,( B) cell viability, ( C) Glutamine ( D) lactate and ( E) glucose consumption profiles.

    Techniques Used: Expressing

    Cell pool generation and cell line development for PYC2 engineering. (A-D) Schematic diagram of two-phase selection of pMPYC transfected CHO cells with varying concentration of MTX and puromycin. ( B-D) Cell density, viability and lactate production profiles for stable pool 1B, pool 1C and pool 2D in fed-batch culture mode as a function of time. (E-F ) Flow diagram depicts the steps followed to obtain a clonal CHO cell line from the heterogeneous population of a stable pool. Flow-cytometry used to gate the healthy cells population and sort the single CHO cell in a 384 well plate. Live cell imager is used to monitor the clonality and growth of the colony/cell in the consecutive days. Single clones are expanded from 384 well plates to T25 flask.
    Figure Legend Snippet: Cell pool generation and cell line development for PYC2 engineering. (A-D) Schematic diagram of two-phase selection of pMPYC transfected CHO cells with varying concentration of MTX and puromycin. ( B-D) Cell density, viability and lactate production profiles for stable pool 1B, pool 1C and pool 2D in fed-batch culture mode as a function of time. (E-F ) Flow diagram depicts the steps followed to obtain a clonal CHO cell line from the heterogeneous population of a stable pool. Flow-cytometry used to gate the healthy cells population and sort the single CHO cell in a 384 well plate. Live cell imager is used to monitor the clonality and growth of the colony/cell in the consecutive days. Single clones are expanded from 384 well plates to T25 flask.

    Techniques Used: Selection, Transfection, Concentration Assay, Flow Cytometry, Cytometry, Clone Assay

    Gene copy number and mRNA expression analysis of the PYC2 expressing clones. (A) Fold expression of the PYC2 m-RNA relative to the house keeping β-actin gene (cDNA used as a template). ( B) Calculation of gene copies relative to house-keeping β-actin gene for selected PYC2 clone#12 (genomic DNA used as a template).
    Figure Legend Snippet: Gene copy number and mRNA expression analysis of the PYC2 expressing clones. (A) Fold expression of the PYC2 m-RNA relative to the house keeping β-actin gene (cDNA used as a template). ( B) Calculation of gene copies relative to house-keeping β-actin gene for selected PYC2 clone#12 (genomic DNA used as a template).

    Techniques Used: Expressing

    13) Product Images from "De novo transcriptome assembly: a new laccase multigene family from the marine-derived basidiomycete Peniophora sp. CBMAI 1063"

    Article Title: De novo transcriptome assembly: a new laccase multigene family from the marine-derived basidiomycete Peniophora sp. CBMAI 1063

    Journal: AMB Express

    doi: 10.1186/s13568-017-0526-7

    Predicted amino acid sequence alignments of all 10 putative laccases from  Peniophora  sp. CBMAI 1063. Amino acids with 100% matches are highlighted in black. Numbers above the amino acids indicate that they are copper ion-bound. Dots below the amino acids indicate conserved regions in the fungal laccases (L1, L2, L3 and L4)
    Figure Legend Snippet: Predicted amino acid sequence alignments of all 10 putative laccases from Peniophora sp. CBMAI 1063. Amino acids with 100% matches are highlighted in black. Numbers above the amino acids indicate that they are copper ion-bound. Dots below the amino acids indicate conserved regions in the fungal laccases (L1, L2, L3 and L4)

    Techniques Used: Sequencing

    14) Product Images from "Ultra-sensitive chemiluminescence imaging DNA hybridization method in the detection of mosquito-borne viruses and parasites"

    Article Title: Ultra-sensitive chemiluminescence imaging DNA hybridization method in the detection of mosquito-borne viruses and parasites

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-017-1975-1

    Determination of in vitro transcribed RNAs and VLPs by agarose gel electrophoresis. a Agarose gel electrophoresis of in vitro transcribed RNAs. The size of the in vitro transcribed RNAs were marked above each lane and compared with a RNA marker (Thermo Fisher). b RNase A and DNase I digestion of VLPs. Lane 1: PET-MS2 (RNase A); Lane 2: PET-MS2 (DNase I); Lane 3: PET-MS2 (RNase A + DNase I); Lane 4: PET-MS2; Lane 5: YFV VLP(RNase A); Lane 6: YFV VLP (DNase I); Lane 7: YFV VLP (RNase A + DNase I); Lane 8: YFV VLP; Lane 9: EEEV VLP (RNase A); Lane 10: EEEV VLP (DNase I); Lane 11: EEEV VLP (RNase A + DNase I); Lane 12: EEEV VLP. The size of VLPs are compared to a DNA marker (TaKaRa). The nucleic acids between 1,000–2,000 bp are resistant to both RNase A and DNase I due to their packaging in the internal section of the VLPs
    Figure Legend Snippet: Determination of in vitro transcribed RNAs and VLPs by agarose gel electrophoresis. a Agarose gel electrophoresis of in vitro transcribed RNAs. The size of the in vitro transcribed RNAs were marked above each lane and compared with a RNA marker (Thermo Fisher). b RNase A and DNase I digestion of VLPs. Lane 1: PET-MS2 (RNase A); Lane 2: PET-MS2 (DNase I); Lane 3: PET-MS2 (RNase A + DNase I); Lane 4: PET-MS2; Lane 5: YFV VLP(RNase A); Lane 6: YFV VLP (DNase I); Lane 7: YFV VLP (RNase A + DNase I); Lane 8: YFV VLP; Lane 9: EEEV VLP (RNase A); Lane 10: EEEV VLP (DNase I); Lane 11: EEEV VLP (RNase A + DNase I); Lane 12: EEEV VLP. The size of VLPs are compared to a DNA marker (TaKaRa). The nucleic acids between 1,000–2,000 bp are resistant to both RNase A and DNase I due to their packaging in the internal section of the VLPs

    Techniques Used: In Vitro, Agarose Gel Electrophoresis, Marker, Positron Emission Tomography

    15) Product Images from "Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize"

    Article Title: Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize

    Journal: Plant Methods

    doi: 10.1186/s13007-016-0152-4

    Agarose gel electrophoresis showing genomic DNA preparation of two Z. mays hybrids M10 ( lanes 1 – 4 ) and M321 ( lanes 5 – 8 ). DNA extractions using the Mericon extraction method with different agarose concentrations, 1% ( a ), 1.5% ( b ) and 2% g agarose ( c ), lane− empty, lane+ positive Probe NTC. M A: λ DNA- HindIII marker, M B and C: one Kb Marker
    Figure Legend Snippet: Agarose gel electrophoresis showing genomic DNA preparation of two Z. mays hybrids M10 ( lanes 1 – 4 ) and M321 ( lanes 5 – 8 ). DNA extractions using the Mericon extraction method with different agarose concentrations, 1% ( a ), 1.5% ( b ) and 2% g agarose ( c ), lane− empty, lane+ positive Probe NTC. M A: λ DNA- HindIII marker, M B and C: one Kb Marker

    Techniques Used: Agarose Gel Electrophoresis, Marker

    Nano-Drop measurement profile of genomic DNA extractions from Z. mays . DNA extractions using a Qiagen extraction method. Probe = Sample
    Figure Legend Snippet: Nano-Drop measurement profile of genomic DNA extractions from Z. mays . DNA extractions using a Qiagen extraction method. Probe = Sample

    Techniques Used:

    Nano-Drop measurement profile of genomic DNA extractions from Z. mays . DNA extractions using Mericon extraction method. Probe = Sample
    Figure Legend Snippet: Nano-Drop measurement profile of genomic DNA extractions from Z. mays . DNA extractions using Mericon extraction method. Probe = Sample

    Techniques Used:

    16) Product Images from "Goatpox outbreak at a high altitude goat farm of Mizoram: possibility of wild life spill over to domestic goat population"

    Article Title: Goatpox outbreak at a high altitude goat farm of Mizoram: possibility of wild life spill over to domestic goat population

    Journal: VirusDisease

    doi: 10.1007/s13337-018-0482-0

    Phylogenetic tree constructed based on the based on the P32 gene sequences of Capripox virus. Red colour bullet indicate the virus isolated in the present study and Blue coloured bullets indicated the goatpox viruses reported from India earlier (colour figure online)
    Figure Legend Snippet: Phylogenetic tree constructed based on the based on the P32 gene sequences of Capripox virus. Red colour bullet indicate the virus isolated in the present study and Blue coloured bullets indicated the goatpox viruses reported from India earlier (colour figure online)

    Techniques Used: Construct, Isolation

    17) Product Images from "Separating DNA with different topologies by atomic force microscopy in comparison with gel electrophoresis"

    Article Title: Separating DNA with different topologies by atomic force microscopy in comparison with gel electrophoresis

    Journal: The journal of physical chemistry. B

    doi: 10.1021/jp105603k

    (A) An AFM image shows the mixture of supercoiled and linear pUC18 DNA with the same weight ratio. Scan size is 1 × 1 μm 2 and color codes are the same as in Figure 1. (B) A photograph of the agarose gel shows the separated supercoiled
    Figure Legend Snippet: (A) An AFM image shows the mixture of supercoiled and linear pUC18 DNA with the same weight ratio. Scan size is 1 × 1 μm 2 and color codes are the same as in Figure 1. (B) A photograph of the agarose gel shows the separated supercoiled

    Techniques Used: Agarose Gel Electrophoresis

    (A) A photograph of the agarose gel shows separated supercoiled and relaxed pUC18 bands after supercoiled pUC18 DNA was subjected 1.5 MJ/m 2 UVA radiation and T4 Endonuclease V treatment. (B and C) are AFM images of pUC18 DNAs that were extracted from
    Figure Legend Snippet: (A) A photograph of the agarose gel shows separated supercoiled and relaxed pUC18 bands after supercoiled pUC18 DNA was subjected 1.5 MJ/m 2 UVA radiation and T4 Endonuclease V treatment. (B and C) are AFM images of pUC18 DNAs that were extracted from

    Techniques Used: Agarose Gel Electrophoresis

    18) Product Images from "Epigenetic transcriptional repression of cellular genes by a viral SET protein"

    Article Title: Epigenetic transcriptional repression of cellular genes by a viral SET protein

    Journal: Nature cell biology

    doi:

    Presence of vSET in PBCV-1 virions
    Figure Legend Snippet: Presence of vSET in PBCV-1 virions

    Techniques Used:

    19) Product Images from "Nuclear import of cutaneous beta genus HPV8 E7 oncoprotein is mediated by hydrophobic interactions between its zinc-binding domain and FG nucleoporins"

    Article Title: Nuclear import of cutaneous beta genus HPV8 E7 oncoprotein is mediated by hydrophobic interactions between its zinc-binding domain and FG nucleoporins

    Journal: Virology

    doi: 10.1016/j.virol.2013.11.020

    Kap β2 in excess competes with GST-8E7 for nuclear import Digitonin-permeabilized HeLa cells were incubated with GST-8E7 (panels A and C), M9-GST (panels E and G) or GST (panels I and K) in the presence of HeLa cytosol, or Hela cytosol plus Kap β2 in excess. Panels A, C, E, G, I and K show the protein localization and panels B, D, F, H, J and L the DAPI staining of the nuclei. Note the nuclear import of GST-8E7 in panel A and its inhibition in panel C.
    Figure Legend Snippet: Kap β2 in excess competes with GST-8E7 for nuclear import Digitonin-permeabilized HeLa cells were incubated with GST-8E7 (panels A and C), M9-GST (panels E and G) or GST (panels I and K) in the presence of HeLa cytosol, or Hela cytosol plus Kap β2 in excess. Panels A, C, E, G, I and K show the protein localization and panels B, D, F, H, J and L the DAPI staining of the nuclei. Note the nuclear import of GST-8E7 in panel A and its inhibition in panel C.

    Techniques Used: Incubation, Staining, Inhibition

    An antibody to Nup62 FG repeats domain inhibits the nuclear import of HPV8 E7 Digitonin-permeabilized HeLa cells were incubated with GST-8E7 in the presence of only transport buffer (panel A), or HeLa cytosol (panel B) or HeLa cytosol plus anti-Nup62 antibody (panel C), or with GST in the presence of HeLa cytosol (panel D). Note the nuclear import of GST-8E7 in panel B and its inhibition in panel C. The negative control GST was not imported into the nucleus in the presence of HeLa cytosol (panel D). Panels E-H represent the DAPI staining of the nuclei.
    Figure Legend Snippet: An antibody to Nup62 FG repeats domain inhibits the nuclear import of HPV8 E7 Digitonin-permeabilized HeLa cells were incubated with GST-8E7 in the presence of only transport buffer (panel A), or HeLa cytosol (panel B) or HeLa cytosol plus anti-Nup62 antibody (panel C), or with GST in the presence of HeLa cytosol (panel D). Note the nuclear import of GST-8E7 in panel B and its inhibition in panel C. The negative control GST was not imported into the nucleus in the presence of HeLa cytosol (panel D). Panels E-H represent the DAPI staining of the nuclei.

    Techniques Used: Incubation, Inhibition, Negative Control, Staining

    Mutation of hydrophobic residues within the zinc-binding domain disrupts the nuclear import of HPV8 E7 oncoprotein Digitonin-permeabilized HeLa cells were incubated with GST-8E7 (panels A and C), GST-8E7 LRLFV65AAAAA (panels E and G), GST-8E7 R66A (panels I and K) and GST (panels M and O), in the presence of only transport buffer (panels A, E, I, and M) or HeLa cytosol and energy mix (panels C, G, K and O). Panels A, C, E, G, I, K, M and O show the protein localization and panels B, D, F, H, J, L, N and P the DAPI staining of the nuclei.
    Figure Legend Snippet: Mutation of hydrophobic residues within the zinc-binding domain disrupts the nuclear import of HPV8 E7 oncoprotein Digitonin-permeabilized HeLa cells were incubated with GST-8E7 (panels A and C), GST-8E7 LRLFV65AAAAA (panels E and G), GST-8E7 R66A (panels I and K) and GST (panels M and O), in the presence of only transport buffer (panels A, E, I, and M) or HeLa cytosol and energy mix (panels C, G, K and O). Panels A, C, E, G, I, K, M and O show the protein localization and panels B, D, F, H, J, L, N and P the DAPI staining of the nuclei.

    Techniques Used: Mutagenesis, Binding Assay, Incubation, Staining

    GST-8E7 and GST-8cE7 are imported into the nuclei of digitonin-permeabilized HeLa cells Digitonin-permeabilized HeLa cells were incubated with GST-8E7 (panels A and C), GST-8nE7 (panels E and G), GST-8cE7 (panels I and K), M9-GST (panels M and O) and GST (panels Q and S) in the presence of either transport buffer (A, E, I, M, and Q) or exogenous HeLa cytosol (panels C, G, K, O and S). Protein localization was detected with an anti-GST antibody. Panels A, C, E, G, I, K, M, O, Q and S show the protein localization and panels B, D, F, H, J, L, N, P, R and T the DAPI staining of the nuclei.
    Figure Legend Snippet: GST-8E7 and GST-8cE7 are imported into the nuclei of digitonin-permeabilized HeLa cells Digitonin-permeabilized HeLa cells were incubated with GST-8E7 (panels A and C), GST-8nE7 (panels E and G), GST-8cE7 (panels I and K), M9-GST (panels M and O) and GST (panels Q and S) in the presence of either transport buffer (A, E, I, M, and Q) or exogenous HeLa cytosol (panels C, G, K, O and S). Protein localization was detected with an anti-GST antibody. Panels A, C, E, G, I, K, M, O, Q and S show the protein localization and panels B, D, F, H, J, L, N, P, R and T the DAPI staining of the nuclei.

    Techniques Used: Incubation, Staining

    The C91A mutation disrupts the nuclear import of GST-8E7 Digitonin-permeabilized HeLa cells were incubated with GST-8E7 (panels A and C), GST-8E7 C91A (panels E and G), M9-GST (panels I and K) and GST (panels M and O) in the presence of either transport buffer (panels A, E, I and M) or exogenous HeLa cytosol (panels C, G, K and O). Protein localization was detected with an anti-GST antibody. Panels A, C, E, G, I, K, M and O show the protein localization and panels B, D, F, H, J, L, N and P the DAPI staining of the nuclei.
    Figure Legend Snippet: The C91A mutation disrupts the nuclear import of GST-8E7 Digitonin-permeabilized HeLa cells were incubated with GST-8E7 (panels A and C), GST-8E7 C91A (panels E and G), M9-GST (panels I and K) and GST (panels M and O) in the presence of either transport buffer (panels A, E, I and M) or exogenous HeLa cytosol (panels C, G, K and O). Protein localization was detected with an anti-GST antibody. Panels A, C, E, G, I, K, M and O show the protein localization and panels B, D, F, H, J, L, N and P the DAPI staining of the nuclei.

    Techniques Used: Mutagenesis, Incubation, Staining

    20) Product Images from "CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci"

    Article Title: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky501

    CRISPR/Cas9-mediated knock-in of an optimized TetO repeat using linear donor DNA with short homology arms (HAs). ( A ) Schematic of the strategy to create linear donor DNA with short HAs via PCR. The final plasmid used as a template is shown together with the primers used for amplification of the linear donor (black arrows). Blasticidin resistance ( BlaR ) gene under EFS promoter is for positive selection. Selection cassette is flanked with loxP sites. ( B ) Schematic of the complete initial cassette containing three TetO repeats (green), four random 10 bp (light blue) sequences, three restriction sites StyI, SpeI and BamHI (all of them are in red), and primer binding sites (dark blue) used for synthesis of the complementary strand and amplification of the product. ( C ) Schematic of the insertion of 3-mer TetO cassette into pSP2 vector through StyI and BamHI sites. Each TetO sequence is indicated with a green box. ( D ) Schematic of the assembly of the TetO repeat multimers. StyI/SpeI site, created by insertion of the TetO cassette, cannot be cut by either of these two enzymes. ( E ) Results of the PCRs to amplify 48-mer and 96-mer TetO repeat donors for the HSP70 locus. The expected band sizes were around 3 kb for the 48-mer and around 4.6 kb for the 96-mer TetO donor. NEB 1 kb ladder (M) was used. ( F ) Schematic of the knock-in strategy. HAs are shown in purple. sgRNA target site is indicated with a red triangle. DSB: double strand break.
    Figure Legend Snippet: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat using linear donor DNA with short homology arms (HAs). ( A ) Schematic of the strategy to create linear donor DNA with short HAs via PCR. The final plasmid used as a template is shown together with the primers used for amplification of the linear donor (black arrows). Blasticidin resistance ( BlaR ) gene under EFS promoter is for positive selection. Selection cassette is flanked with loxP sites. ( B ) Schematic of the complete initial cassette containing three TetO repeats (green), four random 10 bp (light blue) sequences, three restriction sites StyI, SpeI and BamHI (all of them are in red), and primer binding sites (dark blue) used for synthesis of the complementary strand and amplification of the product. ( C ) Schematic of the insertion of 3-mer TetO cassette into pSP2 vector through StyI and BamHI sites. Each TetO sequence is indicated with a green box. ( D ) Schematic of the assembly of the TetO repeat multimers. StyI/SpeI site, created by insertion of the TetO cassette, cannot be cut by either of these two enzymes. ( E ) Results of the PCRs to amplify 48-mer and 96-mer TetO repeat donors for the HSP70 locus. The expected band sizes were around 3 kb for the 48-mer and around 4.6 kb for the 96-mer TetO donor. NEB 1 kb ladder (M) was used. ( F ) Schematic of the knock-in strategy. HAs are shown in purple. sgRNA target site is indicated with a red triangle. DSB: double strand break.

    Techniques Used: CRISPR, Knock-In, Polymerase Chain Reaction, Plasmid Preparation, Amplification, Selection, Binding Assay, Sequencing

    21) Product Images from "Improved reverse transcription-polymerase chain reaction assay for the detection of flaviviruses with semi-nested primers for discrimination between dengue virus serotypes and Zika virus"

    Article Title: Improved reverse transcription-polymerase chain reaction assay for the detection of flaviviruses with semi-nested primers for discrimination between dengue virus serotypes and Zika virus

    Journal: Memórias do Instituto Oswaldo Cruz

    doi: 10.1590/0074-02760170393

    : evaluation of the semi-nested polymerase chain reaction (PCR) for identification of dengue virus (DENV)1-4 serotypes and Zika virus (ZIKV). The efficiency of the semi-nested reaction using the primers DENV1F6.2, DENV2F10, DENV3F6.1, DENV4F3, ZIKVF8, and CRNS5_7NR6 was evaluated under different conditions. (A) Size resolution of each amplicon in a 2% agarose gel. The letters above each lane indicate the templates containing RNA of DENV serotypes (D1, D2, D3, and D4) or ZIKV (Z). The results of semi-nested reactions containing each primer individually and a template positive for Chikungunya virus are presented (below). (B) Tests using a mixture of reverse-transcribed RNAs as the template from two types of viruses. All possible combinations among the five viruses have been evaluated and are shown above each their respective gel lanes. The size of each amplicon in the 2% agarose gel are indicated (black arrows). (C) Dilution test to verify the sensitivity of the semi-nested reaction. L = 100 bp size marker.
    Figure Legend Snippet: : evaluation of the semi-nested polymerase chain reaction (PCR) for identification of dengue virus (DENV)1-4 serotypes and Zika virus (ZIKV). The efficiency of the semi-nested reaction using the primers DENV1F6.2, DENV2F10, DENV3F6.1, DENV4F3, ZIKVF8, and CRNS5_7NR6 was evaluated under different conditions. (A) Size resolution of each amplicon in a 2% agarose gel. The letters above each lane indicate the templates containing RNA of DENV serotypes (D1, D2, D3, and D4) or ZIKV (Z). The results of semi-nested reactions containing each primer individually and a template positive for Chikungunya virus are presented (below). (B) Tests using a mixture of reverse-transcribed RNAs as the template from two types of viruses. All possible combinations among the five viruses have been evaluated and are shown above each their respective gel lanes. The size of each amplicon in the 2% agarose gel are indicated (black arrows). (C) Dilution test to verify the sensitivity of the semi-nested reaction. L = 100 bp size marker.

    Techniques Used: Nested PCR, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Marker

    22) Product Images from "TCTN2: a novel tumor marker with oncogenic properties"

    Article Title: TCTN2: a novel tumor marker with oncogenic properties

    Journal: Oncotarget

    doi: 10.18632/oncotarget.20438

    TCTN2 is expressed in cancer cell lines and localizes in the inner side of plasma membrane (A) Total proteins were extracted from breast (SKBR3 and MCF7), lung (H226 and HOP92), colon (HCT15, HT29 and Colo205) and ovarian (SKOV3 and OVCAR8) cancer cell lines. Proteins were resolved by SDS-polyacrylamide gel electrophoresis and analyzed by Western blot with anti-TCTN2 monoclonal antibody. Anti-β-Actin monoclonal antibody was used as a loading control. (B) TCTN2 was silenced in HOP92, OVCAR8 and HCT15 cancer cell lines with four commercially available (QIAGEN) TCTN2-specific siRNAs at 10 nM concentration or irrelevant siRNA (AllStars Negative Control siRNA, QIAGEN) using the Hi-Perfect transfection reagent (QIAGEN). TCTN2 expression was assayed 48 hours later by Western blot. Anti-β-Actin monoclonal antibody was used as a loading control. (C) TCTN2 localizes in the inner side of plasma membrane in cancer cells lines. HCT15, HOP92 and HT29 cells were analyzed by confocal microscopy. Cells were incubated with the anti-TCTN2 monoclonal antibody, with (lower panels) or without (upper panels) permeabilization pre-treatment with 0.01% BriJ96 ® . Cells were subsequently stained with Alexafluor 488-labeled goat anti-mouse antibodies to detect TCTN2 (green) and DAPI to visualize nuclei (blue).
    Figure Legend Snippet: TCTN2 is expressed in cancer cell lines and localizes in the inner side of plasma membrane (A) Total proteins were extracted from breast (SKBR3 and MCF7), lung (H226 and HOP92), colon (HCT15, HT29 and Colo205) and ovarian (SKOV3 and OVCAR8) cancer cell lines. Proteins were resolved by SDS-polyacrylamide gel electrophoresis and analyzed by Western blot with anti-TCTN2 monoclonal antibody. Anti-β-Actin monoclonal antibody was used as a loading control. (B) TCTN2 was silenced in HOP92, OVCAR8 and HCT15 cancer cell lines with four commercially available (QIAGEN) TCTN2-specific siRNAs at 10 nM concentration or irrelevant siRNA (AllStars Negative Control siRNA, QIAGEN) using the Hi-Perfect transfection reagent (QIAGEN). TCTN2 expression was assayed 48 hours later by Western blot. Anti-β-Actin monoclonal antibody was used as a loading control. (C) TCTN2 localizes in the inner side of plasma membrane in cancer cells lines. HCT15, HOP92 and HT29 cells were analyzed by confocal microscopy. Cells were incubated with the anti-TCTN2 monoclonal antibody, with (lower panels) or without (upper panels) permeabilization pre-treatment with 0.01% BriJ96 ® . Cells were subsequently stained with Alexafluor 488-labeled goat anti-mouse antibodies to detect TCTN2 (green) and DAPI to visualize nuclei (blue).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Western Blot, Concentration Assay, Negative Control, Transfection, Expressing, Confocal Microscopy, Incubation, Staining, Labeling

    TCTN2 is overexpressed in primary tumors of colon lung and ovary (A) TCTN2 is overexpressed in tumor samples and localizes at the cell membrane. Cancerous and matched normal samples from colon, lung and ovary were stained with the anti-TCTN2 monoclonal antibody, arrayed in parallel on the same TMA slides and analyzed simultaneously. (B) Specific recognition of TCTN2 antibodies - TCTN2 expressed in mammalian cells is a glycosylated protein. Western blot on total protein extracts from HeLa cells transfected with the empty plasmid or full length TCTN2, or recombinant TCTN2 domain (amino acids 171-444) expressed in E. coli, using the polyclonal (pAb291-YOM, left panel) and monoclonal antibody (anti-TCTN2 mAb, right panel). The recognition of the mAb anti TCTN2 was also assessed after treatment with Peptide-N-Glycosidase F (PNGaseF).
    Figure Legend Snippet: TCTN2 is overexpressed in primary tumors of colon lung and ovary (A) TCTN2 is overexpressed in tumor samples and localizes at the cell membrane. Cancerous and matched normal samples from colon, lung and ovary were stained with the anti-TCTN2 monoclonal antibody, arrayed in parallel on the same TMA slides and analyzed simultaneously. (B) Specific recognition of TCTN2 antibodies - TCTN2 expressed in mammalian cells is a glycosylated protein. Western blot on total protein extracts from HeLa cells transfected with the empty plasmid or full length TCTN2, or recombinant TCTN2 domain (amino acids 171-444) expressed in E. coli, using the polyclonal (pAb291-YOM, left panel) and monoclonal antibody (anti-TCTN2 mAb, right panel). The recognition of the mAb anti TCTN2 was also assessed after treatment with Peptide-N-Glycosidase F (PNGaseF).

    Techniques Used: Staining, Western Blot, Transfection, Plasmid Preparation, Recombinant

    TCTN2 downregulation, by different platforms, alters growth phenotypes in four cell lines (A) Representation of TCTN2 gene promoter and first exon, transcription start site (TSS); A and B mark the region targeted. (B) TCTN2 mRNA downregulation using CRISPR-dCas9 sgRNA A and B regions alone. (C) TCTN2 mRNA downregulation using ZF A and B regions fused to a transcriptional repressor (SKD). (D) Apoptosis was measured using the 1, 1′,3,3,3′,3′-Hexamethylindodicarbocyanine iodide (DilC) assay in cells treated with different ZFs after TCTN2 downregulation. (E) Colony-forming assay in cells after TCTN2 downregulation. (F) Visual representation of the colony-forming assay from HT-29 cells. Data represent the mean value of two independent experiments, run in triplicate, and are shown as mean ± s.e.m. Statistical significance was assessed by non paired two-tailed Student's t-test ( * P
    Figure Legend Snippet: TCTN2 downregulation, by different platforms, alters growth phenotypes in four cell lines (A) Representation of TCTN2 gene promoter and first exon, transcription start site (TSS); A and B mark the region targeted. (B) TCTN2 mRNA downregulation using CRISPR-dCas9 sgRNA A and B regions alone. (C) TCTN2 mRNA downregulation using ZF A and B regions fused to a transcriptional repressor (SKD). (D) Apoptosis was measured using the 1, 1′,3,3,3′,3′-Hexamethylindodicarbocyanine iodide (DilC) assay in cells treated with different ZFs after TCTN2 downregulation. (E) Colony-forming assay in cells after TCTN2 downregulation. (F) Visual representation of the colony-forming assay from HT-29 cells. Data represent the mean value of two independent experiments, run in triplicate, and are shown as mean ± s.e.m. Statistical significance was assessed by non paired two-tailed Student's t-test ( * P

    Techniques Used: CRISPR, Two Tailed Test

    TCTN2 act as an oncogene: evidences collected by means of epigenetic editing and overexpression (A) Stable inducible HT-29 cells were generated and TCTN2 downregulation was addressed over time, after short-term induction of ZFP fusions. (B) Boyden invasiveness chamber assay in cells after TCTN2 downregulation induced by Dox addition. Cells migrated towards the lower surface of the chamber filters were fixed and counted after Diff-Quick staining. Images of the visual counting of each sample are reported below the graphs. (C) Colony forming assay in cells after TCTN2 downregulation induced by Dox. Lower panel: visual representation of the colony-forming assay from HT-29 cells without and with Dox treatment. (D) Anchorage-independent growth of HCT15 cells after transfection with a plasmid encoding full-length TCTN2. Data represent the mean value of two independent experiments, run in triplicate, and are shown as mean ± s.e.m Statistical significance was assessed by nonpaired two-tailed Student's t-test, ( * P
    Figure Legend Snippet: TCTN2 act as an oncogene: evidences collected by means of epigenetic editing and overexpression (A) Stable inducible HT-29 cells were generated and TCTN2 downregulation was addressed over time, after short-term induction of ZFP fusions. (B) Boyden invasiveness chamber assay in cells after TCTN2 downregulation induced by Dox addition. Cells migrated towards the lower surface of the chamber filters were fixed and counted after Diff-Quick staining. Images of the visual counting of each sample are reported below the graphs. (C) Colony forming assay in cells after TCTN2 downregulation induced by Dox. Lower panel: visual representation of the colony-forming assay from HT-29 cells without and with Dox treatment. (D) Anchorage-independent growth of HCT15 cells after transfection with a plasmid encoding full-length TCTN2. Data represent the mean value of two independent experiments, run in triplicate, and are shown as mean ± s.e.m Statistical significance was assessed by nonpaired two-tailed Student's t-test, ( * P

    Techniques Used: Activated Clotting Time Assay, Over Expression, Generated, Boyden Chamber Assay, Diff-Quik, Staining, Transfection, Plasmid Preparation, Two Tailed Test

    23) Product Images from "Gene Capture by Helitron Transposons Reshuffles the Transcriptome of Maize"

    Article Title: Gene Capture by Helitron Transposons Reshuffles the Transcriptome of Maize

    Journal: Genetics

    doi: 10.1534/genetics.111.136176

    Genomic and RT–PCR analysis of Helitron Hel1-331 . (A) PCR product amplified from genomic DNA extracted from different maize inbred lines using primers, H31-1F and H31-1R, flanking the 5′ and 3′ sequence of the Helitron insertion, respectively. (B) RT–PCR products amplified from root and shoot tissues of maize inbred lines B73 and Mo17 using primers, H31E1F and H31E7R. (C) Splice alignment of the sequences of the RT–PCR products shown in B with the Helitron Hel1-331 sequence. The exons of a captured hypothetical gene, gi: 212721678, and an uncharacterized gene, are color coded in orange and yellow, respectively. In the alignment, boxes and lines denote exons and introns, respectively. Alternative donor and acceptor splice sites are joined by dashed lines and * marks the position of the retained introns. The size of the transcripts and the A and T nucleotides flanking the insertion site of the Helitron are indicated.
    Figure Legend Snippet: Genomic and RT–PCR analysis of Helitron Hel1-331 . (A) PCR product amplified from genomic DNA extracted from different maize inbred lines using primers, H31-1F and H31-1R, flanking the 5′ and 3′ sequence of the Helitron insertion, respectively. (B) RT–PCR products amplified from root and shoot tissues of maize inbred lines B73 and Mo17 using primers, H31E1F and H31E7R. (C) Splice alignment of the sequences of the RT–PCR products shown in B with the Helitron Hel1-331 sequence. The exons of a captured hypothetical gene, gi: 212721678, and an uncharacterized gene, are color coded in orange and yellow, respectively. In the alignment, boxes and lines denote exons and introns, respectively. Alternative donor and acceptor splice sites are joined by dashed lines and * marks the position of the retained introns. The size of the transcripts and the A and T nucleotides flanking the insertion site of the Helitron are indicated.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Sequencing

    24) Product Images from "Development and comprehensive characterization of porcine hepatocellular carcinoma for translational liver cancer investigation"

    Article Title: Development and comprehensive characterization of porcine hepatocellular carcinoma for translational liver cancer investigation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.27647

    Oncopig intrahepatic HCC tumor formation. ( A ) Liver ultrasound depicting a hypoechoic 1 cm round intrahepatic HCC tumor (circled, L = liver, GB = gallbladder). ( B ) Contrast enhanced liver CT depicts same HCC tumor (circled). ( C ) Photograph of transected intrahepatic HCC tumor. ( D ) H E (20×) of Oncopig intrahepatic HCC tumor reveals architectural distortion characterized by expansion of liver cords, nuclear pleomorphism, anisonucleosis, and nodular fibrosis. Masson’s trichrome of adjacent non-tumorous liver demonstrates dense collagen bands (arrows) consistent with METAVIR grade 2-3 fibrosis. ( E ) Arginase-1 IHC (20×) shows patchy arginase-1 expression (brown) consistent with hepatocellular differentiation. KRAS G12D IHC (20×) confirms KRAS G12D expression (brown) consistent with malignancy.
    Figure Legend Snippet: Oncopig intrahepatic HCC tumor formation. ( A ) Liver ultrasound depicting a hypoechoic 1 cm round intrahepatic HCC tumor (circled, L = liver, GB = gallbladder). ( B ) Contrast enhanced liver CT depicts same HCC tumor (circled). ( C ) Photograph of transected intrahepatic HCC tumor. ( D ) H E (20×) of Oncopig intrahepatic HCC tumor reveals architectural distortion characterized by expansion of liver cords, nuclear pleomorphism, anisonucleosis, and nodular fibrosis. Masson’s trichrome of adjacent non-tumorous liver demonstrates dense collagen bands (arrows) consistent with METAVIR grade 2-3 fibrosis. ( E ) Arginase-1 IHC (20×) shows patchy arginase-1 expression (brown) consistent with hepatocellular differentiation. KRAS G12D IHC (20×) confirms KRAS G12D expression (brown) consistent with malignancy.

    Techniques Used: Immunohistochemistry, Expressing

    CRISPR/Cas9-mediated disruption of Oncopig KRAS G12D and TP53 R167H transgenes. ( A ) Schematic representation of the Oncopig transgene showing gRNA target sites and primers used for PCR. IRES, Internal ribosome entry site. ( B ) KRAS G12D and TP53 R167H editing efficiencies at multiple time points post transfection with Cas9 and gRNAs. ( C ) Frameshift mutations resulting in protein truncation for 2 Oncopig TP53 R167H KO HCC cell lines developed via single cell clone isolation and screening. Dashed line marks the cleavage position, and dashed grey boxes represent nucleotide deletions. Dotted regions represent frameshifts in predicted protein sequences. ( D ) Positive arginase-1 staining (brown) of parental and TP53 R167H KO cell lines (scale bar, 300 μm). ( E ) Cellular proliferation of Oncopig parental and TP53 R167H KO HCC cell lines. Values represent mean ± S. D. ( n ≥ 3). ** indicates P
    Figure Legend Snippet: CRISPR/Cas9-mediated disruption of Oncopig KRAS G12D and TP53 R167H transgenes. ( A ) Schematic representation of the Oncopig transgene showing gRNA target sites and primers used for PCR. IRES, Internal ribosome entry site. ( B ) KRAS G12D and TP53 R167H editing efficiencies at multiple time points post transfection with Cas9 and gRNAs. ( C ) Frameshift mutations resulting in protein truncation for 2 Oncopig TP53 R167H KO HCC cell lines developed via single cell clone isolation and screening. Dashed line marks the cleavage position, and dashed grey boxes represent nucleotide deletions. Dotted regions represent frameshifts in predicted protein sequences. ( D ) Positive arginase-1 staining (brown) of parental and TP53 R167H KO cell lines (scale bar, 300 μm). ( E ) Cellular proliferation of Oncopig parental and TP53 R167H KO HCC cell lines. Values represent mean ± S. D. ( n ≥ 3). ** indicates P

    Techniques Used: CRISPR, Polymerase Chain Reaction, Transfection, Isolation, Staining

    Oncopig, human, and murine HCC in vitro chemotherapeutic susceptibility. ( A ) Gene expression levels in Oncopig ( n = 3 cell lines) and human HCC cells (HepG2, Huh7, and Hep3B). ( B – F ) Correlation analysis of logIC 50 values demonstrating more similar in vitro chemotherapeutic responses between Oncopig and human compared to murine Hepa1-6 and human HCC cells. Chemotherapeutic response of each HCC cell line towards sorafenib, doxorubicin, cisplatin, mitomycin C, and 5-FU was determined. Pearson correlation between logIC 50 in Oncopig HCC cells or murine Hepa1-6 cells and the following human HCC cells was analyzed: (B) HepG2, (C) Hep3B, (D) Huh7, (E) SNU-387, and (F) SNU-475. * denotes P
    Figure Legend Snippet: Oncopig, human, and murine HCC in vitro chemotherapeutic susceptibility. ( A ) Gene expression levels in Oncopig ( n = 3 cell lines) and human HCC cells (HepG2, Huh7, and Hep3B). ( B – F ) Correlation analysis of logIC 50 values demonstrating more similar in vitro chemotherapeutic responses between Oncopig and human compared to murine Hepa1-6 and human HCC cells. Chemotherapeutic response of each HCC cell line towards sorafenib, doxorubicin, cisplatin, mitomycin C, and 5-FU was determined. Pearson correlation between logIC 50 in Oncopig HCC cells or murine Hepa1-6 cells and the following human HCC cells was analyzed: (B) HepG2, (C) Hep3B, (D) Huh7, (E) SNU-387, and (F) SNU-475. * denotes P

    Techniques Used: In Vitro, Expressing

    Oncopig HCC xenograft tumor development. ( A ) Representative SQ Oncopig HCC xenograft tumor. ( B ) Excised Oncopig HCC xenograft tumor. ( C ) H E (20×) of Oncopig HCC xenograft tumor reveals densely cellular subcutaneous nodule with interspersed fat cells. Intervening fibrous vascular septae noted. ( D ) On arginase-1 IHC (20×), epithelial cells show focal arginase-1 expression (brown) consistent with hepatocellular differentiation. ( E ) AFP expression across Oncopig HCC xenograft tumors ( n = 10).
    Figure Legend Snippet: Oncopig HCC xenograft tumor development. ( A ) Representative SQ Oncopig HCC xenograft tumor. ( B ) Excised Oncopig HCC xenograft tumor. ( C ) H E (20×) of Oncopig HCC xenograft tumor reveals densely cellular subcutaneous nodule with interspersed fat cells. Intervening fibrous vascular septae noted. ( D ) On arginase-1 IHC (20×), epithelial cells show focal arginase-1 expression (brown) consistent with hepatocellular differentiation. ( E ) AFP expression across Oncopig HCC xenograft tumors ( n = 10).

    Techniques Used: Immunohistochemistry, Expressing

    Oncopig SQ HCC autograft formation. ( A ) Photograph of visible SQ HCC tumor (circled) in Oncopig flank. ( B ) Excision of 2.0 cm SQ HCC tumor. ( C ) Excised and transected SQ HCC tumor. ( D ) H E (20×) of Oncopig SQ HCC tumor demonstrates prominent, dispersed, pleomorphic large atypical cells, 5–10× the size of lymphocytes, flanking fibrous vascular septae, surrounded by dense mixed immune cell infiltrates. Arginase-1 IHC (20×) shows that these atypical cells show patchy arginase-1 expression (brown) consistent with hepatocellular differentiation. KRAS G12D IHC (20×) confirms KRAS G12D expression (brown) consistent with malignancy. ( E ) AFP expression across Oncopig SQ HCC tumors ( n = 6).
    Figure Legend Snippet: Oncopig SQ HCC autograft formation. ( A ) Photograph of visible SQ HCC tumor (circled) in Oncopig flank. ( B ) Excision of 2.0 cm SQ HCC tumor. ( C ) Excised and transected SQ HCC tumor. ( D ) H E (20×) of Oncopig SQ HCC tumor demonstrates prominent, dispersed, pleomorphic large atypical cells, 5–10× the size of lymphocytes, flanking fibrous vascular septae, surrounded by dense mixed immune cell infiltrates. Arginase-1 IHC (20×) shows that these atypical cells show patchy arginase-1 expression (brown) consistent with hepatocellular differentiation. KRAS G12D IHC (20×) confirms KRAS G12D expression (brown) consistent with malignancy. ( E ) AFP expression across Oncopig SQ HCC tumors ( n = 6).

    Techniques Used: Immunohistochemistry, Expressing

    Genomic signatures of Oncopig HCC. ( A ) Somatic copy-number calling reveals a largely copy-neutral tumor in line with the young age of the tumor. ( B ) Representative venn diagram showing distribution of SNVs in the cell line and 2 out of 5 tumor samples. ( C ) Mutational signatures identified resemble signatures observed in human HCC tumors (Signatures 1, 12, and 17).
    Figure Legend Snippet: Genomic signatures of Oncopig HCC. ( A ) Somatic copy-number calling reveals a largely copy-neutral tumor in line with the young age of the tumor. ( B ) Representative venn diagram showing distribution of SNVs in the cell line and 2 out of 5 tumor samples. ( C ) Mutational signatures identified resemble signatures observed in human HCC tumors (Signatures 1, 12, and 17).

    Techniques Used:

    Oncopig and human HCC in vitro phenotypes. ( A ) Schematic of Oncopig transgene construct and agarose gel electrophoresis of RT-PCR products confirming Oncopig transgene ( KRAS G12D and TP53 R167H ) expression following exposure to AdCre. ( B ) Positive arginase-1 and KRAS G12D staining (brown) of cultured Oncopig HCC cell lines (20×). ( C ) Oncopig and human HCC cell cycle lengths. ( D ) Representative cell migration images depicting faster gap closure in Oncopig compared to HepG2 and half gap closure rates for Oncopig ( n = 15 cell lines) and human HCC cells. ( E ) AFP secretion from Oncopig ( n = 15 cell lines) and human HCC cells. Huh7, SNU-387, and SNU475 are known non-AFP producing cell lines. ns = non-significant, * denotes p -value
    Figure Legend Snippet: Oncopig and human HCC in vitro phenotypes. ( A ) Schematic of Oncopig transgene construct and agarose gel electrophoresis of RT-PCR products confirming Oncopig transgene ( KRAS G12D and TP53 R167H ) expression following exposure to AdCre. ( B ) Positive arginase-1 and KRAS G12D staining (brown) of cultured Oncopig HCC cell lines (20×). ( C ) Oncopig and human HCC cell cycle lengths. ( D ) Representative cell migration images depicting faster gap closure in Oncopig compared to HepG2 and half gap closure rates for Oncopig ( n = 15 cell lines) and human HCC cells. ( E ) AFP secretion from Oncopig ( n = 15 cell lines) and human HCC cells. Huh7, SNU-387, and SNU475 are known non-AFP producing cell lines. ns = non-significant, * denotes p -value

    Techniques Used: In Vitro, Construct, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Cell Culture, Migration

    25) Product Images from "Isolation and characterization of a novelSphingobium yanoikuyae strain variant that uses biohazardous saturated hydrocarbons and aromatic compounds as sole carbon sources"

    Article Title: Isolation and characterization of a novelSphingobium yanoikuyae strain variant that uses biohazardous saturated hydrocarbons and aromatic compounds as sole carbon sources

    Journal: F1000Research

    doi: 10.12688/f1000research.25284.1

    PCR amplification of the 16S rRNA partial gene sequence of CC4533 and NCBI-BLAST analyses. ( A ) A schematic diagram showing the conserved and hypervariable regions in the 16S rRNA gene. The interspersed conserved regions (C1–C9) are shown in gray, and the hypervariable regions (V1–V9) are depicted in white. The black box within the C4 region represents 11 nucleotides (788 -798 base pairs) that are invariant in bacteria. PCR primers are shown in thick black arrows. Forward primer is in the C2 region and the reverse primer is in the C4 region. The figure is based on the 16S rRNA gene sequence of E. coli . ( B ) A DNA agarose gel showing the results of PCR with the primers shown in Figure 13A . Lane 1 represents PCR with water (zero DNA control) and Lane 2 shows the PCR product from a bacterial strain LMJ 41 isolated by our lab and, Lane 3 shows the CC45333 PCR product. 1kb plus DNA ladder was used as a DNA molecular size ladder on the agarose gel. ( C ) A schematic diagram showing the nucleotide changes in CC4533 in the 16S rRNA region spanning the C2 and C4 regions in comparison to the best NCBI- BLAST hit (score of 802; E-value 0 and percent identity of 99.55%): Sphingobium yanoikuyae strain PR86 16S ribosomal RNA gene, partial sequence (GenBank Accession #: MN232173.1 ). Black nucleotides show the native nucleotides in the BLAST hit Sphingobium yanoikuyae strain PR86 that were substituted by the depicted red nucleotides in CC4533 16S rRNA gene sequence. The black bold numbers within the parenthesis beside the nucleotides show the specific nucleotide position where the nucleotide changes have occurred. Nucleotide positions shown in the figures have been assigned according to that of the 16S rRNA gene sequence of E. coli . DNA sequencing data is available as Underlying data 52 .
    Figure Legend Snippet: PCR amplification of the 16S rRNA partial gene sequence of CC4533 and NCBI-BLAST analyses. ( A ) A schematic diagram showing the conserved and hypervariable regions in the 16S rRNA gene. The interspersed conserved regions (C1–C9) are shown in gray, and the hypervariable regions (V1–V9) are depicted in white. The black box within the C4 region represents 11 nucleotides (788 -798 base pairs) that are invariant in bacteria. PCR primers are shown in thick black arrows. Forward primer is in the C2 region and the reverse primer is in the C4 region. The figure is based on the 16S rRNA gene sequence of E. coli . ( B ) A DNA agarose gel showing the results of PCR with the primers shown in Figure 13A . Lane 1 represents PCR with water (zero DNA control) and Lane 2 shows the PCR product from a bacterial strain LMJ 41 isolated by our lab and, Lane 3 shows the CC45333 PCR product. 1kb plus DNA ladder was used as a DNA molecular size ladder on the agarose gel. ( C ) A schematic diagram showing the nucleotide changes in CC4533 in the 16S rRNA region spanning the C2 and C4 regions in comparison to the best NCBI- BLAST hit (score of 802; E-value 0 and percent identity of 99.55%): Sphingobium yanoikuyae strain PR86 16S ribosomal RNA gene, partial sequence (GenBank Accession #: MN232173.1 ). Black nucleotides show the native nucleotides in the BLAST hit Sphingobium yanoikuyae strain PR86 that were substituted by the depicted red nucleotides in CC4533 16S rRNA gene sequence. The black bold numbers within the parenthesis beside the nucleotides show the specific nucleotide position where the nucleotide changes have occurred. Nucleotide positions shown in the figures have been assigned according to that of the 16S rRNA gene sequence of E. coli . DNA sequencing data is available as Underlying data 52 .

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Agarose Gel Electrophoresis, Isolation, DNA Sequencing

    26) Product Images from "Filter paper-based spin column method for cost-efficient DNA or RNA purification"

    Article Title: Filter paper-based spin column method for cost-efficient DNA or RNA purification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0203011

    The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin miniprep kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.
    Figure Legend Snippet: The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin miniprep kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.

    Techniques Used: Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Gel Extraction, Plasmid Preparation

    Evaluation of purification of tobacco genomic DNA and total RNA using filter paper-based spin columns with respective Qiagen kit buffers and homemade buffers. (A) Agarose gel electrophoresis for 2.5 μl tobacco genomic DNAs elution from purification experiments using Qiagen DNeasy plant mini kit buffers with Qiagen original spin column (Lane Q/Q), filter paper recharged used spin column (Lane Q/R) and filter paper-based homemade spin column (Lane Q/H*), followed by tobacco genomic DNAs purified using homemade buffer with Qiagen original spin column (Lane H/Q), filter paper recharged used spin column (Lane H/R) and filter paper-based homemade spin column (Lane H/H*). (B) UV spectrum curve of tobacco DNAs purified using Qiagen kit (Q/Q, black curve), filter paper recharged spin columns with Qiagen kit buffers (Q/R, blue curve) or homemade buffers (H/R, red curve) from the same amount leaf tissue. Y-axis is UV absorbance, and X-axis is wavelength (nM). (C) Amplification plots for three duplicated qPCR reactions contain 20 ng DNA purified using Qiagen kit (Q/Q, Blue curves) or DNA purified from filter paper recharged spin column with homemade buffer (H/R, Red curves) respectively. The x-axis is PCR cycle numbers, Y-axis is the level of SYBR fluorescence, and the green line is an arbitrary threshold to determine the Cq value (the fractional cycle number at which amplification curve meet threshold level). (D) MOPS-formaldehyde denaturing agarose gel electrophoresis separated 5 μl RNA purified using Qiagen RNeasy plant mini kit buffers with a Qiagen original spin column (Lane Q/Q), filter paper recharged used spin column (Lane Q/R) and homemade filter paper-based spin column (Lane Q/H*), followed total tobacco RNAs purified by using homemade buffer with Qiagen original spin column (Lane H/Q), filter paper recharged used spin column (Lane H/R) and filter paper-based homemade spin column (Lane H/H*). (E) UV spectrum of tobacco total RNA purified using Qiagen kit (Q/Q, black curve), filter paper recharged spin column with Qiagen RNeasy plant mini kit buffers (Q/R, blue curve) or homemade buffers (H/R, red curve). Y-axis is UV absorbance, and the X-axis is wavelength. (F) Amplification plots of three duplicated qRT-PCR reactions for 2.5 ng RNA purified using Qiagen kit (Q/Q, Blue curves) or RNA purified using filter paper recharged spin column with homemade buffer (H/R, Red curves) respectively. Note: * The starting material amount is 100 mg tobacco leaf tissue for experiments using a Qiagen spin column or filter paper recharged spin column, and half amount of plant sample (50 mg) used for homemade spin column purification. All DNAs or RNAs were eluted using 100 ul elution solution.
    Figure Legend Snippet: Evaluation of purification of tobacco genomic DNA and total RNA using filter paper-based spin columns with respective Qiagen kit buffers and homemade buffers. (A) Agarose gel electrophoresis for 2.5 μl tobacco genomic DNAs elution from purification experiments using Qiagen DNeasy plant mini kit buffers with Qiagen original spin column (Lane Q/Q), filter paper recharged used spin column (Lane Q/R) and filter paper-based homemade spin column (Lane Q/H*), followed by tobacco genomic DNAs purified using homemade buffer with Qiagen original spin column (Lane H/Q), filter paper recharged used spin column (Lane H/R) and filter paper-based homemade spin column (Lane H/H*). (B) UV spectrum curve of tobacco DNAs purified using Qiagen kit (Q/Q, black curve), filter paper recharged spin columns with Qiagen kit buffers (Q/R, blue curve) or homemade buffers (H/R, red curve) from the same amount leaf tissue. Y-axis is UV absorbance, and X-axis is wavelength (nM). (C) Amplification plots for three duplicated qPCR reactions contain 20 ng DNA purified using Qiagen kit (Q/Q, Blue curves) or DNA purified from filter paper recharged spin column with homemade buffer (H/R, Red curves) respectively. The x-axis is PCR cycle numbers, Y-axis is the level of SYBR fluorescence, and the green line is an arbitrary threshold to determine the Cq value (the fractional cycle number at which amplification curve meet threshold level). (D) MOPS-formaldehyde denaturing agarose gel electrophoresis separated 5 μl RNA purified using Qiagen RNeasy plant mini kit buffers with a Qiagen original spin column (Lane Q/Q), filter paper recharged used spin column (Lane Q/R) and homemade filter paper-based spin column (Lane Q/H*), followed total tobacco RNAs purified by using homemade buffer with Qiagen original spin column (Lane H/Q), filter paper recharged used spin column (Lane H/R) and filter paper-based homemade spin column (Lane H/H*). (E) UV spectrum of tobacco total RNA purified using Qiagen kit (Q/Q, black curve), filter paper recharged spin column with Qiagen RNeasy plant mini kit buffers (Q/R, blue curve) or homemade buffers (H/R, red curve). Y-axis is UV absorbance, and the X-axis is wavelength. (F) Amplification plots of three duplicated qRT-PCR reactions for 2.5 ng RNA purified using Qiagen kit (Q/Q, Blue curves) or RNA purified using filter paper recharged spin column with homemade buffer (H/R, Red curves) respectively. Note: * The starting material amount is 100 mg tobacco leaf tissue for experiments using a Qiagen spin column or filter paper recharged spin column, and half amount of plant sample (50 mg) used for homemade spin column purification. All DNAs or RNAs were eluted using 100 ul elution solution.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Amplification, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Fluorescence, Quantitative RT-PCR

    27) Product Images from "Requisite Chromatin Remodeling for Myeloid and Erythroid Lineage Differentiation from Erythromyeloid Progenitors"

    Article Title: Requisite Chromatin Remodeling for Myeloid and Erythroid Lineage Differentiation from Erythromyeloid Progenitors

    Journal: Cell reports

    doi: 10.1016/j.celrep.2020.108395

    BAF155 Interacts with PU.1 and Is Recruited to Its Target Genes (A) qRT-PCR analysis of Pu.1 expression (top) and epigenome browser view of the Spi1/Pu.1 locus (bottom) from WT and Baf155 CKO YSs. Data are from at least two biological replicates for either genotype, with each replicate consisting of an individual YS. Data are presented as mean ± SD. Student’s t test; **p
    Figure Legend Snippet: BAF155 Interacts with PU.1 and Is Recruited to Its Target Genes (A) qRT-PCR analysis of Pu.1 expression (top) and epigenome browser view of the Spi1/Pu.1 locus (bottom) from WT and Baf155 CKO YSs. Data are from at least two biological replicates for either genotype, with each replicate consisting of an individual YS. Data are presented as mean ± SD. Student’s t test; **p

    Techniques Used: Quantitative RT-PCR, Expressing

    28) Product Images from "Techniques for the Isolation of High-Quality RNA from Cells Encapsulated in Chitosan Hydrogels"

    Article Title: Techniques for the Isolation of High-Quality RNA from Cells Encapsulated in Chitosan Hydrogels

    Journal: Tissue Engineering. Part C, Methods

    doi: 10.1089/ten.tec.2012.0693

    Representative end-point RT-PCR gene expression results for (a) 18S and (b) TfR from RNA isolated using the freeze grind+CTAB+RNeasy ® (FCR) method, the mince+CTAB+RNeasy ® (MCR) method, and the lysozyme+CTAB+RNeasy ® (LCR) method. Genomic contamination was detected following agarose gel electrophoresis in all minus-RT controls. In addition, as shown in the TfR results, where the primers were designed to span an intron–exon boundary, two products were formed during the PCR, corresponding to a genomic product size of 270 bp and an mRNA product size of 62 bp.
    Figure Legend Snippet: Representative end-point RT-PCR gene expression results for (a) 18S and (b) TfR from RNA isolated using the freeze grind+CTAB+RNeasy ® (FCR) method, the mince+CTAB+RNeasy ® (MCR) method, and the lysozyme+CTAB+RNeasy ® (LCR) method. Genomic contamination was detected following agarose gel electrophoresis in all minus-RT controls. In addition, as shown in the TfR results, where the primers were designed to span an intron–exon boundary, two products were formed during the PCR, corresponding to a genomic product size of 270 bp and an mRNA product size of 62 bp.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    29) Product Images from "Genome-Wide Analysis of Off-Target CRISPR/Cas9 Activity in Single-Cell-Derived Human Hematopoietic Stem and Progenitor Cell Clones"

    Article Title: Genome-Wide Analysis of Off-Target CRISPR/Cas9 Activity in Single-Cell-Derived Human Hematopoietic Stem and Progenitor Cell Clones

    Journal: Genes

    doi: 10.3390/genes11121501

    Characterization of CRISPR/Cas9 genome editing by T7 endonuclease I (T7E1) assay. ( A ) T7E1 analysis of CRISPR/Cas9-mediated gene editing in bulk HSPCs. For analysis of bulk (i.e., pre-clonal) HSPC gene editing efficiency, genomic DNA (gDNA) was isolated from gene-edited and control HSPC samples at 3 days post-treatment. Isolated gDNA was subjected to PCR amplification using either CXCR4 (left panel) or AAVS1 (right panel) target site-specific primer sets as described in Materials and Methods. Where indicated, PCR amplicons were digested with T7E1, and enzymatic cleavage products were resolved by electrophoresis on a native 4–20% polyacrylamide gradient gel. Amplicon substrates are indicated by an asterisk (*), T7E1 cleavage products are indicated by arrowheads. Clonal isolates selected for WGS and variant analysis are indicated by a color-coded dot. ( B ) T7E1 analysis of CRISPR/Cas9-mediated gene editing in HSPC clonal isolates. For analysis of gene editing efficiency in HSPC clonal isolates, gDNA was extracted from cellular colonies approximately 2–4 weeks post-treatment. Isolated gDNA was subjected to PCR amplification using either CXCR4 (left panel) or AAVS1 (right panel) target site-specific primer sets. Where indicated, PCR amplicons were digested with T7E1, and enzymatic cleavage products were resolved by native polyacrylamide gel electrophoresis (4–20% gradient gel). Amplicon substrates are indicated by an asterisk (*), T7E1 cleavage products are indicated by arrowheads. Clonal isolates selected for whole genome sequencing (WGS) and variant analysis are indicated by a color-coded dot. EP, electroporation; kb, kilobase pairs; sgRNA, single guide RNA. ( C ) WGS read depth. Violin plot showing read depth for each sample. The median (40.3×) is indicated by a black line.
    Figure Legend Snippet: Characterization of CRISPR/Cas9 genome editing by T7 endonuclease I (T7E1) assay. ( A ) T7E1 analysis of CRISPR/Cas9-mediated gene editing in bulk HSPCs. For analysis of bulk (i.e., pre-clonal) HSPC gene editing efficiency, genomic DNA (gDNA) was isolated from gene-edited and control HSPC samples at 3 days post-treatment. Isolated gDNA was subjected to PCR amplification using either CXCR4 (left panel) or AAVS1 (right panel) target site-specific primer sets as described in Materials and Methods. Where indicated, PCR amplicons were digested with T7E1, and enzymatic cleavage products were resolved by electrophoresis on a native 4–20% polyacrylamide gradient gel. Amplicon substrates are indicated by an asterisk (*), T7E1 cleavage products are indicated by arrowheads. Clonal isolates selected for WGS and variant analysis are indicated by a color-coded dot. ( B ) T7E1 analysis of CRISPR/Cas9-mediated gene editing in HSPC clonal isolates. For analysis of gene editing efficiency in HSPC clonal isolates, gDNA was extracted from cellular colonies approximately 2–4 weeks post-treatment. Isolated gDNA was subjected to PCR amplification using either CXCR4 (left panel) or AAVS1 (right panel) target site-specific primer sets. Where indicated, PCR amplicons were digested with T7E1, and enzymatic cleavage products were resolved by native polyacrylamide gel electrophoresis (4–20% gradient gel). Amplicon substrates are indicated by an asterisk (*), T7E1 cleavage products are indicated by arrowheads. Clonal isolates selected for whole genome sequencing (WGS) and variant analysis are indicated by a color-coded dot. EP, electroporation; kb, kilobase pairs; sgRNA, single guide RNA. ( C ) WGS read depth. Violin plot showing read depth for each sample. The median (40.3×) is indicated by a black line.

    Techniques Used: CRISPR, Isolation, Polymerase Chain Reaction, Amplification, Electrophoresis, Variant Assay, Polyacrylamide Gel Electrophoresis, Sequencing, Electroporation

    30) Product Images from "Genetic diversity of Trichoderma atroviride strains collected in Poland and identification of loci useful in detection of within-species diversity"

    Article Title: Genetic diversity of Trichoderma atroviride strains collected in Poland and identification of loci useful in detection of within-species diversity

    Journal: Folia Microbiologica

    doi: 10.1007/s12223-015-0385-z

    Dendrogram generated using Jaccard’s coefficient and the UPGMA clustering method. Binary matrices for T. atroviride strains were constructed based on evaluation of RAPD amplicons generated using 55 primers. Cluster analysis of the binary data was performed using NTSYS-pc 2.1 software. Similarity matrices were generated using Jaccard’s coefficient and an unweighted pair-group method using arithmetic averages (UPGMA) was used to generate the dendrogram
    Figure Legend Snippet: Dendrogram generated using Jaccard’s coefficient and the UPGMA clustering method. Binary matrices for T. atroviride strains were constructed based on evaluation of RAPD amplicons generated using 55 primers. Cluster analysis of the binary data was performed using NTSYS-pc 2.1 software. Similarity matrices were generated using Jaccard’s coefficient and an unweighted pair-group method using arithmetic averages (UPGMA) was used to generate the dendrogram

    Techniques Used: Generated, Construct, Software

    31) Product Images from "APOBEC3B can impair genomic stability by inducing base substitutions in genomic DNA in human cells"

    Article Title: APOBEC3B can impair genomic stability by inducing base substitutions in genomic DNA in human cells

    Journal: Scientific Reports

    doi: 10.1038/srep00806

    Foreign DNA editing by A3A, A3B, and AID. (A) Agarose gel analyses of 3D-PCR products from HEK293 cells. Cells were transfected with expression vector for A3A, A3B wild-type or mutant, or AID together with pEGFP-N3 and pEF-UGI. Total DNA was recovered 2 days after transfection, and EGFP gene was amplified by 3D-PCR at the indicated denaturation temperatures (Td). (B) Mutation matrices of hyperedited EGFP sequences derived from cloned amplicons at 83.8°C of Td. “n” indicates the number of bases sequenced. We sequenced 5 clones (2,225 base pairs in total) in each group. (C) Frequencies of C/G to T/A transitions in hyperedited EGFP genes. C/G to T/A transitions per 1,000 sequenced base pairs are shown. (D) Dinucleotide contexts in foreign DNA editing. The rates of indicated dinucleotide sequence at the C to T transitions are shown. Asterisks indicate statistical significance in a χ 2 test (p
    Figure Legend Snippet: Foreign DNA editing by A3A, A3B, and AID. (A) Agarose gel analyses of 3D-PCR products from HEK293 cells. Cells were transfected with expression vector for A3A, A3B wild-type or mutant, or AID together with pEGFP-N3 and pEF-UGI. Total DNA was recovered 2 days after transfection, and EGFP gene was amplified by 3D-PCR at the indicated denaturation temperatures (Td). (B) Mutation matrices of hyperedited EGFP sequences derived from cloned amplicons at 83.8°C of Td. “n” indicates the number of bases sequenced. We sequenced 5 clones (2,225 base pairs in total) in each group. (C) Frequencies of C/G to T/A transitions in hyperedited EGFP genes. C/G to T/A transitions per 1,000 sequenced base pairs are shown. (D) Dinucleotide contexts in foreign DNA editing. The rates of indicated dinucleotide sequence at the C to T transitions are shown. Asterisks indicate statistical significance in a χ 2 test (p

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Amplification, Derivative Assay, Clone Assay, Sequencing

    Expression of A3B and somatic mutations in oncogenes in human lymphoma cell lines. (A) Quantitative RT-PCR for A3A , A3B , and AID in lymphoma cell lines. The levels of target cDNA were normalized to the endogenous hypoxanthine phosphoribosyl transferase 1 ( HPRT1 ) and then compared to those in peripheral blood lymphocytes. (B) Mutational analyses of C-myc , Pax5 , and A20 in SUDH6 and KIS1 cells. We recovered total DNA from the cells and amplified the sequence between exon1 and intron1 of C-myc , Pax5 and A20 by PCR and performed direct sequencing of the amplicons. Locations of somatic mutations are shown below the loci with their positions. (C) The expression levels of transcripts of C-myc , Pax5 , and A20 in KIS1 and SUDHL6 cells. Quantitative RT-PCR was similarly performed with (a).
    Figure Legend Snippet: Expression of A3B and somatic mutations in oncogenes in human lymphoma cell lines. (A) Quantitative RT-PCR for A3A , A3B , and AID in lymphoma cell lines. The levels of target cDNA were normalized to the endogenous hypoxanthine phosphoribosyl transferase 1 ( HPRT1 ) and then compared to those in peripheral blood lymphocytes. (B) Mutational analyses of C-myc , Pax5 , and A20 in SUDH6 and KIS1 cells. We recovered total DNA from the cells and amplified the sequence between exon1 and intron1 of C-myc , Pax5 and A20 by PCR and performed direct sequencing of the amplicons. Locations of somatic mutations are shown below the loci with their positions. (C) The expression levels of transcripts of C-myc , Pax5 , and A20 in KIS1 and SUDHL6 cells. Quantitative RT-PCR was similarly performed with (a).

    Techniques Used: Expressing, Quantitative RT-PCR, Amplification, Sequencing, Polymerase Chain Reaction

    Deep sequencing of EGFP genes in genomic DNA. (A) The distributions of C/G to T/A substitutions in the EGFP sequences. Total DNA was recovered form HEK293/EGFP cells 7 days after transfection with expression vector for A3A, A3B wild type or H66/253R or AID. We amplified a portion of EGFP sequence from thymine 47 to cytidine 504 (top schematic) by PCR with high-fidelity polymerase and sequenced the amplicons by GS-junior bench top system (Roche). Sequence data were analyzed with equipped software. “Coverage” indicates the total numbers of sequenced reads. (B) Frequencies of base substitutions in hyperedited EGFP genes. Base substitutions were classified to 6 groups and substituted base number of each group per 1,000 sequenced base pairs are show. (C) Dinucleotide contexts in genomic DNA editing. The rates of indicated dinucleotide sequence at the C to T transitions are shown. Deviations in the editing contexts do not reach statistical significance (p
    Figure Legend Snippet: Deep sequencing of EGFP genes in genomic DNA. (A) The distributions of C/G to T/A substitutions in the EGFP sequences. Total DNA was recovered form HEK293/EGFP cells 7 days after transfection with expression vector for A3A, A3B wild type or H66/253R or AID. We amplified a portion of EGFP sequence from thymine 47 to cytidine 504 (top schematic) by PCR with high-fidelity polymerase and sequenced the amplicons by GS-junior bench top system (Roche). Sequence data were analyzed with equipped software. “Coverage” indicates the total numbers of sequenced reads. (B) Frequencies of base substitutions in hyperedited EGFP genes. Base substitutions were classified to 6 groups and substituted base number of each group per 1,000 sequenced base pairs are show. (C) Dinucleotide contexts in genomic DNA editing. The rates of indicated dinucleotide sequence at the C to T transitions are shown. Deviations in the editing contexts do not reach statistical significance (p

    Techniques Used: Sequencing, Transfection, Expressing, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Software

    A3B induced somatic mutations into c-myc gene in human lymphoma cells. (A) Agarose gel analyses of 3D-PCR products of c-Myc genes in SUDHL6. We transfected expression vector for A3B wild-type or H66/253R or empty vector and recovered total DNA 7 days after transfection. C-myc genes were amplified by 3D-PCR at the indicated denaturation temperatures (Td). (B) Clonal sequencing of amplicons from A3B-WT expressing SUDHL6 cells. We sequenced 11 clones (5104 base pairs in total). Seventy six bases from thymine 310 to adenine 385 in which mutations are concentrated among sequenced 464 base pairs are shown. The numbers of C/G to T/A substitutions in sequenced 464 base pair length are shown at the right end. (C) Dinucleotide contexts of somatic mutations in c-Myc gene by A3B. The rates of indicated dinucleotide sequence at the C to T transitions are shown. Asterisks indicate statistical significance in a χ 2 test (p
    Figure Legend Snippet: A3B induced somatic mutations into c-myc gene in human lymphoma cells. (A) Agarose gel analyses of 3D-PCR products of c-Myc genes in SUDHL6. We transfected expression vector for A3B wild-type or H66/253R or empty vector and recovered total DNA 7 days after transfection. C-myc genes were amplified by 3D-PCR at the indicated denaturation temperatures (Td). (B) Clonal sequencing of amplicons from A3B-WT expressing SUDHL6 cells. We sequenced 11 clones (5104 base pairs in total). Seventy six bases from thymine 310 to adenine 385 in which mutations are concentrated among sequenced 464 base pairs are shown. The numbers of C/G to T/A substitutions in sequenced 464 base pair length are shown at the right end. (C) Dinucleotide contexts of somatic mutations in c-Myc gene by A3B. The rates of indicated dinucleotide sequence at the C to T transitions are shown. Asterisks indicate statistical significance in a χ 2 test (p

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Transfection, Expressing, Plasmid Preparation, Amplification, Sequencing, Clone Assay

    32) Product Images from "A functional metagenomic approach for expanding the synthetic biology toolbox for biomass conversion"

    Article Title: A functional metagenomic approach for expanding the synthetic biology toolbox for biomass conversion

    Journal: Molecular Systems Biology

    doi: 10.1038/msb.2010.16

    Functional metagenomic platform for discovery of novel functional genetic elements from diverse environmental microbiomes. Shown is a schematic detailing the key steps required for selecting functional genetic elements from diverse environments that confer a desired selective advantage to a microbial catalyst. Metagenomic DNA is directly extracted from arbitrary environmental samples without earlier culturing steps, purified, and transformed into a microbial host of interest. The entire library of putative functional genetic elements is subjected to a selection pressure (e.g. chemicals at inhibitory concentrations or recalcitrant substrates) that only allows survival of hosts containing functional genetic elements, which counteract the selection pressure (e.g. by allowing usage of the recalcitrant substrates or by conferring tolerance by intracellular or extracellular inactivation or efflux of the inhibitory compound). This scheme is ideally suited for discovery of novel functional genetic elements for biomass conversion to biofuels.
    Figure Legend Snippet: Functional metagenomic platform for discovery of novel functional genetic elements from diverse environmental microbiomes. Shown is a schematic detailing the key steps required for selecting functional genetic elements from diverse environments that confer a desired selective advantage to a microbial catalyst. Metagenomic DNA is directly extracted from arbitrary environmental samples without earlier culturing steps, purified, and transformed into a microbial host of interest. The entire library of putative functional genetic elements is subjected to a selection pressure (e.g. chemicals at inhibitory concentrations or recalcitrant substrates) that only allows survival of hosts containing functional genetic elements, which counteract the selection pressure (e.g. by allowing usage of the recalcitrant substrates or by conferring tolerance by intracellular or extracellular inactivation or efflux of the inhibitory compound). This scheme is ideally suited for discovery of novel functional genetic elements for biomass conversion to biofuels.

    Techniques Used: Functional Assay, Environmental Sampling, Purification, Transformation Assay, Selection

    33) Product Images from "Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR"

    Article Title: Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011337

    Determination of lysis speed and burst sizes for bacteriophages ϕA1122, L-413C, and P2 vir1 on Y. pestis CO92 pgm − . Phage burst sizes (an average phage progeny produced by one bacterial cell) correspond to plateaus on the curves.
    Figure Legend Snippet: Determination of lysis speed and burst sizes for bacteriophages ϕA1122, L-413C, and P2 vir1 on Y. pestis CO92 pgm − . Phage burst sizes (an average phage progeny produced by one bacterial cell) correspond to plateaus on the curves.

    Techniques Used: Lysis, Produced

    Dynamics of growth of phages ϕA1122 and L-413C on different concentrations of Y. pestis cells detected by qPCR. The starting points of phage infection correspond to 100 PFU per 1 µl sample and are normalized to 1. A. The titer rise of ϕA1122. B. L-413C amplification.
    Figure Legend Snippet: Dynamics of growth of phages ϕA1122 and L-413C on different concentrations of Y. pestis cells detected by qPCR. The starting points of phage infection correspond to 100 PFU per 1 µl sample and are normalized to 1. A. The titer rise of ϕA1122. B. L-413C amplification.

    Techniques Used: Real-time Polymerase Chain Reaction, Infection, Amplification

    Parameters of ϕA1122- and L-413C-based qPCR tests for phage DNA and live phage particles determined by linear regression method. A and B, standard curves plotted for DNA concentrations of ϕA1122 and L-413C, respectively. C and D, standard curves plotted for live phage particles of ϕA1122 and L-413C, respectively.
    Figure Legend Snippet: Parameters of ϕA1122- and L-413C-based qPCR tests for phage DNA and live phage particles determined by linear regression method. A and B, standard curves plotted for DNA concentrations of ϕA1122 and L-413C, respectively. C and D, standard curves plotted for live phage particles of ϕA1122 and L-413C, respectively.

    Techniques Used: Real-time Polymerase Chain Reaction

    Lytic properties of bacteriophages ϕA1122, L-413C, and P2 vir1 towards Y. pestis CO92 pgm − . The dynamics of lysis was determined in BHI broth at multiplicity of infection of 0.1. Optical density was normalized to the start of infection (1 on the Y axis corresponds to the initial OD 600 = 0.2).
    Figure Legend Snippet: Lytic properties of bacteriophages ϕA1122, L-413C, and P2 vir1 towards Y. pestis CO92 pgm − . The dynamics of lysis was determined in BHI broth at multiplicity of infection of 0.1. Optical density was normalized to the start of infection (1 on the Y axis corresponds to the initial OD 600 = 0.2).

    Techniques Used: Lysis, Infection

    34) Product Images from "Encapsidation of Viral RNA in Picornavirales: Studies on Cowpea Mosaic Virus Demonstrate Dependence on Viral Replication"

    Article Title: Encapsidation of Viral RNA in Picornavirales: Studies on Cowpea Mosaic Virus Demonstrate Dependence on Viral Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.01520-18

    A replication-competent version of RNA-1 is necessary for RNA encapsidation. CPMV particles were purified from N. benthamiana leaves agroinfiltrated with pBinPS1NT and pBinPS2NT (lanes 1 in each gel), pBinPS1NT and pEAQ- HT -VP60 (lanes 2), pBinP32E and pBinPS2NT (lanes 3), and pBinP32E and pEAQ- HT -VP60 (lanes 4) or with pEAQ- HT -VP60 alone (lanes 5). In each case, the CPMV RNAs expressed within the leaves are indicated. The purified particles were examined by either denaturing SDS-PAGE followed by staining with Instant Blue (a) or electrophoresis on a nondenaturing agarose gel (b) followed by staining with either Instant Blue (left) to visualize protein or ethidium bromide (EtBr) (right) to visualize nucleic acid. The positions of the large (L) coat protein and two forms of the small [S (slow) and S (fast)] coat protein are indicated to the left of the gel in panel a. Note that in panel b, CPMV particles separate into distinct electrophoretic populations based on the presence or absence of the labile 24 amino acids at the C terminus of the small coat protein ( 2 ) as seen in panel a.
    Figure Legend Snippet: A replication-competent version of RNA-1 is necessary for RNA encapsidation. CPMV particles were purified from N. benthamiana leaves agroinfiltrated with pBinPS1NT and pBinPS2NT (lanes 1 in each gel), pBinPS1NT and pEAQ- HT -VP60 (lanes 2), pBinP32E and pBinPS2NT (lanes 3), and pBinP32E and pEAQ- HT -VP60 (lanes 4) or with pEAQ- HT -VP60 alone (lanes 5). In each case, the CPMV RNAs expressed within the leaves are indicated. The purified particles were examined by either denaturing SDS-PAGE followed by staining with Instant Blue (a) or electrophoresis on a nondenaturing agarose gel (b) followed by staining with either Instant Blue (left) to visualize protein or ethidium bromide (EtBr) (right) to visualize nucleic acid. The positions of the large (L) coat protein and two forms of the small [S (slow) and S (fast)] coat protein are indicated to the left of the gel in panel a. Note that in panel b, CPMV particles separate into distinct electrophoretic populations based on the presence or absence of the labile 24 amino acids at the C terminus of the small coat protein ( 2 ) as seen in panel a.

    Techniques Used: Purification, SDS Page, Staining, Electrophoresis, Agarose Gel Electrophoresis

    GDD mutants of RNA-1 are encapsidation deficient. Particles were purified from leaves agroinfiltrated with pEAQ-RNA-2 and an RNA-1-based construct, as indicated. The same preparation of purified particles was used for the gels shown here. (a) Protein content of particles visualized by SDS-PAGE and Instant Blue staining. The correctly processed large (L) coat protein and two electrophoretic forms of the small (S) coat protein present in all samples are indicated. (b) Purified particles from the same preparations as in panel a were analyzed in duplicate on a native agarose gel. (Left) Half of the gel was stained with Instant Blue protein stain to reveal equal loading of particles; (right) the other half of the gel was stained with ethidium bromide to visualize encapsidated nucleic acid. The positions of the L coat protein and two forms of the S coat protein are indicated to the left of the gel in panel a. Note that in panel b, CPMV particles separate into distinct electrophoretic populations based on the presence or absence of the labile 24 amino acids at the C terminus of the small coat protein ( 2 ) as seen in panel a. (c) RNA extracted from equal amounts of purified particles analyzed on an ethidium bromide-stained denaturing agarose RNA gel to reveal encapsidated RNA-1 and RNA-2. (d) Nucleic acid sequencing was carried out on the encapsidated RNA-1-Int-GAD seen in lane 2 in panel c, and this revealed that the encapsidated RNA has preserved the GAD mutation and has not reverted to wild-type GDD. Duplicate sequencing chromatograms are aligned to the wild-type sequence encoding GDD to highlight the point mutation, with the relevant amino acids indicated above. Sequencing was carried out by Eurofins Scientific, and sequence alignment was carried out using Vector NTI Advance 11.5.3.
    Figure Legend Snippet: GDD mutants of RNA-1 are encapsidation deficient. Particles were purified from leaves agroinfiltrated with pEAQ-RNA-2 and an RNA-1-based construct, as indicated. The same preparation of purified particles was used for the gels shown here. (a) Protein content of particles visualized by SDS-PAGE and Instant Blue staining. The correctly processed large (L) coat protein and two electrophoretic forms of the small (S) coat protein present in all samples are indicated. (b) Purified particles from the same preparations as in panel a were analyzed in duplicate on a native agarose gel. (Left) Half of the gel was stained with Instant Blue protein stain to reveal equal loading of particles; (right) the other half of the gel was stained with ethidium bromide to visualize encapsidated nucleic acid. The positions of the L coat protein and two forms of the S coat protein are indicated to the left of the gel in panel a. Note that in panel b, CPMV particles separate into distinct electrophoretic populations based on the presence or absence of the labile 24 amino acids at the C terminus of the small coat protein ( 2 ) as seen in panel a. (c) RNA extracted from equal amounts of purified particles analyzed on an ethidium bromide-stained denaturing agarose RNA gel to reveal encapsidated RNA-1 and RNA-2. (d) Nucleic acid sequencing was carried out on the encapsidated RNA-1-Int-GAD seen in lane 2 in panel c, and this revealed that the encapsidated RNA has preserved the GAD mutation and has not reverted to wild-type GDD. Duplicate sequencing chromatograms are aligned to the wild-type sequence encoding GDD to highlight the point mutation, with the relevant amino acids indicated above. Sequencing was carried out by Eurofins Scientific, and sequence alignment was carried out using Vector NTI Advance 11.5.3.

    Techniques Used: Purification, Construct, SDS Page, Staining, Agarose Gel Electrophoresis, Sequencing, Mutagenesis, Plasmid Preparation

    Heterologous RNA can be encapsidated when bordered by RNA-2 UTRs. (a) Expression of GFP following agroinfiltration of N. benthamiana leaves with the constructs indicated. GFP fluorescence was visualized under UV light at 7 dpi, with the empty pEAQ vector (e.v.) agroinfiltrated as a negative control. (b) RNA extracted from particles purified from plants agroinfiltrated with pEAQ-HT-VP60-24K together with GFP constructs flanked by different 5′ UTRs, as indicated, in the presence or absence of pBinPS1NT. In each case, RNA was extracted from 1.5 mg purified particles, and the resulting RNA was analyzed by denaturing agarose gel electrophoresis and subsequent Northern blotting. (Top left) Northern blotting using a probe specific for the coding region of GFP; (top right) agarose gel before transfer to nylon membranes stained with ethidium bromide; (bottom) Instant Blue-stained SDS-PAGE gel used to reveal processing of VP60 and protein content of the particle preparations. (c) Sequence analysis of the RNA-2 5′ UTR at the positions of the HT mutations. Sequencing was performed on HT -GFP RNA shown in lane 2 in panel b, from particles from leaves coinfiltrated with pBinPS1NT, pEAQ-HT-VP60-24K, and pEAQ-HT-GFP. The duplicate chromatograms show that the HT mutations have not reverted to the wild type prior to encapsidation and are still HT .
    Figure Legend Snippet: Heterologous RNA can be encapsidated when bordered by RNA-2 UTRs. (a) Expression of GFP following agroinfiltration of N. benthamiana leaves with the constructs indicated. GFP fluorescence was visualized under UV light at 7 dpi, with the empty pEAQ vector (e.v.) agroinfiltrated as a negative control. (b) RNA extracted from particles purified from plants agroinfiltrated with pEAQ-HT-VP60-24K together with GFP constructs flanked by different 5′ UTRs, as indicated, in the presence or absence of pBinPS1NT. In each case, RNA was extracted from 1.5 mg purified particles, and the resulting RNA was analyzed by denaturing agarose gel electrophoresis and subsequent Northern blotting. (Top left) Northern blotting using a probe specific for the coding region of GFP; (top right) agarose gel before transfer to nylon membranes stained with ethidium bromide; (bottom) Instant Blue-stained SDS-PAGE gel used to reveal processing of VP60 and protein content of the particle preparations. (c) Sequence analysis of the RNA-2 5′ UTR at the positions of the HT mutations. Sequencing was performed on HT -GFP RNA shown in lane 2 in panel b, from particles from leaves coinfiltrated with pBinPS1NT, pEAQ-HT-VP60-24K, and pEAQ-HT-GFP. The duplicate chromatograms show that the HT mutations have not reverted to the wild type prior to encapsidation and are still HT .

    Techniques Used: Expressing, Construct, Fluorescence, Plasmid Preparation, Negative Control, Purification, Agarose Gel Electrophoresis, Northern Blot, Staining, SDS Page, Sequencing

    RNA-2 is abundant in the cell even in the absence of RNA-1. (Top) Northern blot of total RNA extracted from agroinfiltrated leaf material probed with an RNA probe specific for RNA-2 positive strands. Leaves were either agroinfiltrated with pEAQ-RNA-2 alone or coinfiltrated with both pBinPS1NT and pEAQ-RNA-2 and were harvested at 1, 4, and 5 days postinfiltration (dpi). (Bottom) Ethidium bromide-stained denaturing agarose gel prior to transfer of the RNAs to the membrane, showing the levels of 25S rRNA present in each sample as a loading control.
    Figure Legend Snippet: RNA-2 is abundant in the cell even in the absence of RNA-1. (Top) Northern blot of total RNA extracted from agroinfiltrated leaf material probed with an RNA probe specific for RNA-2 positive strands. Leaves were either agroinfiltrated with pEAQ-RNA-2 alone or coinfiltrated with both pBinPS1NT and pEAQ-RNA-2 and were harvested at 1, 4, and 5 days postinfiltration (dpi). (Bottom) Ethidium bromide-stained denaturing agarose gel prior to transfer of the RNAs to the membrane, showing the levels of 25S rRNA present in each sample as a loading control.

    Techniques Used: Northern Blot, Staining, Agarose Gel Electrophoresis

    The 87K GAD mutation reduces replication efficiency, while the AAA mutation abolishes it. Total RNA was extracted from leaves agroinfiltrated with pEAQ-RNA-2 and an RNA-1-based construct, as indicated. Gene-specific qRT-PCR was carried out to quantify negative (-ve)-stranded RNA-1 and RNA-2 (replication intermediates) in the different samples. Data from three replicate experiments were analyzed using Bio-Rad CFX software to show normalized expression of negative-stranded RNA-1 (left) and RNA-2 (right) relative to RNA-1-32E. Error bars represent standard errors of the means.
    Figure Legend Snippet: The 87K GAD mutation reduces replication efficiency, while the AAA mutation abolishes it. Total RNA was extracted from leaves agroinfiltrated with pEAQ-RNA-2 and an RNA-1-based construct, as indicated. Gene-specific qRT-PCR was carried out to quantify negative (-ve)-stranded RNA-1 and RNA-2 (replication intermediates) in the different samples. Data from three replicate experiments were analyzed using Bio-Rad CFX software to show normalized expression of negative-stranded RNA-1 (left) and RNA-2 (right) relative to RNA-1-32E. Error bars represent standard errors of the means.

    Techniques Used: Mutagenesis, Construct, Quantitative RT-PCR, Software, Expressing

    Truncated RNA-2 constructs can be encapsidated in the presence of RNA-1. (a) Northern blots of RNA packaged in particles produced with different versions of RNA-2 with and without RNA-1. RNA was extracted from particles purified from leaves agroinfiltrated with HT -VP60-24K and an RNA-2 construct, as indicated, in the presence or absence of pBinPS1NT. In each case, the RNA was extracted from 3 mg purified particles, and the resulting RNA was split equally on two duplicate denaturing agarose gels for subsequent Northern blotting. (Left) Detection of RNA-1 (top) or RNA-2 (middle) with probes annealing to the 32K ProC sequence of RNA-1 or the RNA-2 5′ UTR, respectively; (right) denaturing agarose gels before transfer to nylon membranes; (bottom) purified particle preparations visualized on an Instant Blue-stained denaturing SDS-PAGE gel serving as a control for processing of VP60 and the use of equal amounts of particles for each RNA extraction. (b) HT -VP60 is encapsidated in the presence of RNA-1. The presence of encapsidated RNA was analyzed in purified particles extracted from plants transiently expressing pEAQ- HT -VP60-24K with or without pEAQ-XhoI-RNA-2 and with or without pBinPS1NT. RNA was extracted from 2 mg purified particles, and the resulting RNA was loaded onto a denaturing agarose gel for subsequent Northern blotting. (Left) Immunoblot detection of VP60 with the probe annealing to a sequence within the VP60 coding region that is partially removed in the construct resulting from XhoI digestion; (middle) denaturing agarose gel before transfer to a nylon membrane; (right) Instant Blue-stained protein on a denaturing SDS-PAGE gel serving as a control for processing of the VP60 protein precursor and the use of equal amounts of particles in each RNA extraction.
    Figure Legend Snippet: Truncated RNA-2 constructs can be encapsidated in the presence of RNA-1. (a) Northern blots of RNA packaged in particles produced with different versions of RNA-2 with and without RNA-1. RNA was extracted from particles purified from leaves agroinfiltrated with HT -VP60-24K and an RNA-2 construct, as indicated, in the presence or absence of pBinPS1NT. In each case, the RNA was extracted from 3 mg purified particles, and the resulting RNA was split equally on two duplicate denaturing agarose gels for subsequent Northern blotting. (Left) Detection of RNA-1 (top) or RNA-2 (middle) with probes annealing to the 32K ProC sequence of RNA-1 or the RNA-2 5′ UTR, respectively; (right) denaturing agarose gels before transfer to nylon membranes; (bottom) purified particle preparations visualized on an Instant Blue-stained denaturing SDS-PAGE gel serving as a control for processing of VP60 and the use of equal amounts of particles for each RNA extraction. (b) HT -VP60 is encapsidated in the presence of RNA-1. The presence of encapsidated RNA was analyzed in purified particles extracted from plants transiently expressing pEAQ- HT -VP60-24K with or without pEAQ-XhoI-RNA-2 and with or without pBinPS1NT. RNA was extracted from 2 mg purified particles, and the resulting RNA was loaded onto a denaturing agarose gel for subsequent Northern blotting. (Left) Immunoblot detection of VP60 with the probe annealing to a sequence within the VP60 coding region that is partially removed in the construct resulting from XhoI digestion; (middle) denaturing agarose gel before transfer to a nylon membrane; (right) Instant Blue-stained protein on a denaturing SDS-PAGE gel serving as a control for processing of the VP60 protein precursor and the use of equal amounts of particles in each RNA extraction.

    Techniques Used: Construct, Northern Blot, Produced, Purification, Sequencing, Staining, SDS Page, RNA Extraction, Expressing, Agarose Gel Electrophoresis

    35) Product Images from "A Versatile Nanowire Platform for Highly Efficient Isolation and Direct PCR-free Colorimetric Detection of Human Papillomavirus DNA from Unprocessed Urine"

    Article Title: A Versatile Nanowire Platform for Highly Efficient Isolation and Direct PCR-free Colorimetric Detection of Human Papillomavirus DNA from Unprocessed Urine

    Journal: Theranostics

    doi: 10.7150/thno.21696

    (a) The concentration of cfDNA isolated from urine samples of HPV-negative healthy controls and HPV-positive cervical cancer patients using PEI-mPpy NWs. (b) Type-specific concordance of HPV DNA genotypes of the results from cervical swabs and PCR-free urinary HPV detection by PEI-mPpy NWs.
    Figure Legend Snippet: (a) The concentration of cfDNA isolated from urine samples of HPV-negative healthy controls and HPV-positive cervical cancer patients using PEI-mPpy NWs. (b) Type-specific concordance of HPV DNA genotypes of the results from cervical swabs and PCR-free urinary HPV detection by PEI-mPpy NWs.

    Techniques Used: Concentration Assay, Isolation, Polymerase Chain Reaction

    (A) Magnetic nanowire-based colorimetric assay for HPV DNA detection and genotyping in urine samples. Colorimetric detection was performed on the nanowire-DNA complexes that contained cfDNA isolated from the urine samples using PEI-mPpy NWs. The biotin-labeled capture and detector probes were specifically designed to recognize the corresponding target HPV DNAs attached to the nanowire, even without a PCR amplification step. After hybridization of the target with type-specific probes, multiple horseradish peroxidase (HRP)- and streptavidin-labeled polypyrrole nanoparticles (HRP/st-tagged NPs) were added to produce amplified colorimetric signals that are even visible to the naked eye. (B) UV-Vis absorption spectra of nanowire-DNA complexes that are hybridized specifically with their complementary capture/detector probes followed by the addition of HRP/st-tagged NPs. Known concentrations of genomic DNA from HPV-positive SiHa cells (Left; HPV-16; 0, 0.16, 0.52, 1.3, 2.6, 5.2, 26 pg/µL) and that from HPV-positive HeLa cells (Right; HPV-18; 0, 0.12, 0.4, 1.8, 3.9, 19 pg/µL) were spiked into HPV-negative urine pool ex vivo , for HPV DNA isolation and colorimetric detection. The insets show plots of the corresponding absorbance at 650 nm versus various concentrations of genomic DNA from HPV-positive SiHa cells (Left; HPV-16) and HeLa cells (Right; HPV-18) that were extracted by PEI-mPpy NWs. The error bars represent the standard deviations from five independent measurements.
    Figure Legend Snippet: (A) Magnetic nanowire-based colorimetric assay for HPV DNA detection and genotyping in urine samples. Colorimetric detection was performed on the nanowire-DNA complexes that contained cfDNA isolated from the urine samples using PEI-mPpy NWs. The biotin-labeled capture and detector probes were specifically designed to recognize the corresponding target HPV DNAs attached to the nanowire, even without a PCR amplification step. After hybridization of the target with type-specific probes, multiple horseradish peroxidase (HRP)- and streptavidin-labeled polypyrrole nanoparticles (HRP/st-tagged NPs) were added to produce amplified colorimetric signals that are even visible to the naked eye. (B) UV-Vis absorption spectra of nanowire-DNA complexes that are hybridized specifically with their complementary capture/detector probes followed by the addition of HRP/st-tagged NPs. Known concentrations of genomic DNA from HPV-positive SiHa cells (Left; HPV-16; 0, 0.16, 0.52, 1.3, 2.6, 5.2, 26 pg/µL) and that from HPV-positive HeLa cells (Right; HPV-18; 0, 0.12, 0.4, 1.8, 3.9, 19 pg/µL) were spiked into HPV-negative urine pool ex vivo , for HPV DNA isolation and colorimetric detection. The insets show plots of the corresponding absorbance at 650 nm versus various concentrations of genomic DNA from HPV-positive SiHa cells (Left; HPV-16) and HeLa cells (Right; HPV-18) that were extracted by PEI-mPpy NWs. The error bars represent the standard deviations from five independent measurements.

    Techniques Used: Colorimetric Assay, Isolation, Labeling, Polymerase Chain Reaction, Amplification, Hybridization, Ex Vivo, DNA Extraction

    (a) Assessment of clinical performance of the proposed PCR-free colorimetric assay by evaluating urine samples of HPV-positive cervical cancer patients (HPV16(+) and HPV18(+)), HPV-negative healthy controls (HPV(-)), and PBS. The photographs display the color change of nanowire-DNA complexes after hybridization with biotin-labelled capture and detector probes with the corresponding target HPV DNA isolated using the nanowire, followed by the addition of HRP/st-tagged NPs. (b) The UV-Vis spectra of nanowire-DNA complexes that contain cfDNA isolated from HPV18-positive urine by PEI-mPpy NWs. With the addition of HPV16/HPV18 probes and HRP/st-tagged NPs, type-specific HPVs can be specifically detected. (c) Average absorbance values of circulating cfDNA isolated from urine samples of HPV-positive cervical cancer patients (HPV16(+) and HPV18(+)), HPV-negative healthy controls (HPV(-)), and PBS after the reaction with different probe types specific for HPV16 or HPV18. A total of 24 HPV-positive and HPV-negative urine samples were collected and tested. The error bars represent the standard deviations from five independent measurements.
    Figure Legend Snippet: (a) Assessment of clinical performance of the proposed PCR-free colorimetric assay by evaluating urine samples of HPV-positive cervical cancer patients (HPV16(+) and HPV18(+)), HPV-negative healthy controls (HPV(-)), and PBS. The photographs display the color change of nanowire-DNA complexes after hybridization with biotin-labelled capture and detector probes with the corresponding target HPV DNA isolated using the nanowire, followed by the addition of HRP/st-tagged NPs. (b) The UV-Vis spectra of nanowire-DNA complexes that contain cfDNA isolated from HPV18-positive urine by PEI-mPpy NWs. With the addition of HPV16/HPV18 probes and HRP/st-tagged NPs, type-specific HPVs can be specifically detected. (c) Average absorbance values of circulating cfDNA isolated from urine samples of HPV-positive cervical cancer patients (HPV16(+) and HPV18(+)), HPV-negative healthy controls (HPV(-)), and PBS after the reaction with different probe types specific for HPV16 or HPV18. A total of 24 HPV-positive and HPV-negative urine samples were collected and tested. The error bars represent the standard deviations from five independent measurements.

    Techniques Used: Polymerase Chain Reaction, Colorimetric Assay, Hybridization, Isolation

    (a) A novel approach of PEI-mPpy NWs in the extraction, identification, and PCR-free sequential detection of multiple HPV genotypes from urine specimens of cervical cancer patients. (b) The UV-Vis colorimetric results of cfDNA isolated by PEI-mPpy NWs from urine of cervical cancer patients who were found to be positive for both HPV16 and HPV18, demonstrating multiple uses of the same nanowire-DNA complexes for the detection of HPV with different genetic variations. However, no response was observed for non-HPV probes (EGFR19 and EGFR21). The photographs indicate the color change as a result of type-specific hybridization between target HPVs and their complementary probes.
    Figure Legend Snippet: (a) A novel approach of PEI-mPpy NWs in the extraction, identification, and PCR-free sequential detection of multiple HPV genotypes from urine specimens of cervical cancer patients. (b) The UV-Vis colorimetric results of cfDNA isolated by PEI-mPpy NWs from urine of cervical cancer patients who were found to be positive for both HPV16 and HPV18, demonstrating multiple uses of the same nanowire-DNA complexes for the detection of HPV with different genetic variations. However, no response was observed for non-HPV probes (EGFR19 and EGFR21). The photographs indicate the color change as a result of type-specific hybridization between target HPVs and their complementary probes.

    Techniques Used: Polymerase Chain Reaction, Isolation, Hybridization

    36) Product Images from "A Versatile Nanowire Platform for Highly Efficient Isolation and Direct PCR-free Colorimetric Detection of Human Papillomavirus DNA from Unprocessed Urine"

    Article Title: A Versatile Nanowire Platform for Highly Efficient Isolation and Direct PCR-free Colorimetric Detection of Human Papillomavirus DNA from Unprocessed Urine

    Journal: Theranostics

    doi: 10.7150/thno.21696

    (a) The concentration of cfDNA isolated from urine samples of HPV-negative healthy controls and HPV-positive cervical cancer patients using PEI-mPpy NWs. (b) Type-specific concordance of HPV DNA genotypes of the results from cervical swabs and PCR-free urinary HPV detection by PEI-mPpy NWs.
    Figure Legend Snippet: (a) The concentration of cfDNA isolated from urine samples of HPV-negative healthy controls and HPV-positive cervical cancer patients using PEI-mPpy NWs. (b) Type-specific concordance of HPV DNA genotypes of the results from cervical swabs and PCR-free urinary HPV detection by PEI-mPpy NWs.

    Techniques Used: Concentration Assay, Isolation, Polymerase Chain Reaction

    Validation of PEI-mPpy NWs in the extraction of cfDNA in urine samples of cervical cancer patients. Comparisons of the concentration of cfDNA isolated from urine samples of ten representative cervical cancer patients by using PEI-mPpy NWs and a Qiagen DNA extraction kit. PEI-mPpy NWs and a Qiagen kit were employed for the extraction of cfDNA from urine.
    Figure Legend Snippet: Validation of PEI-mPpy NWs in the extraction of cfDNA in urine samples of cervical cancer patients. Comparisons of the concentration of cfDNA isolated from urine samples of ten representative cervical cancer patients by using PEI-mPpy NWs and a Qiagen DNA extraction kit. PEI-mPpy NWs and a Qiagen kit were employed for the extraction of cfDNA from urine.

    Techniques Used: Concentration Assay, Isolation, DNA Extraction

    (A) Magnetic nanowire-based colorimetric assay for HPV DNA detection and genotyping in urine samples. Colorimetric detection was performed on the nanowire-DNA complexes that contained cfDNA isolated from the urine samples using PEI-mPpy NWs. The biotin-labeled capture and detector probes were specifically designed to recognize the corresponding target HPV DNAs attached to the nanowire, even without a PCR amplification step. After hybridization of the target with type-specific probes, multiple horseradish peroxidase (HRP)- and streptavidin-labeled polypyrrole nanoparticles (HRP/st-tagged NPs) were added to produce amplified colorimetric signals that are even visible to the naked eye. (B) UV-Vis absorption spectra of nanowire-DNA complexes that are hybridized specifically with their complementary capture/detector probes followed by the addition of HRP/st-tagged NPs. Known concentrations of genomic DNA from HPV-positive SiHa cells (Left; HPV-16; 0, 0.16, 0.52, 1.3, 2.6, 5.2, 26 pg/µL) and that from HPV-positive HeLa cells (Right; HPV-18; 0, 0.12, 0.4, 1.8, 3.9, 19 pg/µL) were spiked into HPV-negative urine pool ex vivo , for HPV DNA isolation and colorimetric detection. The insets show plots of the corresponding absorbance at 650 nm versus various concentrations of genomic DNA from HPV-positive SiHa cells (Left; HPV-16) and HeLa cells (Right; HPV-18) that were extracted by PEI-mPpy NWs. The error bars represent the standard deviations from five independent measurements.
    Figure Legend Snippet: (A) Magnetic nanowire-based colorimetric assay for HPV DNA detection and genotyping in urine samples. Colorimetric detection was performed on the nanowire-DNA complexes that contained cfDNA isolated from the urine samples using PEI-mPpy NWs. The biotin-labeled capture and detector probes were specifically designed to recognize the corresponding target HPV DNAs attached to the nanowire, even without a PCR amplification step. After hybridization of the target with type-specific probes, multiple horseradish peroxidase (HRP)- and streptavidin-labeled polypyrrole nanoparticles (HRP/st-tagged NPs) were added to produce amplified colorimetric signals that are even visible to the naked eye. (B) UV-Vis absorption spectra of nanowire-DNA complexes that are hybridized specifically with their complementary capture/detector probes followed by the addition of HRP/st-tagged NPs. Known concentrations of genomic DNA from HPV-positive SiHa cells (Left; HPV-16; 0, 0.16, 0.52, 1.3, 2.6, 5.2, 26 pg/µL) and that from HPV-positive HeLa cells (Right; HPV-18; 0, 0.12, 0.4, 1.8, 3.9, 19 pg/µL) were spiked into HPV-negative urine pool ex vivo , for HPV DNA isolation and colorimetric detection. The insets show plots of the corresponding absorbance at 650 nm versus various concentrations of genomic DNA from HPV-positive SiHa cells (Left; HPV-16) and HeLa cells (Right; HPV-18) that were extracted by PEI-mPpy NWs. The error bars represent the standard deviations from five independent measurements.

    Techniques Used: Colorimetric Assay, Isolation, Labeling, Polymerase Chain Reaction, Amplification, Hybridization, Ex Vivo, DNA Extraction

    (a) Assessment of clinical performance of the proposed PCR-free colorimetric assay by evaluating urine samples of HPV-positive cervical cancer patients (HPV16(+) and HPV18(+)), HPV-negative healthy controls (HPV(-)), and PBS. The photographs display the color change of nanowire-DNA complexes after hybridization with biotin-labelled capture and detector probes with the corresponding target HPV DNA isolated using the nanowire, followed by the addition of HRP/st-tagged NPs. (b) The UV-Vis spectra of nanowire-DNA complexes that contain cfDNA isolated from HPV18-positive urine by PEI-mPpy NWs. With the addition of HPV16/HPV18 probes and HRP/st-tagged NPs, type-specific HPVs can be specifically detected. (c) Average absorbance values of circulating cfDNA isolated from urine samples of HPV-positive cervical cancer patients (HPV16(+) and HPV18(+)), HPV-negative healthy controls (HPV(-)), and PBS after the reaction with different probe types specific for HPV16 or HPV18. A total of 24 HPV-positive and HPV-negative urine samples were collected and tested. The error bars represent the standard deviations from five independent measurements.
    Figure Legend Snippet: (a) Assessment of clinical performance of the proposed PCR-free colorimetric assay by evaluating urine samples of HPV-positive cervical cancer patients (HPV16(+) and HPV18(+)), HPV-negative healthy controls (HPV(-)), and PBS. The photographs display the color change of nanowire-DNA complexes after hybridization with biotin-labelled capture and detector probes with the corresponding target HPV DNA isolated using the nanowire, followed by the addition of HRP/st-tagged NPs. (b) The UV-Vis spectra of nanowire-DNA complexes that contain cfDNA isolated from HPV18-positive urine by PEI-mPpy NWs. With the addition of HPV16/HPV18 probes and HRP/st-tagged NPs, type-specific HPVs can be specifically detected. (c) Average absorbance values of circulating cfDNA isolated from urine samples of HPV-positive cervical cancer patients (HPV16(+) and HPV18(+)), HPV-negative healthy controls (HPV(-)), and PBS after the reaction with different probe types specific for HPV16 or HPV18. A total of 24 HPV-positive and HPV-negative urine samples were collected and tested. The error bars represent the standard deviations from five independent measurements.

    Techniques Used: Polymerase Chain Reaction, Colorimetric Assay, Hybridization, Isolation

    (a) A novel approach of PEI-mPpy NWs in the extraction, identification, and PCR-free sequential detection of multiple HPV genotypes from urine specimens of cervical cancer patients. (b) The UV-Vis colorimetric results of cfDNA isolated by PEI-mPpy NWs from urine of cervical cancer patients who were found to be positive for both HPV16 and HPV18, demonstrating multiple uses of the same nanowire-DNA complexes for the detection of HPV with different genetic variations. However, no response was observed for non-HPV probes (EGFR19 and EGFR21). The photographs indicate the color change as a result of type-specific hybridization between target HPVs and their complementary probes.
    Figure Legend Snippet: (a) A novel approach of PEI-mPpy NWs in the extraction, identification, and PCR-free sequential detection of multiple HPV genotypes from urine specimens of cervical cancer patients. (b) The UV-Vis colorimetric results of cfDNA isolated by PEI-mPpy NWs from urine of cervical cancer patients who were found to be positive for both HPV16 and HPV18, demonstrating multiple uses of the same nanowire-DNA complexes for the detection of HPV with different genetic variations. However, no response was observed for non-HPV probes (EGFR19 and EGFR21). The photographs indicate the color change as a result of type-specific hybridization between target HPVs and their complementary probes.

    Techniques Used: Polymerase Chain Reaction, Isolation, Hybridization

    (a) Schematic diagram of highly efficient isolation of urinary cfDNA using polyethyleneimine-conjugated magnetic nanowires (PEI-mPpy NWs) and direct PCR-free colorimetric detection of target HPVs via sequential addition of complementary probes and multiple horseradish peroxidase (HRP)-/streptavidin-labelled polypyrrole nanoparticles (HRP/st-tagged NPs) to dramatically amplify colorimetric signals. (b) SEM image of PEI-mPpy NWs (Left, Scale bar = 10 µm). Inset is a TEM image of the nanowires (scale bar = 1 µm). TEM image at high magnification showing the presence of a large quantity of magnetic nanoparticles (MNPs; 10 nm) doped within the nanowire (Middle, Scale bar = 50 nm). SEM image of HRP/streptavidin-conjugated nanoparticles (Right, scale bar = 200 µm)
    Figure Legend Snippet: (a) Schematic diagram of highly efficient isolation of urinary cfDNA using polyethyleneimine-conjugated magnetic nanowires (PEI-mPpy NWs) and direct PCR-free colorimetric detection of target HPVs via sequential addition of complementary probes and multiple horseradish peroxidase (HRP)-/streptavidin-labelled polypyrrole nanoparticles (HRP/st-tagged NPs) to dramatically amplify colorimetric signals. (b) SEM image of PEI-mPpy NWs (Left, Scale bar = 10 µm). Inset is a TEM image of the nanowires (scale bar = 1 µm). TEM image at high magnification showing the presence of a large quantity of magnetic nanoparticles (MNPs; 10 nm) doped within the nanowire (Middle, Scale bar = 50 nm). SEM image of HRP/streptavidin-conjugated nanoparticles (Right, scale bar = 200 µm)

    Techniques Used: Isolation, Polymerase Chain Reaction, Transmission Electron Microscopy

    37) Product Images from "Proteomic analysis of Biomphalaria glabrata plasma proteins with binding affinity to those expressed by early developing larval Schistosoma mansoni"

    Article Title: Proteomic analysis of Biomphalaria glabrata plasma proteins with binding affinity to those expressed by early developing larval Schistosoma mansoni

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006081

    PCR amplification of NMRI and BS-90 B . glabrata galectin-related protein (GREP) transcripts. Complementary DNA synthesized from whole body RNA extracts of 10 individual NMRI and 10 BS-90 B . glabrata snails were used to generate amplification products of the near-complete coding region of the BS-90 GREP sequence. GREP amplicons for each snail sample (1–10) are shown. Primers to B . glabrata α-actinin served as a loading control. Note that GREP amplicons were generated using cDNA from all BS-90 samples tested, while only 4/10 NMRI snails produced amplicons, demonstrating differential GREP gene expression in the NMRI snail population.
    Figure Legend Snippet: PCR amplification of NMRI and BS-90 B . glabrata galectin-related protein (GREP) transcripts. Complementary DNA synthesized from whole body RNA extracts of 10 individual NMRI and 10 BS-90 B . glabrata snails were used to generate amplification products of the near-complete coding region of the BS-90 GREP sequence. GREP amplicons for each snail sample (1–10) are shown. Primers to B . glabrata α-actinin served as a loading control. Note that GREP amplicons were generated using cDNA from all BS-90 samples tested, while only 4/10 NMRI snails produced amplicons, demonstrating differential GREP gene expression in the NMRI snail population.

    Techniques Used: Polymerase Chain Reaction, Amplification, Synthesized, Sequencing, Generated, Produced, Expressing

    PCR amplification of NMRI and BS-90 B . glabrata ADAM-TS, FREP12, and CREP2 transcripts. Whole body total RNA from 10 individual NMRI and 10 BS-90 B . glabrata snails were subjected to cDNA synthesis and used in PCR analysis of the ADAM-TS metalloproteinase, FREP12 and CREP2 transcript expression. Amplicons of the predicted size are shown for each snail sample (1–10). Primers to B . glabrata α-actinin served as a loading control.
    Figure Legend Snippet: PCR amplification of NMRI and BS-90 B . glabrata ADAM-TS, FREP12, and CREP2 transcripts. Whole body total RNA from 10 individual NMRI and 10 BS-90 B . glabrata snails were subjected to cDNA synthesis and used in PCR analysis of the ADAM-TS metalloproteinase, FREP12 and CREP2 transcript expression. Amplicons of the predicted size are shown for each snail sample (1–10). Primers to B . glabrata α-actinin served as a loading control.

    Techniques Used: Polymerase Chain Reaction, Amplification, Expressing

    38) Product Images from "Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data"

    Article Title: Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data

    Journal: Genome Biology

    doi: 10.1186/s13059-018-1408-2

    Effect of conversion artefacts on the biases in WGBS. a Presence of unconverted cytosines as percentage of total cytosine content, measured by LC-MS for three different BS-conversion protocols. The three protocols differ by denaturation method (Heat or Alkaline) or molarity of bisulfite (4.5 vs 9 M for Am-BS) but not by BS incubation temperature (65–70 °C). Averaged fold differences in quantity are shown above horizontal brackets , and a dotted line shows the usual level of genomic 5mC for reference of scale. For conversion differences between methods with 50 and 65 °C incubation temperatures, see Additional file 2 : Figure S10a. b A theoretical sum of 5mC and unconverted C as measured by LC-MS for J1 WT mESCs for three BS conversion protocols. Both 5mC and unconverted C will be interpreted as 5mC after amplification of WGBS libraries, boosting the overall levels of methylation, depending on the BS treatment protocol. c Absolute quantification of unconverted cytosines in the unmethylated TKO mESC line, as measured by Heat and Alkaline BS-seq. d Context distribution of BS conversion artefacts; the value is the same for Heat and Alkaline and therefore plotted as an average. e CH methylation on both strands of the mouse major satellite repeat as measured by pre- and post-bisulfite WGBS methods. 5mC percentage from the BS cloning from Additional file 2 : Figure S5a is plotted in both panels for reference. Positive y-axis values indicate the top strand and negative the bottom strand. Statistical analyses in a – c were performed for matched experimental pairs with unpaired two-tailed t -tests against Heat in a and c , and WT ES in b . Error bars in a – c represent standard error of the mean, * p
    Figure Legend Snippet: Effect of conversion artefacts on the biases in WGBS. a Presence of unconverted cytosines as percentage of total cytosine content, measured by LC-MS for three different BS-conversion protocols. The three protocols differ by denaturation method (Heat or Alkaline) or molarity of bisulfite (4.5 vs 9 M for Am-BS) but not by BS incubation temperature (65–70 °C). Averaged fold differences in quantity are shown above horizontal brackets , and a dotted line shows the usual level of genomic 5mC for reference of scale. For conversion differences between methods with 50 and 65 °C incubation temperatures, see Additional file 2 : Figure S10a. b A theoretical sum of 5mC and unconverted C as measured by LC-MS for J1 WT mESCs for three BS conversion protocols. Both 5mC and unconverted C will be interpreted as 5mC after amplification of WGBS libraries, boosting the overall levels of methylation, depending on the BS treatment protocol. c Absolute quantification of unconverted cytosines in the unmethylated TKO mESC line, as measured by Heat and Alkaline BS-seq. d Context distribution of BS conversion artefacts; the value is the same for Heat and Alkaline and therefore plotted as an average. e CH methylation on both strands of the mouse major satellite repeat as measured by pre- and post-bisulfite WGBS methods. 5mC percentage from the BS cloning from Additional file 2 : Figure S5a is plotted in both panels for reference. Positive y-axis values indicate the top strand and negative the bottom strand. Statistical analyses in a – c were performed for matched experimental pairs with unpaired two-tailed t -tests against Heat in a and c , and WT ES in b . Error bars in a – c represent standard error of the mean, * p

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Incubation, Amplification, Methylation, Clone Assay, Two Tailed Test

    39) Product Images from "Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data"

    Article Title: Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data

    Journal: Genome Biology

    doi: 10.1186/s13059-018-1408-2

    Effect of DNA methylation status on the degradation and amplification biases. a Coverage of dinucleotides in WGBS datasets from unmethylated and in vitro M.CviPI-methylated TKO DNA prepared with the Heat BS-seq protocol. For direct comparison, the increase in coverage is expressed as fold difference from the genomic average and normalised to the AA dinucleotide. The dinucleotides are grouped as derived from C, G or A/T only and presented in the box-plot panel (right) as total percentage increase in coverage; crosses mark mean values and error bars represent minimum and maximum values. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test against the AT-only dinucleotides; **** p
    Figure Legend Snippet: Effect of DNA methylation status on the degradation and amplification biases. a Coverage of dinucleotides in WGBS datasets from unmethylated and in vitro M.CviPI-methylated TKO DNA prepared with the Heat BS-seq protocol. For direct comparison, the increase in coverage is expressed as fold difference from the genomic average and normalised to the AA dinucleotide. The dinucleotides are grouped as derived from C, G or A/T only and presented in the box-plot panel (right) as total percentage increase in coverage; crosses mark mean values and error bars represent minimum and maximum values. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test against the AT-only dinucleotides; **** p

    Techniques Used: DNA Methylation Assay, Amplification, In Vitro, Methylation, Derivative Assay

    40) Product Images from "Blockade of voltage-gated sodium channels inhibits invasion of endocrine-resistant breast cancer cells"

    Article Title: Blockade of voltage-gated sodium channels inhibits invasion of endocrine-resistant breast cancer cells

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2015.3239

    VGSC knockdown by siRNA transfection. (A) pII cells were seeded into 12-well plates, allowed to attach overnight and then either transfected with a scrambled sequence (open bar) or SCN5A siRNA (solid bar). RNA was extracted from the cells, converted to cDNA and PCR amplified. Ct values were converted to ratios as described in Materials and methods. Histobars represent mean ± SEM of 3 independent determinations. * Significant difference from control with p=0.026. (B and C) Cells were seeded into a μ-dish 35mm, high and incubated at 37°C/5% CO 2 for 24 h, then transfected with scrambled sequence (B) or SCN5A siRNA (C). After 48 h, cells were fixed and stained with Na v 1.5 antibody (red), phallotoxin (green) or DAPI (blue).
    Figure Legend Snippet: VGSC knockdown by siRNA transfection. (A) pII cells were seeded into 12-well plates, allowed to attach overnight and then either transfected with a scrambled sequence (open bar) or SCN5A siRNA (solid bar). RNA was extracted from the cells, converted to cDNA and PCR amplified. Ct values were converted to ratios as described in Materials and methods. Histobars represent mean ± SEM of 3 independent determinations. * Significant difference from control with p=0.026. (B and C) Cells were seeded into a μ-dish 35mm, high and incubated at 37°C/5% CO 2 for 24 h, then transfected with scrambled sequence (B) or SCN5A siRNA (C). After 48 h, cells were fixed and stained with Na v 1.5 antibody (red), phallotoxin (green) or DAPI (blue).

    Techniques Used: Transfection, Sequencing, Polymerase Chain Reaction, Amplification, Incubation, Staining

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    Agarose Gel Electrophoresis:

    Article Title: Gene Capture by Helitron Transposons Reshuffles the Transcriptome of Maize
    Article Snippet: .. The amplified PCR products were resolved on 1% agarose gels, excised, and purified using DNA agarose gel purification kit, QIAquick Gel Extraction kit (Qiagen). .. The purified DNA was cloned and sequenced in both directions by either ABI Prism Dye Terminator sequencing protocol provided by Applied Biosystem (Foster City, CA) or done by the University of Florida Interdisciplinary Center for Biotechnology Research DNA Sequencing Core Laboratory.

    Pull Down Assay:

    Article Title: LncRNA MAGI2-AS3 Overexpression Sensitizes Esophageal Cancer Cells to Irradiation Through Down-Regulation of HOXB7 via EZH2
    Article Snippet: .. RNA Pull-Down Assay T7 RNA polymerase (Ambion, United States) was used to transcribe the MAGI2-AS3 fragment in vitro , which were then treated with RNeasy Plus Mini kit (Qiagen, Germany), DNase I (Qiagen, Germany), and purified by RNeasy Mini Kit. .. The 3′end of the purified RNA was biotinylated with a biotin RNA labeling mixture (Ambion, United States).

    Polymerase Chain Reaction:

    Article Title: TLR2 Deficiency Exacerbates Imiquimod-Induced Psoriasis-Like Skin Inflammation through Decrease in Regulatory T Cells and Impaired IL-10 Production
    Article Snippet: .. RNA Isolation and Quantitative Reverse-Transcription PCR Analysis RNA was obtained from the back skin with RNeasy Fibrous Tissue Mini Kit (QIAGEN, Valencia, CA, USA). .. Complementary DNA was synthesized using Rever Tra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan).

    Article Title: Requisite Chromatin Remodeling for Myeloid and Erythroid Lineage Differentiation from Erythromyeloid Progenitors
    Article Snippet: .. Quantitative real-time reverse transcription PCR (qRT-PCR)Total RNA from YS was prepared with RNeasy Micro/Mini Kit (QIAGEN), and reverse-transcribed into cDNA with qScript cDNA SuperMix (101414–106, Quanta) according to the manufacturer’s protocol. .. Gene expression was measured by quantitative real-time PCR with primers indicated in .

    Article Title: Gene Capture by Helitron Transposons Reshuffles the Transcriptome of Maize
    Article Snippet: .. The amplified PCR products were resolved on 1% agarose gels, excised, and purified using DNA agarose gel purification kit, QIAquick Gel Extraction kit (Qiagen). .. The purified DNA was cloned and sequenced in both directions by either ABI Prism Dye Terminator sequencing protocol provided by Applied Biosystem (Foster City, CA) or done by the University of Florida Interdisciplinary Center for Biotechnology Research DNA Sequencing Core Laboratory.

    Isolation:

    Article Title: TLR2 Deficiency Exacerbates Imiquimod-Induced Psoriasis-Like Skin Inflammation through Decrease in Regulatory T Cells and Impaired IL-10 Production
    Article Snippet: .. RNA Isolation and Quantitative Reverse-Transcription PCR Analysis RNA was obtained from the back skin with RNeasy Fibrous Tissue Mini Kit (QIAGEN, Valencia, CA, USA). .. Complementary DNA was synthesized using Rever Tra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan).

    Quantitative RT-PCR:

    Article Title: Changes in microRNA Expression in the Cochlear Nucleus and Inferior Colliculus after Acute Noise-Induced Hearing Loss
    Article Snippet: .. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Using an miScript® II RT kit (Qiagen, Hilden, Germany), 2 µg of RNA was mixed with reverse-transcription master mix and incubated for 60 min at 37 °C. .. To inactivate the miScript reverse transcriptase, the mixture was incubated for 5 min at 95 °C and then placed on ice.

    Purification:

    Article Title: LncRNA MAGI2-AS3 Overexpression Sensitizes Esophageal Cancer Cells to Irradiation Through Down-Regulation of HOXB7 via EZH2
    Article Snippet: .. RNA Pull-Down Assay T7 RNA polymerase (Ambion, United States) was used to transcribe the MAGI2-AS3 fragment in vitro , which were then treated with RNeasy Plus Mini kit (Qiagen, Germany), DNase I (Qiagen, Germany), and purified by RNeasy Mini Kit. .. The 3′end of the purified RNA was biotinylated with a biotin RNA labeling mixture (Ambion, United States).

    Article Title: Gene Capture by Helitron Transposons Reshuffles the Transcriptome of Maize
    Article Snippet: .. The amplified PCR products were resolved on 1% agarose gels, excised, and purified using DNA agarose gel purification kit, QIAquick Gel Extraction kit (Qiagen). .. The purified DNA was cloned and sequenced in both directions by either ABI Prism Dye Terminator sequencing protocol provided by Applied Biosystem (Foster City, CA) or done by the University of Florida Interdisciplinary Center for Biotechnology Research DNA Sequencing Core Laboratory.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Cnp Promoter-Driven Sustained ERK1/2 Activation Increases B-Cell Activation and Suppresses Experimental Autoimmune Encephalomyelitis
    Article Snippet: .. Quantitative Reverse Transcription Polymerase Chain Reaction Stimulated B-cells were collected from individual wells on 96-well plates and centrifuged at 450 g for 5 min. Supernatant was removed and cells resuspended in 350 uL of RLT buffer from the Qiagen RNeasy Mini Prep kit (74104, Qiagen), then frozen at -80°C for a minimum of 24 hrs. before additional processing. .. After thawing on ice, samples were prepared with the QiaShredder columns (79656, Qiagen) for homogenization followed by isolation with the Qiagen RNA Mini Prep kit.

    Article Title: Changes in microRNA Expression in the Cochlear Nucleus and Inferior Colliculus after Acute Noise-Induced Hearing Loss
    Article Snippet: .. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Using an miScript® II RT kit (Qiagen, Hilden, Germany), 2 µg of RNA was mixed with reverse-transcription master mix and incubated for 60 min at 37 °C. .. To inactivate the miScript reverse transcriptase, the mixture was incubated for 5 min at 95 °C and then placed on ice.

    Modification:

    Article Title: Putative degraders of low‐density polyethylene‐derived compounds are ubiquitous members of plastic‐associated bacterial communities in the marine environment
    Article Snippet: .. DNA extraction and 16S rRNA sequencing DNA extraction of all samples was performed using the modified bead‐beating approach in combination with the Puregene Tissue DNA extraction kit (Qiagen, Valencia, CA) (Debeljak et al., ). ..

    Incubation:

    Article Title: Changes in microRNA Expression in the Cochlear Nucleus and Inferior Colliculus after Acute Noise-Induced Hearing Loss
    Article Snippet: .. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Using an miScript® II RT kit (Qiagen, Hilden, Germany), 2 µg of RNA was mixed with reverse-transcription master mix and incubated for 60 min at 37 °C. .. To inactivate the miScript reverse transcriptase, the mixture was incubated for 5 min at 95 °C and then placed on ice.

    Sequencing:

    Article Title: Putative degraders of low‐density polyethylene‐derived compounds are ubiquitous members of plastic‐associated bacterial communities in the marine environment
    Article Snippet: .. DNA extraction and 16S rRNA sequencing DNA extraction of all samples was performed using the modified bead‐beating approach in combination with the Puregene Tissue DNA extraction kit (Qiagen, Valencia, CA) (Debeljak et al., ). ..

    Gel Extraction:

    Article Title: Gene Capture by Helitron Transposons Reshuffles the Transcriptome of Maize
    Article Snippet: .. The amplified PCR products were resolved on 1% agarose gels, excised, and purified using DNA agarose gel purification kit, QIAquick Gel Extraction kit (Qiagen). .. The purified DNA was cloned and sequenced in both directions by either ABI Prism Dye Terminator sequencing protocol provided by Applied Biosystem (Foster City, CA) or done by the University of Florida Interdisciplinary Center for Biotechnology Research DNA Sequencing Core Laboratory.

    In Vitro:

    Article Title: LncRNA MAGI2-AS3 Overexpression Sensitizes Esophageal Cancer Cells to Irradiation Through Down-Regulation of HOXB7 via EZH2
    Article Snippet: .. RNA Pull-Down Assay T7 RNA polymerase (Ambion, United States) was used to transcribe the MAGI2-AS3 fragment in vitro , which were then treated with RNeasy Plus Mini kit (Qiagen, Germany), DNase I (Qiagen, Germany), and purified by RNeasy Mini Kit. .. The 3′end of the purified RNA was biotinylated with a biotin RNA labeling mixture (Ambion, United States).

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    Qiagen qiaquik gel extraction protocol
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