dna  (Thermo Fisher)


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    KingFisher Cell and Tissue DNA Kit
    Description:
    Rapidly purify high-quality, reproducible DNA from cell and tissue samples, as well as cultured bacteria, with the Thermo Scientific KingFisher Cell and Tissue DNA Kit. This unique nucleic acid purification workflow provides an optimized, high-throughput method for extreme flexibility.
    Catalog Number:
    97030196
    Price:
    None
    Applications:
    DNA & RNA Purification & Analysis|Automated Nucleic Acid Purification
    Size:
    Each
    Category:
    Instruments and Equipment, DNA⁄RNA Purification Instruments & Accessories, DNA⁄RNA Purification Instruments
    Score:
    85
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    Structured Review

    Thermo Fisher dna
    APOL1 polymorphism regulates HIV-1 persistence in CIHPs. a Statistical histogram graph showing HIV-1 accumulation in CIHPs transiently transfected with a APOL1-WT expressing vector compared to the control vector (Ctrl) following a time course incubation (3 and 6 h) with the virus. Results are expressed as relative fold change of HIV-1 <t>DNA</t> RU5 in APOL1-WT transfected cells compared to Ctrl and normalized to <t>GAPDH</t> gene ( N = 4). b Time-course experiments showing the HIV-1 accumulation in CIHPs stable transfected with the control vector (Ctrl) or the specific APOL1-WT or APOL1-G1/G2 expressing vectors. Results are expressed as relative fold change of HIV-1 DNA RU5 in HIV-1 incubated CIHPs versus non HIV-1 treated cells and normalized to GAPDH gene ( N = 4). c Rescue of infectious HIV-1 by CD4 pos T lymphocytes co-cultured in time-course experiments (1, 3, 5 and 7 days) with HIV-1 pulsed CIHPs stable transfected with APOL1-WT or APOL1-G1 or APOL1-G2 compared with cells transfected with control vector (Ctrl). Since HIV-1 internalization in human podocytes is characterized by an abortive HIV-1 infection, cell lysates were collected and analyzed by qPCR for HIV-1 Gag gene not present in podocytes and indicative of a productive infection in CD4 pos T cells. Results are expressed as fold increased of HIV-1 DNA Gag gene in PBMC co-cultured with HIV-1-pulsed CIHPs transfected with APOL1-WT or APOL1-G1 or APOL1-G2 or an empty vector (Ctrl) compared to their HIV-1 untreated counterparts and normalized to the amount of GAPDH gene ( N = 4). d Statistical histogram graph showing TFEB gene expression in CIHPs transiently transfected with control vector (Ctrl) or APOL1-WT or APOL1-G1 or APOL1-G2 expressing vector. Results are expressed as fold change of transfected cells compared to Ctrl and normalized to the expression of GAPDH gene ( N = 4). P values: *
    Rapidly purify high-quality, reproducible DNA from cell and tissue samples, as well as cultured bacteria, with the Thermo Scientific KingFisher Cell and Tissue DNA Kit. This unique nucleic acid purification workflow provides an optimized, high-throughput method for extreme flexibility.
    https://www.bioz.com/result/dna/product/Thermo Fisher
    Average 98 stars, based on 0 article reviews
    Price from $9.99 to $1999.99
    dna - by Bioz Stars, 2019-12
    98/100 stars

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    Images

    1) Product Images from "Impact of APOL1 polymorphism and IL-1β priming in the entry and persistence of HIV-1 in human podocytes"

    Article Title: Impact of APOL1 polymorphism and IL-1β priming in the entry and persistence of HIV-1 in human podocytes

    Journal: Retrovirology

    doi: 10.1186/s12977-016-0296-3

    APOL1 polymorphism regulates HIV-1 persistence in CIHPs. a Statistical histogram graph showing HIV-1 accumulation in CIHPs transiently transfected with a APOL1-WT expressing vector compared to the control vector (Ctrl) following a time course incubation (3 and 6 h) with the virus. Results are expressed as relative fold change of HIV-1 DNA RU5 in APOL1-WT transfected cells compared to Ctrl and normalized to GAPDH gene ( N = 4). b Time-course experiments showing the HIV-1 accumulation in CIHPs stable transfected with the control vector (Ctrl) or the specific APOL1-WT or APOL1-G1/G2 expressing vectors. Results are expressed as relative fold change of HIV-1 DNA RU5 in HIV-1 incubated CIHPs versus non HIV-1 treated cells and normalized to GAPDH gene ( N = 4). c Rescue of infectious HIV-1 by CD4 pos T lymphocytes co-cultured in time-course experiments (1, 3, 5 and 7 days) with HIV-1 pulsed CIHPs stable transfected with APOL1-WT or APOL1-G1 or APOL1-G2 compared with cells transfected with control vector (Ctrl). Since HIV-1 internalization in human podocytes is characterized by an abortive HIV-1 infection, cell lysates were collected and analyzed by qPCR for HIV-1 Gag gene not present in podocytes and indicative of a productive infection in CD4 pos T cells. Results are expressed as fold increased of HIV-1 DNA Gag gene in PBMC co-cultured with HIV-1-pulsed CIHPs transfected with APOL1-WT or APOL1-G1 or APOL1-G2 or an empty vector (Ctrl) compared to their HIV-1 untreated counterparts and normalized to the amount of GAPDH gene ( N = 4). d Statistical histogram graph showing TFEB gene expression in CIHPs transiently transfected with control vector (Ctrl) or APOL1-WT or APOL1-G1 or APOL1-G2 expressing vector. Results are expressed as fold change of transfected cells compared to Ctrl and normalized to the expression of GAPDH gene ( N = 4). P values: *
    Figure Legend Snippet: APOL1 polymorphism regulates HIV-1 persistence in CIHPs. a Statistical histogram graph showing HIV-1 accumulation in CIHPs transiently transfected with a APOL1-WT expressing vector compared to the control vector (Ctrl) following a time course incubation (3 and 6 h) with the virus. Results are expressed as relative fold change of HIV-1 DNA RU5 in APOL1-WT transfected cells compared to Ctrl and normalized to GAPDH gene ( N = 4). b Time-course experiments showing the HIV-1 accumulation in CIHPs stable transfected with the control vector (Ctrl) or the specific APOL1-WT or APOL1-G1/G2 expressing vectors. Results are expressed as relative fold change of HIV-1 DNA RU5 in HIV-1 incubated CIHPs versus non HIV-1 treated cells and normalized to GAPDH gene ( N = 4). c Rescue of infectious HIV-1 by CD4 pos T lymphocytes co-cultured in time-course experiments (1, 3, 5 and 7 days) with HIV-1 pulsed CIHPs stable transfected with APOL1-WT or APOL1-G1 or APOL1-G2 compared with cells transfected with control vector (Ctrl). Since HIV-1 internalization in human podocytes is characterized by an abortive HIV-1 infection, cell lysates were collected and analyzed by qPCR for HIV-1 Gag gene not present in podocytes and indicative of a productive infection in CD4 pos T cells. Results are expressed as fold increased of HIV-1 DNA Gag gene in PBMC co-cultured with HIV-1-pulsed CIHPs transfected with APOL1-WT or APOL1-G1 or APOL1-G2 or an empty vector (Ctrl) compared to their HIV-1 untreated counterparts and normalized to the amount of GAPDH gene ( N = 4). d Statistical histogram graph showing TFEB gene expression in CIHPs transiently transfected with control vector (Ctrl) or APOL1-WT or APOL1-G1 or APOL1-G2 expressing vector. Results are expressed as fold change of transfected cells compared to Ctrl and normalized to the expression of GAPDH gene ( N = 4). P values: *

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Incubation, Cell Culture, Infection, Real-time Polymerase Chain Reaction

    APOL1 polymorphism affects IL-1β-dependent persistence of HIV-1 in CIHPs. a Time course experiments showing DC-SIGN gene expression in CIHPs after stimulation with rhIL-1β (25 ng/mL) compared to untreated cells (Ctrl) and normalized to GAPDH gene expression ( N = 3). b Statistical histogram graph showing DC-SIGN gene expression after 18 h of rhIL-1β (25 ng/mL) treatment in CIHPs stably transfected with either empty vector (Ctrl) or WT or APOL1 risk Vs. Results are expressed as relative fold change in rhIL-1β treated cells versus non treated cells (Mock) and normalized to GAPDH gene expression ( N = 3). c Statistical histogram graph showing HIV-1 accumulation after rhIL-1β stimulation in CIHPs stably transfected with the control vector (Ctrl) or the specific APOL1-WT or APOL1-G1/G2 expressing vectors. Results are expressed as relative fold change of HIV-1 DNA RU5 in HIV-1 incubated CIHPs versus non HIV-1-pulsed cells (Mock) and normalized to GAPDH gene ( N = 3). d Rescue of infectious HIV-1 by CD4 pos T lymphocytes co-cultured in time-course experiments (0, 5 and 7 days) with HIV-1 pulsed CIHPs stable transfected with control vector (Ctrl), APOL1-WT or APOL1-G1/G2 Vs and pretreated or not (Mock) with rhIL-1β. Results are expressed as fold increased of HIV-1 DNA Gag gene in PBMC co-cultured with HIV-1 pulsed CIHPs compared to their HIV-1 untreated counterparts and normalized to the amount of GAPDH gene ( N = 3). Horizontal lines represent Mock and rhIL - 1 β in the Ctrl sample depicting the fold change in HIV-1 replication. e APOL1 gene expression in CIHPs incubated with rhIL-1β (25 ng/mL) and subsequently treated with HIV-1 for 6 h compared to their counterparts incubated with rhIL-1β alone, HIV-1 alone or untreated (Ctrl) Results are expressed as relative fold change normalized to GAPDH gene expression ( N = 5). f Statistical histogram graph showing the APOL1 gene expression in CIHPs incubated with IL-1β (25 ng/mL) and subsequently treated with HIV-1 for 6 h either in pre absence (Ctrl) or in the presence of blocking anti-DC-SIGN mAb. Results are expressed as relative fold change in treated cells normalized to GAPDH gene expression ( N = 3). P values *
    Figure Legend Snippet: APOL1 polymorphism affects IL-1β-dependent persistence of HIV-1 in CIHPs. a Time course experiments showing DC-SIGN gene expression in CIHPs after stimulation with rhIL-1β (25 ng/mL) compared to untreated cells (Ctrl) and normalized to GAPDH gene expression ( N = 3). b Statistical histogram graph showing DC-SIGN gene expression after 18 h of rhIL-1β (25 ng/mL) treatment in CIHPs stably transfected with either empty vector (Ctrl) or WT or APOL1 risk Vs. Results are expressed as relative fold change in rhIL-1β treated cells versus non treated cells (Mock) and normalized to GAPDH gene expression ( N = 3). c Statistical histogram graph showing HIV-1 accumulation after rhIL-1β stimulation in CIHPs stably transfected with the control vector (Ctrl) or the specific APOL1-WT or APOL1-G1/G2 expressing vectors. Results are expressed as relative fold change of HIV-1 DNA RU5 in HIV-1 incubated CIHPs versus non HIV-1-pulsed cells (Mock) and normalized to GAPDH gene ( N = 3). d Rescue of infectious HIV-1 by CD4 pos T lymphocytes co-cultured in time-course experiments (0, 5 and 7 days) with HIV-1 pulsed CIHPs stable transfected with control vector (Ctrl), APOL1-WT or APOL1-G1/G2 Vs and pretreated or not (Mock) with rhIL-1β. Results are expressed as fold increased of HIV-1 DNA Gag gene in PBMC co-cultured with HIV-1 pulsed CIHPs compared to their HIV-1 untreated counterparts and normalized to the amount of GAPDH gene ( N = 3). Horizontal lines represent Mock and rhIL - 1 β in the Ctrl sample depicting the fold change in HIV-1 replication. e APOL1 gene expression in CIHPs incubated with rhIL-1β (25 ng/mL) and subsequently treated with HIV-1 for 6 h compared to their counterparts incubated with rhIL-1β alone, HIV-1 alone or untreated (Ctrl) Results are expressed as relative fold change normalized to GAPDH gene expression ( N = 5). f Statistical histogram graph showing the APOL1 gene expression in CIHPs incubated with IL-1β (25 ng/mL) and subsequently treated with HIV-1 for 6 h either in pre absence (Ctrl) or in the presence of blocking anti-DC-SIGN mAb. Results are expressed as relative fold change in treated cells normalized to GAPDH gene expression ( N = 3). P values *

    Techniques Used: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Incubation, Cell Culture, Blocking Assay

    IL-1β increases HIV-1 accumulation in CIHPs. a Time and b dose response of IL-1β gene expression in unstimulated (Ctrl) and rhIL-1β treated CIHPs. Results are expressed as relative fold change in rhIL-1β treated cells versus Ctrl and normalized to GAPDH gene expression ( N = 3). c Statistical histogram graph showing the IL-1β gene expression in CIHPs untreated (Ctrl), treated with rhIL-1β alone (25 ng/mL) for 16 h or incubated with HIV-1 either alone (6, 24 h) or with IL-1β (6, 24 h). Results are expressed as fold change of IL-1β gene expression in treated cells versus Ctrl and normalized to the GAPDH gene expression ( N = 5). d Statistical histogram graph showing the amount of IL-1β protein secretion by CIHPs primed with rhIL-1β (25 ng/mL) and subsequently incubated with HIV-1 for 6 h compared to their counterparts incubated with rhIL-1β alone, HIV-1 alone or untreated cells (Ctrl) ( N = 5). e Time course experiments showing HIV-1 accumulation in CIHPs either untreated (Ctrl) or pre-stimulated with rhIL-1β (25 ng/mL) for 16 h and followed by incubation with HIV-1 at different time points (3, 6, 24, 48, 72 h). Results are expressed as relative fold change of HIV-1 DNA RU5 in HIV-1 treated cells compared to their HIV-1 untreated counterparts for both experimental settings and normalized to GAPDH gene ( N = 5). f Statistical histogram graph showing the rescue of infectious HIV-1 by CD4 pos T lymphocytes co-cultured for 3 and 5 days with HIV-1-pulsed CIHPs either primed or none (Ctrl) with rhIL-1β. Since HIV-1 internalization in human podocytes is characterized by an abortive HIV-1 infection, cell lysates were collected and analyzed by qPCR for HIV-1 Gag gene not present in podocytes and indicative of a productive infection in CD4 pos T cells. Results are expressed as relative fold change of DNA HIV-1 Gag copies in rhIL-1β treated compared to Ctrl and normalized to DNA copies of GAPDH gene ( N = 5 ). P values: *
    Figure Legend Snippet: IL-1β increases HIV-1 accumulation in CIHPs. a Time and b dose response of IL-1β gene expression in unstimulated (Ctrl) and rhIL-1β treated CIHPs. Results are expressed as relative fold change in rhIL-1β treated cells versus Ctrl and normalized to GAPDH gene expression ( N = 3). c Statistical histogram graph showing the IL-1β gene expression in CIHPs untreated (Ctrl), treated with rhIL-1β alone (25 ng/mL) for 16 h or incubated with HIV-1 either alone (6, 24 h) or with IL-1β (6, 24 h). Results are expressed as fold change of IL-1β gene expression in treated cells versus Ctrl and normalized to the GAPDH gene expression ( N = 5). d Statistical histogram graph showing the amount of IL-1β protein secretion by CIHPs primed with rhIL-1β (25 ng/mL) and subsequently incubated with HIV-1 for 6 h compared to their counterparts incubated with rhIL-1β alone, HIV-1 alone or untreated cells (Ctrl) ( N = 5). e Time course experiments showing HIV-1 accumulation in CIHPs either untreated (Ctrl) or pre-stimulated with rhIL-1β (25 ng/mL) for 16 h and followed by incubation with HIV-1 at different time points (3, 6, 24, 48, 72 h). Results are expressed as relative fold change of HIV-1 DNA RU5 in HIV-1 treated cells compared to their HIV-1 untreated counterparts for both experimental settings and normalized to GAPDH gene ( N = 5). f Statistical histogram graph showing the rescue of infectious HIV-1 by CD4 pos T lymphocytes co-cultured for 3 and 5 days with HIV-1-pulsed CIHPs either primed or none (Ctrl) with rhIL-1β. Since HIV-1 internalization in human podocytes is characterized by an abortive HIV-1 infection, cell lysates were collected and analyzed by qPCR for HIV-1 Gag gene not present in podocytes and indicative of a productive infection in CD4 pos T cells. Results are expressed as relative fold change of DNA HIV-1 Gag copies in rhIL-1β treated compared to Ctrl and normalized to DNA copies of GAPDH gene ( N = 5 ). P values: *

    Techniques Used: Expressing, Incubation, Cell Culture, Infection, Real-time Polymerase Chain Reaction

    Increased expression of WT APOL1 inversely correlates to HIV-1 persistence in CIHPs. a Summary graph of statistical dot plots ( left panel ) showing the co-localization off WT APOL1 with Rab5, Rab7, EEA1 and LAMP1 as an average measured by Pearson’s Coefficient on data analyzed with the Olympus FLUOVIEW FV1000 confocal microscope software ( N = 3). Representative fluorescent microscopic images ( right panel ) showing the co-localization of WT APOL1 ( red ) with Rab5, Rab7, EEA1 and LAMP1 ( green ) marked vesicles and plasma membrane in CIHPs. Nucleuses are stained with DAPI in blue . b Statistical histogram graph showing the dose-dependent response of APOL1 gene expression after IFN-γ stimulation (16 h). Results are expressed as relative fold change in IFN-γ treated CIHPs versus untreated controls (Ctrl) and normalized to GAPDH gene expression ( N = 4). c Statistical histogram graph showing HIV-1 accumulation in CIHPs incubated for 16 h with IFN-γ (10 ng/mL) compared to their untreated counterparts (Ctrl). Results are expressed as relative fold change of HIV-1 DNA RU5 in IFN-γ treated cells versus Ctrl and normalized to GAPDH gene ( N = 4). P values *
    Figure Legend Snippet: Increased expression of WT APOL1 inversely correlates to HIV-1 persistence in CIHPs. a Summary graph of statistical dot plots ( left panel ) showing the co-localization off WT APOL1 with Rab5, Rab7, EEA1 and LAMP1 as an average measured by Pearson’s Coefficient on data analyzed with the Olympus FLUOVIEW FV1000 confocal microscope software ( N = 3). Representative fluorescent microscopic images ( right panel ) showing the co-localization of WT APOL1 ( red ) with Rab5, Rab7, EEA1 and LAMP1 ( green ) marked vesicles and plasma membrane in CIHPs. Nucleuses are stained with DAPI in blue . b Statistical histogram graph showing the dose-dependent response of APOL1 gene expression after IFN-γ stimulation (16 h). Results are expressed as relative fold change in IFN-γ treated CIHPs versus untreated controls (Ctrl) and normalized to GAPDH gene expression ( N = 4). c Statistical histogram graph showing HIV-1 accumulation in CIHPs incubated for 16 h with IFN-γ (10 ng/mL) compared to their untreated counterparts (Ctrl). Results are expressed as relative fold change of HIV-1 DNA RU5 in IFN-γ treated cells versus Ctrl and normalized to GAPDH gene ( N = 4). P values *

    Techniques Used: Expressing, Microscopy, Software, Staining, Incubation

    2) Product Images from "Absence of XMRV in Peripheral Blood Mononuclear Cells of ARV-Treatment Na?ve HIV-1 Infected and HIV-1/HCV Coinfected Individuals and Blood Donors"

    Article Title: Absence of XMRV in Peripheral Blood Mononuclear Cells of ARV-Treatment Na?ve HIV-1 Infected and HIV-1/HCV Coinfected Individuals and Blood Donors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031398

    Detecting murine DNA by IAP PCR. PCR products were analyzed on 1.5% agarose gels containing ethidium bromide. (A) Sensitivity of the IAP PCR assay was determined by performing PCRs on titrations of EL4 murine cell line DNA in a background of 200 ng LNCaP DNA. One murine cell equivalent (1 eq) indicates 6 pg of EL4 DNA. XMRV-infected LNCaP (iLNCaP) and uninfected LNCaP (uLNCaP) were included as controls. (B) Screening results for 17 HIV-1 + patient samples. Arrow points to sample 103219, which tested positive for XMRV by non-nested gag PCR. (m) 100 base pair molecular weight marker, (EL4) 6 pg of murine EL4 cell line DNA without a background of human DNA.
    Figure Legend Snippet: Detecting murine DNA by IAP PCR. PCR products were analyzed on 1.5% agarose gels containing ethidium bromide. (A) Sensitivity of the IAP PCR assay was determined by performing PCRs on titrations of EL4 murine cell line DNA in a background of 200 ng LNCaP DNA. One murine cell equivalent (1 eq) indicates 6 pg of EL4 DNA. XMRV-infected LNCaP (iLNCaP) and uninfected LNCaP (uLNCaP) were included as controls. (B) Screening results for 17 HIV-1 + patient samples. Arrow points to sample 103219, which tested positive for XMRV by non-nested gag PCR. (m) 100 base pair molecular weight marker, (EL4) 6 pg of murine EL4 cell line DNA without a background of human DNA.

    Techniques Used: Polymerase Chain Reaction, Infection, Molecular Weight, Marker

    Sensitivity analysis of XMRV PCR assays. PCR products were analyzed on agarose gels containing ethidium bromide. (A) Non-nested PCR assays targeting the XMRV env gene (top panel) and the gag gene (bottom panel), and (B) a nested PCR assay targeting the XMRV env gene were evaluated for their ability to detect either (A) provirus in XMRV-infected PNT1A cell DNA or (B) provirus in XMRV-infected LNCaP cell DNA diluted in uninfected cell DNA. Dilutions of infected cells in uninfected cells are indicated by ratios, i.e. 1∶10 4 indicates one infected cell diluted in 10 4 uninfected cells. (m) 100 base pair molecular weight marker, (H 2 O) water used in place of DNA template as a negative control, (u) uninfected PNT1A DNA used as template for negative control.
    Figure Legend Snippet: Sensitivity analysis of XMRV PCR assays. PCR products were analyzed on agarose gels containing ethidium bromide. (A) Non-nested PCR assays targeting the XMRV env gene (top panel) and the gag gene (bottom panel), and (B) a nested PCR assay targeting the XMRV env gene were evaluated for their ability to detect either (A) provirus in XMRV-infected PNT1A cell DNA or (B) provirus in XMRV-infected LNCaP cell DNA diluted in uninfected cell DNA. Dilutions of infected cells in uninfected cells are indicated by ratios, i.e. 1∶10 4 indicates one infected cell diluted in 10 4 uninfected cells. (m) 100 base pair molecular weight marker, (H 2 O) water used in place of DNA template as a negative control, (u) uninfected PNT1A DNA used as template for negative control.

    Techniques Used: Polymerase Chain Reaction, Nested PCR, Infection, Molecular Weight, Marker, Negative Control

    Screening for XMRV in patient PBMCs by PCR. PCR products were analyzed on agarose gels containing ethidium bromide. (A) Representative gels for non-nested env (top panel) and non-nested gag (bottom panel) PCRs are shown containing a set of three replicates for each of 5 HIV-1 + patient samples. A yellow arrow indicates the sole PCR band, from patient 103219, found to be comprised of XMRV DNA by sequencing. (B) A representative gel for nested env PCR is shown for the same 5 HIV-1 + patient samples depicted in (A). Vertical black arrows in (A) and (B) indicate lanes from patient 103219 containing either (A, bottom panel) a band comprised of XMRV sequence or (B) a band of the expected mobility for the target sequence. (m) 100 base pair molecular weight marker, (1∶10 4 ) DNA from one infected cell diluted in DNA from 10 4 uninfected cells used as template for positive control.
    Figure Legend Snippet: Screening for XMRV in patient PBMCs by PCR. PCR products were analyzed on agarose gels containing ethidium bromide. (A) Representative gels for non-nested env (top panel) and non-nested gag (bottom panel) PCRs are shown containing a set of three replicates for each of 5 HIV-1 + patient samples. A yellow arrow indicates the sole PCR band, from patient 103219, found to be comprised of XMRV DNA by sequencing. (B) A representative gel for nested env PCR is shown for the same 5 HIV-1 + patient samples depicted in (A). Vertical black arrows in (A) and (B) indicate lanes from patient 103219 containing either (A, bottom panel) a band comprised of XMRV sequence or (B) a band of the expected mobility for the target sequence. (m) 100 base pair molecular weight marker, (1∶10 4 ) DNA from one infected cell diluted in DNA from 10 4 uninfected cells used as template for positive control.

    Techniques Used: Polymerase Chain Reaction, DNA Sequencing, Sequencing, Molecular Weight, Marker, Infection, Positive Control

    3) Product Images from "Effect of black tea extract on herpes simplex virus-1 infection of cultured cells"

    Article Title: Effect of black tea extract on herpes simplex virus-1 infection of cultured cells

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/1472-6882-13-139

    Gel electrophoresis of PCR products. A . PCR products extracted from HSV-1 infected A549 cells either treated with 1.4 mM BTE (columns 2–4) or untreated (columns 5–7). Column 1 contains the DNA ladder, with visible bands identified to the left in base pairs (bp). Columns 2 and 5, 3 and 6, 4 and 7 contain DNA amplified with primers for the HSV-1 gD, GFP and pUL46 genes, respectively. B . Gel electrophoresis of HSV-1 GFP PCR products extracted from HSV-1 infected Vero cells either untreated (column 2) or treated with 0.14 μM, 1.4 μM, or 1.4 mM BTE (columns 3 – 5, respectively). Column 1 contains the DNA ladder, with visible bands identified to the left in base pairs (bp).
    Figure Legend Snippet: Gel electrophoresis of PCR products. A . PCR products extracted from HSV-1 infected A549 cells either treated with 1.4 mM BTE (columns 2–4) or untreated (columns 5–7). Column 1 contains the DNA ladder, with visible bands identified to the left in base pairs (bp). Columns 2 and 5, 3 and 6, 4 and 7 contain DNA amplified with primers for the HSV-1 gD, GFP and pUL46 genes, respectively. B . Gel electrophoresis of HSV-1 GFP PCR products extracted from HSV-1 infected Vero cells either untreated (column 2) or treated with 0.14 μM, 1.4 μM, or 1.4 mM BTE (columns 3 – 5, respectively). Column 1 contains the DNA ladder, with visible bands identified to the left in base pairs (bp).

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Infection, Amplification

    4) Product Images from "Fenamate NSAIDs inhibit the NLRP3 inflammasome and protect against Alzheimer's disease in rodent models"

    Article Title: Fenamate NSAIDs inhibit the NLRP3 inflammasome and protect against Alzheimer's disease in rodent models

    Journal: Nature Communications

    doi: 10.1038/ncomms12504

    Fenamate NSAIDs inhibit IL-1β processing and release. ( a ) iBMDMs were primed with LPS (1 μg ml −1 , 2 h) then pre-treated with NSAID at indicated concentration before stimulating with ATP (5 mM, 1 h). ( b – d ) Murine primary BMDMs from WT ( b ) or NLRP3 −/− ( c , d ) mice were primed with LPS (1 μg ml −1 , 4 h) and pre-treated with NSAID (100 μM, 15 min) before stimulating with monosodium urate (MSU) crystals (250 μg ml −1 , 4 h) ( b ), transfected ultrapure flagellin from Salmonella typhimurium (1 ng per 1,000 cells, 2 h) ( c ), or transfected DNA (0.66 ng per 1,000 cells, 4 h) ( d ). Supernatants were analysed by ELISA. Data are presented as mean % IL-1β release versus vehicle (DMSO) control+s.e.m ( n =3 or 4). NS, not significantly different, * P
    Figure Legend Snippet: Fenamate NSAIDs inhibit IL-1β processing and release. ( a ) iBMDMs were primed with LPS (1 μg ml −1 , 2 h) then pre-treated with NSAID at indicated concentration before stimulating with ATP (5 mM, 1 h). ( b – d ) Murine primary BMDMs from WT ( b ) or NLRP3 −/− ( c , d ) mice were primed with LPS (1 μg ml −1 , 4 h) and pre-treated with NSAID (100 μM, 15 min) before stimulating with monosodium urate (MSU) crystals (250 μg ml −1 , 4 h) ( b ), transfected ultrapure flagellin from Salmonella typhimurium (1 ng per 1,000 cells, 2 h) ( c ), or transfected DNA (0.66 ng per 1,000 cells, 4 h) ( d ). Supernatants were analysed by ELISA. Data are presented as mean % IL-1β release versus vehicle (DMSO) control+s.e.m ( n =3 or 4). NS, not significantly different, * P

    Techniques Used: Concentration Assay, Mouse Assay, Transfection, Enzyme-linked Immunosorbent Assay

    5) Product Images from "DNA Methyltransferase Deficiency Modifies Cancer Susceptibility in Mice Lacking DNA Mismatch Repair"

    Article Title: DNA Methyltransferase Deficiency Modifies Cancer Susceptibility in Mice Lacking DNA Mismatch Repair

    Journal:

    doi: 10.1128/MCB.22.9.2906-2917.2002

    Hypomorphic alleles of the mouse Dnmt1 gene. (A) Schematic map (drawn approximately to scale) showing the positions of the Dnmt1R and Dnmt1N mutations in the 5′ region of the Dnmt1 genomic locus. The first four exons shown are represented as numbered black boxes, and intervening introns are represented as solid lines. The 320-bp insertion ( , ) containing three copies of the lac operator ( lacO ) sequence from E. coli is located just upstream of an Eco RV site (R) in intron 3 and is depicted as an open box (O). H, Hin dIII. The Dnmt1N allele, which has been described previously ( , ), contains a neomycin cassette that replaces a 900-bp Nae I (N) fragment. This mutation deletes part of exon 4 and causes an almost full disruption of Dnmt1 expression. (B) Dnmt1 expression in mutant mice as determined by real-time RT-PCR analysis. RNA was isolated from two thymus tissues of each genotype, and expression was normalized to expression of Gapdh , histone H4 , and Pcna . The bars represent the mean value obtained for each of the normalized measurements shown on a relative scale. The error bars represent the standard error of the mean. The slope of the solid triangle below the graph schematically represents the relative Dnmt1 expression for each genotype. (C) DNA methylation of centromeric minor satellite repeats in Dnmt1 mutant mice. Genomic DNA from tail biopsies of Mlh1−/− mice was digested with methylation-sensitive Hpa II (H) or with a methylation-insensitive isoschizomer, Msp I (M), as a control and then probed with a fragment derived from pMR150 . Results for DNA from a Dnmt1C/C complete-knockout embryonic stem cell line are shown as a control . Lower-molecular-weight bands in the Hpa II lanes indicate DNA hypomethylation. The slope of the solid triangle above the gels schematically represents the relative Dnmt1 expression for each genotype.
    Figure Legend Snippet: Hypomorphic alleles of the mouse Dnmt1 gene. (A) Schematic map (drawn approximately to scale) showing the positions of the Dnmt1R and Dnmt1N mutations in the 5′ region of the Dnmt1 genomic locus. The first four exons shown are represented as numbered black boxes, and intervening introns are represented as solid lines. The 320-bp insertion ( , ) containing three copies of the lac operator ( lacO ) sequence from E. coli is located just upstream of an Eco RV site (R) in intron 3 and is depicted as an open box (O). H, Hin dIII. The Dnmt1N allele, which has been described previously ( , ), contains a neomycin cassette that replaces a 900-bp Nae I (N) fragment. This mutation deletes part of exon 4 and causes an almost full disruption of Dnmt1 expression. (B) Dnmt1 expression in mutant mice as determined by real-time RT-PCR analysis. RNA was isolated from two thymus tissues of each genotype, and expression was normalized to expression of Gapdh , histone H4 , and Pcna . The bars represent the mean value obtained for each of the normalized measurements shown on a relative scale. The error bars represent the standard error of the mean. The slope of the solid triangle below the graph schematically represents the relative Dnmt1 expression for each genotype. (C) DNA methylation of centromeric minor satellite repeats in Dnmt1 mutant mice. Genomic DNA from tail biopsies of Mlh1−/− mice was digested with methylation-sensitive Hpa II (H) or with a methylation-insensitive isoschizomer, Msp I (M), as a control and then probed with a fragment derived from pMR150 . Results for DNA from a Dnmt1C/C complete-knockout embryonic stem cell line are shown as a control . Lower-molecular-weight bands in the Hpa II lanes indicate DNA hypomethylation. The slope of the solid triangle above the gels schematically represents the relative Dnmt1 expression for each genotype.

    Techniques Used: Sequencing, Mutagenesis, Expressing, Mouse Assay, Quantitative RT-PCR, Isolation, DNA Methylation Assay, Methylation, Derivative Assay, Knock-Out, Molecular Weight

    6) Product Images from "A FRET-Based Real-Time PCR Assay to Identify the Main Causal Agents of New World Tegumentary Leishmaniasis"

    Article Title: A FRET-Based Real-Time PCR Assay to Identify the Main Causal Agents of New World Tegumentary Leishmaniasis

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001956

    Melting curve analysis for the MPI -based real-time PCR assay on Leishmania reference strains. Five nanograms of DNA (references) or 5 ul of nested-PCR products (samples) were used as template to run the assay. The species designation is given based on the Tm calculated for each melting peak (point in the x axis for the peak of the curve). FRET probes on L. ( L. ) amazonensis ( ama ) or L. ( L. ) mexicana ( mex ) DNA are melted at lower temperature (53°C) while on L. ( V. ) braziliensis ( bra ) DNA are melted at the highest temperature (74°C). This assay could not differentiate between L. ( V. ) guyanensis ( guy ) and L. ( V. ) panamensis ( pan ) due to their overlapping Tm (72°C). All assessed Leishmania species yielded a melting curve. A straight dotted line corresponds to the negative controls ( neg ), DNA free or parasite-free human DNA. inf = L. infantum ; lain = L. ( V. ) lainsoni ; per = L. ( V. ) peruviana .
    Figure Legend Snippet: Melting curve analysis for the MPI -based real-time PCR assay on Leishmania reference strains. Five nanograms of DNA (references) or 5 ul of nested-PCR products (samples) were used as template to run the assay. The species designation is given based on the Tm calculated for each melting peak (point in the x axis for the peak of the curve). FRET probes on L. ( L. ) amazonensis ( ama ) or L. ( L. ) mexicana ( mex ) DNA are melted at lower temperature (53°C) while on L. ( V. ) braziliensis ( bra ) DNA are melted at the highest temperature (74°C). This assay could not differentiate between L. ( V. ) guyanensis ( guy ) and L. ( V. ) panamensis ( pan ) due to their overlapping Tm (72°C). All assessed Leishmania species yielded a melting curve. A straight dotted line corresponds to the negative controls ( neg ), DNA free or parasite-free human DNA. inf = L. infantum ; lain = L. ( V. ) lainsoni ; per = L. ( V. ) peruviana .

    Techniques Used: Real-time Polymerase Chain Reaction, Nested PCR

    Melting curve analysis for the 6PGD -based real-time PCR assay on Leishmania reference strains. Five nanograms of DNA (references) or 5 ul of nested-PCR products (samples) were used as template to run the assay. The species designation is given based on the Tm calculated for each melting peak (point in the x axis for the peak of the curve). FRET probes on L. ( V. ) panamensis ( pan ) DNA are melted at the lowest temperature (56°C) while on L. ( V. ) braziliensis ( bra ) or L. ( V. ) peruviana ( per ) DNA are melted at the highest temperature (68°C). Due to their overlapping Tm , this assay could not differentiate these two species. L. ( L. ) amazonensis , L. ( L. ) mexicana and L. infantum did not yield a melting curve. A straight dotted line corresponds to the negative controls ( neg ), DNA free or parasite-free human DNA. guy = L. ( V. ) guyanensis ; lain = L. ( V. ) lainsoni .
    Figure Legend Snippet: Melting curve analysis for the 6PGD -based real-time PCR assay on Leishmania reference strains. Five nanograms of DNA (references) or 5 ul of nested-PCR products (samples) were used as template to run the assay. The species designation is given based on the Tm calculated for each melting peak (point in the x axis for the peak of the curve). FRET probes on L. ( V. ) panamensis ( pan ) DNA are melted at the lowest temperature (56°C) while on L. ( V. ) braziliensis ( bra ) or L. ( V. ) peruviana ( per ) DNA are melted at the highest temperature (68°C). Due to their overlapping Tm , this assay could not differentiate these two species. L. ( L. ) amazonensis , L. ( L. ) mexicana and L. infantum did not yield a melting curve. A straight dotted line corresponds to the negative controls ( neg ), DNA free or parasite-free human DNA. guy = L. ( V. ) guyanensis ; lain = L. ( V. ) lainsoni .

    Techniques Used: Real-time Polymerase Chain Reaction, Nested PCR

    7) Product Images from "Association of interferon gamma gene polymorphism and susceptibility to hepatitis C virus infection in Egyptian patients: A multicenter, family‐based study"

    Article Title: Association of interferon gamma gene polymorphism and susceptibility to hepatitis C virus infection in Egyptian patients: A multicenter, family‐based study

    Journal: JGH Open: An Open Access Journal of Gastroenterology and Hepatology

    doi: 10.1002/jgh3.12024

    +874 IFN‐γ amplification‐refractory mutation system‐polymerase chain reaction (ARMS‐PCR) gel. Part I: Ethidium bromide‐stained 2% agarose gel for single‐nucleotide polymorphism (SNP) IFN‐γ (+874 A/T). PCR‐ARMS product: (A) First well is a DNA marker of 100 bp. (B) Second and third wells are blank samples for forward 01 and forward 02 that has deionized water instead of the sample template. (C) Sample (1) has only two DNA bands for one well, one of them at 426 bp of human growth hormone (HGH) internal control and the other band at 261 bp of +874 A allele; however, the well of the T allele did not work, so these samples are negatives for the +874 T allele. (D) Sample (2) has one DNA band of each well at 426 bp of (HGH) internal control and only one band at 261 bp of +874 A allele, with the absence of T allele band, so these samples are negatives for the +874 T allele. (E) Samples (3,7,8) have two bands for each well, one of which is of 261 bp, indicating the presence of the +874 A allele or +874 T allele, so these samples are positives for A and T alleles. The second band is the internal control (HGH), which is of 426 bp. (F) Samples (4,5,6) have one DNA band of each well at 426 bp of (HGH) internal control and only one band at 261 bp of +874 T allele with the absence of A allele band, so these samples are negatives for the +874 A allele. Part II: IFN‐γ (+874 A/T) SNP using sequence‐based typing technique. Lane (a): Representative sequence chromatographs of IFN‐γ (+874T) intron 1 of homozygote sample TT. Lane (b): Representative sequence chromatographs of IFN‐γ (+874A) intron 1 of homozygote sample AA. Lane (c): Representative sequence chromatographs of IFN‐γ (+874T/A) intron 1 of heterozygote sample TA. IFN‐γ, interferon gamma.
    Figure Legend Snippet: +874 IFN‐γ amplification‐refractory mutation system‐polymerase chain reaction (ARMS‐PCR) gel. Part I: Ethidium bromide‐stained 2% agarose gel for single‐nucleotide polymorphism (SNP) IFN‐γ (+874 A/T). PCR‐ARMS product: (A) First well is a DNA marker of 100 bp. (B) Second and third wells are blank samples for forward 01 and forward 02 that has deionized water instead of the sample template. (C) Sample (1) has only two DNA bands for one well, one of them at 426 bp of human growth hormone (HGH) internal control and the other band at 261 bp of +874 A allele; however, the well of the T allele did not work, so these samples are negatives for the +874 T allele. (D) Sample (2) has one DNA band of each well at 426 bp of (HGH) internal control and only one band at 261 bp of +874 A allele, with the absence of T allele band, so these samples are negatives for the +874 T allele. (E) Samples (3,7,8) have two bands for each well, one of which is of 261 bp, indicating the presence of the +874 A allele or +874 T allele, so these samples are positives for A and T alleles. The second band is the internal control (HGH), which is of 426 bp. (F) Samples (4,5,6) have one DNA band of each well at 426 bp of (HGH) internal control and only one band at 261 bp of +874 T allele with the absence of A allele band, so these samples are negatives for the +874 A allele. Part II: IFN‐γ (+874 A/T) SNP using sequence‐based typing technique. Lane (a): Representative sequence chromatographs of IFN‐γ (+874T) intron 1 of homozygote sample TT. Lane (b): Representative sequence chromatographs of IFN‐γ (+874A) intron 1 of homozygote sample AA. Lane (c): Representative sequence chromatographs of IFN‐γ (+874T/A) intron 1 of heterozygote sample TA. IFN‐γ, interferon gamma.

    Techniques Used: Amplification, Mutagenesis, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Marker, Sequencing

    8) Product Images from ""

    Article Title:

    Journal:

    doi: 10.1074/jbc.M112.380949

    A HIF-1 binding site is located within nucleotides −278/−250 of the proximal hGH1 promoter region. A , alignment and comparison of human (nucleotides −327/−1) and mouse (nucleotides −355/−1) growth hormone gene proximal promoter regions is shown. Conserved TATA box and Pit-1 binding sites are indicated. The hGH but not the mGH promoter region contains a palindromic hexanucleotide ( bold ) that includes the core HBS at nucleotide position −263/−259 ( underlined ). Asterisks are used to indicate the boundaries of the −279/−250 hGHp-HBS fragment used for EMSA. B , EMSA was done with a combination of recombinant HIF-1α ( rHIF-1 α) and HIF-1β protein together with radiolabeled probes: hGHp-HBS, EPO-HBS (positive control), and RF1 (negative control), with sequences as indicated. A single complex was seen in the presence of recombinant protein with both hGHp-HBS and EPO-HBS probes by autoradiography but required prolonged exposure to detect the EPO-HBS-protein complex. Competition of the hGHp-HBS-protein complex with a 2.5-, 5-, and 50-fold molar excess of unlabeled hGHp-HBS and EPO-HBS oligonucleotide as well as a 50-fold molar excess of RF-1 oligonucleotides was also performed to assess affinity/specificity. Competition was detected with hGHp-HBS and EPO-HBS but was more evident with a 5-fold molar excess of unlabeled hGHp-HBS than EPO-HBS; no competition with RF1 was observed. C , protein immunoblotting was done to assess HIF-1α protein in nuclear extracts (100 μg) of primary pituitary cells treated with 15 n m insulin for 24 h. Nuclear proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoreactive proteins were detected by chemiluminescence. The HIF-1α protein band (∼120 kDa) is indicated by black arrowhead . Upstream stimulatory factor 1 ( USF-1 ; 43 kDa) was used as a loading control for nuclear proteins. D , a ChIP assay was performed with an anti-HIF1α antibody on chromatin isolated from primary pituitary cells treated with 15 n m insulin for 24 h. Binding events were calculated based on the signals obtained from the immunoprecipitated/input DNA amplification using specific primers to hGHp-HBS and the control untranscribed region ( Untr6 ) by qPCR. The results are expressed as relative mean change plus or minus S.E. of the mean compared with the control (0 n m insulin) Untr6 value, which is arbitrarily set to 1. Significant differences are indicated by *, p < 0.05.
    Figure Legend Snippet: A HIF-1 binding site is located within nucleotides −278/−250 of the proximal hGH1 promoter region. A , alignment and comparison of human (nucleotides −327/−1) and mouse (nucleotides −355/−1) growth hormone gene proximal promoter regions is shown. Conserved TATA box and Pit-1 binding sites are indicated. The hGH but not the mGH promoter region contains a palindromic hexanucleotide ( bold ) that includes the core HBS at nucleotide position −263/−259 ( underlined ). Asterisks are used to indicate the boundaries of the −279/−250 hGHp-HBS fragment used for EMSA. B , EMSA was done with a combination of recombinant HIF-1α ( rHIF-1 α) and HIF-1β protein together with radiolabeled probes: hGHp-HBS, EPO-HBS (positive control), and RF1 (negative control), with sequences as indicated. A single complex was seen in the presence of recombinant protein with both hGHp-HBS and EPO-HBS probes by autoradiography but required prolonged exposure to detect the EPO-HBS-protein complex. Competition of the hGHp-HBS-protein complex with a 2.5-, 5-, and 50-fold molar excess of unlabeled hGHp-HBS and EPO-HBS oligonucleotide as well as a 50-fold molar excess of RF-1 oligonucleotides was also performed to assess affinity/specificity. Competition was detected with hGHp-HBS and EPO-HBS but was more evident with a 5-fold molar excess of unlabeled hGHp-HBS than EPO-HBS; no competition with RF1 was observed. C , protein immunoblotting was done to assess HIF-1α protein in nuclear extracts (100 μg) of primary pituitary cells treated with 15 n m insulin for 24 h. Nuclear proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoreactive proteins were detected by chemiluminescence. The HIF-1α protein band (∼120 kDa) is indicated by black arrowhead . Upstream stimulatory factor 1 ( USF-1 ; 43 kDa) was used as a loading control for nuclear proteins. D , a ChIP assay was performed with an anti-HIF1α antibody on chromatin isolated from primary pituitary cells treated with 15 n m insulin for 24 h. Binding events were calculated based on the signals obtained from the immunoprecipitated/input DNA amplification using specific primers to hGHp-HBS and the control untranscribed region ( Untr6 ) by qPCR. The results are expressed as relative mean change plus or minus S.E. of the mean compared with the control (0 n m insulin) Untr6 value, which is arbitrarily set to 1. Significant differences are indicated by *, p < 0.05.

    Techniques Used: Binding Assay, Recombinant, Positive Control, Negative Control, Autoradiography, Polyacrylamide Gel Electrophoresis, Chromatin Immunoprecipitation, Isolation, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    9) Product Images from "HIV-1 capsids bind and exploit the kinesin-1 adaptor FEZ1 for inward movement to the nucleus"

    Article Title: HIV-1 capsids bind and exploit the kinesin-1 adaptor FEZ1 for inward movement to the nucleus

    Journal: Nature communications

    doi: 10.1038/ncomms7660

    Kinesin-1 regulates nuclear entry of HIV-1 DNA (a–g) Kinesin-1 depletion affects early HIV-1 infection regardless of the route of viral entry. NHDF (a–b), CHME3 (c–d,g) or Jurkat (e–f) cells were transfected with control (ctrl), Kif5A or Kif5B siRNAs. 48h post-transfection cells were infected with HIV-1-VSV-luc (a, c, e) or HIV-1-Ampho-luc (g) followed by measurements of luciferase activity in NHDF (a), CHME3 (c, g) or Jurkat (e) cells. (b, d and f). WB analysis demonstrated kinesin-1 depletion in samples from a, c and e using antibodies against kinesin-1 (Kif5A/B), Kif5B or β-actin (loading control). (h) Kinesin-1 knockdown does not inhibit VSV infection. NHDF cells treated with control, Kif5A or Kif5B siRNAs were infected 48h post-transfection with VSV at m.o.i. 10. 8h after infection cells were lysed and analyzed by WB using anti-VSV-G or anti-VSV-N antibodies. eIF4E was used as loading control. (i) Effects of kinesin-1 depletion on fusion of HIV-1 cores into the cytosol. NHDFs were treated with control, Kif5A or Kif5B siRNAs and then either mock infected (upper panels) or infected with HIV-1-VSV-luc containing BlaM-Vpr (lower panels). FACS analysis of cells showed ~14–18% shift from green (uncleaved CCF2) to blue (cleaved CCF2) cells in control and kinesin-1-depleted cultures. (j–l) Kinesin-1 regulates nuclear entry of HIV-1 DNA. (j) CHME3 cells treated with control (Ctrl), Kif5A or Kif5B siRNAs were infected with HIV-1-VSV-puro 48h post-transfection. Low molecular Hirt DNA was isolated 24h post-infection and levels of viral MSS-DNA, total viral DNA and 2-LTRs in samples were measured by qPCR using specific primers to MSS, puromycin or 2-LTRs, respectively. Copy numbers were calculated and normalized to input DNA in each sample. Data are presented as mean +/- SEM. (k–l) CHME3 cells were transfected with plasmids expressing either GFP or GFP-tagged dominant negative Kif5B (Kif5B-GFP-DN). 48h post-transfection cells were infected (k) and levels of MSS DNA, total DN and 2-LTRs were measured as described in j. or (l) cells were lysed in Laemmli buffer and analyzed by WB using anti-GFP antibody to detect GFP or dominant-negative GFP-Kif5 (Kif5B-GFP-DN). Molecular weight markers (in kDa) are shown to the right of WBs.
    Figure Legend Snippet: Kinesin-1 regulates nuclear entry of HIV-1 DNA (a–g) Kinesin-1 depletion affects early HIV-1 infection regardless of the route of viral entry. NHDF (a–b), CHME3 (c–d,g) or Jurkat (e–f) cells were transfected with control (ctrl), Kif5A or Kif5B siRNAs. 48h post-transfection cells were infected with HIV-1-VSV-luc (a, c, e) or HIV-1-Ampho-luc (g) followed by measurements of luciferase activity in NHDF (a), CHME3 (c, g) or Jurkat (e) cells. (b, d and f). WB analysis demonstrated kinesin-1 depletion in samples from a, c and e using antibodies against kinesin-1 (Kif5A/B), Kif5B or β-actin (loading control). (h) Kinesin-1 knockdown does not inhibit VSV infection. NHDF cells treated with control, Kif5A or Kif5B siRNAs were infected 48h post-transfection with VSV at m.o.i. 10. 8h after infection cells were lysed and analyzed by WB using anti-VSV-G or anti-VSV-N antibodies. eIF4E was used as loading control. (i) Effects of kinesin-1 depletion on fusion of HIV-1 cores into the cytosol. NHDFs were treated with control, Kif5A or Kif5B siRNAs and then either mock infected (upper panels) or infected with HIV-1-VSV-luc containing BlaM-Vpr (lower panels). FACS analysis of cells showed ~14–18% shift from green (uncleaved CCF2) to blue (cleaved CCF2) cells in control and kinesin-1-depleted cultures. (j–l) Kinesin-1 regulates nuclear entry of HIV-1 DNA. (j) CHME3 cells treated with control (Ctrl), Kif5A or Kif5B siRNAs were infected with HIV-1-VSV-puro 48h post-transfection. Low molecular Hirt DNA was isolated 24h post-infection and levels of viral MSS-DNA, total viral DNA and 2-LTRs in samples were measured by qPCR using specific primers to MSS, puromycin or 2-LTRs, respectively. Copy numbers were calculated and normalized to input DNA in each sample. Data are presented as mean +/- SEM. (k–l) CHME3 cells were transfected with plasmids expressing either GFP or GFP-tagged dominant negative Kif5B (Kif5B-GFP-DN). 48h post-transfection cells were infected (k) and levels of MSS DNA, total DN and 2-LTRs were measured as described in j. or (l) cells were lysed in Laemmli buffer and analyzed by WB using anti-GFP antibody to detect GFP or dominant-negative GFP-Kif5 (Kif5B-GFP-DN). Molecular weight markers (in kDa) are shown to the right of WBs.

    Techniques Used: Infection, Transfection, Luciferase, Activity Assay, Western Blot, FACS, Isolation, Real-time Polymerase Chain Reaction, Expressing, Dominant Negative Mutation, Molecular Weight

    10) Product Images from "Epigenetic regulation of lateralized fetal spinal gene expression underlies hemispheric asymmetries"

    Article Title: Epigenetic regulation of lateralized fetal spinal gene expression underlies hemispheric asymmetries

    Journal: eLife

    doi: 10.7554/eLife.22784

    Epigenetic regulation of gene expression asymmetries in human fetal spinal cord. ( A ) Asymmetrically expressed miRNA transcripts at 8, 10 and 12 weeks PC. The extent of expression asymmetries is measured in log 2 (fold change). Red bars show rightward asymmetrically expressed microRNA transcripts, blue bars show leftward asymmetrically expressed miRNA transcripts. ( B ) Number of CpG sites showing differential DNA methylation per chromosome, compared between the left and right spinal cord for 8 and 10 weeks PC. Depicted are only CpG sites with methylation asymmetries in both samples. Red bars represent the number of CpG sites that showed significantly higher DNA methylation on the right side, blue bars show the number of CpG sites that showed significantly more DNA methylation on the left side. ( C ) Percentage of differential DNA methylation in leftward (blue) and rightward (red) asymmetrically methylated CpG sites as a function of p-value. ( D ) Percentage of gene expression asymmetries on each chromosome at 8 weeks PC that can be explained by regulation via asymmetrically expressed miRNAs or asymmetric DNA methylation of CpG sites within and 1500 nucleotides upstream of the expressed genes. The source files of asymmetrically expressed miRNAs, asymmetrically expressed targets of miRNAs, enriched KEGG pathways and differentially methylated CpG sites are available in Figure 3—source data 1 , Figure 3—source data 2 , Figure 3—source data 3 , and Figure 3—source data 4 respectively. DOI: http://dx.doi.org/10.7554/eLife.22784.006 10.7554/eLife.22784.007 Asymmetrically expressed miRNAs per week. DOI: http://dx.doi.org/10.7554/eLife.22784.007 10.7554/eLife.22784.008 Asymmetrically expressed RNA targets of asymmetrically expressed miRNAs per week. DOI: http://dx.doi.org/10.7554/eLife.22784.008 10.7554/eLife.22784.009 Enriched KEGG pathways per week and side of the spinal cord. DOI: http://dx.doi.org/10.7554/eLife.22784.009 10.7554/eLife.22784.010 Asymmetrically methylated CpG sites per week and side of the spinal cord. DOI: http://dx.doi.org/10.7554/eLife.22784.010
    Figure Legend Snippet: Epigenetic regulation of gene expression asymmetries in human fetal spinal cord. ( A ) Asymmetrically expressed miRNA transcripts at 8, 10 and 12 weeks PC. The extent of expression asymmetries is measured in log 2 (fold change). Red bars show rightward asymmetrically expressed microRNA transcripts, blue bars show leftward asymmetrically expressed miRNA transcripts. ( B ) Number of CpG sites showing differential DNA methylation per chromosome, compared between the left and right spinal cord for 8 and 10 weeks PC. Depicted are only CpG sites with methylation asymmetries in both samples. Red bars represent the number of CpG sites that showed significantly higher DNA methylation on the right side, blue bars show the number of CpG sites that showed significantly more DNA methylation on the left side. ( C ) Percentage of differential DNA methylation in leftward (blue) and rightward (red) asymmetrically methylated CpG sites as a function of p-value. ( D ) Percentage of gene expression asymmetries on each chromosome at 8 weeks PC that can be explained by regulation via asymmetrically expressed miRNAs or asymmetric DNA methylation of CpG sites within and 1500 nucleotides upstream of the expressed genes. The source files of asymmetrically expressed miRNAs, asymmetrically expressed targets of miRNAs, enriched KEGG pathways and differentially methylated CpG sites are available in Figure 3—source data 1 , Figure 3—source data 2 , Figure 3—source data 3 , and Figure 3—source data 4 respectively. DOI: http://dx.doi.org/10.7554/eLife.22784.006 10.7554/eLife.22784.007 Asymmetrically expressed miRNAs per week. DOI: http://dx.doi.org/10.7554/eLife.22784.007 10.7554/eLife.22784.008 Asymmetrically expressed RNA targets of asymmetrically expressed miRNAs per week. DOI: http://dx.doi.org/10.7554/eLife.22784.008 10.7554/eLife.22784.009 Enriched KEGG pathways per week and side of the spinal cord. DOI: http://dx.doi.org/10.7554/eLife.22784.009 10.7554/eLife.22784.010 Asymmetrically methylated CpG sites per week and side of the spinal cord. DOI: http://dx.doi.org/10.7554/eLife.22784.010

    Techniques Used: Expressing, DNA Methylation Assay, Methylation

    11) Product Images from "Actin and myosin contribute to mammalian mitochondrial DNA maintenance"

    Article Title: Actin and myosin contribute to mammalian mitochondrial DNA maintenance

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr052

    β-Actin siRNA perturbs mtDNA mass, organization and mitochondrial morphology. ( A ) HOS cell DNA was analysed by Q-PCR at intervals after transfection of cells with an siRNA targeting β-actin. A 40% decrease in mtDNA copy number was observed 48 72 and 96 h after transfection, which was statistically significant only at the 96 h time point ( P = 0.0087); however, when results for the three time points were combined, the consistent decrease in mtDNA copy number was highly significant (*** P
    Figure Legend Snippet: β-Actin siRNA perturbs mtDNA mass, organization and mitochondrial morphology. ( A ) HOS cell DNA was analysed by Q-PCR at intervals after transfection of cells with an siRNA targeting β-actin. A 40% decrease in mtDNA copy number was observed 48 72 and 96 h after transfection, which was statistically significant only at the 96 h time point ( P = 0.0087); however, when results for the three time points were combined, the consistent decrease in mtDNA copy number was highly significant (*** P

    Techniques Used: Polymerase Chain Reaction, Transfection

    A protease-resistant population of β-actin and NM-IIA co-fractionates with human mitochondrial DNA. ( A ) Mitochondria isolated from HEK cells by differential and sucrose gradient centrifugation were treated without (−) or with (+) trypsin, prior to lysis and fractionation on an iodixanol gradient. Protein and DNA were recovered from the different fractions (15 in total) and analysed by immuno or Southern blotting. (Fractions 1–6 were blank in all cases and do not appear as part of the figure.) The DNA probe was specific for human mtDNA, whereas the antibodies applied were designed to detect the outer mitochondrial membrane protein TOM20, the cytoskeletal proteins β-actin, NM-IIA, β-tubullin and vimentin, the mitochondrial matrix chaperone HSP60, the mtDNA binding protein TFAM, the candidate nucleoid protein ATAD3, an inner mitochondrial membrane protein COX2 and CYC, which is located between the outer and inner mitochondrial membranes in the intermembrane space. (Another IMS protein AK2 showed an identical distribution to CYC, data not shown.) Boxes demarcate the fractions where the mtDNA was concentrated. The amount of protein present in the iodixanol gradient fractions containing mtDNA ( 8–10 ), where β-actin survived trypsin treatment, was lower than other fractions (14 and 15), where it did not, ( Supplementary Figure S4 ); and none of the protein recovered from iodixanol gradients was inherently trypsin-resistant ( Supplementary Figure S5 ). ( B ) β-actin, but not β-actin-like 2 is imported into mitochondria. In vitro synthesised human β-actin and β-actin-like 2, before and after incubation with isolated rat liver mitochondria, separated by 12% SDS–PAGE (‘Materials and Methods’).
    Figure Legend Snippet: A protease-resistant population of β-actin and NM-IIA co-fractionates with human mitochondrial DNA. ( A ) Mitochondria isolated from HEK cells by differential and sucrose gradient centrifugation were treated without (−) or with (+) trypsin, prior to lysis and fractionation on an iodixanol gradient. Protein and DNA were recovered from the different fractions (15 in total) and analysed by immuno or Southern blotting. (Fractions 1–6 were blank in all cases and do not appear as part of the figure.) The DNA probe was specific for human mtDNA, whereas the antibodies applied were designed to detect the outer mitochondrial membrane protein TOM20, the cytoskeletal proteins β-actin, NM-IIA, β-tubullin and vimentin, the mitochondrial matrix chaperone HSP60, the mtDNA binding protein TFAM, the candidate nucleoid protein ATAD3, an inner mitochondrial membrane protein COX2 and CYC, which is located between the outer and inner mitochondrial membranes in the intermembrane space. (Another IMS protein AK2 showed an identical distribution to CYC, data not shown.) Boxes demarcate the fractions where the mtDNA was concentrated. The amount of protein present in the iodixanol gradient fractions containing mtDNA ( 8–10 ), where β-actin survived trypsin treatment, was lower than other fractions (14 and 15), where it did not, ( Supplementary Figure S4 ); and none of the protein recovered from iodixanol gradients was inherently trypsin-resistant ( Supplementary Figure S5 ). ( B ) β-actin, but not β-actin-like 2 is imported into mitochondria. In vitro synthesised human β-actin and β-actin-like 2, before and after incubation with isolated rat liver mitochondria, separated by 12% SDS–PAGE (‘Materials and Methods’).

    Techniques Used: Isolation, Gradient Centrifugation, Lysis, Fractionation, Southern Blot, Binding Assay, In Vitro, Incubation, SDS Page

    Possible arrangement of intra-mitochondrial actomyosin in relation to mtDNA and nucleoid proteins. The most abundant proteins in enriched rat mtDNA preparations identified in this report ( Figure 1 C) and an earlier study ( 11 ) were TFAM, ATAD3, NIPSNAP1, NM-IIA, β-actin, TUFM and Prohibitin. Furthermore, TFAM, ATAD3, NM-IIA and β-actin co-fractionate with mitochondrial nucleoprotein complexes resolved on iodixanol gradients ( Figure 5 A and inset). Given the known cytoskeletal properties of NM-IIA and β-actin, they are more likely to provide structural support to, and facilitate movement of mitochondrial nucleoids, than function as DNA packaging proteins (dark grey hexagons). Although a substantial majority of ATAD3 co-fractionates with mtDNA ( Figure 5 A), ATAD3 is more widely distributed in the mitochondrial network than mtDNA ( 11 ), and so many molecules of ATAD3 must be linked indirectly to mtDNA, presumably via protein–protein interactions (light grey hexagons). The depiction of nucleoprotein complexes bound to the mitochondrial inner membrane is based on the earlier demonstration of a tight association between mitochondrial membranes and ATAD3 and mtDNA ( 11 ). This model does not exclude that in Figure 1 F, as cytoskeletal NM-IIA and β-actin may also contribute to mtDNA stability. Nor does the model obviate the need for other proteins, such as mitofilin, to provide structural support to mitochondria. OMM—outer mitochondrial membrane; IMM—inner mitochondrial membrane; NM-IIA—non-muscle myosin heavy chain IIA.
    Figure Legend Snippet: Possible arrangement of intra-mitochondrial actomyosin in relation to mtDNA and nucleoid proteins. The most abundant proteins in enriched rat mtDNA preparations identified in this report ( Figure 1 C) and an earlier study ( 11 ) were TFAM, ATAD3, NIPSNAP1, NM-IIA, β-actin, TUFM and Prohibitin. Furthermore, TFAM, ATAD3, NM-IIA and β-actin co-fractionate with mitochondrial nucleoprotein complexes resolved on iodixanol gradients ( Figure 5 A and inset). Given the known cytoskeletal properties of NM-IIA and β-actin, they are more likely to provide structural support to, and facilitate movement of mitochondrial nucleoids, than function as DNA packaging proteins (dark grey hexagons). Although a substantial majority of ATAD3 co-fractionates with mtDNA ( Figure 5 A), ATAD3 is more widely distributed in the mitochondrial network than mtDNA ( 11 ), and so many molecules of ATAD3 must be linked indirectly to mtDNA, presumably via protein–protein interactions (light grey hexagons). The depiction of nucleoprotein complexes bound to the mitochondrial inner membrane is based on the earlier demonstration of a tight association between mitochondrial membranes and ATAD3 and mtDNA ( 11 ). This model does not exclude that in Figure 1 F, as cytoskeletal NM-IIA and β-actin may also contribute to mtDNA stability. Nor does the model obviate the need for other proteins, such as mitofilin, to provide structural support to mitochondria. OMM—outer mitochondrial membrane; IMM—inner mitochondrial membrane; NM-IIA—non-muscle myosin heavy chain IIA.

    Techniques Used:

    12) Product Images from "Overexpression of Endoglin Modulates TGF-?1-Signalling Pathways in a Novel Immortalized Mouse Hepatic Stellate Cell Line"

    Article Title: Overexpression of Endoglin Modulates TGF-?1-Signalling Pathways in a Novel Immortalized Mouse Hepatic Stellate Cell Line

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056116

    Endogenous and heterologous expression of endoglin. ( A ) The indicated mouse cells were cultured in growth medium (see Fig. 2F) and cellular proteins were analyzed by Western blot using specific antibodies to Collagen I, Endoglin (mouse specific), vimentin, CTGF, α-SMA, Id2, and GFP. To validate antibody specificity proteins of COS-7 cells transiently transfected with a mouse endoglin cDNA (mEng) or empty pcDNA vector as a control (Co.) were analyzed in parallel. The experiment was repeated three times. ( B ) Col-GFP or COS-7 cells were transiently transfected with a cDNA coding for rat endoglin (rEng) using the indicated transfection reagents and DNA to reagent ratios. Cellular proteins of the corresponding cells were prepared and analyzed by Western blot using a specific antibody to rat endoglin (PPabE2, [24] . Membranes ( A, B ) were incubated with an antibody to β-actin to monitor equal protein loading.
    Figure Legend Snippet: Endogenous and heterologous expression of endoglin. ( A ) The indicated mouse cells were cultured in growth medium (see Fig. 2F) and cellular proteins were analyzed by Western blot using specific antibodies to Collagen I, Endoglin (mouse specific), vimentin, CTGF, α-SMA, Id2, and GFP. To validate antibody specificity proteins of COS-7 cells transiently transfected with a mouse endoglin cDNA (mEng) or empty pcDNA vector as a control (Co.) were analyzed in parallel. The experiment was repeated three times. ( B ) Col-GFP or COS-7 cells were transiently transfected with a cDNA coding for rat endoglin (rEng) using the indicated transfection reagents and DNA to reagent ratios. Cellular proteins of the corresponding cells were prepared and analyzed by Western blot using a specific antibody to rat endoglin (PPabE2, [24] . Membranes ( A, B ) were incubated with an antibody to β-actin to monitor equal protein loading.

    Techniques Used: Expressing, Cell Culture, Western Blot, Transfection, Plasmid Preparation, Incubation

    13) Product Images from "DNA Qualification Workflow for Next Generation Sequencing of Histopathological Samples"

    Article Title: DNA Qualification Workflow for Next Generation Sequencing of Histopathological Samples

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0062692

    Significant discrepancies in DNA quantification by NanoDrop and Qubit. A total of 100 ng of DNA based on NanoDrop (N, black bars) or Qubit (Q, grey bars) measurements was analyzed by electrophoresis on 0.8% agarose gel. Sample ID is indicated at the bottom. Lane L contains 200 ng of DNA as the reference for normalization. Densitometric analysis (bar chart) was performed by ImageJ software [20] . It is clear from the electrophoretic bands and their densitometric charts that NanoDrop overestimates DNA concentration.
    Figure Legend Snippet: Significant discrepancies in DNA quantification by NanoDrop and Qubit. A total of 100 ng of DNA based on NanoDrop (N, black bars) or Qubit (Q, grey bars) measurements was analyzed by electrophoresis on 0.8% agarose gel. Sample ID is indicated at the bottom. Lane L contains 200 ng of DNA as the reference for normalization. Densitometric analysis (bar chart) was performed by ImageJ software [20] . It is clear from the electrophoretic bands and their densitometric charts that NanoDrop overestimates DNA concentration.

    Techniques Used: Electrophoresis, Agarose Gel Electrophoresis, Software, Concentration Assay

    DNA qualification for next-generation sequencing applications. Effect of low-quality DNA on next-generation sequencing (NGS) workflow. Three FFPE samples were tested for construction of NGS amplicon libraries (Ion Torrent Ampliseq Cancer Panel). Qubit: 40 ng of DNA according to Qubit measurement were processed using the Ampliseq library construction kit (multiplex PCR amplification of 191 DNA regions from 46 cancer-related genes). NanoDrop: absorption spectra of samples showed different degrees of organic contamination (230 nm spike, A260/A230 ratio). Agilent: quality and quantity of the obtained libraries were evaluated by Agilent high sensitivity assay on-chip electrophoresis, where the library is represented by the large band between 150 and 200 bp. Fragments test: histogram showing length and abundance of produced sequences. Sample FFPE 5 did not produce a good library due to high organic contamination; this is revealed by the remarkable spike at 230 nm that concurs to the low 260/230 ratio, and explains the faint electrophoretic band and the almost flat fragments test histogram.
    Figure Legend Snippet: DNA qualification for next-generation sequencing applications. Effect of low-quality DNA on next-generation sequencing (NGS) workflow. Three FFPE samples were tested for construction of NGS amplicon libraries (Ion Torrent Ampliseq Cancer Panel). Qubit: 40 ng of DNA according to Qubit measurement were processed using the Ampliseq library construction kit (multiplex PCR amplification of 191 DNA regions from 46 cancer-related genes). NanoDrop: absorption spectra of samples showed different degrees of organic contamination (230 nm spike, A260/A230 ratio). Agilent: quality and quantity of the obtained libraries were evaluated by Agilent high sensitivity assay on-chip electrophoresis, where the library is represented by the large band between 150 and 200 bp. Fragments test: histogram showing length and abundance of produced sequences. Sample FFPE 5 did not produce a good library due to high organic contamination; this is revealed by the remarkable spike at 230 nm that concurs to the low 260/230 ratio, and explains the faint electrophoretic band and the almost flat fragments test histogram.

    Techniques Used: Next-Generation Sequencing, Formalin-fixed Paraffin-Embedded, Amplification, Multiplex Assay, Polymerase Chain Reaction, Sensitive Assay, Chromatin Immunoprecipitation, Electrophoresis, Produced

    Influence of RNA contamination on DNA quantification. DNA quantifications (n = 5) by NanoDrop and Qubit in the presence of RNA contamination. A DNA sample with a concentration of 38 ng/µl was mixed with different volumes of total RNA at 33 ng/µl extracted from the same tissue sample to obtain the indicated ratios; bars and brackets indicate mean and 95% confidence interval; asterisks show measurements significantly different from pure DNA (* p
    Figure Legend Snippet: Influence of RNA contamination on DNA quantification. DNA quantifications (n = 5) by NanoDrop and Qubit in the presence of RNA contamination. A DNA sample with a concentration of 38 ng/µl was mixed with different volumes of total RNA at 33 ng/µl extracted from the same tissue sample to obtain the indicated ratios; bars and brackets indicate mean and 95% confidence interval; asterisks show measurements significantly different from pure DNA (* p

    Techniques Used: Concentration Assay

    Cross-validation of DNA samples quantification by qPCR. Bland-Altman plots for inter-technology (NanoDrop or Qubit vs. qPCR) comparison of all samples (A), and according to the different sample sources, as indicated (B, C). A) Qubit measurements show high correlation (mean measured/expected ratio = 0.92; SD = 0.69; Wilcoxon signed rank test p = 0.07) with the measurements obtained by qPCR (x-axis), whereas NanoDrop measurements tend to overestimate samples concentration (mean measured/expected ratio = 3.8; SD = 6.4; Wilcoxon signed rank test p
    Figure Legend Snippet: Cross-validation of DNA samples quantification by qPCR. Bland-Altman plots for inter-technology (NanoDrop or Qubit vs. qPCR) comparison of all samples (A), and according to the different sample sources, as indicated (B, C). A) Qubit measurements show high correlation (mean measured/expected ratio = 0.92; SD = 0.69; Wilcoxon signed rank test p = 0.07) with the measurements obtained by qPCR (x-axis), whereas NanoDrop measurements tend to overestimate samples concentration (mean measured/expected ratio = 3.8; SD = 6.4; Wilcoxon signed rank test p

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

    Intra- and inter-method accuracy and precision. Distribution of DNA sample concentration (dispersion chart) was estimated by both NanoDrop (black) and Qubit (gray) on repeated (n = 20) measurements of two commercial human genomic DNA preparations (Sample L 200 ng/µl; Sample G 5 ng/µl). For both samples, NanoDrop overestimated the DNA concentration (+8.8% for L and +24.0% for G, p
    Figure Legend Snippet: Intra- and inter-method accuracy and precision. Distribution of DNA sample concentration (dispersion chart) was estimated by both NanoDrop (black) and Qubit (gray) on repeated (n = 20) measurements of two commercial human genomic DNA preparations (Sample L 200 ng/µl; Sample G 5 ng/µl). For both samples, NanoDrop overestimated the DNA concentration (+8.8% for L and +24.0% for G, p

    Techniques Used: Concentration Assay

    14) Product Images from "Genome sequence of Perigonia lusca single nucleopolyhedrovirus: insights into the evolution of a nucleotide metabolism enzyme in the family Baculoviridae"

    Article Title: Genome sequence of Perigonia lusca single nucleopolyhedrovirus: insights into the evolution of a nucleotide metabolism enzyme in the family Baculoviridae

    Journal: Scientific Reports

    doi: 10.1038/srep24612

    Expression of HA-Pelu112 or HA-Erel005 accelerated AcMNPV replication, viral DNA synthesis, and viral protein expression. ( A ) Analysis of BV production by endpoint dilution assays. Titers were determined from supernatants of cells infected with parental Ac-PG (Control), Ac- ha-pelu112 -PG (Pelu), or Ac- ha-erel005 -PG (Erel) (MOI of 0.01) at the designated time points in triplicate. Statistical differences at 24 and 48 h p.i. obtained by unpaired T-test are shown ( p values: * p ≤ 0.01; ** p ≤ 0.001). ( B ) Yields of occlusion bodies (OB) were increased 2.5-fold in the recombinant viruses. OBs were purified from Sf9 cells infected with the respective viruses (MOI of 5) at 120 h p.i. Bar heights indicate the averages of four repeats, and the error bars represent the standard deviations. Statistical differences by unpaired T-test are shown ( p values: ***p ≤ 0.0001; * p ≤ 0.01). ( C ) Cells were infected (MOI of 10) with the indicated viruses and at 0, 12, 24, 36, and 48 h p.i. total intracellular DNA was purified and analyzed by real-time PCR in three repeats. Statistical difference by unpaired T-test are shown by letters above the bar heights. Different letters indicate that statistical difference exists. ( D ) The fused genes accelerated both IE-1 and GP64 expression during in vitro virus infection when compared to the control virus. Lysates obtained from the same number of cells was loaded in each lane. Cells were infected with the indicated viruses (MOI of 0.01) and at 0, 12, 24, 36, and 48 h p.i. total cellular proteins were analyzed by immunoblotting with specific anti-IE-1 or anti-GP64 antibodies.
    Figure Legend Snippet: Expression of HA-Pelu112 or HA-Erel005 accelerated AcMNPV replication, viral DNA synthesis, and viral protein expression. ( A ) Analysis of BV production by endpoint dilution assays. Titers were determined from supernatants of cells infected with parental Ac-PG (Control), Ac- ha-pelu112 -PG (Pelu), or Ac- ha-erel005 -PG (Erel) (MOI of 0.01) at the designated time points in triplicate. Statistical differences at 24 and 48 h p.i. obtained by unpaired T-test are shown ( p values: * p ≤ 0.01; ** p ≤ 0.001). ( B ) Yields of occlusion bodies (OB) were increased 2.5-fold in the recombinant viruses. OBs were purified from Sf9 cells infected with the respective viruses (MOI of 5) at 120 h p.i. Bar heights indicate the averages of four repeats, and the error bars represent the standard deviations. Statistical differences by unpaired T-test are shown ( p values: ***p ≤ 0.0001; * p ≤ 0.01). ( C ) Cells were infected (MOI of 10) with the indicated viruses and at 0, 12, 24, 36, and 48 h p.i. total intracellular DNA was purified and analyzed by real-time PCR in three repeats. Statistical difference by unpaired T-test are shown by letters above the bar heights. Different letters indicate that statistical difference exists. ( D ) The fused genes accelerated both IE-1 and GP64 expression during in vitro virus infection when compared to the control virus. Lysates obtained from the same number of cells was loaded in each lane. Cells were infected with the indicated viruses (MOI of 0.01) and at 0, 12, 24, 36, and 48 h p.i. total cellular proteins were analyzed by immunoblotting with specific anti-IE-1 or anti-GP64 antibodies.

    Techniques Used: Expressing, Hemagglutination Assay, DNA Synthesis, Infection, Recombinant, Purification, Real-time Polymerase Chain Reaction, In Vitro

    15) Product Images from "A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein"

    Article Title: A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein

    Journal: Nature methods

    doi: 10.1038/nmeth.3935

    SmURFP+BV expressed in vivo and smURFP fusions in mammalian cells. ( a ) Representative image of smURFP expressed in two HT1080 tumor xenografts without exogenous BV. Fluorescence only (left) and overlay of fluorescence and mouse body (right). Three additional mice are shown in Supplementary Fig. 14 . Scale bar = 0.5 cm. ( b–d ) PC3 cells were transfected with DNA and FP fusions were imaged 48 h later after incubation with 25 µM BV for 4 h. #aa is linker length in amino acids and in parentheses: (protein origin, protein name, and cellular location). Fusions at the smURFP N-terminus: ( b ) ManII-10aa-smURFP+BV (mouse, mannosidase II, and Golgi complex) and ( c ) PDHA1-10aa-smURFP+BV (human, pyruvate dehydrogenase, and mitochondria). Fusions at the smURFP C-terminus: ( d ) SmURFP+BV-18aa-αTub (human, α-tubulin, and microtubules) and ( e ) SmURFP+BV-10aa-LamB1 (human, lamin B1, and nuclear envelope). ( b–e ) Cell images are representative of > 20 imaged cells. Similar images were obtained with incubation of 1 µM BVMe 2 . Scale bar = 10 µm.
    Figure Legend Snippet: SmURFP+BV expressed in vivo and smURFP fusions in mammalian cells. ( a ) Representative image of smURFP expressed in two HT1080 tumor xenografts without exogenous BV. Fluorescence only (left) and overlay of fluorescence and mouse body (right). Three additional mice are shown in Supplementary Fig. 14 . Scale bar = 0.5 cm. ( b–d ) PC3 cells were transfected with DNA and FP fusions were imaged 48 h later after incubation with 25 µM BV for 4 h. #aa is linker length in amino acids and in parentheses: (protein origin, protein name, and cellular location). Fusions at the smURFP N-terminus: ( b ) ManII-10aa-smURFP+BV (mouse, mannosidase II, and Golgi complex) and ( c ) PDHA1-10aa-smURFP+BV (human, pyruvate dehydrogenase, and mitochondria). Fusions at the smURFP C-terminus: ( d ) SmURFP+BV-18aa-αTub (human, α-tubulin, and microtubules) and ( e ) SmURFP+BV-10aa-LamB1 (human, lamin B1, and nuclear envelope). ( b–e ) Cell images are representative of > 20 imaged cells. Similar images were obtained with incubation of 1 µM BVMe 2 . Scale bar = 10 µm.

    Techniques Used: In Vivo, Fluorescence, Mouse Assay, Transfection, Incubation

    16) Product Images from "Elevated Levels of DNA Strand Breaks Induced by a Base Analog in the Human Cell Line with the P32T ITPA Variant"

    Article Title: Elevated Levels of DNA Strand Breaks Induced by a Base Analog in the Human Cell Line with the P32T ITPA Variant

    Journal: Journal of Nucleic Acids

    doi: 10.4061/2010/872180

    Verification of the genotype of the P32T cell line. Electropherogram of the DNA sequence of the region with C94A mutation is shown. The experiment was performed as described in Section 2 .
    Figure Legend Snippet: Verification of the genotype of the P32T cell line. Electropherogram of the DNA sequence of the region with C94A mutation is shown. The experiment was performed as described in Section 2 .

    Techniques Used: Sequencing, Mutagenesis

    HAP induces DNA breaks in human cells. The effect of HAP and 100 μ M hydrogen peroxide on the frequency of single-stranded breaks recorded at the end of treatment in WI-38, HCT116 and P32T cell cultures by single-cell electrophoresis at pH > 13. (a) Analysis of the concentration-dependent effects of HAP and hydrogen peroxide on the frequency of DNA strand breaks in WI-38, HCT116 and P32T cells. The damaging DNA-agent dose responses are plotted on the x -axis. The mean percent DNA in the comet tail is plotted on the y -axis as a ratio of the amount of DNA in the comet tail/the amount of DNA in the whole comet. The insert in the upper part of the plot is a linear plot of this correlation for each culture. Results are shown as means ± standard error of results of six different experiments. The level of statistical significance was set at P
    Figure Legend Snippet: HAP induces DNA breaks in human cells. The effect of HAP and 100 μ M hydrogen peroxide on the frequency of single-stranded breaks recorded at the end of treatment in WI-38, HCT116 and P32T cell cultures by single-cell electrophoresis at pH > 13. (a) Analysis of the concentration-dependent effects of HAP and hydrogen peroxide on the frequency of DNA strand breaks in WI-38, HCT116 and P32T cells. The damaging DNA-agent dose responses are plotted on the x -axis. The mean percent DNA in the comet tail is plotted on the y -axis as a ratio of the amount of DNA in the comet tail/the amount of DNA in the whole comet. The insert in the upper part of the plot is a linear plot of this correlation for each culture. Results are shown as means ± standard error of results of six different experiments. The level of statistical significance was set at P

    Techniques Used: Electrophoresis, Concentration Assay

    17) Product Images from "Variable reduction in Norrin signaling activity caused by novel mutations in FZD4 identified in patients with familial exudative vitreoretinopathy"

    Article Title: Variable reduction in Norrin signaling activity caused by novel mutations in FZD4 identified in patients with familial exudative vitreoretinopathy

    Journal: Molecular Vision

    doi:

    Analysis of mutations in FZD4 on its biological activity in the Norrin/β-catenin signaling pathway. A : SuperTopFlash (STF) cells/well were cotransfected with 700 ng DNA (200 ng of Norrin, 200 ng of LRP5, 100 ng of pSV-β-galactosidase control vector, and 200 ng of FZD4 [wild-type or mutation]) and 1.1 μl Lipofectamine 2000 transfection reagent. Forty-eight hours after transfection, the cells were harvested and washed twice with PBS. Luciferase activities were measured with a dual-luciferase assay kit. Reporter activity was normalized to the coexpressed β-galactosidase activity in each well. The positive control (LRP+/FZD4+/Norrin+) was normalized to 1. Each test was performed in triplicate. The reporter assay was repeated three times, and a representative result was obtained. Statistical analyses were performed with a two-tailed unpaired Student t test. B : Human embryonic kidney (HEK) 293 cells were transfected with 600 ng FLAG-FZD4 WT or mutant plasmids, respectively. Forty-eight hours post-transfection, cell lysates were immunoblotted (IB) with FLAG or actin. P, positive control. C : Protein quantification: the gray value of FLAG-FZD4/actin and normalized wild-type to 1 (1#:c.1188_1192del/p.F396fs; 2#: c.1220delC/p.A407Vfs; 3#: c.905G > A/p.C302Y; 4#: c.1325T > A/p.V442E).
    Figure Legend Snippet: Analysis of mutations in FZD4 on its biological activity in the Norrin/β-catenin signaling pathway. A : SuperTopFlash (STF) cells/well were cotransfected with 700 ng DNA (200 ng of Norrin, 200 ng of LRP5, 100 ng of pSV-β-galactosidase control vector, and 200 ng of FZD4 [wild-type or mutation]) and 1.1 μl Lipofectamine 2000 transfection reagent. Forty-eight hours after transfection, the cells were harvested and washed twice with PBS. Luciferase activities were measured with a dual-luciferase assay kit. Reporter activity was normalized to the coexpressed β-galactosidase activity in each well. The positive control (LRP+/FZD4+/Norrin+) was normalized to 1. Each test was performed in triplicate. The reporter assay was repeated three times, and a representative result was obtained. Statistical analyses were performed with a two-tailed unpaired Student t test. B : Human embryonic kidney (HEK) 293 cells were transfected with 600 ng FLAG-FZD4 WT or mutant plasmids, respectively. Forty-eight hours post-transfection, cell lysates were immunoblotted (IB) with FLAG or actin. P, positive control. C : Protein quantification: the gray value of FLAG-FZD4/actin and normalized wild-type to 1 (1#:c.1188_1192del/p.F396fs; 2#: c.1220delC/p.A407Vfs; 3#: c.905G > A/p.C302Y; 4#: c.1325T > A/p.V442E).

    Techniques Used: Activity Assay, Plasmid Preparation, Mutagenesis, Transfection, Luciferase, Positive Control, Reporter Assay, Two Tailed Test

    18) Product Images from "Components of a new gene family of ferroxidases involved in virulence are functionally specialized in fungal dimorphism"

    Article Title: Components of a new gene family of ferroxidases involved in virulence are functionally specialized in fungal dimorphism

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26051-x

    Disruption of genes fet3a, fet3b and fet3c . ( A ) Schematic representation of wild-type and mutant loci after homologous recombination with the disruption fragments of genes fet3a (left), fet3b (middle) and fet3c (right). The position of the probes used (A, B and C) and the expected sizes of the restriction fragments are indicated. Dashed lines are genomic sequences not included in the disruption fragment. ( B ) Southern blot analysis of the wild-type recipient strain (WT) and transformants obtained with the disruption fragments after ten vegetative cycles in selective medium. Genomic DNA (1 μg) was digested with Hind III and hybridized with probes A, B and C, which recognized wild type and disrupted alleles, but could discriminate between them. The positions and sizes of the GeneRuler DNA ladder mixture (M) (Fermentas) are indicated. The cropped blots are displayed in the main figure, the black lines surrounding blots indicate the cropping lines. The scanned full blots are presented in Supplementary Fig. 3 . Asterisk (*) indicated that marker M(Kb) was revealed after a second re-hibridization of the same membrane.
    Figure Legend Snippet: Disruption of genes fet3a, fet3b and fet3c . ( A ) Schematic representation of wild-type and mutant loci after homologous recombination with the disruption fragments of genes fet3a (left), fet3b (middle) and fet3c (right). The position of the probes used (A, B and C) and the expected sizes of the restriction fragments are indicated. Dashed lines are genomic sequences not included in the disruption fragment. ( B ) Southern blot analysis of the wild-type recipient strain (WT) and transformants obtained with the disruption fragments after ten vegetative cycles in selective medium. Genomic DNA (1 μg) was digested with Hind III and hybridized with probes A, B and C, which recognized wild type and disrupted alleles, but could discriminate between them. The positions and sizes of the GeneRuler DNA ladder mixture (M) (Fermentas) are indicated. The cropped blots are displayed in the main figure, the black lines surrounding blots indicate the cropping lines. The scanned full blots are presented in Supplementary Fig. 3 . Asterisk (*) indicated that marker M(Kb) was revealed after a second re-hibridization of the same membrane.

    Techniques Used: Mutagenesis, Homologous Recombination, Genomic Sequencing, Southern Blot, Marker

    Generation of double deletion mutants Δ fet3a /Δ fet3b , Δ fet3c/ Δ fet3b and Δ fet3a/ Δ fet3c . ( A ) Schematic representation of wild type and mutant loci after homologous recombination with the disruption fragments of genes fet3b (left) and fet3c (right). The position of the probes used (B and C) and the expected sizes of the restriction fragments are indicated. Dashed lines, sequences not included in the disruption fragment. ( B ) Southern blot analysis of the wild-type recipient strain (WT) and transformants obtained with the disruption fragments after ten vegetative cycles in selective medium. Genomic DNA (1 μg) was digested with Hind III (transformants for fet3b , left and right) or Xba I (transformants for fet3c , middle) and hybridized with probes B and C, which recognized wild-type and disrupted alleles, but could discriminate between them. The positions and sizes of the GeneRuler DNA ladder mixture (M) (Fermentas) are indicated. The cropped blots are displayed in the main figure, the black lines surrounding blots indicate the cropping lines. The scanned full blots are presented in Supplementary Fig. 3 .
    Figure Legend Snippet: Generation of double deletion mutants Δ fet3a /Δ fet3b , Δ fet3c/ Δ fet3b and Δ fet3a/ Δ fet3c . ( A ) Schematic representation of wild type and mutant loci after homologous recombination with the disruption fragments of genes fet3b (left) and fet3c (right). The position of the probes used (B and C) and the expected sizes of the restriction fragments are indicated. Dashed lines, sequences not included in the disruption fragment. ( B ) Southern blot analysis of the wild-type recipient strain (WT) and transformants obtained with the disruption fragments after ten vegetative cycles in selective medium. Genomic DNA (1 μg) was digested with Hind III (transformants for fet3b , left and right) or Xba I (transformants for fet3c , middle) and hybridized with probes B and C, which recognized wild-type and disrupted alleles, but could discriminate between them. The positions and sizes of the GeneRuler DNA ladder mixture (M) (Fermentas) are indicated. The cropped blots are displayed in the main figure, the black lines surrounding blots indicate the cropping lines. The scanned full blots are presented in Supplementary Fig. 3 .

    Techniques Used: Mutagenesis, Homologous Recombination, Southern Blot

    19) Product Images from "Electrostatic confinement and manipulation of DNA molecules for genome analysis"

    Article Title: Electrostatic confinement and manipulation of DNA molecules for genome analysis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1711069114

    Electrostatic confinement and manipulation of DNA: device considerations. ( A ) Microchannel/nanoslit device schematic (top view): 1.6-μm height × 20-μm width microchannels (molecule bus) connecting 100-nm-high × 1-μm-wide × 28.3-μm-long nanoslits. Entire device, 1.0 cm × 1.0 cm square, comprises 126 microchannels, each one harboring 1,100 nanoslits bounded by molecular gates. ( B ) Electrostatic potential determined by FE simulation of the entire device within the buffer chamber. Such simulations guided electrode locations for producing the appropriate field lines within the microchannel/nanoslit device. ( C ) Microchannel/nanoslit device (imaged by DIC microscopy) is superimposed with arrows showing the direction and magnitude of field lines within device microchannel and nanoslit features (70 V applied). ( D ) Cartoon (top view) shows the direction and magnitude of the electrokinetic forces for low and high ionic strength conditions. ( Inset ) SEM micrograph (top view) of a patterned silicon master detailing nanoslits and molecular gates. Micrographs of DNA dumbbells bearing nanocoded labels (red punctates) are shown placed within the device. At low I , EO (blue arrows) guides molecules along the microchannel, while EP (yellow arrows) drives them toward the molecular gates. At high I , both directions are dominated by EP. Molecular trajectories (dotted line) are also drawn. ( E ) SEM of cup-like molecular gate features and dimensions (top view) of a silicon master. Illustration below shows DNA molecules (green) within a microchannel (1.6 μm high). Several molecular gates are shown bearing DNAs threaded into nanoslits (100 nm high), which pass through to the other side to form dumbbells. Note small 1-μm × 100-nm slit openings at the bottom of molecular gates. Cross-sectional view ( Inset ) depicts intersecting ion distributions (green) surrounding DNA and the nanoslit walls (red). ( F ) Perspective drawing showing DNA molecules (green balls/threads) within a microchannel. Ion clouds surround DNA and device walls. Lateral cross-sectional view within a nanoslit (see E ; view A–A), showing ion clouds, under low and high ionic strength surrounding a DNA molecule (green) and nanoslit (red). At low I , an “electrostatic bottle” is created because ion clouds overlap, electrostatically confining the now-stiffened (increased persistence length) DNA molecule. In contrast, high I engenders a short Debye length, allowing the molecule to more freely diffuse throughout the entire height of the nanoslit. Furthermore, ionic strength conditions collectively affect the profile of the EO flow fields, illustrated by arrows, where the maximum velocity depends directly on the ratio between confinement dimensions and Debye length.
    Figure Legend Snippet: Electrostatic confinement and manipulation of DNA: device considerations. ( A ) Microchannel/nanoslit device schematic (top view): 1.6-μm height × 20-μm width microchannels (molecule bus) connecting 100-nm-high × 1-μm-wide × 28.3-μm-long nanoslits. Entire device, 1.0 cm × 1.0 cm square, comprises 126 microchannels, each one harboring 1,100 nanoslits bounded by molecular gates. ( B ) Electrostatic potential determined by FE simulation of the entire device within the buffer chamber. Such simulations guided electrode locations for producing the appropriate field lines within the microchannel/nanoslit device. ( C ) Microchannel/nanoslit device (imaged by DIC microscopy) is superimposed with arrows showing the direction and magnitude of field lines within device microchannel and nanoslit features (70 V applied). ( D ) Cartoon (top view) shows the direction and magnitude of the electrokinetic forces for low and high ionic strength conditions. ( Inset ) SEM micrograph (top view) of a patterned silicon master detailing nanoslits and molecular gates. Micrographs of DNA dumbbells bearing nanocoded labels (red punctates) are shown placed within the device. At low I , EO (blue arrows) guides molecules along the microchannel, while EP (yellow arrows) drives them toward the molecular gates. At high I , both directions are dominated by EP. Molecular trajectories (dotted line) are also drawn. ( E ) SEM of cup-like molecular gate features and dimensions (top view) of a silicon master. Illustration below shows DNA molecules (green) within a microchannel (1.6 μm high). Several molecular gates are shown bearing DNAs threaded into nanoslits (100 nm high), which pass through to the other side to form dumbbells. Note small 1-μm × 100-nm slit openings at the bottom of molecular gates. Cross-sectional view ( Inset ) depicts intersecting ion distributions (green) surrounding DNA and the nanoslit walls (red). ( F ) Perspective drawing showing DNA molecules (green balls/threads) within a microchannel. Ion clouds surround DNA and device walls. Lateral cross-sectional view within a nanoslit (see E ; view A–A), showing ion clouds, under low and high ionic strength surrounding a DNA molecule (green) and nanoslit (red). At low I , an “electrostatic bottle” is created because ion clouds overlap, electrostatically confining the now-stiffened (increased persistence length) DNA molecule. In contrast, high I engenders a short Debye length, allowing the molecule to more freely diffuse throughout the entire height of the nanoslit. Furthermore, ionic strength conditions collectively affect the profile of the EO flow fields, illustrated by arrows, where the maximum velocity depends directly on the ratio between confinement dimensions and Debye length.

    Techniques Used: Microscopy, Flow Cytometry

    Parking, loading, and synchronized formation of T4 DNA dumbbells. Schematic of electrical signal triggering synchronized loading and dumbbell formation of parked DNA molecules into nanoslits. Micrographs (superimposed fluorescence and DIC), accompanied by cartoons, show T4 (165.6 kb) DNA molecules within several microchannels ( t = 0 s) migrating toward the molecular gates ( V p = 20 V) for parking. (Parking: 1–2) A portion of these DNA molecules now resides ( V p = 20 V; t = 70.10 s) within molecular gates, and are now parked. (Loading: 3–4) Parked molecules are triggered ( t = 70.32 s) to synchronously load into the adjoining slits by a short, higher-voltage pulse (1.0 s; V L = 70 V) to form an array of dumbbells (Dumbbells: 5) ( t = 74.01 s).
    Figure Legend Snippet: Parking, loading, and synchronized formation of T4 DNA dumbbells. Schematic of electrical signal triggering synchronized loading and dumbbell formation of parked DNA molecules into nanoslits. Micrographs (superimposed fluorescence and DIC), accompanied by cartoons, show T4 (165.6 kb) DNA molecules within several microchannels ( t = 0 s) migrating toward the molecular gates ( V p = 20 V) for parking. (Parking: 1–2) A portion of these DNA molecules now resides ( V p = 20 V; t = 70.10 s) within molecular gates, and are now parked. (Loading: 3–4) Parked molecules are triggered ( t = 70.32 s) to synchronously load into the adjoining slits by a short, higher-voltage pulse (1.0 s; V L = 70 V) to form an array of dumbbells (Dumbbells: 5) ( t = 74.01 s).

    Techniques Used: Fluorescence

    20) Product Images from "Ms1, a novel sRNA interacting with the RNA polymerase core in mycobacteria"

    Article Title: Ms1, a novel sRNA interacting with the RNA polymerase core in mycobacteria

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku793

    Mycobacterial Ms1 sRNA is expressed in amounts comparable to 6S RNAs. ( A ) Total RNA was isolated from Bacillus subtilis ( B.s. ), Escherichia coli ( E.c. ) and Mycobacterium smegmatis ( M.s. ) in exponential (EX) or stationary (ST) phase. RNAs were resolved on denaturing polyacrylamide gels and stained with GelRed. In M. smegmatis , an ∼300 nt sRNA was present in stationary phase cells in amounts comparable to B. subtilis or E. coli 6S RNAs. ( B ) Before loading onto the gel, total RNA from M. smegmatis stationary phase was incubated either with a complementary DNA oligonucleotide (anti-Ms1 oligo) or nonspecific control oligonucleotides (ns-oligo 1 and ns-oligo 2) and treated with RNase H. ( C ) The first nucleotide of Ms1 is adenine transcribed from position 6 242 368 in the genome. The putative −10 and −35 promoter sequences (framed) are perfectly conserved in M. smegmatis , Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium avium . The 5′ end sequences of previously identified Ms1 homologs in these species are highlighted in bold. The consensus promoter sequence was adopted from ( 36 ). ( D ) The flanking genes of Ms1 in M. smegmatis are shown. ( E ) Scheme of Ms1's position in the genome of M. smegmatis with respect to the origin of replication (ori).
    Figure Legend Snippet: Mycobacterial Ms1 sRNA is expressed in amounts comparable to 6S RNAs. ( A ) Total RNA was isolated from Bacillus subtilis ( B.s. ), Escherichia coli ( E.c. ) and Mycobacterium smegmatis ( M.s. ) in exponential (EX) or stationary (ST) phase. RNAs were resolved on denaturing polyacrylamide gels and stained with GelRed. In M. smegmatis , an ∼300 nt sRNA was present in stationary phase cells in amounts comparable to B. subtilis or E. coli 6S RNAs. ( B ) Before loading onto the gel, total RNA from M. smegmatis stationary phase was incubated either with a complementary DNA oligonucleotide (anti-Ms1 oligo) or nonspecific control oligonucleotides (ns-oligo 1 and ns-oligo 2) and treated with RNase H. ( C ) The first nucleotide of Ms1 is adenine transcribed from position 6 242 368 in the genome. The putative −10 and −35 promoter sequences (framed) are perfectly conserved in M. smegmatis , Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium avium . The 5′ end sequences of previously identified Ms1 homologs in these species are highlighted in bold. The consensus promoter sequence was adopted from ( 36 ). ( D ) The flanking genes of Ms1 in M. smegmatis are shown. ( E ) Scheme of Ms1's position in the genome of M. smegmatis with respect to the origin of replication (ori).

    Techniques Used: Isolation, Staining, Incubation, Sequencing

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Impact of APOL1 polymorphism and IL-1β priming in the entry and persistence of HIV-1 in human podocytes
    Article Snippet: Cell lysates were prepared as described in Malnati et al. [ ] and subjected to qPCR TaqMan analysis by using HIV-1 specific primers: Gag Fw-5′-ACATCAAGCAGCCATGCAAAT-3′, Rev-5′-ATCTGGCCTGGTGCAATAGG-3′ and probe 5′-CATCAATGAGGAAGCTGCAGAATGGGATAGA-3′, LTR RU5 Fw-5′-GGCTAACTAGGGAACCCACTG-3′, Rev-5′-CTGCTAGAGATTTTCCACACTGAC-3′ and probe 5′-TGTGTGCCCGTCTGTTGTGTG-3′ [ , ]. .. Amplification of GAPDH gene as a reference was used to control the amount of DNA in each sample (Applied Biosystem).

    Incubation:

    Article Title: Impact of APOL1 polymorphism and IL-1β priming in the entry and persistence of HIV-1 in human podocytes
    Article Snippet: Viral stock before incubation with podocytes were treated (1 h at room temperature) with 200 U/mL of RNase-free DNase (Roche, Basel, Switzerland). .. Amplification of GAPDH gene as a reference was used to control the amount of DNA in each sample (Applied Biosystem).

    Amplification:

    Article Title: Impact of APOL1 polymorphism and IL-1β priming in the entry and persistence of HIV-1 in human podocytes
    Article Snippet: Cell lysates were prepared as described in Malnati et al. [ ] and subjected to qPCR TaqMan analysis by using HIV-1 specific primers: Gag Fw-5′-ACATCAAGCAGCCATGCAAAT-3′, Rev-5′-ATCTGGCCTGGTGCAATAGG-3′ and probe 5′-CATCAATGAGGAAGCTGCAGAATGGGATAGA-3′, LTR RU5 Fw-5′-GGCTAACTAGGGAACCCACTG-3′, Rev-5′-CTGCTAGAGATTTTCCACACTGAC-3′ and probe 5′-TGTGTGCCCGTCTGTTGTGTG-3′ [ , ]. .. Amplification of GAPDH gene as a reference was used to control the amount of DNA in each sample (Applied Biosystem). .. CIHPs were growth on the coverslip’s coated with human collagen IV then were fixed with 4 % paraformaldehyde, followed by permeabilization and blocking with 0.3 % Triton X-100 and 10 % goat serum.

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    Thermo Fisher linear sheared salmon sperm dna
    Design and production of a FRET pair-labeled topo VI to report on B-subunit conformation. ( A ) Three different conformations of the topo VI holoenzyme, an open SAXS model of S. shibatae topo VI, a partially closed crystallographic model of M. mazei topo VI, and a fully closed SAXS model of Ss topo VI are shown with Thr155 displayed in purple sticks and the distance between Thr155 dimer pairs shown by pink dashed arrows. Native cysteine residues mutated to alanine or serine residues to remove off-target labeling sites are shown as yellow-green sticks on the Mm topo VI crystal structure. ( B ) Purified topo VI cyslite-155C -labeled with Alexa555 and Alexa647, separated on SDS-PAGE and stained for protein with coomassie blue ( left) after scanning for Alexa555 emission ( middle ), and Alexa647 emission ( right ). A dilution series of enzyme (1 μg, 0.1 μg, 0.01 μg) was run, along with a standards ladder (lane marked L, molecular weights in kilodaltons labeled on left). Positions of Mm Top6B and Mm Top6A are labeled on the right. ( C ) Supercoil relaxation activity of FRET pair-labeled topo VI compared to wild type as a function of enzyme concentration. For the enzyme titrations (0.3–20 nM in two-fold steps, marked above), each experiment proceeded for 30 min prior to quenching and contained 3.5 nM plasmid (10.2 μM bp <t>DNA).</t> Topoisomer products were separated on agarose gel with the position of <t>supercoiled</t> and relaxed topoisomers marked on the left. Cartoons denote the construct used in each activity assay. This enzyme was used for Top6B dimerization experiments reported in Figure 2B–D .
    Linear Sheared Salmon Sperm Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mouse anti dna pkcs
    The PL motif of <t>DNA-PKcs</t> is required for robust DNA replication. ( A ) Sequence comparison of human DNA-PKcs amino acid 337–350 with the PIP box motif of other PIPs (left panel) and homology with other vertebrates (right panel). Consensus sequence of the PIP box, QxxΨxxϑϑ. Ψ = M, L, I; ϑ = F, Y. The DNA-PKcs PL mutant (mPL) carries alanine substitutions at F345 and Y346. ( B ) Expression of DNA-PKcs in wild-type CHO AA8 cells, DNA-PKcs deficient V3 cells and V3 cells complemented with empty vector (V3), wild-type DNA-PKcs (WT) and mutants carrying alanine substitutions at the Thr2609 cluster (V3–6A) or the PL motif (V3-mPL). ( C ) Growth curve of V3 and derived cell lines. Cell numbers were analyzed by a cell counter (Beckman Coulter Z2). Data are presented as mean ± s.d., N = 2. ( D ) V3-WT and V3-mPL cells were pulse-labeled with iododeoxyuridine (IdU, 10 min) and chlorodeoxyuridine (CldU, 20 min) sequentially, and were analyzed by the DNA fiber assay. The lengths of IdU (red) and CldU (green) tracks were analyzed from > 100 ongoing DNA replication tracks.
    Mouse Anti Dna Pkcs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti dna pkcs
    TRAIL inhibited P-gp efflux function in MDR cells by P-gp cleavage via caspase-3 activation . (A) The cell lysates obtained from CEM/VLB 100 cells treated with or without TRAIL (10 ng/ml) for 3 ~ 9 h ( left ) and the cell lysates of the MDR cells treated with TRAIL (5 ng/ml) for 6 h or pretreated with 50 μM Z-DEVD-FMK, a specific caspase-3 inhibitor, for 3 h and then with TRAIL for 6 h ( right ) were subjected to western blot analysis to monitor levels of P-gp, <t>DNA-PKcs,</t> caspase-3 and PARP and their its cleavage fragment (CF). (B) flow cytometric assay of P-gp efflux activity in TRAIL-treated MDR cells was based on extrusion of the fluorescent P-gp substrate, rhodamine123 (Rho123). The efflux activity of P-gp is highly temperature sensitive because functions optimally 37°C but is inactive at 4°C. Cell suspension from CEM and CEM/VLB 100 cells treated with or without 10 ng/ml TRAIL for 6 h was incubated with Rho 123 and further incubated at 37°C for 3 h (TREATED 37°C as TRAIL-treated cells or CTRL37°C as TRAIL-untreated control) to allow P-gp-mediated drug efflux or on ice as control (CTRL 4°C).
    Anti Dna Pkcs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Design and production of a FRET pair-labeled topo VI to report on B-subunit conformation. ( A ) Three different conformations of the topo VI holoenzyme, an open SAXS model of S. shibatae topo VI, a partially closed crystallographic model of M. mazei topo VI, and a fully closed SAXS model of Ss topo VI are shown with Thr155 displayed in purple sticks and the distance between Thr155 dimer pairs shown by pink dashed arrows. Native cysteine residues mutated to alanine or serine residues to remove off-target labeling sites are shown as yellow-green sticks on the Mm topo VI crystal structure. ( B ) Purified topo VI cyslite-155C -labeled with Alexa555 and Alexa647, separated on SDS-PAGE and stained for protein with coomassie blue ( left) after scanning for Alexa555 emission ( middle ), and Alexa647 emission ( right ). A dilution series of enzyme (1 μg, 0.1 μg, 0.01 μg) was run, along with a standards ladder (lane marked L, molecular weights in kilodaltons labeled on left). Positions of Mm Top6B and Mm Top6A are labeled on the right. ( C ) Supercoil relaxation activity of FRET pair-labeled topo VI compared to wild type as a function of enzyme concentration. For the enzyme titrations (0.3–20 nM in two-fold steps, marked above), each experiment proceeded for 30 min prior to quenching and contained 3.5 nM plasmid (10.2 μM bp DNA). Topoisomer products were separated on agarose gel with the position of supercoiled and relaxed topoisomers marked on the left. Cartoons denote the construct used in each activity assay. This enzyme was used for Top6B dimerization experiments reported in Figure 2B–D .

    Journal: eLife

    Article Title: Topoisomerase VI senses and exploits both DNA crossings and bends to facilitate strand passage

    doi: 10.7554/eLife.31724

    Figure Lengend Snippet: Design and production of a FRET pair-labeled topo VI to report on B-subunit conformation. ( A ) Three different conformations of the topo VI holoenzyme, an open SAXS model of S. shibatae topo VI, a partially closed crystallographic model of M. mazei topo VI, and a fully closed SAXS model of Ss topo VI are shown with Thr155 displayed in purple sticks and the distance between Thr155 dimer pairs shown by pink dashed arrows. Native cysteine residues mutated to alanine or serine residues to remove off-target labeling sites are shown as yellow-green sticks on the Mm topo VI crystal structure. ( B ) Purified topo VI cyslite-155C -labeled with Alexa555 and Alexa647, separated on SDS-PAGE and stained for protein with coomassie blue ( left) after scanning for Alexa555 emission ( middle ), and Alexa647 emission ( right ). A dilution series of enzyme (1 μg, 0.1 μg, 0.01 μg) was run, along with a standards ladder (lane marked L, molecular weights in kilodaltons labeled on left). Positions of Mm Top6B and Mm Top6A are labeled on the right. ( C ) Supercoil relaxation activity of FRET pair-labeled topo VI compared to wild type as a function of enzyme concentration. For the enzyme titrations (0.3–20 nM in two-fold steps, marked above), each experiment proceeded for 30 min prior to quenching and contained 3.5 nM plasmid (10.2 μM bp DNA). Topoisomer products were separated on agarose gel with the position of supercoiled and relaxed topoisomers marked on the left. Cartoons denote the construct used in each activity assay. This enzyme was used for Top6B dimerization experiments reported in Figure 2B–D .

    Article Snippet: Competition assays were carried out similarly to binding assays, with protein diluted in binding assay dilution buffer and incubated with the 70 bp fluorescein-labeled duplex and either negatively supercoiled pSG483 plasmid DNA (pBluescript SK derivative, 2927 bp) or linear sheared salmon-sperm DNA (ThermoFisher Scientific, Waltham, MA).

    Techniques: Labeling, Purification, SDS Page, Staining, Activity Assay, Concentration Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Construct

    AMPPNP is sufficient to close the ATP gate of Sc Top2. ( A ) Supercoil relaxation activity of the FRET pair-labeled Sc Top2 ΔCTR-cyslite-180C was compared to wild type as a function of enzyme concentration. For the enzyme titrations (0.3–20 nM in two-fold steps, marked above), each experiment proceeded for 30 min prior to quenching and contained 3.5 nM plasmid (10.2 μM bp DNA). Topoisomer products were separated on agarose gel with the position of supercoiled and relaxed topoisomers marked on the left. Cartoons denote the construct used. ( B ) Fluorescence emission spectra produced by 530 nm excitation of Alexa555/Alexa647-labeled Sc Top2 ΔCTR-cyslite-180C show that in contrast to the behavior of topo VI reported in Figure 2B–D , addition of AMPPNP (dashed line spectra), is sufficient to dimerize the ATP gate as assessed by the relative increase in acceptor emission and decrease in donor emission as compared to controls without nucleotide (solid line spectra). Presence ( orange spectra ) or absence ( goldenrod spectra ) of supercoiled DNA does not appreciably modify the conformational state of the ATPase domain as accessed by FRET. Spectral emission was normalized by total emission from 545 nm to 700 nm.

    Journal: eLife

    Article Title: Topoisomerase VI senses and exploits both DNA crossings and bends to facilitate strand passage

    doi: 10.7554/eLife.31724

    Figure Lengend Snippet: AMPPNP is sufficient to close the ATP gate of Sc Top2. ( A ) Supercoil relaxation activity of the FRET pair-labeled Sc Top2 ΔCTR-cyslite-180C was compared to wild type as a function of enzyme concentration. For the enzyme titrations (0.3–20 nM in two-fold steps, marked above), each experiment proceeded for 30 min prior to quenching and contained 3.5 nM plasmid (10.2 μM bp DNA). Topoisomer products were separated on agarose gel with the position of supercoiled and relaxed topoisomers marked on the left. Cartoons denote the construct used. ( B ) Fluorescence emission spectra produced by 530 nm excitation of Alexa555/Alexa647-labeled Sc Top2 ΔCTR-cyslite-180C show that in contrast to the behavior of topo VI reported in Figure 2B–D , addition of AMPPNP (dashed line spectra), is sufficient to dimerize the ATP gate as assessed by the relative increase in acceptor emission and decrease in donor emission as compared to controls without nucleotide (solid line spectra). Presence ( orange spectra ) or absence ( goldenrod spectra ) of supercoiled DNA does not appreciably modify the conformational state of the ATPase domain as accessed by FRET. Spectral emission was normalized by total emission from 545 nm to 700 nm.

    Article Snippet: Competition assays were carried out similarly to binding assays, with protein diluted in binding assay dilution buffer and incubated with the 70 bp fluorescein-labeled duplex and either negatively supercoiled pSG483 plasmid DNA (pBluescript SK derivative, 2927 bp) or linear sheared salmon-sperm DNA (ThermoFisher Scientific, Waltham, MA).

    Techniques: Activity Assay, Labeling, Concentration Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Construct, Fluorescence, Produced

    Topo II processively relaxes supercoiled DNA as compared to topo VI. An example of processive supercoil relaxation by Sc Top2 ΔCTR , as compared to topo VI in Figure 1C–D . Sc Top2 ΔCTR was pre-incubated in a 1:1.4 ratio to a 2.9 kb negatively supercoiled plasmid (6.7 ng/uL in assay). Reactions were started by addition of either ( A ) ATP or ( B ) ATP and a 6.5 kb ‘chase’ plasmid (6.7 ng/uL in assay) to compete for unbound enzyme. Samples were quenched at 0, 1, 2, 4, 6, 8, 10, 15, and 20 min. Each condition was also incubated without ATP for 20 min as a negative control. Topoisomer species and identification of each plasmid are indicated on the left of each gel.

    Journal: eLife

    Article Title: Topoisomerase VI senses and exploits both DNA crossings and bends to facilitate strand passage

    doi: 10.7554/eLife.31724

    Figure Lengend Snippet: Topo II processively relaxes supercoiled DNA as compared to topo VI. An example of processive supercoil relaxation by Sc Top2 ΔCTR , as compared to topo VI in Figure 1C–D . Sc Top2 ΔCTR was pre-incubated in a 1:1.4 ratio to a 2.9 kb negatively supercoiled plasmid (6.7 ng/uL in assay). Reactions were started by addition of either ( A ) ATP or ( B ) ATP and a 6.5 kb ‘chase’ plasmid (6.7 ng/uL in assay) to compete for unbound enzyme. Samples were quenched at 0, 1, 2, 4, 6, 8, 10, 15, and 20 min. Each condition was also incubated without ATP for 20 min as a negative control. Topoisomer species and identification of each plasmid are indicated on the left of each gel.

    Article Snippet: Competition assays were carried out similarly to binding assays, with protein diluted in binding assay dilution buffer and incubated with the 70 bp fluorescein-labeled duplex and either negatively supercoiled pSG483 plasmid DNA (pBluescript SK derivative, 2927 bp) or linear sheared salmon-sperm DNA (ThermoFisher Scientific, Waltham, MA).

    Techniques: Incubation, Plasmid Preparation, Negative Control

    Supercoil relaxation activity of topo VI mutants as a function of time. Strand passage activity of mutant topo VI constructs for relaxing supercoiled DNA as compared to wild type are shown as a function of time and in relation to enzyme titrations reported in Figure 4A . In each time course experiment, 2.5 nM of enzyme processed 3.5 nM of negatively supercoiled plasmid (10.2 μM bp) and was quenched with EDTA and SDS at the indicated time (0–120 min). A no-ATP control (-ATP) was taken after a 120 min incubation period for each variant. A no-enzyme control (-topo) was also taken and run with the timecourse experiments for the KGRR constructs. The placement and nature of mutations in each construct are depicted in the cartoons above each timecourse (‘•••' - AAA; ‘---' – EEE).

    Journal: eLife

    Article Title: Topoisomerase VI senses and exploits both DNA crossings and bends to facilitate strand passage

    doi: 10.7554/eLife.31724

    Figure Lengend Snippet: Supercoil relaxation activity of topo VI mutants as a function of time. Strand passage activity of mutant topo VI constructs for relaxing supercoiled DNA as compared to wild type are shown as a function of time and in relation to enzyme titrations reported in Figure 4A . In each time course experiment, 2.5 nM of enzyme processed 3.5 nM of negatively supercoiled plasmid (10.2 μM bp) and was quenched with EDTA and SDS at the indicated time (0–120 min). A no-ATP control (-ATP) was taken after a 120 min incubation period for each variant. A no-enzyme control (-topo) was also taken and run with the timecourse experiments for the KGRR constructs. The placement and nature of mutations in each construct are depicted in the cartoons above each timecourse (‘•••' - AAA; ‘---' – EEE).

    Article Snippet: Competition assays were carried out similarly to binding assays, with protein diluted in binding assay dilution buffer and incubated with the 70 bp fluorescein-labeled duplex and either negatively supercoiled pSG483 plasmid DNA (pBluescript SK derivative, 2927 bp) or linear sheared salmon-sperm DNA (ThermoFisher Scientific, Waltham, MA).

    Techniques: Activity Assay, Mutagenesis, Construct, Plasmid Preparation, Incubation, Variant Assay

    AMPPNP-dependent conformational response of KGRR AAA in bending assay conditions bound to excess 70 bp duplex or supercoiled DNA. Change in ratiometric FRET efficiency for the Alexa555/647-labeled KGRR AAA construct incubated with either excess 70 bp duplex or supercoiled DNA was monitored over time after the addition of AMPPNP. AMPPNP promotes Top6B dimerization when KGRR AAA is bound to the 70 bp duplex, but does not stabilize gate closure to the extent of supercoiled DNA alone, reconciling the Top6B dimerization ( Figure 6B ) and bending assay ( Figure 7D ) data for this mutant. Numerical data are reported in Figure 7—source data 1 .

    Journal: eLife

    Article Title: Topoisomerase VI senses and exploits both DNA crossings and bends to facilitate strand passage

    doi: 10.7554/eLife.31724

    Figure Lengend Snippet: AMPPNP-dependent conformational response of KGRR AAA in bending assay conditions bound to excess 70 bp duplex or supercoiled DNA. Change in ratiometric FRET efficiency for the Alexa555/647-labeled KGRR AAA construct incubated with either excess 70 bp duplex or supercoiled DNA was monitored over time after the addition of AMPPNP. AMPPNP promotes Top6B dimerization when KGRR AAA is bound to the 70 bp duplex, but does not stabilize gate closure to the extent of supercoiled DNA alone, reconciling the Top6B dimerization ( Figure 6B ) and bending assay ( Figure 7D ) data for this mutant. Numerical data are reported in Figure 7—source data 1 .

    Article Snippet: Competition assays were carried out similarly to binding assays, with protein diluted in binding assay dilution buffer and incubated with the 70 bp fluorescein-labeled duplex and either negatively supercoiled pSG483 plasmid DNA (pBluescript SK derivative, 2927 bp) or linear sheared salmon-sperm DNA (ThermoFisher Scientific, Waltham, MA).

    Techniques: Labeling, Construct, Incubation, Mutagenesis

    Conformational response of H2TH AAA and KGRR AAA constructs on different substrates as determined by FRET in the absence of nucleotide. Comparison of the fluorescence emission spectra produced by 530 nm excitation of Alexa555/Alexa647-labeled ( A ) TopoVI cyslite-155C , ( B ) KGRR AAA, cyslite-155C and ( C ) H2TH AAA, cyslite-155C alone and on supercoiled DNA, as related to Figure 6D . For all three constructs, supercoiled DNA modulates the conformation of the Topo6B ATPase domain in the absence of nucleotide as assessed by FRET. Spectral emission was normalized by total emission from 545 nm to 700 nm.

    Journal: eLife

    Article Title: Topoisomerase VI senses and exploits both DNA crossings and bends to facilitate strand passage

    doi: 10.7554/eLife.31724

    Figure Lengend Snippet: Conformational response of H2TH AAA and KGRR AAA constructs on different substrates as determined by FRET in the absence of nucleotide. Comparison of the fluorescence emission spectra produced by 530 nm excitation of Alexa555/Alexa647-labeled ( A ) TopoVI cyslite-155C , ( B ) KGRR AAA, cyslite-155C and ( C ) H2TH AAA, cyslite-155C alone and on supercoiled DNA, as related to Figure 6D . For all three constructs, supercoiled DNA modulates the conformation of the Topo6B ATPase domain in the absence of nucleotide as assessed by FRET. Spectral emission was normalized by total emission from 545 nm to 700 nm.

    Article Snippet: Competition assays were carried out similarly to binding assays, with protein diluted in binding assay dilution buffer and incubated with the 70 bp fluorescein-labeled duplex and either negatively supercoiled pSG483 plasmid DNA (pBluescript SK derivative, 2927 bp) or linear sheared salmon-sperm DNA (ThermoFisher Scientific, Waltham, MA).

    Techniques: Construct, Fluorescence, Produced, Labeling

    Determination of contaminating ATPase activity levels present in topo VI preparations using a hydrolysis-deficient mutant. ( A ) Rate of steady-state ATP hydrolysis measured as a function of ATP concentration for topo VI alone and an ATPase-deficient construct of topo VI (Top6B E44A ). Rates were determined spectroscopically using an NADH-coupled assay (Materials and methods). Points and error bars correspond to the mean and standard deviation of three independent experiments. Measured rates for Top6B E44A were used to estimate the topo VI-specific ATPase rates presented in Figure 2A . ( B ) Stimulation of ATP hydrolysis above basal levels catalyzed by wildtype topo VI ( left ) or the Top6B E44A ATPase-deficient construct ( right ) as a function of the basepair concentration (µM) of sheared salmon-sperm, DNA (pink), or a 2.9 kb supercoiled plasmid (orange). ATP was held at 2 mM, and rates were determined spectroscopically using an NADH coupled assay. Points and error bars correspond to the mean and standard deviation of three independent experiments. Numerical data for ( A–B ) are reported in Figure 2—source data 1 .

    Journal: eLife

    Article Title: Topoisomerase VI senses and exploits both DNA crossings and bends to facilitate strand passage

    doi: 10.7554/eLife.31724

    Figure Lengend Snippet: Determination of contaminating ATPase activity levels present in topo VI preparations using a hydrolysis-deficient mutant. ( A ) Rate of steady-state ATP hydrolysis measured as a function of ATP concentration for topo VI alone and an ATPase-deficient construct of topo VI (Top6B E44A ). Rates were determined spectroscopically using an NADH-coupled assay (Materials and methods). Points and error bars correspond to the mean and standard deviation of three independent experiments. Measured rates for Top6B E44A were used to estimate the topo VI-specific ATPase rates presented in Figure 2A . ( B ) Stimulation of ATP hydrolysis above basal levels catalyzed by wildtype topo VI ( left ) or the Top6B E44A ATPase-deficient construct ( right ) as a function of the basepair concentration (µM) of sheared salmon-sperm, DNA (pink), or a 2.9 kb supercoiled plasmid (orange). ATP was held at 2 mM, and rates were determined spectroscopically using an NADH coupled assay. Points and error bars correspond to the mean and standard deviation of three independent experiments. Numerical data for ( A–B ) are reported in Figure 2—source data 1 .

    Article Snippet: Competition assays were carried out similarly to binding assays, with protein diluted in binding assay dilution buffer and incubated with the 70 bp fluorescein-labeled duplex and either negatively supercoiled pSG483 plasmid DNA (pBluescript SK derivative, 2927 bp) or linear sheared salmon-sperm DNA (ThermoFisher Scientific, Waltham, MA).

    Techniques: Activity Assay, Mutagenesis, Concentration Assay, Construct, Standard Deviation, Plasmid Preparation

    The PL motif of DNA-PKcs is required for robust DNA replication. ( A ) Sequence comparison of human DNA-PKcs amino acid 337–350 with the PIP box motif of other PIPs (left panel) and homology with other vertebrates (right panel). Consensus sequence of the PIP box, QxxΨxxϑϑ. Ψ = M, L, I; ϑ = F, Y. The DNA-PKcs PL mutant (mPL) carries alanine substitutions at F345 and Y346. ( B ) Expression of DNA-PKcs in wild-type CHO AA8 cells, DNA-PKcs deficient V3 cells and V3 cells complemented with empty vector (V3), wild-type DNA-PKcs (WT) and mutants carrying alanine substitutions at the Thr2609 cluster (V3–6A) or the PL motif (V3-mPL). ( C ) Growth curve of V3 and derived cell lines. Cell numbers were analyzed by a cell counter (Beckman Coulter Z2). Data are presented as mean ± s.d., N = 2. ( D ) V3-WT and V3-mPL cells were pulse-labeled with iododeoxyuridine (IdU, 10 min) and chlorodeoxyuridine (CldU, 20 min) sequentially, and were analyzed by the DNA fiber assay. The lengths of IdU (red) and CldU (green) tracks were analyzed from > 100 ongoing DNA replication tracks.

    Journal: Nucleic Acids Research

    Article Title: PIDD mediates the association of DNA-PKcs and ATR at stalled replication forks to facilitate the ATR signaling pathway

    doi: 10.1093/nar/gkx1298

    Figure Lengend Snippet: The PL motif of DNA-PKcs is required for robust DNA replication. ( A ) Sequence comparison of human DNA-PKcs amino acid 337–350 with the PIP box motif of other PIPs (left panel) and homology with other vertebrates (right panel). Consensus sequence of the PIP box, QxxΨxxϑϑ. Ψ = M, L, I; ϑ = F, Y. The DNA-PKcs PL mutant (mPL) carries alanine substitutions at F345 and Y346. ( B ) Expression of DNA-PKcs in wild-type CHO AA8 cells, DNA-PKcs deficient V3 cells and V3 cells complemented with empty vector (V3), wild-type DNA-PKcs (WT) and mutants carrying alanine substitutions at the Thr2609 cluster (V3–6A) or the PL motif (V3-mPL). ( C ) Growth curve of V3 and derived cell lines. Cell numbers were analyzed by a cell counter (Beckman Coulter Z2). Data are presented as mean ± s.d., N = 2. ( D ) V3-WT and V3-mPL cells were pulse-labeled with iododeoxyuridine (IdU, 10 min) and chlorodeoxyuridine (CldU, 20 min) sequentially, and were analyzed by the DNA fiber assay. The lengths of IdU (red) and CldU (green) tracks were analyzed from > 100 ongoing DNA replication tracks.

    Article Snippet: Mouse anti-DNA-PKcs (Thermo Fisher Scientific, 1:100 dilution), rabbit anti-ATR (Bethyl, 1:100), mouse anti-RPA2 (EMD Millipore, 1:50) and rabbit anti-FLAG (Sigma, 1:100) antibodies were commercially available at the indicated vendors.

    Techniques: Sequencing, Mutagenesis, Expressing, Plasmid Preparation, Derivative Assay, Labeling

    The DNA-PKcs PL motif mediates the DNA-PKcs and PIDD association in vitro and in vivo . ( A ) Recombinant His-tagged PIDD death domain (a.a. 778–873) was pulled down by the GST fusion protein carrying the wild-type DNA-PKcs N’ terminal fragment (a.a. 1–403) but not the PL mutant fragments (2A or 4A), as indicated. The loading of the GST fusions was demonstrated by Ponceau S staining. ( B ) V3-WT or V3-mPL1 cells were transfected with FLAG-tagged PIDD-C (a.a. 446–910) or PIDD-CC (a.a. 588–910) constructs. Whole cell lysates were subjected to co-IP with anti-DNA-PKcs antibody and western blotted against anti-DNA-PKcs or anti-FLAG antibodies. ( C ) V3-WT and V3-mPL1 cells transfected with the FLAG-PIDD construct were UV (20 J/m 2 ) irradiated and harvested at 30 min for PLA (green) using anti-DNA-PKcs and anti-FLAG antibodies. The right panel shows the quantification of PLA spots per nucleus. N > 200. **** P

    Journal: Nucleic Acids Research

    Article Title: PIDD mediates the association of DNA-PKcs and ATR at stalled replication forks to facilitate the ATR signaling pathway

    doi: 10.1093/nar/gkx1298

    Figure Lengend Snippet: The DNA-PKcs PL motif mediates the DNA-PKcs and PIDD association in vitro and in vivo . ( A ) Recombinant His-tagged PIDD death domain (a.a. 778–873) was pulled down by the GST fusion protein carrying the wild-type DNA-PKcs N’ terminal fragment (a.a. 1–403) but not the PL mutant fragments (2A or 4A), as indicated. The loading of the GST fusions was demonstrated by Ponceau S staining. ( B ) V3-WT or V3-mPL1 cells were transfected with FLAG-tagged PIDD-C (a.a. 446–910) or PIDD-CC (a.a. 588–910) constructs. Whole cell lysates were subjected to co-IP with anti-DNA-PKcs antibody and western blotted against anti-DNA-PKcs or anti-FLAG antibodies. ( C ) V3-WT and V3-mPL1 cells transfected with the FLAG-PIDD construct were UV (20 J/m 2 ) irradiated and harvested at 30 min for PLA (green) using anti-DNA-PKcs and anti-FLAG antibodies. The right panel shows the quantification of PLA spots per nucleus. N > 200. **** P

    Article Snippet: Mouse anti-DNA-PKcs (Thermo Fisher Scientific, 1:100 dilution), rabbit anti-ATR (Bethyl, 1:100), mouse anti-RPA2 (EMD Millipore, 1:50) and rabbit anti-FLAG (Sigma, 1:100) antibodies were commercially available at the indicated vendors.

    Techniques: In Vitro, In Vivo, Recombinant, Mutagenesis, Staining, Transfection, Construct, Co-Immunoprecipitation Assay, Western Blot, Irradiation, Proximity Ligation Assay

    PIDD bridges DNA-PKcs and ATR upon replication stress. ( A ) 293FT cells were transfected with control (siCon) and siRNA against PIDD (siPIDD), and analyzed for PIDD protein expression. ( B ) Control and siPIDD-transfected 293FT cells were exposed to UV (20 J/m 2 ) and harvested at the indicated time points. Whole cell lysates were subjected to western blot. ( C and D ) Control and siPIDD-transfected 293FT cells were exposed to UV and harvested at 30 min for PLA (green) using anti-ATR in combination with (C) anti-DNA-PKcs or (D) anti-RPA2 antibodies. The right panel shows the quantification of the PLA spots per nucleus. N > 300. **** P

    Journal: Nucleic Acids Research

    Article Title: PIDD mediates the association of DNA-PKcs and ATR at stalled replication forks to facilitate the ATR signaling pathway

    doi: 10.1093/nar/gkx1298

    Figure Lengend Snippet: PIDD bridges DNA-PKcs and ATR upon replication stress. ( A ) 293FT cells were transfected with control (siCon) and siRNA against PIDD (siPIDD), and analyzed for PIDD protein expression. ( B ) Control and siPIDD-transfected 293FT cells were exposed to UV (20 J/m 2 ) and harvested at the indicated time points. Whole cell lysates were subjected to western blot. ( C and D ) Control and siPIDD-transfected 293FT cells were exposed to UV and harvested at 30 min for PLA (green) using anti-ATR in combination with (C) anti-DNA-PKcs or (D) anti-RPA2 antibodies. The right panel shows the quantification of the PLA spots per nucleus. N > 300. **** P

    Article Snippet: Mouse anti-DNA-PKcs (Thermo Fisher Scientific, 1:100 dilution), rabbit anti-ATR (Bethyl, 1:100), mouse anti-RPA2 (EMD Millipore, 1:50) and rabbit anti-FLAG (Sigma, 1:100) antibodies were commercially available at the indicated vendors.

    Techniques: Transfection, Expressing, Western Blot, Proximity Ligation Assay

    DNA-PKcs PL motif participates in the ATR signaling pathway and intra-S checkpoint. ( A ) V3-WT and V3-mPL1 cells were exposed to UV (20 J/m 2 ) and harvested at the indicated time points for WB using regular and phospho-specific antibodies against DNA-PKcs. ( B ) V3-WT and V3-mPL1 cells were pulse-labeled with 50 μM EdU, exposed to UV and then stained against EdU (red) and anti-pT2647 antibody (green). ( C and D ) Cells were treated with UV or HU and harvested at the indicated time points. Whole cell lysates were analyzed with indicated antibodies. ( E ) V3-WT and V3-mPL1 cells were sequentially labeled with IdU (100 μM, 10 min) and CldU (100 μM, 20 min) with or without UV exposure in between labels, and then analyzed by DNA fiber assay. The length of DNA tracks labeled with IdU (red) and CldU (green) were measured. The ratios of CldU to IdU in length were calculated from ongoing replication tracks (red-green, N ≥ 100).

    Journal: Nucleic Acids Research

    Article Title: PIDD mediates the association of DNA-PKcs and ATR at stalled replication forks to facilitate the ATR signaling pathway

    doi: 10.1093/nar/gkx1298

    Figure Lengend Snippet: DNA-PKcs PL motif participates in the ATR signaling pathway and intra-S checkpoint. ( A ) V3-WT and V3-mPL1 cells were exposed to UV (20 J/m 2 ) and harvested at the indicated time points for WB using regular and phospho-specific antibodies against DNA-PKcs. ( B ) V3-WT and V3-mPL1 cells were pulse-labeled with 50 μM EdU, exposed to UV and then stained against EdU (red) and anti-pT2647 antibody (green). ( C and D ) Cells were treated with UV or HU and harvested at the indicated time points. Whole cell lysates were analyzed with indicated antibodies. ( E ) V3-WT and V3-mPL1 cells were sequentially labeled with IdU (100 μM, 10 min) and CldU (100 μM, 20 min) with or without UV exposure in between labels, and then analyzed by DNA fiber assay. The length of DNA tracks labeled with IdU (red) and CldU (green) were measured. The ratios of CldU to IdU in length were calculated from ongoing replication tracks (red-green, N ≥ 100).

    Article Snippet: Mouse anti-DNA-PKcs (Thermo Fisher Scientific, 1:100 dilution), rabbit anti-ATR (Bethyl, 1:100), mouse anti-RPA2 (EMD Millipore, 1:50) and rabbit anti-FLAG (Sigma, 1:100) antibodies were commercially available at the indicated vendors.

    Techniques: Western Blot, Labeling, Staining

    The PL motif of DNA-PKcs is required for DNA replication stress response. ( A – C ) V3 derivative cell lines were analyzed for their colony forming ability against IR (A), UV (B) and HU (C). Data are presented as mean ± s.e.m., N = 3. ( D ) V3 derived cells were pulse-labeled with 50 μM EdU, treated with HU (2 mM) for 2 h, pre-extracted with 0.1% TX-100, and stained against anti-RPA2 antibody and EdU. Data are presented as RPA2 densities in EdU+ nuclei ( N > 150). **** P

    Journal: Nucleic Acids Research

    Article Title: PIDD mediates the association of DNA-PKcs and ATR at stalled replication forks to facilitate the ATR signaling pathway

    doi: 10.1093/nar/gkx1298

    Figure Lengend Snippet: The PL motif of DNA-PKcs is required for DNA replication stress response. ( A – C ) V3 derivative cell lines were analyzed for their colony forming ability against IR (A), UV (B) and HU (C). Data are presented as mean ± s.e.m., N = 3. ( D ) V3 derived cells were pulse-labeled with 50 μM EdU, treated with HU (2 mM) for 2 h, pre-extracted with 0.1% TX-100, and stained against anti-RPA2 antibody and EdU. Data are presented as RPA2 densities in EdU+ nuclei ( N > 150). **** P

    Article Snippet: Mouse anti-DNA-PKcs (Thermo Fisher Scientific, 1:100 dilution), rabbit anti-ATR (Bethyl, 1:100), mouse anti-RPA2 (EMD Millipore, 1:50) and rabbit anti-FLAG (Sigma, 1:100) antibodies were commercially available at the indicated vendors.

    Techniques: Derivative Assay, Labeling, Staining

    Ku80 is dispensable for DNA-PKcs and ATR association in response to UV irradiation. HCT116 Ku86 Flox/− cells were sham treated or incubated with Ad-Cre adenovirus expressing Cre recombinase for 4 days. ( A ) Cells were exposed to UV (20 J/m 2 ) and analyzed for DNA-PKcs phosphorylation by western blot. ( B ) Cells were pulse-labeled with EdU, exposed to UV, followed by immunostaining against EdU (red) and anti-pT2647 (green) antibodies. ( C ) Cells were exposed to UV and analyzed by PLA using anti-ATR and anti-DNA-PKcs antibodies. Right, quantification. Ku86 Flox/− sham, N > 300. **** P

    Journal: Nucleic Acids Research

    Article Title: PIDD mediates the association of DNA-PKcs and ATR at stalled replication forks to facilitate the ATR signaling pathway

    doi: 10.1093/nar/gkx1298

    Figure Lengend Snippet: Ku80 is dispensable for DNA-PKcs and ATR association in response to UV irradiation. HCT116 Ku86 Flox/− cells were sham treated or incubated with Ad-Cre adenovirus expressing Cre recombinase for 4 days. ( A ) Cells were exposed to UV (20 J/m 2 ) and analyzed for DNA-PKcs phosphorylation by western blot. ( B ) Cells were pulse-labeled with EdU, exposed to UV, followed by immunostaining against EdU (red) and anti-pT2647 (green) antibodies. ( C ) Cells were exposed to UV and analyzed by PLA using anti-ATR and anti-DNA-PKcs antibodies. Right, quantification. Ku86 Flox/− sham, N > 300. **** P

    Article Snippet: Mouse anti-DNA-PKcs (Thermo Fisher Scientific, 1:100 dilution), rabbit anti-ATR (Bethyl, 1:100), mouse anti-RPA2 (EMD Millipore, 1:50) and rabbit anti-FLAG (Sigma, 1:100) antibodies were commercially available at the indicated vendors.

    Techniques: Irradiation, Incubation, Expressing, Western Blot, Labeling, Immunostaining, Proximity Ligation Assay

    TRAIL inhibited P-gp efflux function in MDR cells by P-gp cleavage via caspase-3 activation . (A) The cell lysates obtained from CEM/VLB 100 cells treated with or without TRAIL (10 ng/ml) for 3 ~ 9 h ( left ) and the cell lysates of the MDR cells treated with TRAIL (5 ng/ml) for 6 h or pretreated with 50 μM Z-DEVD-FMK, a specific caspase-3 inhibitor, for 3 h and then with TRAIL for 6 h ( right ) were subjected to western blot analysis to monitor levels of P-gp, DNA-PKcs, caspase-3 and PARP and their its cleavage fragment (CF). (B) flow cytometric assay of P-gp efflux activity in TRAIL-treated MDR cells was based on extrusion of the fluorescent P-gp substrate, rhodamine123 (Rho123). The efflux activity of P-gp is highly temperature sensitive because functions optimally 37°C but is inactive at 4°C. Cell suspension from CEM and CEM/VLB 100 cells treated with or without 10 ng/ml TRAIL for 6 h was incubated with Rho 123 and further incubated at 37°C for 3 h (TREATED 37°C as TRAIL-treated cells or CTRL37°C as TRAIL-untreated control) to allow P-gp-mediated drug efflux or on ice as control (CTRL 4°C).

    Journal: Molecular Cancer

    Article Title: TRAIL sensitize MDR cells to MDR-related drugs by down-regulation of P-glycoprotein through inhibition of DNA-PKcs/Akt/GSK-3? pathway and activation of caspases

    doi: 10.1186/1476-4598-9-199

    Figure Lengend Snippet: TRAIL inhibited P-gp efflux function in MDR cells by P-gp cleavage via caspase-3 activation . (A) The cell lysates obtained from CEM/VLB 100 cells treated with or without TRAIL (10 ng/ml) for 3 ~ 9 h ( left ) and the cell lysates of the MDR cells treated with TRAIL (5 ng/ml) for 6 h or pretreated with 50 μM Z-DEVD-FMK, a specific caspase-3 inhibitor, for 3 h and then with TRAIL for 6 h ( right ) were subjected to western blot analysis to monitor levels of P-gp, DNA-PKcs, caspase-3 and PARP and their its cleavage fragment (CF). (B) flow cytometric assay of P-gp efflux activity in TRAIL-treated MDR cells was based on extrusion of the fluorescent P-gp substrate, rhodamine123 (Rho123). The efflux activity of P-gp is highly temperature sensitive because functions optimally 37°C but is inactive at 4°C. Cell suspension from CEM and CEM/VLB 100 cells treated with or without 10 ng/ml TRAIL for 6 h was incubated with Rho 123 and further incubated at 37°C for 3 h (TREATED 37°C as TRAIL-treated cells or CTRL37°C as TRAIL-untreated control) to allow P-gp-mediated drug efflux or on ice as control (CTRL 4°C).

    Article Snippet: Western blot analysis was performed with the following antibodies: anti-Bax, anti-caspase-3, anti-PARP, anti-Bcl-2 (Santa Cruz Biotechnology, CA), anti-Akt, anti-phospho-Akt (Ser 473), anti-caspase-8, anti-caspase-9 (Cell signal, Danvers, MA), anti-DNA-PKcs (Thermo Fisher Scientific, CA), anti-DR5, anti-caspase-10 (Calbiochem, Germany), anti-pGSK-3β (Ser8), anti-GSK-3β, anti-GSK-3α, anti-c-Myc (Epitomics, CA), anti-DR4 (R & D Systems, MN) and anti β-actin (Sigma-Aldrich) antibodies, Secondary antibodies were obtained from GE Healthcare.

    Techniques: Activation Assay, Western Blot, Flow Cytometry, Activity Assay, Incubation

    Potentiated suppression of DNA-PKcs expression by TRAIL leads to a severe inhibition of both Akt/GSK-3β phosphorylation and P-gp and c-FLIPs expression and enhances the cytotoxicity of TRAIL and of MDR-related drug in MDR cells . (A) CEM/VLB 100 cells were transfected with a siRNA against DNA-PKcs or scrambled siRNA as a control. After 48 h, the transfectant was treated with or without TRAIL (1- and 10 ng/ml for 24 h), and the changed level of DNA-PKcs, pAkt (Ser473), tAkt, pGSK3β (Ser9), GSK-3α/β, P-gp, PARP, c-FLIP L/S and actin was performed by western blot analysis. (B) Each transfectant was treated with indicated doses of TRAIL or vincristine (VCR). After 5 days, the percentage of growth inhibition was determined incubation using the MTT assay. Data represent means ± S.D. of triplicate experiments. ** p

    Journal: Molecular Cancer

    Article Title: TRAIL sensitize MDR cells to MDR-related drugs by down-regulation of P-glycoprotein through inhibition of DNA-PKcs/Akt/GSK-3? pathway and activation of caspases

    doi: 10.1186/1476-4598-9-199

    Figure Lengend Snippet: Potentiated suppression of DNA-PKcs expression by TRAIL leads to a severe inhibition of both Akt/GSK-3β phosphorylation and P-gp and c-FLIPs expression and enhances the cytotoxicity of TRAIL and of MDR-related drug in MDR cells . (A) CEM/VLB 100 cells were transfected with a siRNA against DNA-PKcs or scrambled siRNA as a control. After 48 h, the transfectant was treated with or without TRAIL (1- and 10 ng/ml for 24 h), and the changed level of DNA-PKcs, pAkt (Ser473), tAkt, pGSK3β (Ser9), GSK-3α/β, P-gp, PARP, c-FLIP L/S and actin was performed by western blot analysis. (B) Each transfectant was treated with indicated doses of TRAIL or vincristine (VCR). After 5 days, the percentage of growth inhibition was determined incubation using the MTT assay. Data represent means ± S.D. of triplicate experiments. ** p

    Article Snippet: Western blot analysis was performed with the following antibodies: anti-Bax, anti-caspase-3, anti-PARP, anti-Bcl-2 (Santa Cruz Biotechnology, CA), anti-Akt, anti-phospho-Akt (Ser 473), anti-caspase-8, anti-caspase-9 (Cell signal, Danvers, MA), anti-DNA-PKcs (Thermo Fisher Scientific, CA), anti-DR5, anti-caspase-10 (Calbiochem, Germany), anti-pGSK-3β (Ser8), anti-GSK-3β, anti-GSK-3α, anti-c-Myc (Epitomics, CA), anti-DR4 (R & D Systems, MN) and anti β-actin (Sigma-Aldrich) antibodies, Secondary antibodies were obtained from GE Healthcare.

    Techniques: Expressing, Inhibition, Transfection, Western Blot, Incubation, MTT Assay

    Supprerssion of DNA-PKcs up-regulated surface expression of DR5 and down-regulated the expression of c-FLIPs . (A) CEM/VLB 100 cells were transfected with a siRNA against DNA-PKcs or scrambled siRNA as a control. After 48 h, the total RNA extracted from transfectant of CEM/VLB 100 cells performed RT-PCR analysis to monitor the mRNA levels of DNA-PKcs, DR4/5, c-FLIP L/S , MDR1, and actin as a loading control. (B) The transfectant incubated with an anti-DR4 or -DR5 (1:500), and subsequently labeled with FITC-conjugated secondary antibodies (1:1000) to determine the surface expression of DR4 and DR5. Goat IgG2a was also used as control isotype antibody.

    Journal: Molecular Cancer

    Article Title: TRAIL sensitize MDR cells to MDR-related drugs by down-regulation of P-glycoprotein through inhibition of DNA-PKcs/Akt/GSK-3? pathway and activation of caspases

    doi: 10.1186/1476-4598-9-199

    Figure Lengend Snippet: Supprerssion of DNA-PKcs up-regulated surface expression of DR5 and down-regulated the expression of c-FLIPs . (A) CEM/VLB 100 cells were transfected with a siRNA against DNA-PKcs or scrambled siRNA as a control. After 48 h, the total RNA extracted from transfectant of CEM/VLB 100 cells performed RT-PCR analysis to monitor the mRNA levels of DNA-PKcs, DR4/5, c-FLIP L/S , MDR1, and actin as a loading control. (B) The transfectant incubated with an anti-DR4 or -DR5 (1:500), and subsequently labeled with FITC-conjugated secondary antibodies (1:1000) to determine the surface expression of DR4 and DR5. Goat IgG2a was also used as control isotype antibody.

    Article Snippet: Western blot analysis was performed with the following antibodies: anti-Bax, anti-caspase-3, anti-PARP, anti-Bcl-2 (Santa Cruz Biotechnology, CA), anti-Akt, anti-phospho-Akt (Ser 473), anti-caspase-8, anti-caspase-9 (Cell signal, Danvers, MA), anti-DNA-PKcs (Thermo Fisher Scientific, CA), anti-DR5, anti-caspase-10 (Calbiochem, Germany), anti-pGSK-3β (Ser8), anti-GSK-3β, anti-GSK-3α, anti-c-Myc (Epitomics, CA), anti-DR4 (R & D Systems, MN) and anti β-actin (Sigma-Aldrich) antibodies, Secondary antibodies were obtained from GE Healthcare.

    Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Incubation, Labeling

    Expression of p-gp in MDR cells is associated with the enhanced expression of c-Myc and DR5 and activation of DNA-PKcs/Akt/GSK-3β pathway . The protein levels of P-gp, c-Myc, DNA-PKcs, pAkt, GSK-3β, phosphorylated Akt (pAkt) at Ser 473, total Akt (tAkt), phosphorylated GSK-3β (pSK3β) at Ser9, total GSK-3α/β, DR4 and DR5 were determined by western analysis. Actin was used as a loading control.

    Journal: Molecular Cancer

    Article Title: TRAIL sensitize MDR cells to MDR-related drugs by down-regulation of P-glycoprotein through inhibition of DNA-PKcs/Akt/GSK-3? pathway and activation of caspases

    doi: 10.1186/1476-4598-9-199

    Figure Lengend Snippet: Expression of p-gp in MDR cells is associated with the enhanced expression of c-Myc and DR5 and activation of DNA-PKcs/Akt/GSK-3β pathway . The protein levels of P-gp, c-Myc, DNA-PKcs, pAkt, GSK-3β, phosphorylated Akt (pAkt) at Ser 473, total Akt (tAkt), phosphorylated GSK-3β (pSK3β) at Ser9, total GSK-3α/β, DR4 and DR5 were determined by western analysis. Actin was used as a loading control.

    Article Snippet: Western blot analysis was performed with the following antibodies: anti-Bax, anti-caspase-3, anti-PARP, anti-Bcl-2 (Santa Cruz Biotechnology, CA), anti-Akt, anti-phospho-Akt (Ser 473), anti-caspase-8, anti-caspase-9 (Cell signal, Danvers, MA), anti-DNA-PKcs (Thermo Fisher Scientific, CA), anti-DR5, anti-caspase-10 (Calbiochem, Germany), anti-pGSK-3β (Ser8), anti-GSK-3β, anti-GSK-3α, anti-c-Myc (Epitomics, CA), anti-DR4 (R & D Systems, MN) and anti β-actin (Sigma-Aldrich) antibodies, Secondary antibodies were obtained from GE Healthcare.

    Techniques: Expressing, Activation Assay, Western Blot

    TRAIL inhibited P-gp expression and DNA-PKcs/Akt/GSK-3β signaling pathway and up-regulation of death receptors and down-regulation of c-FLIP in MDR cells . (A) Cell lysates obtained from CEM or CEM/VLB 100 cells treated with indicated dose of TRAIL for 24 h were subjected to western blot analysis to monitor levels of P-glycoprotein (P-gp), DNA-PKcs, pAkt (Ser473), tAkt, pGSK3β (Ser 9), GSK-3α and -3β, Mcl-1. (B) CEM/VLB 100 cells treated with TRAIL (10 ng/ml) for 24 h were incubated on ice in the presence of DR4- and DR5-specific antibodies (1:500), and subsequently labeled with FITC-conjugated secondary antibody (1:1000). The fluorescence intensity was analyzed with flow cytometry. The thin line indicates that the cells only incubated with Goat IgG2a that was used as a control isotype antibody; the thick line indicates the specific labeling ( left ). The cells treated with or without TRAIL, and changed mRNA level of c-FLIP L/S was determined by RT-PCR analysis ( right ).

    Journal: Molecular Cancer

    Article Title: TRAIL sensitize MDR cells to MDR-related drugs by down-regulation of P-glycoprotein through inhibition of DNA-PKcs/Akt/GSK-3? pathway and activation of caspases

    doi: 10.1186/1476-4598-9-199

    Figure Lengend Snippet: TRAIL inhibited P-gp expression and DNA-PKcs/Akt/GSK-3β signaling pathway and up-regulation of death receptors and down-regulation of c-FLIP in MDR cells . (A) Cell lysates obtained from CEM or CEM/VLB 100 cells treated with indicated dose of TRAIL for 24 h were subjected to western blot analysis to monitor levels of P-glycoprotein (P-gp), DNA-PKcs, pAkt (Ser473), tAkt, pGSK3β (Ser 9), GSK-3α and -3β, Mcl-1. (B) CEM/VLB 100 cells treated with TRAIL (10 ng/ml) for 24 h were incubated on ice in the presence of DR4- and DR5-specific antibodies (1:500), and subsequently labeled with FITC-conjugated secondary antibody (1:1000). The fluorescence intensity was analyzed with flow cytometry. The thin line indicates that the cells only incubated with Goat IgG2a that was used as a control isotype antibody; the thick line indicates the specific labeling ( left ). The cells treated with or without TRAIL, and changed mRNA level of c-FLIP L/S was determined by RT-PCR analysis ( right ).

    Article Snippet: Western blot analysis was performed with the following antibodies: anti-Bax, anti-caspase-3, anti-PARP, anti-Bcl-2 (Santa Cruz Biotechnology, CA), anti-Akt, anti-phospho-Akt (Ser 473), anti-caspase-8, anti-caspase-9 (Cell signal, Danvers, MA), anti-DNA-PKcs (Thermo Fisher Scientific, CA), anti-DR5, anti-caspase-10 (Calbiochem, Germany), anti-pGSK-3β (Ser8), anti-GSK-3β, anti-GSK-3α, anti-c-Myc (Epitomics, CA), anti-DR4 (R & D Systems, MN) and anti β-actin (Sigma-Aldrich) antibodies, Secondary antibodies were obtained from GE Healthcare.

    Techniques: Expressing, Western Blot, Incubation, Labeling, Fluorescence, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction

    TRAIL enhanced the cytotoxicity of MDR-related drug in MDR cells by the down-regulation of DNA-PKcs and P-gp via caspase-3 activation . (A) CEM/VLB 100 cells were treated with graded single doses of doxorubicin (DOX) or vinblastine (VLB) after pretreatment of low dose of TRAIL (1 ng/ml) for 6 h. The percentage of cell survival after combined treatment of TRAIL with MDR-related drug was determined after 5 days incubation using the MTT assay. Each bar represents the mean ± S.D. of triplicate experiments. *** p

    Journal: Molecular Cancer

    Article Title: TRAIL sensitize MDR cells to MDR-related drugs by down-regulation of P-glycoprotein through inhibition of DNA-PKcs/Akt/GSK-3? pathway and activation of caspases

    doi: 10.1186/1476-4598-9-199

    Figure Lengend Snippet: TRAIL enhanced the cytotoxicity of MDR-related drug in MDR cells by the down-regulation of DNA-PKcs and P-gp via caspase-3 activation . (A) CEM/VLB 100 cells were treated with graded single doses of doxorubicin (DOX) or vinblastine (VLB) after pretreatment of low dose of TRAIL (1 ng/ml) for 6 h. The percentage of cell survival after combined treatment of TRAIL with MDR-related drug was determined after 5 days incubation using the MTT assay. Each bar represents the mean ± S.D. of triplicate experiments. *** p

    Article Snippet: Western blot analysis was performed with the following antibodies: anti-Bax, anti-caspase-3, anti-PARP, anti-Bcl-2 (Santa Cruz Biotechnology, CA), anti-Akt, anti-phospho-Akt (Ser 473), anti-caspase-8, anti-caspase-9 (Cell signal, Danvers, MA), anti-DNA-PKcs (Thermo Fisher Scientific, CA), anti-DR5, anti-caspase-10 (Calbiochem, Germany), anti-pGSK-3β (Ser8), anti-GSK-3β, anti-GSK-3α, anti-c-Myc (Epitomics, CA), anti-DR4 (R & D Systems, MN) and anti β-actin (Sigma-Aldrich) antibodies, Secondary antibodies were obtained from GE Healthcare.

    Techniques: Activation Assay, Incubation, MTT Assay