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    Name:
    QIAGEN Proteinase K
    Description:
    For protease digestion during DNA and RNA preparation Kit contents Qiagen Proteinase K 2mL 600mAU mL Activity Ready to use Solution For Protease Digestion During DNA and RNA Preparation Offer Broad Substrate Specificity with High Activity for a Wide Range of Reaction Conditions Used in Most DNA and RNA Isolation
    Catalog Number:
    19131
    Price:
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    QIAGEN Proteinase K
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    Qiagen pbs buffer
    QIAGEN Proteinase K
    For protease digestion during DNA and RNA preparation Kit contents Qiagen Proteinase K 2mL 600mAU mL Activity Ready to use Solution For Protease Digestion During DNA and RNA Preparation Offer Broad Substrate Specificity with High Activity for a Wide Range of Reaction Conditions Used in Most DNA and RNA Isolation
    https://www.bioz.com/result/pbs buffer/product/Qiagen
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    pbs buffer - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Lipopolysaccharide O-antigen delays plant innate immune recognition of Xylella fastidiosa"

    Article Title: Lipopolysaccharide O-antigen delays plant innate immune recognition of Xylella fastidiosa

    Journal: Nature Communications

    doi: 10.1038/s41467-018-02861-5

    O-antigen-modulated ROS production by extracted LPS and intact bacterial cells ex vivo. Discs of V. vinifera ‘Cabernet Sauvignon’ leaves were treated with 20 μL of a 50 μg/mL solution of purified LPS elicitors (either wzy or wild type LPS) equal to a final amount of 10 μg (based on Kdo content) of LPS, 20 μL of a 10 8 CFU/mL suspension of Xf wild type or wzy cells, or diH 2 0 or 1× PBS-inoculated controls, respectively. a The amplitude of ROS production remained similar for both wild type and wzy LPS, reaching max production at ~4 min, and plateaued starting around 30 min. b Total ROS production is reported as area under the curve (AUC) for plot of luminescence intensity over time. Total ROS production was not significantly different between discs treated with wild type or wzy extracted LPS. c Intact wzy cells induced a significantly stronger oxidative burst that persisted nearly 20 min longer than leaves inoculated with wild type bacteria (which contained fully polymerized O-antigens). Graphs represent the mean of 24 replicates per treatment ± standard error of the mean. d Total ROS production is reported as area under the curve (AUC) for plot of luminescence intensity over time. Discs treated with wzy cells produced significantly more ROS than discs treated with wild type cells. Graphs represent the mean of 24 replicates per treatment ± standard error of the mean. Treatments with different letters over the bars are statistically different ( P
    Figure Legend Snippet: O-antigen-modulated ROS production by extracted LPS and intact bacterial cells ex vivo. Discs of V. vinifera ‘Cabernet Sauvignon’ leaves were treated with 20 μL of a 50 μg/mL solution of purified LPS elicitors (either wzy or wild type LPS) equal to a final amount of 10 μg (based on Kdo content) of LPS, 20 μL of a 10 8 CFU/mL suspension of Xf wild type or wzy cells, or diH 2 0 or 1× PBS-inoculated controls, respectively. a The amplitude of ROS production remained similar for both wild type and wzy LPS, reaching max production at ~4 min, and plateaued starting around 30 min. b Total ROS production is reported as area under the curve (AUC) for plot of luminescence intensity over time. Total ROS production was not significantly different between discs treated with wild type or wzy extracted LPS. c Intact wzy cells induced a significantly stronger oxidative burst that persisted nearly 20 min longer than leaves inoculated with wild type bacteria (which contained fully polymerized O-antigens). Graphs represent the mean of 24 replicates per treatment ± standard error of the mean. d Total ROS production is reported as area under the curve (AUC) for plot of luminescence intensity over time. Discs treated with wzy cells produced significantly more ROS than discs treated with wild type cells. Graphs represent the mean of 24 replicates per treatment ± standard error of the mean. Treatments with different letters over the bars are statistically different ( P

    Techniques Used: Ex Vivo, Purification, Produced

    In situ localization of O-antigen-modulated ROS production in the xylem in petioles of inoculated plants. a DAB-mediated tissue printing of petioles at 15 min post-inoculation indicated a strong production of H 2 O 2 specifically in the xylem vessels of grapevines needle inoculated with wzy cells (location of xylem vessels emphasized with dotted outline). Vines inoculated with wild type Xf exhibited H 2 O 2 production predominantly in peripheral collenchyma tissue, with some production in the xylem vessels. Vines inoculated with 1× PBS buffer served as negative controls. b Mean gray value of grayscale-converted DAB-stained images, representing differences in staining intensity. Grayscale intensities vary from 0 to 255; 0 = black, 255 = white, with the values in between representing shades of gray. The mean gray value of DAB-stained images from wzy -inoculated plants is significantly lower than wild type or 1× PBS-inoculated plants, indicating a darker, or more intense stain, and thus higher amounts of H 2 O 2 . Treatments with different letters over the bars are statistically different ( P
    Figure Legend Snippet: In situ localization of O-antigen-modulated ROS production in the xylem in petioles of inoculated plants. a DAB-mediated tissue printing of petioles at 15 min post-inoculation indicated a strong production of H 2 O 2 specifically in the xylem vessels of grapevines needle inoculated with wzy cells (location of xylem vessels emphasized with dotted outline). Vines inoculated with wild type Xf exhibited H 2 O 2 production predominantly in peripheral collenchyma tissue, with some production in the xylem vessels. Vines inoculated with 1× PBS buffer served as negative controls. b Mean gray value of grayscale-converted DAB-stained images, representing differences in staining intensity. Grayscale intensities vary from 0 to 255; 0 = black, 255 = white, with the values in between representing shades of gray. The mean gray value of DAB-stained images from wzy -inoculated plants is significantly lower than wild type or 1× PBS-inoculated plants, indicating a darker, or more intense stain, and thus higher amounts of H 2 O 2 . Treatments with different letters over the bars are statistically different ( P

    Techniques Used: In Situ, Staining

    2) Product Images from "STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-Associated Herpesvirus"

    Article Title: STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-Associated Herpesvirus

    Journal: Journal of Virology

    doi: 10.1128/JVI.02008-15

    Overexpression of STAT3 restrains susceptibility to KSHV lytic activation. (A to D) BCBL-1 cells were transfected with the pEGFPN1 plasmid (GFP) or the pEGFPN1-STAT3 plasmid (STAT3-GFP), exposed to VPA after 12 h, and harvested after another 24 h for determination of relative amounts of transcripts from KSHV lytic genes ORF50 , ORF59 , ORF9 , and K8.1 by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method (A), for determination of relative amounts of cell-associated KSHV DNA by qPCR (B), for determination of K8.1 + cells by flow cytometry (C), or assayed for infectious virions in the cell supernatant by inoculation of HUVECs and staining with anti-LANA antibody and DAPI 48 h later (D). (A and B) Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (C) Numbers indicate the percentages of GFP + (i.e., transfected) cells that were lytic. Lytic (i.e., K8.1 + ) gates were placed based on a 1% cutoff in similarly treated cells that were stained with isotype control antibody. A representative of the results of two experiments is shown. (E) BCBL-1 cells were transfected with GFP plasmid or STAT3-GFP plasmid (as in panels A to D) and harvested at 24 h posttransfection for Western blot analysis using anti-STAT3 and anti-β-actin antibodies.
    Figure Legend Snippet: Overexpression of STAT3 restrains susceptibility to KSHV lytic activation. (A to D) BCBL-1 cells were transfected with the pEGFPN1 plasmid (GFP) or the pEGFPN1-STAT3 plasmid (STAT3-GFP), exposed to VPA after 12 h, and harvested after another 24 h for determination of relative amounts of transcripts from KSHV lytic genes ORF50 , ORF59 , ORF9 , and K8.1 by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method (A), for determination of relative amounts of cell-associated KSHV DNA by qPCR (B), for determination of K8.1 + cells by flow cytometry (C), or assayed for infectious virions in the cell supernatant by inoculation of HUVECs and staining with anti-LANA antibody and DAPI 48 h later (D). (A and B) Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (C) Numbers indicate the percentages of GFP + (i.e., transfected) cells that were lytic. Lytic (i.e., K8.1 + ) gates were placed based on a 1% cutoff in similarly treated cells that were stained with isotype control antibody. A representative of the results of two experiments is shown. (E) BCBL-1 cells were transfected with GFP plasmid or STAT3-GFP plasmid (as in panels A to D) and harvested at 24 h posttransfection for Western blot analysis using anti-STAT3 and anti-β-actin antibodies.

    Techniques Used: Over Expression, Activation Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Staining, Western Blot

    Chemical inhibition of STAT3 results in KSHV lytic activation, increase in the number of lytic cells, and increase in production of infectious virions. BCBL-1 cells were treated with VPA, WP1066, or VPA plus WP1066 or left untreated. Cells were harvested at 1, 6, 12, and 18 h posttreatment (A), at 24 h posttreatment (B), and at 48 h posttreatment (C). (A) RNA was analyzed at different times by qRT-PCR for relative transcript levels of 4 lytic genes, ORF50 , ORF59 , ORF9 , and ORFK8.1 , compared to untreated cells. KSHV-specific transcript levels were normalized to GAPDH , HRPTI , and B2M (β2 microglobulin), and fold changes were determined by the ΔΔ C T method. Data are presented as means and SEM and are representative of the results of two separate experiments with 3 technical replicates. (B) Cells were immunostained for K8.1 and STAT3 and subjected to flow cytometry. Numbers indicate the percent K8.1 + (lytic) cells; these percentages were determined after comparison with similarly treated cells stained with isotype control antibody. (C) Cell supernatants were used to inoculate primary HUVECs. After 48 h, HUVECs were fixed, permeabilized, stained with anti-LANA antibody and DAPI, and visualized at ×40 magnification.
    Figure Legend Snippet: Chemical inhibition of STAT3 results in KSHV lytic activation, increase in the number of lytic cells, and increase in production of infectious virions. BCBL-1 cells were treated with VPA, WP1066, or VPA plus WP1066 or left untreated. Cells were harvested at 1, 6, 12, and 18 h posttreatment (A), at 24 h posttreatment (B), and at 48 h posttreatment (C). (A) RNA was analyzed at different times by qRT-PCR for relative transcript levels of 4 lytic genes, ORF50 , ORF59 , ORF9 , and ORFK8.1 , compared to untreated cells. KSHV-specific transcript levels were normalized to GAPDH , HRPTI , and B2M (β2 microglobulin), and fold changes were determined by the ΔΔ C T method. Data are presented as means and SEM and are representative of the results of two separate experiments with 3 technical replicates. (B) Cells were immunostained for K8.1 and STAT3 and subjected to flow cytometry. Numbers indicate the percent K8.1 + (lytic) cells; these percentages were determined after comparison with similarly treated cells stained with isotype control antibody. (C) Cell supernatants were used to inoculate primary HUVECs. After 48 h, HUVECs were fixed, permeabilized, stained with anti-LANA antibody and DAPI, and visualized at ×40 magnification.

    Techniques Used: Inhibition, Activation Assay, Quantitative RT-PCR, Flow Cytometry, Cytometry, Staining

    Cells expressing high levels of STAT3 protein are refractory to spontaneous and induced KSHV lytic activation. BCBL-1 cells were treated with TPA or VPA or left untreated (U). Cells were harvested at 1, 24, and 48 h (A and B) and at 48 h (C) posttreatment. (A) RNA was isolated and subjected to qRT-PCR to determine the relative levels of lytic ORFK8.1 transcripts. Fold changes were calculated by the ΔΔ C T method, normalized to three housekeeping genes, GAPDH , HPRTI , and B2M (β2 microglobulin). The data are presented as means and standard errors of the mean (SEM) and are representative of the results of two experiments with 3 technical replicates. (B) Cell lysates were immunoblotted using antibodies to K8.1 and β-actin. (C) Cells were immunostained for K8.1 and STAT3 and subjected to flow cytometry. The numbers within the dot plots in the left column indicate the percent K8.1 hi cells under untreated or TPA- or VPA-treated conditions after comparison with similarly treated cells stained with isotype control antibody; the dashed oval gates indicate K8.1 lo cells. The numbers within the dot plots in the three middle columns indicate the percent K8.1 hi cells in subpopulations expressing different levels of STAT3, i.e., STAT3 hi (high), STAT3 int (intermediate), and STAT3 lo (low). Numbers in the dot plots on the right indicate the percent K8.1 lo cells (from the oval gates in the left-hand dot plots) expressing low, intermediate, and high levels of STAT3.
    Figure Legend Snippet: Cells expressing high levels of STAT3 protein are refractory to spontaneous and induced KSHV lytic activation. BCBL-1 cells were treated with TPA or VPA or left untreated (U). Cells were harvested at 1, 24, and 48 h (A and B) and at 48 h (C) posttreatment. (A) RNA was isolated and subjected to qRT-PCR to determine the relative levels of lytic ORFK8.1 transcripts. Fold changes were calculated by the ΔΔ C T method, normalized to three housekeeping genes, GAPDH , HPRTI , and B2M (β2 microglobulin). The data are presented as means and standard errors of the mean (SEM) and are representative of the results of two experiments with 3 technical replicates. (B) Cell lysates were immunoblotted using antibodies to K8.1 and β-actin. (C) Cells were immunostained for K8.1 and STAT3 and subjected to flow cytometry. The numbers within the dot plots in the left column indicate the percent K8.1 hi cells under untreated or TPA- or VPA-treated conditions after comparison with similarly treated cells stained with isotype control antibody; the dashed oval gates indicate K8.1 lo cells. The numbers within the dot plots in the three middle columns indicate the percent K8.1 hi cells in subpopulations expressing different levels of STAT3, i.e., STAT3 hi (high), STAT3 int (intermediate), and STAT3 lo (low). Numbers in the dot plots on the right indicate the percent K8.1 lo cells (from the oval gates in the left-hand dot plots) expressing low, intermediate, and high levels of STAT3.

    Techniques Used: Expressing, Activation Assay, Isolation, Quantitative RT-PCR, Flow Cytometry, Cytometry, Staining

    3) Product Images from "HDAC1 localizes to the mitochondria of cardiac myocytes and contributes to early cardiac reperfusion injury"

    Article Title: HDAC1 localizes to the mitochondria of cardiac myocytes and contributes to early cardiac reperfusion injury

    Journal: Journal of molecular and cellular cardiology

    doi: 10.1016/j.yjmcc.2017.12.004

    Characterization of HDAC localization in tissues and cells. (A) Class I HDAC activity in whole heart lysates and mitochondrial isolates. Acetylated substrate selective for only class I and class IIb HDACs. HDAC6 activity inhibited with 1 μM Tubastatin A (Tub A). Class I HDACs inhibited with 5 μM MS275. Activity is assessed from a 2 h. reaction. (B) Western blotting for class I HDACs in whole heart nuclear, cytoplasmic, and mitochondrial isolates. (C) Proteinase K digestion of whole heart mitochondrial isolates. (D) Western blotting for HDAC1 in skeletal muscle and liver mitochondrial isolates. (E) Staining for HDAC1 and ACAA2 in cardiac myocytes, fibroblasts and endothelial cells. Bar = 10 μm N = 3 per group for all experiments.
    Figure Legend Snippet: Characterization of HDAC localization in tissues and cells. (A) Class I HDAC activity in whole heart lysates and mitochondrial isolates. Acetylated substrate selective for only class I and class IIb HDACs. HDAC6 activity inhibited with 1 μM Tubastatin A (Tub A). Class I HDACs inhibited with 5 μM MS275. Activity is assessed from a 2 h. reaction. (B) Western blotting for class I HDACs in whole heart nuclear, cytoplasmic, and mitochondrial isolates. (C) Proteinase K digestion of whole heart mitochondrial isolates. (D) Western blotting for HDAC1 in skeletal muscle and liver mitochondrial isolates. (E) Staining for HDAC1 and ACAA2 in cardiac myocytes, fibroblasts and endothelial cells. Bar = 10 μm N = 3 per group for all experiments.

    Techniques Used: Activity Assay, Western Blot, Staining

    4) Product Images from "CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing"

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing

    Journal: mBio

    doi: 10.1128/mBio.01376-18

    Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”
    Figure Legend Snippet: Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”

    Techniques Used: Expressing, Confocal Laser Scanning Microscopy, Microscopy, Over Expression, Plasmid Preparation, Staining

    5) Product Images from "BK Polyomavirus Hijacks Extracellular Vesicles for En Bloc Transmission"

    Article Title: BK Polyomavirus Hijacks Extracellular Vesicles for En Bloc Transmission

    Journal: Journal of Virology

    doi: 10.1128/JVI.01834-19

    BKPyV particles are released within EVs. (A) Chronically infected Vero cells were fixed and processed for electron microscopy. Electron micrographs show the presence of viral particles (indicated by arrowheads) in MVBs. The right panel (bar, 100 nm) corresponds to an enlargement of the left panel (bar, 0.2 μm). (B) Vero cells were infected with BKPyV at an MOI of 1. Supernatant was harvested 3 days postinfection, filtered at 0.45 μm, and overlaid on a 20% to 45% (wt/vol) iodixanol gradient. After a 24-h ultracentrifugation, 17 fractions were collected. The density (g/ml) of each fraction was calculated according to the optical density at 340 nm. BKPyV infectivity in each fraction was assessed by immunofluorescence 3 days after infection of naive Vero cells. It is expressed as percentages of infected cells. AChE activity was analyzed to detect the presence of EVs in each fraction. (C) EVs contained in the supernatant of infected cells were concentrated 100× by PEG precipitation and overlaid on a 20% to 45% (wt/vol) iodixanol gradient. The presence of EVs in fractions 1 to 13 was evaluated by the detection of CD9, CD63, and CD81 by Western blotting. The presence of viral capsids was evaluated by the detection of VP1. The presence of contaminating small cellular organelles was evaluated by the detection of GM130 and calnexin. (D) The experiment was performed as for panel B with the supernatant of HRPTE cells, harvested 10 days postinfection. (E) Fractions containing eBKPyV (fraction 6) or naked BKPyV (fraction 12) were treated with different concentrations of proteinase K for 10 min. The volumes of the fractions were adjusted to treat similar amounts of the VP1 protein under both conditions. The sensitivity to proteinase K digestion was then assessed by detection of the VP1 capsid protein by Western blotting (top panel) and evaluated by quantifying the relative amount of VP1 on the Western blots using ImageJ (bottom panel). Results are reported as the means ± standard deviations from two independent experiments. (F) After iodixanol gradient ultracentrifugation, fractions containing eBKPyV or naked BKPyV were treated with chloroform or left untreated and then analyzed for infectivity on naive Vero cells. (G) The supernatant of Vero cells was harvested 4 days postinfection and treated with chloroform or left untreated before performing the buoyant density iodixanol gradient ultracentrifugation. Results presented in panels B, D, F, and G are means from duplicates from representative experiments.
    Figure Legend Snippet: BKPyV particles are released within EVs. (A) Chronically infected Vero cells were fixed and processed for electron microscopy. Electron micrographs show the presence of viral particles (indicated by arrowheads) in MVBs. The right panel (bar, 100 nm) corresponds to an enlargement of the left panel (bar, 0.2 μm). (B) Vero cells were infected with BKPyV at an MOI of 1. Supernatant was harvested 3 days postinfection, filtered at 0.45 μm, and overlaid on a 20% to 45% (wt/vol) iodixanol gradient. After a 24-h ultracentrifugation, 17 fractions were collected. The density (g/ml) of each fraction was calculated according to the optical density at 340 nm. BKPyV infectivity in each fraction was assessed by immunofluorescence 3 days after infection of naive Vero cells. It is expressed as percentages of infected cells. AChE activity was analyzed to detect the presence of EVs in each fraction. (C) EVs contained in the supernatant of infected cells were concentrated 100× by PEG precipitation and overlaid on a 20% to 45% (wt/vol) iodixanol gradient. The presence of EVs in fractions 1 to 13 was evaluated by the detection of CD9, CD63, and CD81 by Western blotting. The presence of viral capsids was evaluated by the detection of VP1. The presence of contaminating small cellular organelles was evaluated by the detection of GM130 and calnexin. (D) The experiment was performed as for panel B with the supernatant of HRPTE cells, harvested 10 days postinfection. (E) Fractions containing eBKPyV (fraction 6) or naked BKPyV (fraction 12) were treated with different concentrations of proteinase K for 10 min. The volumes of the fractions were adjusted to treat similar amounts of the VP1 protein under both conditions. The sensitivity to proteinase K digestion was then assessed by detection of the VP1 capsid protein by Western blotting (top panel) and evaluated by quantifying the relative amount of VP1 on the Western blots using ImageJ (bottom panel). Results are reported as the means ± standard deviations from two independent experiments. (F) After iodixanol gradient ultracentrifugation, fractions containing eBKPyV or naked BKPyV were treated with chloroform or left untreated and then analyzed for infectivity on naive Vero cells. (G) The supernatant of Vero cells was harvested 4 days postinfection and treated with chloroform or left untreated before performing the buoyant density iodixanol gradient ultracentrifugation. Results presented in panels B, D, F, and G are means from duplicates from representative experiments.

    Techniques Used: Infection, Electron Microscopy, Immunofluorescence, Activity Assay, Western Blot

    6) Product Images from "Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity"

    Article Title: Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.02753

    The growth-promoting effect of eDNA on M. bovis . Comparative growth of M. bovis under axenic and cell culture conditions (A) . RM16 proliferation was monitored in SP4 medium (SP4), cell culture medium (DMEM), and cell culture medium supplemented with 10 μg/ml calf thymus DNA (DMEMD). Mycoplasma titers (log CFU/ml) were determined by CFU titrations. The cytopathic effect induced by M. bovis upon co-incubation with host cells (B) . EBL cells (10 4 cells) were inoculated with RM16 at an MOI of 2 (RM16) or mock-infected (Mock). After 72 h of co-incubation, monolayers were stained with crystal violet and survival cells were estimated by measuring the optical density at 590 nm (OD 590). When indicated, DMEM-based medium was supplemented with 10 μg/ml calf thymus DNA (eDNA). Calf thymus DNA was subjected to the following enzymatic treatments: RNase A (RNase), Proteinase K (ProtK) and DNase I (DNase) digestion (see section “Materials and Methods”). The asterisk indicates that polynucleotides were removed from DNase I digestion products (DNase*). Infected and mock-infected samples were treated identically. Data are the means of at least three independent assays. Standard deviations are indicated by error bars. p -values were determined by using two-sided independent sample t tests and comparing OD590 values of RM16-infected samples to those of mock-infected samples ( *** p
    Figure Legend Snippet: The growth-promoting effect of eDNA on M. bovis . Comparative growth of M. bovis under axenic and cell culture conditions (A) . RM16 proliferation was monitored in SP4 medium (SP4), cell culture medium (DMEM), and cell culture medium supplemented with 10 μg/ml calf thymus DNA (DMEMD). Mycoplasma titers (log CFU/ml) were determined by CFU titrations. The cytopathic effect induced by M. bovis upon co-incubation with host cells (B) . EBL cells (10 4 cells) were inoculated with RM16 at an MOI of 2 (RM16) or mock-infected (Mock). After 72 h of co-incubation, monolayers were stained with crystal violet and survival cells were estimated by measuring the optical density at 590 nm (OD 590). When indicated, DMEM-based medium was supplemented with 10 μg/ml calf thymus DNA (eDNA). Calf thymus DNA was subjected to the following enzymatic treatments: RNase A (RNase), Proteinase K (ProtK) and DNase I (DNase) digestion (see section “Materials and Methods”). The asterisk indicates that polynucleotides were removed from DNase I digestion products (DNase*). Infected and mock-infected samples were treated identically. Data are the means of at least three independent assays. Standard deviations are indicated by error bars. p -values were determined by using two-sided independent sample t tests and comparing OD590 values of RM16-infected samples to those of mock-infected samples ( *** p

    Techniques Used: Cell Culture, Incubation, Infection, Staining

    7) Product Images from "Identification of Small Molecule Inhibitors of Pre-mRNA Splicing *"

    Article Title: Identification of Small Molecule Inhibitors of Pre-mRNA Splicing *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.590976

    Secondary screen. A, schematic representation of the RT-PCR in vitro splicing assay. 20-μl splicing reaction was incubated in the presence of 500 μ m compound at 30 °C for 90 min before the splicing reaction was stop by heat inactivation. After proteinase K digestion, the spliced and unspliced RNA is amplified by RT-PCR. B shows examples of compounds that either inhibit pre-mRNA splicing or do not interfere with the splicing reaction. The star indicates splicing reactions that were inhibited. Lane M , marker (hyperladder, 1 kb).
    Figure Legend Snippet: Secondary screen. A, schematic representation of the RT-PCR in vitro splicing assay. 20-μl splicing reaction was incubated in the presence of 500 μ m compound at 30 °C for 90 min before the splicing reaction was stop by heat inactivation. After proteinase K digestion, the spliced and unspliced RNA is amplified by RT-PCR. B shows examples of compounds that either inhibit pre-mRNA splicing or do not interfere with the splicing reaction. The star indicates splicing reactions that were inhibited. Lane M , marker (hyperladder, 1 kb).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Vitro, Splicing Assay, Incubation, Amplification, Marker

    8) Product Images from "Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens"

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02224

    Comparison of DNA extraction protocols in spiked sputum samples. The  M. tuberculosis mc 2 7000  stock suspension was diluted and used to spike negative sputum samples. Box plots with C T  median, 10th, 25th, 75th, and 90th centiles of 10 replicates. Methods are indicated by colors: brown: Chelex ®  method; pink: Guanidium Isothicyanate/Tris-HCl/EDTA + 3 cycles of freeze thawing and boiling; black: Tween 20/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; green: Nonidet P-40/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; blue: Triton X-100/Tris-HCl/EDTA; purple: NaOH + boiling and sonication.
    Figure Legend Snippet: Comparison of DNA extraction protocols in spiked sputum samples. The M. tuberculosis mc 2 7000 stock suspension was diluted and used to spike negative sputum samples. Box plots with C T median, 10th, 25th, 75th, and 90th centiles of 10 replicates. Methods are indicated by colors: brown: Chelex ® method; pink: Guanidium Isothicyanate/Tris-HCl/EDTA + 3 cycles of freeze thawing and boiling; black: Tween 20/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; green: Nonidet P-40/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; blue: Triton X-100/Tris-HCl/EDTA; purple: NaOH + boiling and sonication.

    Techniques Used: DNA Extraction, Sonication

    9) Product Images from "Characterization of the extent of a large outbreak of Legionnaires’ disease by serological assays"

    Article Title: Characterization of the extent of a large outbreak of Legionnaires’ disease by serological assays

    Journal: BMC Infectious Diseases

    doi: 10.1186/s12879-015-0903-2

    IgG and IgM binding to the outbreak strain with sera from UAT negative LD cases. The immunoblots show IgG and IgM antibody binding, respectively, with sera from 25 UAT-negative cases to whole cells of the L. pneumophila serogroup 1 outbreak strain. Individual cases are identified by numbers above the nitrocellulose strips, and the upper and lower arrows to the right show the positions of proteins of molecular masses of approx. 80 kDa and 25 kDa, respectively. Strip a: binding of a monoclonal antibody to serogroup 1 L. pneumophila (Lp 1 from the Dresden panel [ 26 ]); strip b: IgM antibody reactions of serum from case no. 25 from another experiment with proteinase K treated cells, showing the ladder-like LPS antibody responses. IgG and IgM binding intensities of each serum to LPS are rated below the strips as + (strong), (+) (weak), and – none. Each 12% acrylamide gel was loaded with whole cells from the outbreak strain, corresponding to 2 μg protein/strip, and the strips were incubated with 1:200 serum dilutions. UAT: urine antigen test.
    Figure Legend Snippet: IgG and IgM binding to the outbreak strain with sera from UAT negative LD cases. The immunoblots show IgG and IgM antibody binding, respectively, with sera from 25 UAT-negative cases to whole cells of the L. pneumophila serogroup 1 outbreak strain. Individual cases are identified by numbers above the nitrocellulose strips, and the upper and lower arrows to the right show the positions of proteins of molecular masses of approx. 80 kDa and 25 kDa, respectively. Strip a: binding of a monoclonal antibody to serogroup 1 L. pneumophila (Lp 1 from the Dresden panel [ 26 ]); strip b: IgM antibody reactions of serum from case no. 25 from another experiment with proteinase K treated cells, showing the ladder-like LPS antibody responses. IgG and IgM binding intensities of each serum to LPS are rated below the strips as + (strong), (+) (weak), and – none. Each 12% acrylamide gel was loaded with whole cells from the outbreak strain, corresponding to 2 μg protein/strip, and the strips were incubated with 1:200 serum dilutions. UAT: urine antigen test.

    Techniques Used: Binding Assay, Western Blot, Stripping Membranes, Acrylamide Gel Assay, Incubation

    10) Product Images from "RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues"

    Article Title: RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0167336

    Schematic workflow for glycosaminoglycan (GAG) extraction including RNase treatment Cell/tissue lysates are treated overnight with proteinase K (Prot. K), followed by DNase-I and RNase-I treatment and finally chloroform extraction and dialysis to remove contaminating proteins/DNA/RNA. After drying/concentration of GAG extracts, the purity of the preparations is assessed using ethidium bromide (EtBr) agarose gel electrophoresis, or by measuring the absorbance at 260 nm (A260).
    Figure Legend Snippet: Schematic workflow for glycosaminoglycan (GAG) extraction including RNase treatment Cell/tissue lysates are treated overnight with proteinase K (Prot. K), followed by DNase-I and RNase-I treatment and finally chloroform extraction and dialysis to remove contaminating proteins/DNA/RNA. After drying/concentration of GAG extracts, the purity of the preparations is assessed using ethidium bromide (EtBr) agarose gel electrophoresis, or by measuring the absorbance at 260 nm (A260).

    Techniques Used: Concentration Assay, Agarose Gel Electrophoresis

    11) Product Images from "Examination of the Specificity of Tumor Cell Derived Exosomes with Tumor Cells In Vitro"

    Article Title: Examination of the Specificity of Tumor Cell Derived Exosomes with Tumor Cells In Vitro

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2014.07.026

    Effect of proteins on exosome association. PC3-derived exosomes were treated with proteinase K to remove surface protein moieties. PC3-derived exosomes (untreated or subjected to proteinase K) were incubated with PC3 and MCF-7 cells for 4 hrs at 37°C.
    Figure Legend Snippet: Effect of proteins on exosome association. PC3-derived exosomes were treated with proteinase K to remove surface protein moieties. PC3-derived exosomes (untreated or subjected to proteinase K) were incubated with PC3 and MCF-7 cells for 4 hrs at 37°C.

    Techniques Used: Derivative Assay, Incubation

    12) Product Images from "An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis"

    Article Title: An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33541-5

    Fe 0 -corroding activities in filtrates of an OS7 culture. Filtrates of a culture of strain OS7 grown for 10 days under H 2 + CO 2 (80:20) were prepared, and 1 ml of the filtrate was added to 20 ml of basal medium containing Fe 0 granules either without treatment (OS7 filtrate), or with proteinase K treatment at 50 °C for 20 min and at 100 °C for 5 min (OS7 filtrate + K), or with treatment at 50 °C for 20 min (OS7 filtrate + 50 °C). As a control, 21-ml basal medium containing Fe 0 granules was treated with proteinase K at 50 °C for 20 min and at 100 °C for 5 min (Control + K). Duplicate samples of each treatment were incubated at 37 °C for 5, 8, 11 and 14 days, and the amounts of Fe 2+ in basal medium (A) , and amounts of H 2 in headspace gas (B) were determined. The amounts of CH 4 in headspace gas were also determined, and were zero in all samples.
    Figure Legend Snippet: Fe 0 -corroding activities in filtrates of an OS7 culture. Filtrates of a culture of strain OS7 grown for 10 days under H 2 + CO 2 (80:20) were prepared, and 1 ml of the filtrate was added to 20 ml of basal medium containing Fe 0 granules either without treatment (OS7 filtrate), or with proteinase K treatment at 50 °C for 20 min and at 100 °C for 5 min (OS7 filtrate + K), or with treatment at 50 °C for 20 min (OS7 filtrate + 50 °C). As a control, 21-ml basal medium containing Fe 0 granules was treated with proteinase K at 50 °C for 20 min and at 100 °C for 5 min (Control + K). Duplicate samples of each treatment were incubated at 37 °C for 5, 8, 11 and 14 days, and the amounts of Fe 2+ in basal medium (A) , and amounts of H 2 in headspace gas (B) were determined. The amounts of CH 4 in headspace gas were also determined, and were zero in all samples.

    Techniques Used: Incubation

    13) Product Images from "Functional Overlap but Lack of Complete Cross-Complementation of Streptococcus mutans and Escherichia coli YidC Orthologs "

    Article Title: Functional Overlap but Lack of Complete Cross-Complementation of Streptococcus mutans and Escherichia coli YidC Orthologs

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01366-07

    Membrane insertion of F 1 F o ATPase subunits a and c. (A) Membrane insertion of subunit c of the F 1 F o ATPase. Proteinase K digestion of c-10h-tag at the periplasmic side generates a slightly smaller protein. JS7131 expressing 247YidC1, 247YidC2, or YidC1/YidC2
    Figure Legend Snippet: Membrane insertion of F 1 F o ATPase subunits a and c. (A) Membrane insertion of subunit c of the F 1 F o ATPase. Proteinase K digestion of c-10h-tag at the periplasmic side generates a slightly smaller protein. JS7131 expressing 247YidC1, 247YidC2, or YidC1/YidC2

    Techniques Used: Expressing

    14) Product Images from "Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress"

    Article Title: Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress

    Journal: Genome Biology

    doi: 10.1186/gb-2006-7-2-r9

    mRNA abundance in samples treated with or without protease. Unsupervised hierarchical clustering (Pearson's centered, average-linkage) of approximately 3,800 transcripts. Samples were incubated with buffer alone (-) or protease (+): trypsin (T), proteinase K (K), Qiagen protease (P). Results were normalized to untreated samples (lanes 1, 5, 7, 9, 11, or 13). Lanes 1 to 8: samples from stationary phase cultures. Lanes 3 and 4: stationary phase samples 2 minutes after treatment with menadione (+). Lanes 9 to 14: exponential samples treated with or without protease. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.
    Figure Legend Snippet: mRNA abundance in samples treated with or without protease. Unsupervised hierarchical clustering (Pearson's centered, average-linkage) of approximately 3,800 transcripts. Samples were incubated with buffer alone (-) or protease (+): trypsin (T), proteinase K (K), Qiagen protease (P). Results were normalized to untreated samples (lanes 1, 5, 7, 9, 11, or 13). Lanes 1 to 8: samples from stationary phase cultures. Lanes 3 and 4: stationary phase samples 2 minutes after treatment with menadione (+). Lanes 9 to 14: exponential samples treated with or without protease. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.

    Techniques Used: Incubation

    Venn diagram of transcripts that increased by 1 minute after oxidative stress, 30 minutes after temperature upshift, or after proteinase K treatment of T 0 cell lysates. Transcripts used for this analysis were filtered as described in Figure 7.
    Figure Legend Snippet: Venn diagram of transcripts that increased by 1 minute after oxidative stress, 30 minutes after temperature upshift, or after proteinase K treatment of T 0 cell lysates. Transcripts used for this analysis were filtered as described in Figure 7.

    Techniques Used:

    Venn diagram of transcripts that increased after oxidative stress or proteinase K treatment of T 0 cell lysates. Transcripts were evaluated that had a ≥2-fold increase in abundance by 1 and 30 minutes after oxidative stress or after proteinase K treatment. Transcripts were also required to have good spots in 80% of the time points.
    Figure Legend Snippet: Venn diagram of transcripts that increased after oxidative stress or proteinase K treatment of T 0 cell lysates. Transcripts were evaluated that had a ≥2-fold increase in abundance by 1 and 30 minutes after oxidative stress or after proteinase K treatment. Transcripts were also required to have good spots in 80% of the time points.

    Techniques Used:

    mRNA abundance in samples isolated using two different RNA isolation methods or treated with proteinase K. Unsupervised hierarchical clustering (Person's centered, average-linkage) of approximately 4,000 transcripts. RNA was isolated from unstressed cells from stationary phase cultures using the modified Gentra isolation method, hot phenol, or treated with proteinase K. Results were normalized to samples isolated using our RNA isolation method. Biological replicates for each RNA isolation method are shown. The color scale at the bottom represents the log 2  values for changes in mRNA abundance.
    Figure Legend Snippet: mRNA abundance in samples isolated using two different RNA isolation methods or treated with proteinase K. Unsupervised hierarchical clustering (Person's centered, average-linkage) of approximately 4,000 transcripts. RNA was isolated from unstressed cells from stationary phase cultures using the modified Gentra isolation method, hot phenol, or treated with proteinase K. Results were normalized to samples isolated using our RNA isolation method. Biological replicates for each RNA isolation method are shown. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.

    Techniques Used: Isolation, Modification

    15) Product Images from "Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase"

    Article Title: Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01271-19

    STYM1 cellular extracts have inhibitory protein. Extract was spotted onto plates, allowed to dry under aerobic conditions, and overlaid with a soft top agar inoculated with P. gingivalis . Plates were imaged 2 days after anaerobic growth. (A) Agar overlay assay with equal protein amounts of soluble cell extracts from STYM1, SYB13, SYB7, and 11842. (B to D) Agar overlay assays with STYM1 cellular extract either treated with proteinase K (Prot K) or heat-killed proteinase K (Heat-killed Prot K) (B), passage through a 10-kDa-MWCO filter (Flow and Conc.) (C), or heat treatment (95°C for 20 min) (D).
    Figure Legend Snippet: STYM1 cellular extracts have inhibitory protein. Extract was spotted onto plates, allowed to dry under aerobic conditions, and overlaid with a soft top agar inoculated with P. gingivalis . Plates were imaged 2 days after anaerobic growth. (A) Agar overlay assay with equal protein amounts of soluble cell extracts from STYM1, SYB13, SYB7, and 11842. (B to D) Agar overlay assays with STYM1 cellular extract either treated with proteinase K (Prot K) or heat-killed proteinase K (Heat-killed Prot K) (B), passage through a 10-kDa-MWCO filter (Flow and Conc.) (C), or heat treatment (95°C for 20 min) (D).

    Techniques Used: Overlay Assay

    16) Product Images from "An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis"

    Article Title: An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33541-5

    Fe 0 -corroding activities in filtrates of an OS7 culture. Filtrates of a culture of strain OS7 grown for 10 days under H 2 + CO 2 (80:20) were prepared, and 1 ml of the filtrate was added to 20 ml of basal medium containing Fe 0 granules either without treatment (OS7 filtrate), or with proteinase K treatment at 50 °C for 20 min and at 100 °C for 5 min (OS7 filtrate + K), or with treatment at 50 °C for 20 min (OS7 filtrate + 50 °C). As a control, 21-ml basal medium containing Fe 0 granules was treated with proteinase K at 50 °C for 20 min and at 100 °C for 5 min (Control + K). Duplicate samples of each treatment were incubated at 37 °C for 5, 8, 11 and 14 days, and the amounts of Fe 2+ in basal medium (A) , and amounts of H 2 in headspace gas (B) were determined. The amounts of CH 4 in headspace gas were also determined, and were zero in all samples.
    Figure Legend Snippet: Fe 0 -corroding activities in filtrates of an OS7 culture. Filtrates of a culture of strain OS7 grown for 10 days under H 2 + CO 2 (80:20) were prepared, and 1 ml of the filtrate was added to 20 ml of basal medium containing Fe 0 granules either without treatment (OS7 filtrate), or with proteinase K treatment at 50 °C for 20 min and at 100 °C for 5 min (OS7 filtrate + K), or with treatment at 50 °C for 20 min (OS7 filtrate + 50 °C). As a control, 21-ml basal medium containing Fe 0 granules was treated with proteinase K at 50 °C for 20 min and at 100 °C for 5 min (Control + K). Duplicate samples of each treatment were incubated at 37 °C for 5, 8, 11 and 14 days, and the amounts of Fe 2+ in basal medium (A) , and amounts of H 2 in headspace gas (B) were determined. The amounts of CH 4 in headspace gas were also determined, and were zero in all samples.

    Techniques Used: Incubation

    17) Product Images from "Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress"

    Article Title: Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress

    Journal: Genome Biology

    doi: 10.1186/gb-2006-7-2-r9

    mRNA abundance in samples treated with or without protease. Unsupervised hierarchical clustering (Pearson's centered, average-linkage) of approximately 3,800 transcripts. Samples were incubated with buffer alone (-) or protease (+): trypsin (T), proteinase K (K), Qiagen protease (P). Results were normalized to untreated samples (lanes 1, 5, 7, 9, 11, or 13). Lanes 1 to 8: samples from stationary phase cultures. Lanes 3 and 4: stationary phase samples 2 minutes after treatment with menadione (+). Lanes 9 to 14: exponential samples treated with or without protease. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.
    Figure Legend Snippet: mRNA abundance in samples treated with or without protease. Unsupervised hierarchical clustering (Pearson's centered, average-linkage) of approximately 3,800 transcripts. Samples were incubated with buffer alone (-) or protease (+): trypsin (T), proteinase K (K), Qiagen protease (P). Results were normalized to untreated samples (lanes 1, 5, 7, 9, 11, or 13). Lanes 1 to 8: samples from stationary phase cultures. Lanes 3 and 4: stationary phase samples 2 minutes after treatment with menadione (+). Lanes 9 to 14: exponential samples treated with or without protease. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.

    Techniques Used: Incubation

    Venn diagram of transcripts that increased by 1 minute after oxidative stress, 30 minutes after temperature upshift, or after proteinase K treatment of T 0 cell lysates. Transcripts used for this analysis were filtered as described in Figure 7.
    Figure Legend Snippet: Venn diagram of transcripts that increased by 1 minute after oxidative stress, 30 minutes after temperature upshift, or after proteinase K treatment of T 0 cell lysates. Transcripts used for this analysis were filtered as described in Figure 7.

    Techniques Used:

    Venn diagram of transcripts that increased after oxidative stress or proteinase K treatment of T 0 cell lysates. Transcripts were evaluated that had a ≥2-fold increase in abundance by 1 and 30 minutes after oxidative stress or after proteinase K treatment. Transcripts were also required to have good spots in 80% of the time points.
    Figure Legend Snippet: Venn diagram of transcripts that increased after oxidative stress or proteinase K treatment of T 0 cell lysates. Transcripts were evaluated that had a ≥2-fold increase in abundance by 1 and 30 minutes after oxidative stress or after proteinase K treatment. Transcripts were also required to have good spots in 80% of the time points.

    Techniques Used:

    mRNA abundance in samples isolated using two different RNA isolation methods or treated with proteinase K. Unsupervised hierarchical clustering (Person's centered, average-linkage) of approximately 4,000 transcripts. RNA was isolated from unstressed cells from stationary phase cultures using the modified Gentra isolation method, hot phenol, or treated with proteinase K. Results were normalized to samples isolated using our RNA isolation method. Biological replicates for each RNA isolation method are shown. The color scale at the bottom represents the log 2  values for changes in mRNA abundance.
    Figure Legend Snippet: mRNA abundance in samples isolated using two different RNA isolation methods or treated with proteinase K. Unsupervised hierarchical clustering (Person's centered, average-linkage) of approximately 4,000 transcripts. RNA was isolated from unstressed cells from stationary phase cultures using the modified Gentra isolation method, hot phenol, or treated with proteinase K. Results were normalized to samples isolated using our RNA isolation method. Biological replicates for each RNA isolation method are shown. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.

    Techniques Used: Isolation, Modification

    18) Product Images from "Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase"

    Article Title: Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01271-19

    STYM1 cellular extracts have inhibitory protein. Extract was spotted onto plates, allowed to dry under aerobic conditions, and overlaid with a soft top agar inoculated with P. gingivalis . Plates were imaged 2 days after anaerobic growth. (A) Agar overlay assay with equal protein amounts of soluble cell extracts from STYM1, SYB13, SYB7, and 11842. (B to D) Agar overlay assays with STYM1 cellular extract either treated with proteinase K (Prot K) or heat-killed proteinase K (Heat-killed Prot K) (B), passage through a 10-kDa-MWCO filter (Flow and Conc.) (C), or heat treatment (95°C for 20 min) (D).
    Figure Legend Snippet: STYM1 cellular extracts have inhibitory protein. Extract was spotted onto plates, allowed to dry under aerobic conditions, and overlaid with a soft top agar inoculated with P. gingivalis . Plates were imaged 2 days after anaerobic growth. (A) Agar overlay assay with equal protein amounts of soluble cell extracts from STYM1, SYB13, SYB7, and 11842. (B to D) Agar overlay assays with STYM1 cellular extract either treated with proteinase K (Prot K) or heat-killed proteinase K (Heat-killed Prot K) (B), passage through a 10-kDa-MWCO filter (Flow and Conc.) (C), or heat treatment (95°C for 20 min) (D).

    Techniques Used: Overlay Assay

    19) Product Images from "Progressive Rod-Cone Degeneration (PRCD) Protein Requires N-Terminal S-Acylation and Rhodopsin Binding for Photoreceptor Outer Segment Localization and Maintaining Intracellular Stability"

    Article Title: Progressive Rod-Cone Degeneration (PRCD) Protein Requires N-Terminal S-Acylation and Rhodopsin Binding for Photoreceptor Outer Segment Localization and Maintaining Intracellular Stability

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.6b00489

    Membrane topology and post-translational modifications of PRCD. A , Osmotically intact discs were treated with membrane impermeable proteinase K (at 8 μg/ml) followed by Western blotting with antibodies against PRCD and rhodopsin (recognizing its
    Figure Legend Snippet: Membrane topology and post-translational modifications of PRCD. A , Osmotically intact discs were treated with membrane impermeable proteinase K (at 8 μg/ml) followed by Western blotting with antibodies against PRCD and rhodopsin (recognizing its

    Techniques Used: Western Blot

    20) Product Images from "Identification of OprF as a Complement Component C3 Binding Acceptor Molecule on the Surface of Pseudomonas aeruginosa"

    Article Title: Identification of OprF as a Complement Component C3 Binding Acceptor Molecule on the Surface of Pseudomonas aeruginosa

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00081-15

    OprF is a potential C3 binding acceptor on the P. aeruginosa surface. (A) Mid-log-phase P. aeruginosa was incubated with proteinase K (PK) at 37°C, washed with PBS, and incubated with 20% normal human serum. C3 binding to the bacterial surface
    Figure Legend Snippet: OprF is a potential C3 binding acceptor on the P. aeruginosa surface. (A) Mid-log-phase P. aeruginosa was incubated with proteinase K (PK) at 37°C, washed with PBS, and incubated with 20% normal human serum. C3 binding to the bacterial surface

    Techniques Used: Binding Assay, Incubation

    21) Product Images from "The Probiotic Escherichia coli Strain Nissle 1917 Combats Lambdoid Bacteriophages stx and λ"

    Article Title: The Probiotic Escherichia coli Strain Nissle 1917 Combats Lambdoid Bacteriophages stx and λ

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00929

    Phage inactivation studies of EcN and stx -phages. (A) The stx -phage pfus/ml kinetics were analyzed in the presence and absence of EcN in a period of 72 h. Phage titers were determined with the Phage Plaque Assays (PPA) (B) The phage localization PCR with the stx2 primers was performed on the washed bacterial pellet or the sterile filtered supernatant (sn) of EcN or MG1655 after being incubated with stx -phages (EcN stx , MG stx )for 24 h. (C) Heat killed, heat killed and proteinase K (PK) treated or 1% formaldehyde (1% FA) fixed EcN or MG1655 were prepared as described in section Killing of E. coli and their phage inactivation capabilities were compared to the corresponding living E. coli . Bacteria were incubated for 24 h, static before being killed. The heat killed bacteria or the heat killed plus proteinase K treated bacteria were incubated for 24 h with the stx -phages and subsequently the phage titer was determined with the PPA. (D) Heat killed or living SK22D or the commensal control strains SE15 or IAI1 were incubated with the isolated stx -phages for 24 h before the pfus/ml were determined with a PPA. ns, not significant, * p
    Figure Legend Snippet: Phage inactivation studies of EcN and stx -phages. (A) The stx -phage pfus/ml kinetics were analyzed in the presence and absence of EcN in a period of 72 h. Phage titers were determined with the Phage Plaque Assays (PPA) (B) The phage localization PCR with the stx2 primers was performed on the washed bacterial pellet or the sterile filtered supernatant (sn) of EcN or MG1655 after being incubated with stx -phages (EcN stx , MG stx )for 24 h. (C) Heat killed, heat killed and proteinase K (PK) treated or 1% formaldehyde (1% FA) fixed EcN or MG1655 were prepared as described in section Killing of E. coli and their phage inactivation capabilities were compared to the corresponding living E. coli . Bacteria were incubated for 24 h, static before being killed. The heat killed bacteria or the heat killed plus proteinase K treated bacteria were incubated for 24 h with the stx -phages and subsequently the phage titer was determined with the PPA. (D) Heat killed or living SK22D or the commensal control strains SE15 or IAI1 were incubated with the isolated stx -phages for 24 h before the pfus/ml were determined with a PPA. ns, not significant, * p

    Techniques Used: Polymerase Chain Reaction, Incubation, Isolation

    22) Product Images from "Identification of Amino Acid Residues in BK Virus VP1 That Are Critical for Viability and Growth ▿"

    Article Title: Identification of Amino Acid Residues in BK Virus VP1 That Are Critical for Viability and Growth ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01316-07

    Release and infectivity of mutants. Vero cells were transfected with 5 μg of linear WT or mutant DNA. (A) Five days later, medium supernatant was treated with DNase I, proteinase K, and VP1 DNA amplified via PCR. This revealed that these mutants
    Figure Legend Snippet: Release and infectivity of mutants. Vero cells were transfected with 5 μg of linear WT or mutant DNA. (A) Five days later, medium supernatant was treated with DNase I, proteinase K, and VP1 DNA amplified via PCR. This revealed that these mutants

    Techniques Used: Infection, Transfection, Mutagenesis, Amplification, Polymerase Chain Reaction

    23) Product Images from "Capsular Polysaccharide Is a Receptor of a Clostridium perfringens Bacteriophage CPS1"

    Article Title: Capsular Polysaccharide Is a Receptor of a Clostridium perfringens Bacteriophage CPS1

    Journal: Viruses

    doi: 10.3390/v11111002

    CPS1 binds to polysaccharides on the cell surface. ( A ) CPS1 adsorption assay with ATCC 13124. CPS1 was infected at 30 °C. It was found that 95% of CPS1 adsorbed in 40 min. ( B ) CPS1 adsorption assay with various treatment. 1: BHI (No cells), 2: untreated cells, 3: acetate treatment, 4: 10 mM periodate treatment, 5: 100 mM periodate treatment, 6: proteinase K treatment.
    Figure Legend Snippet: CPS1 binds to polysaccharides on the cell surface. ( A ) CPS1 adsorption assay with ATCC 13124. CPS1 was infected at 30 °C. It was found that 95% of CPS1 adsorbed in 40 min. ( B ) CPS1 adsorption assay with various treatment. 1: BHI (No cells), 2: untreated cells, 3: acetate treatment, 4: 10 mM periodate treatment, 5: 100 mM periodate treatment, 6: proteinase K treatment.

    Techniques Used: Adsorption, Infection

    24) Product Images from "DNA Polymerase Beta Participates in Mitochondrial DNA Repair"

    Article Title: DNA Polymerase Beta Participates in Mitochondrial DNA Repair

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00237-17

    DNA polymerase β is detected in the mitochondria. (A) Mouse mitochondria purified from liver and brain were digested with increasing amounts of proteinase K to remove outer membrane-bound proteins. The liver samples (left) show no Polβ in the mitochondrial compartment. The brain (right) mitochondrial sample revealed Polβ after proteinase digestion, evidence that Polβ was inside the mitochondrial membrane. VDAC1 was used as a mitochondrial outer membrane marker and was digested at 0.5 mg/ml proteinase. Lamin A/C was used as a marker for nuclear contamination. GAPDH shows the extent of cytoplasmic contamination. A second antibody was also used to verify the results [Polβ (Rb)]. Fifty micrograms of protein was loaded. (B) The kidney preparations had a clear Polβ band present. Human kidney cells (HEK293T) were used to determine whether Polβ was found in the mitochondria of human cells and whether the levels could be artificially manipulated. Vector control HEK293T preparations had visible amounts of endogenous Polβ present. Overexpression of Polβ caused elevated levels of mitochondrial Polβ. The TFAM mitochondrial control antibody detected only the human form of the protein. (C) To verify Polβ antibody specificity, HEK293T cells were modified for Polβ knockout using CRISPR/cas9. The far left lane shows the molecular mass of purified Polβ protein. Total cellular protein extracts from clones 23 and 24 have no remaining Polβ. The red line indicates a molecular mass marker at 39 kDa. (D) Mitochondrial extracts from HEK293T/Polβ-KO cells compared to parental and parental+Cas9 cells. Extracts were exposed to either 0.05 or 0.1 mg/ml proteinase K or had treatment (NT). Polβ was detected in the extracts from the parental cell lines but were absent in Polβ-KO lines (clones 23 and 24). Red lines indicate molecular mass markers at 39 and 52 kDa. (E) To visualize Polβ in the mitochondria, HEK293T cells were transfected with either a C-terminal GFP or FLAG full-length (FL) Polβ, a positive control with an MLS sequence from the mitochondrial protein SOD2, and the FL Polβ protein with a 17-aa N-terminal deletion (D17N) (see Materials and Methods for a full description). (F) Immunoblot analysis of modified Polβ-GFP constructs to verify protein localization. Samples 1 to 4, mitochondrial extracts. Samples 5 to 8, whole-cell lysates. Lanes 1 and 5, mock-transfected cells. Lanes 2 and 6, FL Polβ-GFP. Lanes 3 and 7, SOD2 MLS–Polβ-GFP. Lanes 4 and 8, D17N-Polβ-GFP. VDAC1 was used to show mitochondrial enrichment of the samples. Tubulin was used as a cytoplasmic marker. Lanes 1 to 4 contained 10 μg mitochondrial extract per lane, and lanes 5 to 8 contained 2 × 10 4 cells per lane. (G) Localization of the C-terminal FLAG constructs depicted in panel E by immunofluorescence. The FL Polβ-FLAG construct is detected in the mitochondria (PDM channel). Addition of the SOD2 MLS signal enhanced the PDM signal; however, only with the deletion of the 17-aa N-terminal region (D17N) was there complete loss of nuclear localization and enhanced mitochondrial localization (also see Fig. S1G in the supplemental material). DAPI was used to visualize the nucleus (magnification, ×63, z-stack; scale bar = 20 μm).
    Figure Legend Snippet: DNA polymerase β is detected in the mitochondria. (A) Mouse mitochondria purified from liver and brain were digested with increasing amounts of proteinase K to remove outer membrane-bound proteins. The liver samples (left) show no Polβ in the mitochondrial compartment. The brain (right) mitochondrial sample revealed Polβ after proteinase digestion, evidence that Polβ was inside the mitochondrial membrane. VDAC1 was used as a mitochondrial outer membrane marker and was digested at 0.5 mg/ml proteinase. Lamin A/C was used as a marker for nuclear contamination. GAPDH shows the extent of cytoplasmic contamination. A second antibody was also used to verify the results [Polβ (Rb)]. Fifty micrograms of protein was loaded. (B) The kidney preparations had a clear Polβ band present. Human kidney cells (HEK293T) were used to determine whether Polβ was found in the mitochondria of human cells and whether the levels could be artificially manipulated. Vector control HEK293T preparations had visible amounts of endogenous Polβ present. Overexpression of Polβ caused elevated levels of mitochondrial Polβ. The TFAM mitochondrial control antibody detected only the human form of the protein. (C) To verify Polβ antibody specificity, HEK293T cells were modified for Polβ knockout using CRISPR/cas9. The far left lane shows the molecular mass of purified Polβ protein. Total cellular protein extracts from clones 23 and 24 have no remaining Polβ. The red line indicates a molecular mass marker at 39 kDa. (D) Mitochondrial extracts from HEK293T/Polβ-KO cells compared to parental and parental+Cas9 cells. Extracts were exposed to either 0.05 or 0.1 mg/ml proteinase K or had treatment (NT). Polβ was detected in the extracts from the parental cell lines but were absent in Polβ-KO lines (clones 23 and 24). Red lines indicate molecular mass markers at 39 and 52 kDa. (E) To visualize Polβ in the mitochondria, HEK293T cells were transfected with either a C-terminal GFP or FLAG full-length (FL) Polβ, a positive control with an MLS sequence from the mitochondrial protein SOD2, and the FL Polβ protein with a 17-aa N-terminal deletion (D17N) (see Materials and Methods for a full description). (F) Immunoblot analysis of modified Polβ-GFP constructs to verify protein localization. Samples 1 to 4, mitochondrial extracts. Samples 5 to 8, whole-cell lysates. Lanes 1 and 5, mock-transfected cells. Lanes 2 and 6, FL Polβ-GFP. Lanes 3 and 7, SOD2 MLS–Polβ-GFP. Lanes 4 and 8, D17N-Polβ-GFP. VDAC1 was used to show mitochondrial enrichment of the samples. Tubulin was used as a cytoplasmic marker. Lanes 1 to 4 contained 10 μg mitochondrial extract per lane, and lanes 5 to 8 contained 2 × 10 4 cells per lane. (G) Localization of the C-terminal FLAG constructs depicted in panel E by immunofluorescence. The FL Polβ-FLAG construct is detected in the mitochondria (PDM channel). Addition of the SOD2 MLS signal enhanced the PDM signal; however, only with the deletion of the 17-aa N-terminal region (D17N) was there complete loss of nuclear localization and enhanced mitochondrial localization (also see Fig. S1G in the supplemental material). DAPI was used to visualize the nucleus (magnification, ×63, z-stack; scale bar = 20 μm).

    Techniques Used: Purification, Marker, Plasmid Preparation, Over Expression, Modification, Knock-Out, CRISPR, Clone Assay, Transfection, Positive Control, Sequencing, Construct, Immunofluorescence

    25) Product Images from "Systematic Comparison and Validation of Quantitative Real-Time PCR Methods for the Quantitation of Adeno-Associated Viral Products"

    Article Title: Systematic Comparison and Validation of Quantitative Real-Time PCR Methods for the Quantitation of Adeno-Associated Viral Products

    Journal: Human Gene Therapy Methods

    doi: 10.1089/hgtb.2015.013

    Effects of enzymatic treatments on qPCR analysis. A lentiviral vector plasmid carrying the  SV40  sequence was used to spike crude ( left ) and HPLC-purified ( middle ) AAV8 samples and AAV2 ( right ) samples before HPLC purification at a vector concentration of 6.25×10 13  copies/ml; qPCR analysis was performed, targeting spike-specific  SV40  (hatched columns) and vector-specific sequences in AAV8 (gray columns) and AAV2 (cross-hatched columns); DNA copy number was determined on the basis of calibration curves generated from circular plasmid DNA standards. NT, no treatment; DT, treatment with Benzonase and DNase I; DT/PK, treatment with Benzonase and DNase I followed by proteinase K. Mean data are shown ( n =3). Asterisks indicate statistical significance between crude and purified samples, where * p
    Figure Legend Snippet: Effects of enzymatic treatments on qPCR analysis. A lentiviral vector plasmid carrying the SV40 sequence was used to spike crude ( left ) and HPLC-purified ( middle ) AAV8 samples and AAV2 ( right ) samples before HPLC purification at a vector concentration of 6.25×10 13 copies/ml; qPCR analysis was performed, targeting spike-specific SV40 (hatched columns) and vector-specific sequences in AAV8 (gray columns) and AAV2 (cross-hatched columns); DNA copy number was determined on the basis of calibration curves generated from circular plasmid DNA standards. NT, no treatment; DT, treatment with Benzonase and DNase I; DT/PK, treatment with Benzonase and DNase I followed by proteinase K. Mean data are shown ( n =3). Asterisks indicate statistical significance between crude and purified samples, where * p

    Techniques Used: Real-time Polymerase Chain Reaction, Plasmid Preparation, Sequencing, High Performance Liquid Chromatography, Purification, Concentration Assay, Generated

    26) Product Images from "Plasmid-normalized quantification of relative mitochondrial DNA copy number"

    Article Title: Plasmid-normalized quantification of relative mitochondrial DNA copy number

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33684-5

    Time curve showing mtDNA content measured in a blood sample extracted by BioRobot EZ1 Advanced after incubation using 4 different lysis buffers. The buffers used were G2 (included in the EZ1 Tissue kit) and Pierce RIPA Buffer Lyse (Thermo Fisher Scientific, Waltham, MA, USA). Both buffers were used pure and in combination with Proteinase K (PK). The baseline is obtained using the standard EZ1 tissue kit protocol without incubation.
    Figure Legend Snippet: Time curve showing mtDNA content measured in a blood sample extracted by BioRobot EZ1 Advanced after incubation using 4 different lysis buffers. The buffers used were G2 (included in the EZ1 Tissue kit) and Pierce RIPA Buffer Lyse (Thermo Fisher Scientific, Waltham, MA, USA). Both buffers were used pure and in combination with Proteinase K (PK). The baseline is obtained using the standard EZ1 tissue kit protocol without incubation.

    Techniques Used: Incubation, Lysis

    27) Product Images from "CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing"

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing

    Journal: mBio

    doi: 10.1128/mBio.01376-18

    Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”
    Figure Legend Snippet: Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”

    Techniques Used: Expressing, Confocal Laser Scanning Microscopy, Microscopy, Over Expression, Plasmid Preparation, Staining

    28) Product Images from "Plasmid-normalized quantification of relative mitochondrial DNA copy number"

    Article Title: Plasmid-normalized quantification of relative mitochondrial DNA copy number

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33684-5

    Time curve showing mtDNA content measured in a blood sample extracted by BioRobot EZ1 Advanced after incubation using 4 different lysis buffers. The buffers used were G2 (included in the EZ1 Tissue kit) and Pierce RIPA Buffer Lyse (Thermo Fisher Scientific, Waltham, MA, USA). Both buffers were used pure and in combination with Proteinase K (PK). The baseline is obtained using the standard EZ1 tissue kit protocol without incubation.
    Figure Legend Snippet: Time curve showing mtDNA content measured in a blood sample extracted by BioRobot EZ1 Advanced after incubation using 4 different lysis buffers. The buffers used were G2 (included in the EZ1 Tissue kit) and Pierce RIPA Buffer Lyse (Thermo Fisher Scientific, Waltham, MA, USA). Both buffers were used pure and in combination with Proteinase K (PK). The baseline is obtained using the standard EZ1 tissue kit protocol without incubation.

    Techniques Used: Incubation, Lysis

    29) Product Images from "Nitric Oxide Mediates Biofilm Formation and Symbiosis in Silicibacter sp. Strain TrichCH4B"

    Article Title: Nitric Oxide Mediates Biofilm Formation and Symbiosis in Silicibacter sp. Strain TrichCH4B

    Journal: mBio

    doi: 10.1128/mBio.00206-15

    Silicibacter sp. strain TrichCH4B biological response to NO and Trichodesmium erythraeum . (A) Exogenous NO increases Silicibacter sp. TrichCH4B biofilm formation. Static biofilm assays were performed as described in Materials and Methods. With increasing amounts of NO, Silicibacter sp. TrichCH4B formed more biofilm, as quantified by crystal violet staining (OD 570 ). Biofilm formation was normalized against growth (OD 600 ). (B) Addition of T. erythraeum spent medium (TSM) increases Silicibacter sp. TrichCH4B NO formation. Silicibacter sp. TrichCH4B was grown anaerobically with TSM as described in Materials and Methods. To fully digest any proteins, TSM was also treated with proteinase K before the addition to Silicibacter sp. TrichCH4B. Only the TSM (high-molecular-weight [MW] fraction, or retentate, from a 5-kDa-MWCO membrane filter) showed stimulation of NO formation by Silicibacter sp. TrichCH4B. (C) TSM addition increases SiliNOS gene expression. Silicibacter sp. TrichCH4B was grown aerobically with various amounts of TSM, and cDNA from Silicibacter sp. TrichCH4B mRNA was prepared as described in Materials and Methods. Expression of the SiliNOS gene (ΔC(t)) was calculated using Bio-Rad CFX Manager software with rpoD as a reference gene. SiliNOS gene expression increased with the amount of TSM added. (D) TSM addition increases Silicibacter sp. TrichCH4B biofilm formation. Static biofilm assays were performed as described in Materials and Methods. Silicibacter sp. TrichCH4B biofilm formation increased with the amount of TSM added, as quantified by crystal violet staining. Values that are significantly different are indicated by bars and asterisks as follows: *, P
    Figure Legend Snippet: Silicibacter sp. strain TrichCH4B biological response to NO and Trichodesmium erythraeum . (A) Exogenous NO increases Silicibacter sp. TrichCH4B biofilm formation. Static biofilm assays were performed as described in Materials and Methods. With increasing amounts of NO, Silicibacter sp. TrichCH4B formed more biofilm, as quantified by crystal violet staining (OD 570 ). Biofilm formation was normalized against growth (OD 600 ). (B) Addition of T. erythraeum spent medium (TSM) increases Silicibacter sp. TrichCH4B NO formation. Silicibacter sp. TrichCH4B was grown anaerobically with TSM as described in Materials and Methods. To fully digest any proteins, TSM was also treated with proteinase K before the addition to Silicibacter sp. TrichCH4B. Only the TSM (high-molecular-weight [MW] fraction, or retentate, from a 5-kDa-MWCO membrane filter) showed stimulation of NO formation by Silicibacter sp. TrichCH4B. (C) TSM addition increases SiliNOS gene expression. Silicibacter sp. TrichCH4B was grown aerobically with various amounts of TSM, and cDNA from Silicibacter sp. TrichCH4B mRNA was prepared as described in Materials and Methods. Expression of the SiliNOS gene (ΔC(t)) was calculated using Bio-Rad CFX Manager software with rpoD as a reference gene. SiliNOS gene expression increased with the amount of TSM added. (D) TSM addition increases Silicibacter sp. TrichCH4B biofilm formation. Static biofilm assays were performed as described in Materials and Methods. Silicibacter sp. TrichCH4B biofilm formation increased with the amount of TSM added, as quantified by crystal violet staining. Values that are significantly different are indicated by bars and asterisks as follows: *, P

    Techniques Used: Staining, Molecular Weight, Expressing, Software

    30) Product Images from "STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-Associated Herpesvirus"

    Article Title: STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-Associated Herpesvirus

    Journal: Journal of Virology

    doi: 10.1128/JVI.02008-15

    Overexpression of STAT3 restrains susceptibility to KSHV lytic activation. (A to D) BCBL-1 cells were transfected with the pEGFPN1 plasmid (GFP) or the pEGFPN1-STAT3 plasmid (STAT3-GFP), exposed to VPA after 12 h, and harvested after another 24 h for determination of relative amounts of transcripts from KSHV lytic genes ORF50 , ORF59 , ORF9 , and K8.1 by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method (A), for determination of relative amounts of cell-associated KSHV DNA by qPCR (B), for determination of K8.1 + cells by flow cytometry (C), or assayed for infectious virions in the cell supernatant by inoculation of HUVECs and staining with anti-LANA antibody and DAPI 48 h later (D). (A and B) Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (C) Numbers indicate the percentages of GFP + (i.e., transfected) cells that were lytic. Lytic (i.e., K8.1 + ) gates were placed based on a 1% cutoff in similarly treated cells that were stained with isotype control antibody. A representative of the results of two experiments is shown. (E) BCBL-1 cells were transfected with GFP plasmid or STAT3-GFP plasmid (as in panels A to D) and harvested at 24 h posttransfection for Western blot analysis using anti-STAT3 and anti-β-actin antibodies.
    Figure Legend Snippet: Overexpression of STAT3 restrains susceptibility to KSHV lytic activation. (A to D) BCBL-1 cells were transfected with the pEGFPN1 plasmid (GFP) or the pEGFPN1-STAT3 plasmid (STAT3-GFP), exposed to VPA after 12 h, and harvested after another 24 h for determination of relative amounts of transcripts from KSHV lytic genes ORF50 , ORF59 , ORF9 , and K8.1 by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method (A), for determination of relative amounts of cell-associated KSHV DNA by qPCR (B), for determination of K8.1 + cells by flow cytometry (C), or assayed for infectious virions in the cell supernatant by inoculation of HUVECs and staining with anti-LANA antibody and DAPI 48 h later (D). (A and B) Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (C) Numbers indicate the percentages of GFP + (i.e., transfected) cells that were lytic. Lytic (i.e., K8.1 + ) gates were placed based on a 1% cutoff in similarly treated cells that were stained with isotype control antibody. A representative of the results of two experiments is shown. (E) BCBL-1 cells were transfected with GFP plasmid or STAT3-GFP plasmid (as in panels A to D) and harvested at 24 h posttransfection for Western blot analysis using anti-STAT3 and anti-β-actin antibodies.

    Techniques Used: Over Expression, Activation Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Staining, Western Blot

    Chemical inhibition of STAT3 results in KSHV lytic activation, increase in the number of lytic cells, and increase in production of infectious virions. BCBL-1 cells were treated with VPA, WP1066, or VPA plus WP1066 or left untreated. Cells were harvested at 1, 6, 12, and 18 h posttreatment (A), at 24 h posttreatment (B), and at 48 h posttreatment (C). (A) RNA was analyzed at different times by qRT-PCR for relative transcript levels of 4 lytic genes, ORF50 , ORF59 , ORF9 , and ORFK8.1 , compared to untreated cells. KSHV-specific transcript levels were normalized to GAPDH , HRPTI , and B2M (β2 microglobulin), and fold changes were determined by the ΔΔ C T method. Data are presented as means and SEM and are representative of the results of two separate experiments with 3 technical replicates. (B) Cells were immunostained for K8.1 and STAT3 and subjected to flow cytometry. Numbers indicate the percent K8.1 + (lytic) cells; these percentages were determined after comparison with similarly treated cells stained with isotype control antibody. (C) Cell supernatants were used to inoculate primary HUVECs. After 48 h, HUVECs were fixed, permeabilized, stained with anti-LANA antibody and DAPI, and visualized at ×40 magnification.
    Figure Legend Snippet: Chemical inhibition of STAT3 results in KSHV lytic activation, increase in the number of lytic cells, and increase in production of infectious virions. BCBL-1 cells were treated with VPA, WP1066, or VPA plus WP1066 or left untreated. Cells were harvested at 1, 6, 12, and 18 h posttreatment (A), at 24 h posttreatment (B), and at 48 h posttreatment (C). (A) RNA was analyzed at different times by qRT-PCR for relative transcript levels of 4 lytic genes, ORF50 , ORF59 , ORF9 , and ORFK8.1 , compared to untreated cells. KSHV-specific transcript levels were normalized to GAPDH , HRPTI , and B2M (β2 microglobulin), and fold changes were determined by the ΔΔ C T method. Data are presented as means and SEM and are representative of the results of two separate experiments with 3 technical replicates. (B) Cells were immunostained for K8.1 and STAT3 and subjected to flow cytometry. Numbers indicate the percent K8.1 + (lytic) cells; these percentages were determined after comparison with similarly treated cells stained with isotype control antibody. (C) Cell supernatants were used to inoculate primary HUVECs. After 48 h, HUVECs were fixed, permeabilized, stained with anti-LANA antibody and DAPI, and visualized at ×40 magnification.

    Techniques Used: Inhibition, Activation Assay, Quantitative RT-PCR, Flow Cytometry, Cytometry, Staining

    Knockdown of endogenous STAT3 results in increased spontaneous KSHV lytic activation. (A) BCBL-1 cells were transfected with scrambled siRNA or two different siRNAs to STAT3 [si STAT3 (1) and si STAT3 (2)] and harvested 18 h and 48 h later for determination of relative amounts of transcripts from KSHV lytic genes ORF50 , ORF59 , ORF9 , and ORFK8.1 by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method. Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (B) BCBL-1 cells were transfected with scrambled siRNA (Sc) or siRNAs to STAT3 (si-1 and si-2) and harvested 48 h later for determination of relative amounts of cell-associated KSHV DNA by qPCR. Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (C) BCBL-1 cells were transfected with either STAT3 siRNA and FITC + scrambled siRNA (to mark transfected cells) at a 3:1 ratio [si STAT3 (1)] or with FITC − and FITC + scrambled siRNA at a 3:1 ratio (Scrambled). After 36 h, the FITC + (i.e., transfected) population was examined by flow cytometry for K8.1 + cells. Numbers indicate percentages of transfected cells that were spontaneously lytic. A representative of the results of two experiments is shown. (D) BCBL-1 cells were treated as for panel B, and cell supernatants were used to inoculate primary HUVECs. After 48 h, HUVECs were fixed, permeabilized, stained with anti-LANA antibody and DAPI, and visualized at ×40 magnification. (E) BCBL-1 cells were transfected with scrambled siRNA or siRNA to STAT3 , harvested at 48 h posttransfection, and subjected to Western blot analysis using anti-STAT3 and anti-β-actin antibodies.
    Figure Legend Snippet: Knockdown of endogenous STAT3 results in increased spontaneous KSHV lytic activation. (A) BCBL-1 cells were transfected with scrambled siRNA or two different siRNAs to STAT3 [si STAT3 (1) and si STAT3 (2)] and harvested 18 h and 48 h later for determination of relative amounts of transcripts from KSHV lytic genes ORF50 , ORF59 , ORF9 , and ORFK8.1 by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method. Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (B) BCBL-1 cells were transfected with scrambled siRNA (Sc) or siRNAs to STAT3 (si-1 and si-2) and harvested 48 h later for determination of relative amounts of cell-associated KSHV DNA by qPCR. Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (C) BCBL-1 cells were transfected with either STAT3 siRNA and FITC + scrambled siRNA (to mark transfected cells) at a 3:1 ratio [si STAT3 (1)] or with FITC − and FITC + scrambled siRNA at a 3:1 ratio (Scrambled). After 36 h, the FITC + (i.e., transfected) population was examined by flow cytometry for K8.1 + cells. Numbers indicate percentages of transfected cells that were spontaneously lytic. A representative of the results of two experiments is shown. (D) BCBL-1 cells were treated as for panel B, and cell supernatants were used to inoculate primary HUVECs. After 48 h, HUVECs were fixed, permeabilized, stained with anti-LANA antibody and DAPI, and visualized at ×40 magnification. (E) BCBL-1 cells were transfected with scrambled siRNA or siRNA to STAT3 , harvested at 48 h posttransfection, and subjected to Western blot analysis using anti-STAT3 and anti-β-actin antibodies.

    Techniques Used: Activation Assay, Transfection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Staining, Western Blot

    Knockdown of STAT3 suppresses KAP1, and suppression of KAP1 causes an increase in the levels of lytic transcripts. BCBL-1 cells were transfected with scrambled siRNA (Sc) or two different siRNAs to STAT3 [ STAT3 (1) and STAT3 (2)] (A and B) or scrambled siRNA (Sc) or siRNA to KAP1 (C and D) and harvested 24 h (C and D) or 48 h (A and B) later for determination of the relative amounts of transcripts from cellular KAP1 (A) and the KSHV lytic genes ORF50 , ORF59 , ORF9 , and ORFK8.1 (C) by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method or subjected to Western blotting using anti-KAP1 and anti-β-actin antibodies (B and D). Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. Numbers in panels B and D indicate relative amounts of KAP1 protein determined by densitometry after normalization to β-actin.
    Figure Legend Snippet: Knockdown of STAT3 suppresses KAP1, and suppression of KAP1 causes an increase in the levels of lytic transcripts. BCBL-1 cells were transfected with scrambled siRNA (Sc) or two different siRNAs to STAT3 [ STAT3 (1) and STAT3 (2)] (A and B) or scrambled siRNA (Sc) or siRNA to KAP1 (C and D) and harvested 24 h (C and D) or 48 h (A and B) later for determination of the relative amounts of transcripts from cellular KAP1 (A) and the KSHV lytic genes ORF50 , ORF59 , ORF9 , and ORFK8.1 (C) by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method or subjected to Western blotting using anti-KAP1 and anti-β-actin antibodies (B and D). Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. Numbers in panels B and D indicate relative amounts of KAP1 protein determined by densitometry after normalization to β-actin.

    Techniques Used: Transfection, Quantitative RT-PCR, Western Blot

    Cells expressing high levels of STAT3 protein are refractory to spontaneous and induced KSHV lytic activation. BCBL-1 cells were treated with TPA or VPA or left untreated (U). Cells were harvested at 1, 24, and 48 h (A and B) and at 48 h (C) posttreatment. (A) RNA was isolated and subjected to qRT-PCR to determine the relative levels of lytic ORFK8.1 transcripts. Fold changes were calculated by the ΔΔ C T method, normalized to three housekeeping genes, GAPDH , HPRTI , and B2M (β2 microglobulin). The data are presented as means and standard errors of the mean (SEM) and are representative of the results of two experiments with 3 technical replicates. (B) Cell lysates were immunoblotted using antibodies to K8.1 and β-actin. (C) Cells were immunostained for K8.1 and STAT3 and subjected to flow cytometry. The numbers within the dot plots in the left column indicate the percent K8.1 hi cells under untreated or TPA- or VPA-treated conditions after comparison with similarly treated cells stained with isotype control antibody; the dashed oval gates indicate K8.1 lo cells. The numbers within the dot plots in the three middle columns indicate the percent K8.1 hi cells in subpopulations expressing different levels of STAT3, i.e., STAT3 hi (high), STAT3 int (intermediate), and STAT3 lo (low). Numbers in the dot plots on the right indicate the percent K8.1 lo cells (from the oval gates in the left-hand dot plots) expressing low, intermediate, and high levels of STAT3.
    Figure Legend Snippet: Cells expressing high levels of STAT3 protein are refractory to spontaneous and induced KSHV lytic activation. BCBL-1 cells were treated with TPA or VPA or left untreated (U). Cells were harvested at 1, 24, and 48 h (A and B) and at 48 h (C) posttreatment. (A) RNA was isolated and subjected to qRT-PCR to determine the relative levels of lytic ORFK8.1 transcripts. Fold changes were calculated by the ΔΔ C T method, normalized to three housekeeping genes, GAPDH , HPRTI , and B2M (β2 microglobulin). The data are presented as means and standard errors of the mean (SEM) and are representative of the results of two experiments with 3 technical replicates. (B) Cell lysates were immunoblotted using antibodies to K8.1 and β-actin. (C) Cells were immunostained for K8.1 and STAT3 and subjected to flow cytometry. The numbers within the dot plots in the left column indicate the percent K8.1 hi cells under untreated or TPA- or VPA-treated conditions after comparison with similarly treated cells stained with isotype control antibody; the dashed oval gates indicate K8.1 lo cells. The numbers within the dot plots in the three middle columns indicate the percent K8.1 hi cells in subpopulations expressing different levels of STAT3, i.e., STAT3 hi (high), STAT3 int (intermediate), and STAT3 lo (low). Numbers in the dot plots on the right indicate the percent K8.1 lo cells (from the oval gates in the left-hand dot plots) expressing low, intermediate, and high levels of STAT3.

    Techniques Used: Expressing, Activation Assay, Isolation, Quantitative RT-PCR, Flow Cytometry, Cytometry, Staining

    31) Product Images from "STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-Associated Herpesvirus"

    Article Title: STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-Associated Herpesvirus

    Journal: Journal of Virology

    doi: 10.1128/JVI.02008-15

    Knockdown of endogenous STAT3 results in increased spontaneous KSHV lytic activation. (A) BCBL-1 cells were transfected with scrambled siRNA or two different siRNAs to STAT3 [si STAT3 (1) and si STAT3 (2)] and harvested 18 h and 48 h later for determination of relative amounts of transcripts from KSHV lytic genes ORF50 , ORF59 , ORF9 , and ORFK8.1 by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method. Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (B) BCBL-1 cells were transfected with scrambled siRNA (Sc) or siRNAs to STAT3 (si-1 and si-2) and harvested 48 h later for determination of relative amounts of cell-associated KSHV DNA by qPCR. Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (C) BCBL-1 cells were transfected with either STAT3 siRNA and FITC + scrambled siRNA (to mark transfected cells) at a 3:1 ratio [si STAT3 (1)] or with FITC − and FITC + scrambled siRNA at a 3:1 ratio (Scrambled). After 36 h, the FITC + (i.e., transfected) population was examined by flow cytometry for K8.1 + cells. Numbers indicate percentages of transfected cells that were spontaneously lytic. A representative of the results of two experiments is shown. (D) BCBL-1 cells were treated as for panel B, and cell supernatants were used to inoculate primary HUVECs. After 48 h, HUVECs were fixed, permeabilized, stained with anti-LANA antibody and DAPI, and visualized at ×40 magnification. (E) BCBL-1 cells were transfected with scrambled siRNA or siRNA to STAT3 , harvested at 48 h posttransfection, and subjected to Western blot analysis using anti-STAT3 and anti-β-actin antibodies.
    Figure Legend Snippet: Knockdown of endogenous STAT3 results in increased spontaneous KSHV lytic activation. (A) BCBL-1 cells were transfected with scrambled siRNA or two different siRNAs to STAT3 [si STAT3 (1) and si STAT3 (2)] and harvested 18 h and 48 h later for determination of relative amounts of transcripts from KSHV lytic genes ORF50 , ORF59 , ORF9 , and ORFK8.1 by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method. Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (B) BCBL-1 cells were transfected with scrambled siRNA (Sc) or siRNAs to STAT3 (si-1 and si-2) and harvested 48 h later for determination of relative amounts of cell-associated KSHV DNA by qPCR. Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (C) BCBL-1 cells were transfected with either STAT3 siRNA and FITC + scrambled siRNA (to mark transfected cells) at a 3:1 ratio [si STAT3 (1)] or with FITC − and FITC + scrambled siRNA at a 3:1 ratio (Scrambled). After 36 h, the FITC + (i.e., transfected) population was examined by flow cytometry for K8.1 + cells. Numbers indicate percentages of transfected cells that were spontaneously lytic. A representative of the results of two experiments is shown. (D) BCBL-1 cells were treated as for panel B, and cell supernatants were used to inoculate primary HUVECs. After 48 h, HUVECs were fixed, permeabilized, stained with anti-LANA antibody and DAPI, and visualized at ×40 magnification. (E) BCBL-1 cells were transfected with scrambled siRNA or siRNA to STAT3 , harvested at 48 h posttransfection, and subjected to Western blot analysis using anti-STAT3 and anti-β-actin antibodies.

    Techniques Used: Activation Assay, Transfection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Staining, Western Blot

    Knockdown of STAT3 suppresses KAP1, and suppression of KAP1 causes an increase in the levels of lytic transcripts. BCBL-1 cells were transfected with scrambled siRNA (Sc) or two different siRNAs to STAT3 [ STAT3 (1) and STAT3 (2)] (A and B) or scrambled siRNA (Sc) or siRNA to KAP1 (C and D) and harvested 24 h (C and D) or 48 h (A and B) later for determination of the relative amounts of transcripts from cellular KAP1 (A) and the KSHV lytic genes ORF50 , ORF59 , ORF9 , and ORFK8.1 (C) by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method or subjected to Western blotting using anti-KAP1 and anti-β-actin antibodies (B and D). Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. Numbers in panels B and D indicate relative amounts of KAP1 protein determined by densitometry after normalization to β-actin.
    Figure Legend Snippet: Knockdown of STAT3 suppresses KAP1, and suppression of KAP1 causes an increase in the levels of lytic transcripts. BCBL-1 cells were transfected with scrambled siRNA (Sc) or two different siRNAs to STAT3 [ STAT3 (1) and STAT3 (2)] (A and B) or scrambled siRNA (Sc) or siRNA to KAP1 (C and D) and harvested 24 h (C and D) or 48 h (A and B) later for determination of the relative amounts of transcripts from cellular KAP1 (A) and the KSHV lytic genes ORF50 , ORF59 , ORF9 , and ORFK8.1 (C) by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method or subjected to Western blotting using anti-KAP1 and anti-β-actin antibodies (B and D). Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. Numbers in panels B and D indicate relative amounts of KAP1 protein determined by densitometry after normalization to β-actin.

    Techniques Used: Transfection, Quantitative RT-PCR, Western Blot

    32) Product Images from "Lipopolysaccharide O-antigen delays plant innate immune recognition of Xylella fastidiosa"

    Article Title: Lipopolysaccharide O-antigen delays plant innate immune recognition of Xylella fastidiosa

    Journal: Nature Communications

    doi: 10.1038/s41467-018-02861-5

    O-antigen-modulated ROS production by extracted LPS and intact bacterial cells ex vivo. Discs of V. vinifera ‘Cabernet Sauvignon’ leaves were treated with 20 μL of a 50 μg/mL solution of purified LPS elicitors (either wzy or wild type LPS) equal to a final amount of 10 μg (based on Kdo content) of LPS, 20 μL of a 10 8 CFU/mL suspension of Xf wild type or wzy cells, or diH 2 0 or 1× PBS-inoculated controls, respectively. a The amplitude of ROS production remained similar for both wild type and wzy LPS, reaching max production at ~4 min, and plateaued starting around 30 min. b Total ROS production is reported as area under the curve (AUC) for plot of luminescence intensity over time. Total ROS production was not significantly different between discs treated with wild type or wzy extracted LPS. c Intact wzy cells induced a significantly stronger oxidative burst that persisted nearly 20 min longer than leaves inoculated with wild type bacteria (which contained fully polymerized O-antigens). Graphs represent the mean of 24 replicates per treatment ± standard error of the mean. d Total ROS production is reported as area under the curve (AUC) for plot of luminescence intensity over time. Discs treated with wzy cells produced significantly more ROS than discs treated with wild type cells. Graphs represent the mean of 24 replicates per treatment ± standard error of the mean. Treatments with different letters over the bars are statistically different ( P
    Figure Legend Snippet: O-antigen-modulated ROS production by extracted LPS and intact bacterial cells ex vivo. Discs of V. vinifera ‘Cabernet Sauvignon’ leaves were treated with 20 μL of a 50 μg/mL solution of purified LPS elicitors (either wzy or wild type LPS) equal to a final amount of 10 μg (based on Kdo content) of LPS, 20 μL of a 10 8 CFU/mL suspension of Xf wild type or wzy cells, or diH 2 0 or 1× PBS-inoculated controls, respectively. a The amplitude of ROS production remained similar for both wild type and wzy LPS, reaching max production at ~4 min, and plateaued starting around 30 min. b Total ROS production is reported as area under the curve (AUC) for plot of luminescence intensity over time. Total ROS production was not significantly different between discs treated with wild type or wzy extracted LPS. c Intact wzy cells induced a significantly stronger oxidative burst that persisted nearly 20 min longer than leaves inoculated with wild type bacteria (which contained fully polymerized O-antigens). Graphs represent the mean of 24 replicates per treatment ± standard error of the mean. d Total ROS production is reported as area under the curve (AUC) for plot of luminescence intensity over time. Discs treated with wzy cells produced significantly more ROS than discs treated with wild type cells. Graphs represent the mean of 24 replicates per treatment ± standard error of the mean. Treatments with different letters over the bars are statistically different ( P

    Techniques Used: Ex Vivo, Purification, Produced

    In situ localization of O-antigen-modulated ROS production in the xylem in petioles of inoculated plants. a DAB-mediated tissue printing of petioles at 15 min post-inoculation indicated a strong production of H 2 O 2 specifically in the xylem vessels of grapevines needle inoculated with wzy cells (location of xylem vessels emphasized with dotted outline). Vines inoculated with wild type Xf exhibited H 2 O 2 production predominantly in peripheral collenchyma tissue, with some production in the xylem vessels. Vines inoculated with 1× PBS buffer served as negative controls. b Mean gray value of grayscale-converted DAB-stained images, representing differences in staining intensity. Grayscale intensities vary from 0 to 255; 0 = black, 255 = white, with the values in between representing shades of gray. The mean gray value of DAB-stained images from wzy -inoculated plants is significantly lower than wild type or 1× PBS-inoculated plants, indicating a darker, or more intense stain, and thus higher amounts of H 2 O 2 . Treatments with different letters over the bars are statistically different ( P
    Figure Legend Snippet: In situ localization of O-antigen-modulated ROS production in the xylem in petioles of inoculated plants. a DAB-mediated tissue printing of petioles at 15 min post-inoculation indicated a strong production of H 2 O 2 specifically in the xylem vessels of grapevines needle inoculated with wzy cells (location of xylem vessels emphasized with dotted outline). Vines inoculated with wild type Xf exhibited H 2 O 2 production predominantly in peripheral collenchyma tissue, with some production in the xylem vessels. Vines inoculated with 1× PBS buffer served as negative controls. b Mean gray value of grayscale-converted DAB-stained images, representing differences in staining intensity. Grayscale intensities vary from 0 to 255; 0 = black, 255 = white, with the values in between representing shades of gray. The mean gray value of DAB-stained images from wzy -inoculated plants is significantly lower than wild type or 1× PBS-inoculated plants, indicating a darker, or more intense stain, and thus higher amounts of H 2 O 2 . Treatments with different letters over the bars are statistically different ( P

    Techniques Used: In Situ, Staining

    33) Product Images from "DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway"

    Article Title: DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv969

    Rescue of the DNA2 depletion defect by the purified DNA2 protein. DNA substrates bearing different types of ends were incubated with the DNA2-depleted extracts supplemented with either buffer or DNA2 protein. Samples were treated with SDS-EDTA-Proteinase K, and separated on an 1% TAE-agarose gel. The gel was dried and exposed to X-ray film.
    Figure Legend Snippet: Rescue of the DNA2 depletion defect by the purified DNA2 protein. DNA substrates bearing different types of ends were incubated with the DNA2-depleted extracts supplemented with either buffer or DNA2 protein. Samples were treated with SDS-EDTA-Proteinase K, and separated on an 1% TAE-agarose gel. The gel was dried and exposed to X-ray film.

    Techniques Used: Purification, Incubation, Agarose Gel Electrophoresis

    Depletion of DNA2 inhibits resection of DNA with 5′ adducts. ( A ) DNA substrates bearing different types of ends were incubated at 1.5 ng/μl with the control or DNA2-depleted extracts. Samples were treated with SDS-EDTA-Proteinase K, and separated on an 1% TAE-agarose gel. The gel was dried and exposed to X-ray film.
    Figure Legend Snippet: Depletion of DNA2 inhibits resection of DNA with 5′ adducts. ( A ) DNA substrates bearing different types of ends were incubated at 1.5 ng/μl with the control or DNA2-depleted extracts. Samples were treated with SDS-EDTA-Proteinase K, and separated on an 1% TAE-agarose gel. The gel was dried and exposed to X-ray film.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    34) Product Images from "A pressure cooking-based DNA extraction from archival formalin fixed, paraffin embedded tissue"

    Article Title: A pressure cooking-based DNA extraction from archival formalin fixed, paraffin embedded tissue

    Journal: Analytical Biochemistry

    doi: 10.1016/j.ab.2012.03.012

    Comparison of DNA extraction yield between a rapid extraction method and a long extraction method. We adopted a classic phenol-chloroform extraction method combined with proteinase-K digestion overnight as a reference DNA extraction method. The DNA extraction
    Figure Legend Snippet: Comparison of DNA extraction yield between a rapid extraction method and a long extraction method. We adopted a classic phenol-chloroform extraction method combined with proteinase-K digestion overnight as a reference DNA extraction method. The DNA extraction

    Techniques Used: DNA Extraction

    35) Product Images from "Matrix Production, Pigment Synthesis, and Sporulation in a Marine Isolated Strain of Bacillus pumilus"

    Article Title: Matrix Production, Pigment Synthesis, and Sporulation in a Marine Isolated Strain of Bacillus pumilus

    Journal: Marine Drugs

    doi: 10.3390/md13106472

    Determination of amount of biofilm produced by B. pumilus SF214. ( A ) Cells were grown at the indicated temperatures in rich (LB; light gray bars) or sporulation-inducing (DS; dark gray bars) media for 48 h. Cells were then removed, wells were stained and washed, and the OD (570 nm) was determined. ( B ) Biofilm formation at 25 °C in LB medium supplemented with proteinase K or DNase I, as previously indicated [ 14 ].
    Figure Legend Snippet: Determination of amount of biofilm produced by B. pumilus SF214. ( A ) Cells were grown at the indicated temperatures in rich (LB; light gray bars) or sporulation-inducing (DS; dark gray bars) media for 48 h. Cells were then removed, wells were stained and washed, and the OD (570 nm) was determined. ( B ) Biofilm formation at 25 °C in LB medium supplemented with proteinase K or DNase I, as previously indicated [ 14 ].

    Techniques Used: Produced, Staining

    36) Product Images from "Pellicle formation in Shewanella oneidensis"

    Article Title: Pellicle formation in Shewanella oneidensis

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-10-291

    EPS analysis . (A) Effects of proteinase K on pellicle formation and developed pellicles. Upper-panel, pellicle formation of the WT in static LB, in which the proteinase K was added at inoculation to 100 mg/ml (final concentration). Lower panel, developed pellicles of the WT (48 h after inoculation) were treated with 100 mg/ml (final concentration). (B) TLC analysis of monosaccharide in pellicles and supernatants. P and S represent pellicle and supernatant, respectively. Man, gal, and glu represent mannose, galactose, and glucose, respectively. Supernatants of the aggA mutant culture were included in the analysis.
    Figure Legend Snippet: EPS analysis . (A) Effects of proteinase K on pellicle formation and developed pellicles. Upper-panel, pellicle formation of the WT in static LB, in which the proteinase K was added at inoculation to 100 mg/ml (final concentration). Lower panel, developed pellicles of the WT (48 h after inoculation) were treated with 100 mg/ml (final concentration). (B) TLC analysis of monosaccharide in pellicles and supernatants. P and S represent pellicle and supernatant, respectively. Man, gal, and glu represent mannose, galactose, and glucose, respectively. Supernatants of the aggA mutant culture were included in the analysis.

    Techniques Used: Concentration Assay, Thin Layer Chromatography, Mutagenesis

    37) Product Images from "The small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties"

    Article Title: The small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties

    Journal: Virology

    doi: 10.1016/j.virol.2006.07.013

    PRRSV particle assembly in the absence of E protein. (A) Radioimmunoprecipitation of virus particles lacking the E protein. BHK-21 cells were transfected with P129-WT or P129-ΔE genomic clone. At 30 h post-transfection, the cells were radiolabeled with [ 35 S]methionine/cysteine for 24 h at 37 °C, and the supernatants and cells were separately collected. Cell lysates were prepared and subjected to immunoprecipitation with N-specific MAb (lower panel). The pellets were prepared from culture supernatants by ultracentrifugation, lysed in RIPA buffer and used for immunoprecipitation with a mixture of anti-M, anti-N and anti-E-specific antibodies, followed by SDS–15% PAGE under reducing conditions (upper panel). Lane 1, mock transfected; lane 2, P129-WT transfected; lane 3, P129-ΔE transfected. (B) Incorporation of genomic RNA in P129-ΔE virus particles. Culture supernatants were pelleted by ultracentrifugation and the pellets were treated with nucleases in the presence (+) or absence (−) of SDS and Triton X-100, followed by proteinase K treatment. RT-PCR was conducted for E gene amplification followed by electrophoresis in 1.5% agarose gel. Lane 1, molecular weight marker; lanes 2 and 3, culture supernatant spiked with the P129-ΔE full-length plasmid; lanes 4 and 5, supernatant from P129-WT-transfected cells; lanes 6 and 7, culture supernatant from P129-ΔE-transfected cells. (C) Electron microscopy of the culture supernatant from P129-ΔE-transfected cells. Particles in the culture supernatants released from BHK-21 cells transfected with P129-WT (upper panel) or P129-ΔE (lower panel) genomic clones were concentrated by ultracentrifugation through a 20% (wt/vol) sucrose cushion. Pellets were negatively stained by sodium phosphotungstate and visualized by electron microscopy. Scale bar, 100 nm.
    Figure Legend Snippet: PRRSV particle assembly in the absence of E protein. (A) Radioimmunoprecipitation of virus particles lacking the E protein. BHK-21 cells were transfected with P129-WT or P129-ΔE genomic clone. At 30 h post-transfection, the cells were radiolabeled with [ 35 S]methionine/cysteine for 24 h at 37 °C, and the supernatants and cells were separately collected. Cell lysates were prepared and subjected to immunoprecipitation with N-specific MAb (lower panel). The pellets were prepared from culture supernatants by ultracentrifugation, lysed in RIPA buffer and used for immunoprecipitation with a mixture of anti-M, anti-N and anti-E-specific antibodies, followed by SDS–15% PAGE under reducing conditions (upper panel). Lane 1, mock transfected; lane 2, P129-WT transfected; lane 3, P129-ΔE transfected. (B) Incorporation of genomic RNA in P129-ΔE virus particles. Culture supernatants were pelleted by ultracentrifugation and the pellets were treated with nucleases in the presence (+) or absence (−) of SDS and Triton X-100, followed by proteinase K treatment. RT-PCR was conducted for E gene amplification followed by electrophoresis in 1.5% agarose gel. Lane 1, molecular weight marker; lanes 2 and 3, culture supernatant spiked with the P129-ΔE full-length plasmid; lanes 4 and 5, supernatant from P129-WT-transfected cells; lanes 6 and 7, culture supernatant from P129-ΔE-transfected cells. (C) Electron microscopy of the culture supernatant from P129-ΔE-transfected cells. Particles in the culture supernatants released from BHK-21 cells transfected with P129-WT (upper panel) or P129-ΔE (lower panel) genomic clones were concentrated by ultracentrifugation through a 20% (wt/vol) sucrose cushion. Pellets were negatively stained by sodium phosphotungstate and visualized by electron microscopy. Scale bar, 100 nm.

    Techniques Used: Transfection, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Electrophoresis, Agarose Gel Electrophoresis, Molecular Weight, Marker, Plasmid Preparation, Electron Microscopy, Clone Assay, Staining

    38) Product Images from "Hair Cell Loss, Spiral Ganglion Degeneration, and Progressive Sensorineural Hearing Loss in Mice with Targeted Deletion of Slc44a2/Ctl2"

    Article Title: Hair Cell Loss, Spiral Ganglion Degeneration, and Progressive Sensorineural Hearing Loss in Mice with Targeted Deletion of Slc44a2/Ctl2

    Journal: JARO: Journal of the Association for Research in Otolaryngology

    doi: 10.1007/s10162-015-0547-3

    PCR and Southern blot results for five positive ES cell subclones. A PCR genotyping of DNA from the five positive ES cell subclones carrying the Slc44a2 targeting construct (3.3-kb PCR product), subclone 78 that lacked the targeting construct, and +/+ (wild-type) mouse DNA (3.1-kb PCR product) without the construct. B Southern blot showing the presence of the Slc44a2 8.4-kb wild-type and the 6.0-kb recombinant construct in the five positive subclones of clone 29.
    Figure Legend Snippet: PCR and Southern blot results for five positive ES cell subclones. A PCR genotyping of DNA from the five positive ES cell subclones carrying the Slc44a2 targeting construct (3.3-kb PCR product), subclone 78 that lacked the targeting construct, and +/+ (wild-type) mouse DNA (3.1-kb PCR product) without the construct. B Southern blot showing the presence of the Slc44a2 8.4-kb wild-type and the 6.0-kb recombinant construct in the five positive subclones of clone 29.

    Techniques Used: Polymerase Chain Reaction, Southern Blot, Construct, Recombinant

    39) Product Images from "Immune stimulation mediated by autoantigen binding sites within small nuclear RNAs involves Toll-like receptors 7 and 8"

    Article Title: Immune stimulation mediated by autoantigen binding sites within small nuclear RNAs involves Toll-like receptors 7 and 8

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20051696

    U1 snRNP induces type I IFN production. (A) Human PBMCs were stimulated with U1 snRNP (concentration given for total RNA plus protein) complexed to DOTAP (DO), 100 ng/ml LPS, or DOTAP alone, and cytokines were measured. Med, medium control. (B) SDS page of U1 snRNP, 4% stacking, 13.5% separation gel under reducing conditions, and Coomassie blue staining. Lane UF, RNP/Sm antigen, 8.8 μg protein; lane M, SigmaMarker, wide molecular weight range. (C) PBMCs were cultured with 20 μg/ml snRNP, 10 μg/ml poly rI:rC (pIC), snRNP, or poly rI:rC pretreated with RNase A or snRNP pretreated with proteinase K complexed to DOTAP. (D) PBMCs were stimulated with U1 snRNP or U1 snRNA complexed to DOTAP or DOTAP alone. The stimulatory properties of U1 snRNP or snRNA required the presence of an uptake enhancer (not depicted). All experiments show mean ± SEM of one representative out of two or more experiments, each with at least three to six donors.
    Figure Legend Snippet: U1 snRNP induces type I IFN production. (A) Human PBMCs were stimulated with U1 snRNP (concentration given for total RNA plus protein) complexed to DOTAP (DO), 100 ng/ml LPS, or DOTAP alone, and cytokines were measured. Med, medium control. (B) SDS page of U1 snRNP, 4% stacking, 13.5% separation gel under reducing conditions, and Coomassie blue staining. Lane UF, RNP/Sm antigen, 8.8 μg protein; lane M, SigmaMarker, wide molecular weight range. (C) PBMCs were cultured with 20 μg/ml snRNP, 10 μg/ml poly rI:rC (pIC), snRNP, or poly rI:rC pretreated with RNase A or snRNP pretreated with proteinase K complexed to DOTAP. (D) PBMCs were stimulated with U1 snRNP or U1 snRNA complexed to DOTAP or DOTAP alone. The stimulatory properties of U1 snRNP or snRNA required the presence of an uptake enhancer (not depicted). All experiments show mean ± SEM of one representative out of two or more experiments, each with at least three to six donors.

    Techniques Used: Concentration Assay, SDS Page, Staining, Molecular Weight, Cell Culture

    40) Product Images from "GtfA and GtfB Are Both Required for Protein O-Glycosylation in Lactobacillus plantarum"

    Article Title: GtfA and GtfB Are Both Required for Protein O-Glycosylation in Lactobacillus plantarum

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01401-13

    sWGA blot of whole-cell extracts derived from the wild type and tagE5E6 deletion mutant with or without proteinase K treatment for 10, 30, or 60 min. On the left side of the blot the protein sizes are indicated based on the Precision Plus protein dual color standard (Bio-Rad) molecular marker.
    Figure Legend Snippet: sWGA blot of whole-cell extracts derived from the wild type and tagE5E6 deletion mutant with or without proteinase K treatment for 10, 30, or 60 min. On the left side of the blot the protein sizes are indicated based on the Precision Plus protein dual color standard (Bio-Rad) molecular marker.

    Techniques Used: Derivative Assay, Mutagenesis, Marker

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    Article Snippet: .. After vortex mixing, boiling at 100°C for 15 min, then placed in an ultrasonic water bath for 15 min. After centrifugation at 14,000 g for 5 min, the supernatant was used for qPCR. (ii) Guanidium Isothicyanate (GTIC) method: incubation with 200 μl of lysis buffer (10 mM Tris–HCl, 1 mM EDTA, 1 M GTIC, 0.5 M NaCl) for 20 min, combined with 3 cycles of freeze-thawing (-80°C for 5 min and 100°C for 5 min) and boiling at 100°C for 15 min. (iii) Tween 20 method: suspension in 200 μl of lysis buffer [0.45% Tween 20, 50 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2 ], containing 70 μl of 10 mg/ml Lysozyme and was incubated at 37°C for 1 h. 30 μl of proteinase K (10 mg/ml, Qiagen, Germany) and 2% SDS were added, followed by incubation for 1 h at 56°C to remove PCR inhibitors and heated for 15 min at 100°C to ensure complete mycobacterial lysis. (iv) Non-idet P-40 method: the cell pellet was suspended and subjected to method (iii) but NP-40 instead of Tween 20. (v) Triton method: incubation with 200 μl of lysis buffer [100 mM NaCl, 10 mM Tris–HCl (pH 8.0); 1 mM EDTA and 1% Triton X-100]; was incubated for 20 min at 95°C. (vi) NaOH method: incubation with 200 μl lysis buffer [10 mM Tris–HCl (pH 8.0); 1 mM EDTA, 50 mM sodium hydroxide (NaOH) and 2% SDS] at 95°C for 5 min. A total 1800 μl of pure water was added, then vortex mixed, followed by boiling for 15 min at 100°C. .. After vortex mixing, the tubes were placed in ultrasonic bath for 15 min. For methods (ii) to (vi), after centrifugation at 14,000 g for 5 min, the supernatant was transferred to a new tube.

    Real-time Polymerase Chain Reaction:

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: .. After vortex mixing, boiling at 100°C for 15 min, then placed in an ultrasonic water bath for 15 min. After centrifugation at 14,000 g for 5 min, the supernatant was used for qPCR. (ii) Guanidium Isothicyanate (GTIC) method: incubation with 200 μl of lysis buffer (10 mM Tris–HCl, 1 mM EDTA, 1 M GTIC, 0.5 M NaCl) for 20 min, combined with 3 cycles of freeze-thawing (-80°C for 5 min and 100°C for 5 min) and boiling at 100°C for 15 min. (iii) Tween 20 method: suspension in 200 μl of lysis buffer [0.45% Tween 20, 50 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2 ], containing 70 μl of 10 mg/ml Lysozyme and was incubated at 37°C for 1 h. 30 μl of proteinase K (10 mg/ml, Qiagen, Germany) and 2% SDS were added, followed by incubation for 1 h at 56°C to remove PCR inhibitors and heated for 15 min at 100°C to ensure complete mycobacterial lysis. (iv) Non-idet P-40 method: the cell pellet was suspended and subjected to method (iii) but NP-40 instead of Tween 20. (v) Triton method: incubation with 200 μl of lysis buffer [100 mM NaCl, 10 mM Tris–HCl (pH 8.0); 1 mM EDTA and 1% Triton X-100]; was incubated for 20 min at 95°C. (vi) NaOH method: incubation with 200 μl lysis buffer [10 mM Tris–HCl (pH 8.0); 1 mM EDTA, 50 mM sodium hydroxide (NaOH) and 2% SDS] at 95°C for 5 min. A total 1800 μl of pure water was added, then vortex mixed, followed by boiling for 15 min at 100°C. .. After vortex mixing, the tubes were placed in ultrasonic bath for 15 min. For methods (ii) to (vi), after centrifugation at 14,000 g for 5 min, the supernatant was transferred to a new tube.

    Concentration Assay:

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    Incubation:

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    Article Title: Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity
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    Confocal Laser Scanning Microscopy:

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    Imaging:

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    Lysis:

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    Characterization of HDAC localization in tissues and cells. (A) Class I HDAC activity in whole heart lysates and mitochondrial isolates. Acetylated substrate selective for only class I and class IIb HDACs. HDAC6 activity inhibited with 1 μM Tubastatin A (Tub A). Class I HDACs inhibited with 5 μM MS275. Activity is assessed from a 2 h. reaction. (B) Western blotting for class I HDACs in whole heart nuclear, cytoplasmic, and mitochondrial isolates. (C) <t>Proteinase</t> K digestion of whole heart mitochondrial isolates. (D) Western blotting for HDAC1 in skeletal muscle and liver mitochondrial isolates. (E) Staining for HDAC1 and ACAA2 in cardiac myocytes, fibroblasts and endothelial cells. Bar = 10 μm N = 3 per group for all experiments.
    Proteinase K, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 2340 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Characterization of HDAC localization in tissues and cells. (A) Class I HDAC activity in whole heart lysates and mitochondrial isolates. Acetylated substrate selective for only class I and class IIb HDACs. HDAC6 activity inhibited with 1 μM Tubastatin A (Tub A). Class I HDACs inhibited with 5 μM MS275. Activity is assessed from a 2 h. reaction. (B) Western blotting for class I HDACs in whole heart nuclear, cytoplasmic, and mitochondrial isolates. (C) Proteinase K digestion of whole heart mitochondrial isolates. (D) Western blotting for HDAC1 in skeletal muscle and liver mitochondrial isolates. (E) Staining for HDAC1 and ACAA2 in cardiac myocytes, fibroblasts and endothelial cells. Bar = 10 μm N = 3 per group for all experiments.

    Journal: Journal of molecular and cellular cardiology

    Article Title: HDAC1 localizes to the mitochondria of cardiac myocytes and contributes to early cardiac reperfusion injury

    doi: 10.1016/j.yjmcc.2017.12.004

    Figure Lengend Snippet: Characterization of HDAC localization in tissues and cells. (A) Class I HDAC activity in whole heart lysates and mitochondrial isolates. Acetylated substrate selective for only class I and class IIb HDACs. HDAC6 activity inhibited with 1 μM Tubastatin A (Tub A). Class I HDACs inhibited with 5 μM MS275. Activity is assessed from a 2 h. reaction. (B) Western blotting for class I HDACs in whole heart nuclear, cytoplasmic, and mitochondrial isolates. (C) Proteinase K digestion of whole heart mitochondrial isolates. (D) Western blotting for HDAC1 in skeletal muscle and liver mitochondrial isolates. (E) Staining for HDAC1 and ACAA2 in cardiac myocytes, fibroblasts and endothelial cells. Bar = 10 μm N = 3 per group for all experiments.

    Article Snippet: Mitochondria were then incubated in the presence of 50 μg/mL proteinase K (Qiagen, Germantown, MD) for 0, 20, 40 or 60 min at 37 °C.

    Techniques: Activity Assay, Western Blot, Staining

    Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”

    Journal: mBio

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing

    doi: 10.1128/mBio.01376-18

    Figure Lengend Snippet: Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”

    Article Snippet: For proteinase K treatment of aggregates, proteinase K (Qiagen) (final concentration, 5 mg/ml) was added to culture aliquots after 2 h 15 min of growth and incubated for 30 min at room temperature with rocking before imaging by confocal laser scanning microscopy was performed.

    Techniques: Expressing, Confocal Laser Scanning Microscopy, Microscopy, Over Expression, Plasmid Preparation, Staining

    BKPyV particles are released within EVs. (A) Chronically infected Vero cells were fixed and processed for electron microscopy. Electron micrographs show the presence of viral particles (indicated by arrowheads) in MVBs. The right panel (bar, 100 nm) corresponds to an enlargement of the left panel (bar, 0.2 μm). (B) Vero cells were infected with BKPyV at an MOI of 1. Supernatant was harvested 3 days postinfection, filtered at 0.45 μm, and overlaid on a 20% to 45% (wt/vol) iodixanol gradient. After a 24-h ultracentrifugation, 17 fractions were collected. The density (g/ml) of each fraction was calculated according to the optical density at 340 nm. BKPyV infectivity in each fraction was assessed by immunofluorescence 3 days after infection of naive Vero cells. It is expressed as percentages of infected cells. AChE activity was analyzed to detect the presence of EVs in each fraction. (C) EVs contained in the supernatant of infected cells were concentrated 100× by PEG precipitation and overlaid on a 20% to 45% (wt/vol) iodixanol gradient. The presence of EVs in fractions 1 to 13 was evaluated by the detection of CD9, CD63, and CD81 by Western blotting. The presence of viral capsids was evaluated by the detection of VP1. The presence of contaminating small cellular organelles was evaluated by the detection of GM130 and calnexin. (D) The experiment was performed as for panel B with the supernatant of HRPTE cells, harvested 10 days postinfection. (E) Fractions containing eBKPyV (fraction 6) or naked BKPyV (fraction 12) were treated with different concentrations of proteinase K for 10 min. The volumes of the fractions were adjusted to treat similar amounts of the VP1 protein under both conditions. The sensitivity to proteinase K digestion was then assessed by detection of the VP1 capsid protein by Western blotting (top panel) and evaluated by quantifying the relative amount of VP1 on the Western blots using ImageJ (bottom panel). Results are reported as the means ± standard deviations from two independent experiments. (F) After iodixanol gradient ultracentrifugation, fractions containing eBKPyV or naked BKPyV were treated with chloroform or left untreated and then analyzed for infectivity on naive Vero cells. (G) The supernatant of Vero cells was harvested 4 days postinfection and treated with chloroform or left untreated before performing the buoyant density iodixanol gradient ultracentrifugation. Results presented in panels B, D, F, and G are means from duplicates from representative experiments.

    Journal: Journal of Virology

    Article Title: BK Polyomavirus Hijacks Extracellular Vesicles for En Bloc Transmission

    doi: 10.1128/JVI.01834-19

    Figure Lengend Snippet: BKPyV particles are released within EVs. (A) Chronically infected Vero cells were fixed and processed for electron microscopy. Electron micrographs show the presence of viral particles (indicated by arrowheads) in MVBs. The right panel (bar, 100 nm) corresponds to an enlargement of the left panel (bar, 0.2 μm). (B) Vero cells were infected with BKPyV at an MOI of 1. Supernatant was harvested 3 days postinfection, filtered at 0.45 μm, and overlaid on a 20% to 45% (wt/vol) iodixanol gradient. After a 24-h ultracentrifugation, 17 fractions were collected. The density (g/ml) of each fraction was calculated according to the optical density at 340 nm. BKPyV infectivity in each fraction was assessed by immunofluorescence 3 days after infection of naive Vero cells. It is expressed as percentages of infected cells. AChE activity was analyzed to detect the presence of EVs in each fraction. (C) EVs contained in the supernatant of infected cells were concentrated 100× by PEG precipitation and overlaid on a 20% to 45% (wt/vol) iodixanol gradient. The presence of EVs in fractions 1 to 13 was evaluated by the detection of CD9, CD63, and CD81 by Western blotting. The presence of viral capsids was evaluated by the detection of VP1. The presence of contaminating small cellular organelles was evaluated by the detection of GM130 and calnexin. (D) The experiment was performed as for panel B with the supernatant of HRPTE cells, harvested 10 days postinfection. (E) Fractions containing eBKPyV (fraction 6) or naked BKPyV (fraction 12) were treated with different concentrations of proteinase K for 10 min. The volumes of the fractions were adjusted to treat similar amounts of the VP1 protein under both conditions. The sensitivity to proteinase K digestion was then assessed by detection of the VP1 capsid protein by Western blotting (top panel) and evaluated by quantifying the relative amount of VP1 on the Western blots using ImageJ (bottom panel). Results are reported as the means ± standard deviations from two independent experiments. (F) After iodixanol gradient ultracentrifugation, fractions containing eBKPyV or naked BKPyV were treated with chloroform or left untreated and then analyzed for infectivity on naive Vero cells. (G) The supernatant of Vero cells was harvested 4 days postinfection and treated with chloroform or left untreated before performing the buoyant density iodixanol gradient ultracentrifugation. Results presented in panels B, D, F, and G are means from duplicates from representative experiments.

    Article Snippet: Fractions of interest were treated with different concentrations of proteinase K (Qiagen) on ice for 10 min.

    Techniques: Infection, Electron Microscopy, Immunofluorescence, Activity Assay, Western Blot

    The growth-promoting effect of eDNA on M. bovis . Comparative growth of M. bovis under axenic and cell culture conditions (A) . RM16 proliferation was monitored in SP4 medium (SP4), cell culture medium (DMEM), and cell culture medium supplemented with 10 μg/ml calf thymus DNA (DMEMD). Mycoplasma titers (log CFU/ml) were determined by CFU titrations. The cytopathic effect induced by M. bovis upon co-incubation with host cells (B) . EBL cells (10 4 cells) were inoculated with RM16 at an MOI of 2 (RM16) or mock-infected (Mock). After 72 h of co-incubation, monolayers were stained with crystal violet and survival cells were estimated by measuring the optical density at 590 nm (OD 590). When indicated, DMEM-based medium was supplemented with 10 μg/ml calf thymus DNA (eDNA). Calf thymus DNA was subjected to the following enzymatic treatments: RNase A (RNase), Proteinase K (ProtK) and DNase I (DNase) digestion (see section “Materials and Methods”). The asterisk indicates that polynucleotides were removed from DNase I digestion products (DNase*). Infected and mock-infected samples were treated identically. Data are the means of at least three independent assays. Standard deviations are indicated by error bars. p -values were determined by using two-sided independent sample t tests and comparing OD590 values of RM16-infected samples to those of mock-infected samples ( *** p

    Journal: Frontiers in Microbiology

    Article Title: Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity

    doi: 10.3389/fmicb.2019.02753

    Figure Lengend Snippet: The growth-promoting effect of eDNA on M. bovis . Comparative growth of M. bovis under axenic and cell culture conditions (A) . RM16 proliferation was monitored in SP4 medium (SP4), cell culture medium (DMEM), and cell culture medium supplemented with 10 μg/ml calf thymus DNA (DMEMD). Mycoplasma titers (log CFU/ml) were determined by CFU titrations. The cytopathic effect induced by M. bovis upon co-incubation with host cells (B) . EBL cells (10 4 cells) were inoculated with RM16 at an MOI of 2 (RM16) or mock-infected (Mock). After 72 h of co-incubation, monolayers were stained with crystal violet and survival cells were estimated by measuring the optical density at 590 nm (OD 590). When indicated, DMEM-based medium was supplemented with 10 μg/ml calf thymus DNA (eDNA). Calf thymus DNA was subjected to the following enzymatic treatments: RNase A (RNase), Proteinase K (ProtK) and DNase I (DNase) digestion (see section “Materials and Methods”). The asterisk indicates that polynucleotides were removed from DNase I digestion products (DNase*). Infected and mock-infected samples were treated identically. Data are the means of at least three independent assays. Standard deviations are indicated by error bars. p -values were determined by using two-sided independent sample t tests and comparing OD590 values of RM16-infected samples to those of mock-infected samples ( *** p

    Article Snippet: Calf thymus DNA treatment with Proteinase K (Qiagen), DNase I (Invitrogen), and RNase A (Invitrogen) was performed by 1 h incubation in DMEM medium and 15 min heating at 96°C.

    Techniques: Cell Culture, Incubation, Infection, Staining