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    Structured Review

    Thermo Fisher cell free dna
    <t>NET</t> <t>DNA</t> activated macrophages are pivotal to the proinflammatory response in AOSD. a THP-1-derived macrophages were cultured with purified neutrophil extracellular trap (NET) DNA. After 4 h the cells were stained for cell surface markers using specific monoclonal antibodies or the corresponding isotype controls (right panels). Cells were analyzed using flow cytometry. The percentages of proinflammatory macrophages (CD68 + CD86 + ) are indicated (left). The levels of mRNA and protein of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in these THP-1-derived macrophages were measured using RT-PCR ( b ) and ELISA analysis ( c ). The histograms show the means ± SD. * P
    Cell Free Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 8617 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Increased neutrophil extracellular traps activate NLRP3 and inflammatory macrophages in adult-onset Still’s disease"

    Article Title: Increased neutrophil extracellular traps activate NLRP3 and inflammatory macrophages in adult-onset Still’s disease

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1800-z

    NET DNA activated macrophages are pivotal to the proinflammatory response in AOSD. a THP-1-derived macrophages were cultured with purified neutrophil extracellular trap (NET) DNA. After 4 h the cells were stained for cell surface markers using specific monoclonal antibodies or the corresponding isotype controls (right panels). Cells were analyzed using flow cytometry. The percentages of proinflammatory macrophages (CD68 + CD86 + ) are indicated (left). The levels of mRNA and protein of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in these THP-1-derived macrophages were measured using RT-PCR ( b ) and ELISA analysis ( c ). The histograms show the means ± SD. * P
    Figure Legend Snippet: NET DNA activated macrophages are pivotal to the proinflammatory response in AOSD. a THP-1-derived macrophages were cultured with purified neutrophil extracellular trap (NET) DNA. After 4 h the cells were stained for cell surface markers using specific monoclonal antibodies or the corresponding isotype controls (right panels). Cells were analyzed using flow cytometry. The percentages of proinflammatory macrophages (CD68 + CD86 + ) are indicated (left). The levels of mRNA and protein of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in these THP-1-derived macrophages were measured using RT-PCR ( b ) and ELISA analysis ( c ). The histograms show the means ± SD. * P

    Techniques Used: Derivative Assay, Cell Culture, Purification, Staining, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Elevated levels of cell-free DNA and NET-DNA complexes in the circulation of patients with active AOSD. a The concentration of cell-free DNA, citrullinated histone 3 (citH3)-DNA, neutrophil elastase (NE)-DNA, and myeloperoxidase (MPO)-DNA complexes in the sera of patients with adult-onset Still’s disease (AOSD; n = 73) or healthy controls (HC; n = 40) were determined using PicoGreen. b Correlation of sera cell-free DNA with citH3-DNA, NE-DNA, and MPO-DNA complexes was analyzed. c Levels of sera cell-free DNA, citH3-DNA, NE-DNA, and MPO-DNA complexes in AOSD patients with active disease and inactive disease were measured using PicoGreen. d Reduced levels of cell-free DNA, citH3-DNA, NE-DNA, and MPO-DNA complexes in 11 AOSD patients after treatment. The results show the means ± SD. * P
    Figure Legend Snippet: Elevated levels of cell-free DNA and NET-DNA complexes in the circulation of patients with active AOSD. a The concentration of cell-free DNA, citrullinated histone 3 (citH3)-DNA, neutrophil elastase (NE)-DNA, and myeloperoxidase (MPO)-DNA complexes in the sera of patients with adult-onset Still’s disease (AOSD; n = 73) or healthy controls (HC; n = 40) were determined using PicoGreen. b Correlation of sera cell-free DNA with citH3-DNA, NE-DNA, and MPO-DNA complexes was analyzed. c Levels of sera cell-free DNA, citH3-DNA, NE-DNA, and MPO-DNA complexes in AOSD patients with active disease and inactive disease were measured using PicoGreen. d Reduced levels of cell-free DNA, citH3-DNA, NE-DNA, and MPO-DNA complexes in 11 AOSD patients after treatment. The results show the means ± SD. * P

    Techniques Used: Concentration Assay

    AOSD NETs are potent triggers of NLRP3 inflammasomes. After stimulation with neutrophil extracellular trap (NET) DNA from adult-onset Still’s disease (AOSD) and healthy controls (HC), mitochondrial DNA (mtDNA), genomic DNA g(DNA), and RNA, the expression ( a ) and secretion ( b ) of interleukin (IL)-1β and IL-18 in THP-1 monocytes were measured using RT-PCR and ELISA, respectively. c Representative immunoblot analysis for NLRP3 inflammasomes in THP-1 monocytes. The images are representative of three independent Western blot experiments. d After stimulation with NET DNA and non-NET source nucleic acids shown above, the expression of IL-1β and IL-18 in PBMC-derived CD14 + monocytes from healthy controls was measured using RT-PCR. e The expression of IL-1β, pro-IL-1β, and NLRP3 in cell lysates (LYS) and IL-1β in supernatants (SN) from THP-1 monocytes after stimulation with NET-DNA in the presence of the NLRP3 inhibitor MCC950 and DNase I was measured by Western blot. The images are representative of three independent Western blot experiments. The histograms show the means ± SD. * P
    Figure Legend Snippet: AOSD NETs are potent triggers of NLRP3 inflammasomes. After stimulation with neutrophil extracellular trap (NET) DNA from adult-onset Still’s disease (AOSD) and healthy controls (HC), mitochondrial DNA (mtDNA), genomic DNA g(DNA), and RNA, the expression ( a ) and secretion ( b ) of interleukin (IL)-1β and IL-18 in THP-1 monocytes were measured using RT-PCR and ELISA, respectively. c Representative immunoblot analysis for NLRP3 inflammasomes in THP-1 monocytes. The images are representative of three independent Western blot experiments. d After stimulation with NET DNA and non-NET source nucleic acids shown above, the expression of IL-1β and IL-18 in PBMC-derived CD14 + monocytes from healthy controls was measured using RT-PCR. e The expression of IL-1β, pro-IL-1β, and NLRP3 in cell lysates (LYS) and IL-1β in supernatants (SN) from THP-1 monocytes after stimulation with NET-DNA in the presence of the NLRP3 inhibitor MCC950 and DNase I was measured by Western blot. The images are representative of three independent Western blot experiments. The histograms show the means ± SD. * P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay

    Enriched mitochondrial DNA in AOSD NETs and plasma. Mitochondrial copy numbers from neutrophil extracellular traps (NETs) ( a ) and plasma ( b ) were determined using RT-PCR. Symbols represent individual samples; horizontal and vertical lines show the means ± SD. * P
    Figure Legend Snippet: Enriched mitochondrial DNA in AOSD NETs and plasma. Mitochondrial copy numbers from neutrophil extracellular traps (NETs) ( a ) and plasma ( b ) were determined using RT-PCR. Symbols represent individual samples; horizontal and vertical lines show the means ± SD. * P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    ROS support enhanced NETosis in AOSD patients [ 39 ]. Cytoplasmic total reactive oxygen species (ROS) stained with DCFH-DA after the activation of neutrophils with 20 nM phorbol myristate acetate (PMA) for 3.5 h was detected using flow plates ( a ) and flow cytometry ( b and c ); the results show the relative fluorescence intensity, ROS-positive neutrophils, and a representative histogram. Mitochondrial ROS production in neutrophils stimulated with 20 nM PMA for 3.5 h and stained with the red fluorescent mitochondrial superoxide indicator, Mito SOX, was determined using flow cytometry ( d ) and immunofluorescence ( e ). Neutrophils from subjects with adult-onset Still’s disease (AOSD; n = 10) were stimulated with 20 nM PMA in the presence of the indicated ROS inhibitor. f , g Sytox green immunofluorescence analysis was performed, and images of neutrophil extracellular trap (NET) formation from each culture were obtained. h The quantification of the DNA concentration was achieved using PicoGreen. The figures are representative of six independent experiments. Scale bars = 20 μm. The histograms show the means ± SD. * P
    Figure Legend Snippet: ROS support enhanced NETosis in AOSD patients [ 39 ]. Cytoplasmic total reactive oxygen species (ROS) stained with DCFH-DA after the activation of neutrophils with 20 nM phorbol myristate acetate (PMA) for 3.5 h was detected using flow plates ( a ) and flow cytometry ( b and c ); the results show the relative fluorescence intensity, ROS-positive neutrophils, and a representative histogram. Mitochondrial ROS production in neutrophils stimulated with 20 nM PMA for 3.5 h and stained with the red fluorescent mitochondrial superoxide indicator, Mito SOX, was determined using flow cytometry ( d ) and immunofluorescence ( e ). Neutrophils from subjects with adult-onset Still’s disease (AOSD; n = 10) were stimulated with 20 nM PMA in the presence of the indicated ROS inhibitor. f , g Sytox green immunofluorescence analysis was performed, and images of neutrophil extracellular trap (NET) formation from each culture were obtained. h The quantification of the DNA concentration was achieved using PicoGreen. The figures are representative of six independent experiments. Scale bars = 20 μm. The histograms show the means ± SD. * P

    Techniques Used: Staining, Activation Assay, Flow Cytometry, Cytometry, Fluorescence, Immunofluorescence, Concentration Assay

    2) Product Images from "RNA Catabolites Contribute to the Nitrogen Pool and Support Growth Recovery of Wheat"

    Article Title: RNA Catabolites Contribute to the Nitrogen Pool and Support Growth Recovery of Wheat

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.01539

    Diagram of the purine pathway characterized in Arabidopsis adapted from Bernard et al. (2011) . The diagram shows the cellular localization of RNA degradation, purine salvage and catabolism. Proteins (enzymes and transporters) that were examined here at the gene transcriptional level are shown in red; RNS2, ribonuclease type II; ENT1, equilibrative nucleoside transporter 1; ENT3, equlibrative nucleoside transporter 3; XDH, xanthine dehydrogenase; ADK, adenosine kinase; ALN, allantoinase and AAH, allantoate amidohydrolase. Additional enzymes shown include NSH, nucleoside hydrolase and APT, adenine phosphoribosyltransferase. Cellular localization of these proteins is not known in wheat.
    Figure Legend Snippet: Diagram of the purine pathway characterized in Arabidopsis adapted from Bernard et al. (2011) . The diagram shows the cellular localization of RNA degradation, purine salvage and catabolism. Proteins (enzymes and transporters) that were examined here at the gene transcriptional level are shown in red; RNS2, ribonuclease type II; ENT1, equilibrative nucleoside transporter 1; ENT3, equlibrative nucleoside transporter 3; XDH, xanthine dehydrogenase; ADK, adenosine kinase; ALN, allantoinase and AAH, allantoate amidohydrolase. Additional enzymes shown include NSH, nucleoside hydrolase and APT, adenine phosphoribosyltransferase. Cellular localization of these proteins is not known in wheat.

    Techniques Used:

    Transcriptional response to N starvation of genes with putative functions in RNA degradation, nucleoside transport and salvage. Plants of the genotypes Mace and RAC875 were grown for 14 days with full nutrition before applying the treatments +N (1 mM N) or –N (0 mM N) for 1 day, 3 days or 5 days. n = 6 with bulking of 3 plants per n. Transcriptional profile of the putative ribonuclease type II gene ( TaRNS2 ), the putative equilibrative nucleoside transporter 1 gene ( TaENT1 ), the putative equilibrative nucleoside transporter 3 gene ( TaENT3 ) and the putative adenosine kinase gene ( TaADK ) from Mace and RAC875 youngest fully expanded leaf (YFEL, A ), oldest leaf (B) and roots (C) expressed as calibrated normalized relative quantities (CNRQ). Statistical significance between treatments within a single time point and a single genotype at ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.02, ∗ p ≤ 0.05 as determined by T -test analysis and post hoc analysis using the Holm-Sidak method.
    Figure Legend Snippet: Transcriptional response to N starvation of genes with putative functions in RNA degradation, nucleoside transport and salvage. Plants of the genotypes Mace and RAC875 were grown for 14 days with full nutrition before applying the treatments +N (1 mM N) or –N (0 mM N) for 1 day, 3 days or 5 days. n = 6 with bulking of 3 plants per n. Transcriptional profile of the putative ribonuclease type II gene ( TaRNS2 ), the putative equilibrative nucleoside transporter 1 gene ( TaENT1 ), the putative equilibrative nucleoside transporter 3 gene ( TaENT3 ) and the putative adenosine kinase gene ( TaADK ) from Mace and RAC875 youngest fully expanded leaf (YFEL, A ), oldest leaf (B) and roots (C) expressed as calibrated normalized relative quantities (CNRQ). Statistical significance between treatments within a single time point and a single genotype at ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.02, ∗ p ≤ 0.05 as determined by T -test analysis and post hoc analysis using the Holm-Sidak method.

    Techniques Used:

    3) Product Images from "NIM811, a Cyclophilin Inhibitor, Exhibits Potent In Vitro Activity against Hepatitis C Virus Alone or in Combination with Alpha Interferon"

    Article Title: NIM811, a Cyclophilin Inhibitor, Exhibits Potent In Vitro Activity against Hepatitis C Virus Alone or in Combination with Alpha Interferon

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00310-06

    Combination of NIM811 with IFN-α facilitated multilog HCV RNA reduction. HCV replicon cells were treated with various concentrations of NIM811 alone, IFN-α alone, or the two in combination for 3, 6, or 9 days. Medium and compounds were refreshed every 3 days. At the end of each treatment, the quantity of HCV RNA was determined by QRT-PCR and was normalized against the amount of total RNA extracted for each sample. Each data point represents the average for five replicates in cell culture. The level of remaining HCV RNA in compound-treated cells was compared to that in untreated cells at the same time point to calculate the log reduction of HCV RNA.
    Figure Legend Snippet: Combination of NIM811 with IFN-α facilitated multilog HCV RNA reduction. HCV replicon cells were treated with various concentrations of NIM811 alone, IFN-α alone, or the two in combination for 3, 6, or 9 days. Medium and compounds were refreshed every 3 days. At the end of each treatment, the quantity of HCV RNA was determined by QRT-PCR and was normalized against the amount of total RNA extracted for each sample. Each data point represents the average for five replicates in cell culture. The level of remaining HCV RNA in compound-treated cells was compared to that in untreated cells at the same time point to calculate the log reduction of HCV RNA.

    Techniques Used: Quantitative RT-PCR, Cell Culture

    Multilog reduction of HCV RNA in the replicon cells over 9 days of treatment with NIM811. HCV replicon cells (clone A) were treated with four different concentrations of NIM811 for 3, 6, or 9 days. Medium and compounds were refreshed every 3 days. At the end of each treatment, the quantity of HCV RNA was determined by QRT-PCR and was normalized against the amount of total RNA extracted for each sample. Each data point represents the average for five replicates in cell culture. The level of remaining HCV RNA in compound-treated cells was compared to that in untreated cells at the same time point to calculate the log reduction of HCV RNA.
    Figure Legend Snippet: Multilog reduction of HCV RNA in the replicon cells over 9 days of treatment with NIM811. HCV replicon cells (clone A) were treated with four different concentrations of NIM811 for 3, 6, or 9 days. Medium and compounds were refreshed every 3 days. At the end of each treatment, the quantity of HCV RNA was determined by QRT-PCR and was normalized against the amount of total RNA extracted for each sample. Each data point represents the average for five replicates in cell culture. The level of remaining HCV RNA in compound-treated cells was compared to that in untreated cells at the same time point to calculate the log reduction of HCV RNA.

    Techniques Used: Quantitative RT-PCR, Cell Culture

    4) Product Images from "Alterations of gene expression and protein synthesis in co-cultured adipose tissue-derived stem cells and squamous cell-carcinoma cells: consequences for clinical applications"

    Article Title: Alterations of gene expression and protein synthesis in co-cultured adipose tissue-derived stem cells and squamous cell-carcinoma cells: consequences for clinical applications

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/scrt454

    Invasion assay. a) Invasive behavior of ADSCs (black bar) and A431-SCCs (red bar) alone and in co-culture, measured as the level of optical density at 560 nm with a standard deviation (SD). When co-cultured with A431-SCCs (black bar with red checkerboard pattern) ADSCs showed a significantly higher invasive capacity as in mono-culture ( P = 0.014). Co-culture of SCCs with ADSCs (red bars with black diagonal slashes) also leads to a significant increase in the invasive behavior of A431-SCCs ( P
    Figure Legend Snippet: Invasion assay. a) Invasive behavior of ADSCs (black bar) and A431-SCCs (red bar) alone and in co-culture, measured as the level of optical density at 560 nm with a standard deviation (SD). When co-cultured with A431-SCCs (black bar with red checkerboard pattern) ADSCs showed a significantly higher invasive capacity as in mono-culture ( P = 0.014). Co-culture of SCCs with ADSCs (red bars with black diagonal slashes) also leads to a significant increase in the invasive behavior of A431-SCCs ( P

    Techniques Used: Invasion Assay, Co-Culture Assay, Standard Deviation, Cell Culture

    Effect of ADSC-SCC-co-culture on cells’ proliferative activity. (a) The growth of ADSCs on the plate significantly increased ( P = 0.038) in co-culture with A431-SCCs in the transwell insert compared to mono-culture. The growth of co-cultured A431-SCCs in the transwell insert was not significantly affected ( P > 0.05). (b) ADSC-SCC-co-culture with A431-SCCs on the six-well plate and ADSCs in the transwell insert did not significantly affect cell growth of both cell types ( P > 0.05). (c) ADSC-SCC-co-culture with ADSCs on the six-well plate and pSCCs in the transwell insert significantly reduced ADSCs’ growth compared to mono-culture ( P = 0.03). The proliferative activity of pSCCs in co-culture was not affected ( P > 0.05). (d) ADSC-SCC-co-culture with pSCCs on the six-well plate and ADSCs in the transwell insert significantly decreased the growth of pSCCs ( P
    Figure Legend Snippet: Effect of ADSC-SCC-co-culture on cells’ proliferative activity. (a) The growth of ADSCs on the plate significantly increased ( P = 0.038) in co-culture with A431-SCCs in the transwell insert compared to mono-culture. The growth of co-cultured A431-SCCs in the transwell insert was not significantly affected ( P > 0.05). (b) ADSC-SCC-co-culture with A431-SCCs on the six-well plate and ADSCs in the transwell insert did not significantly affect cell growth of both cell types ( P > 0.05). (c) ADSC-SCC-co-culture with ADSCs on the six-well plate and pSCCs in the transwell insert significantly reduced ADSCs’ growth compared to mono-culture ( P = 0.03). The proliferative activity of pSCCs in co-culture was not affected ( P > 0.05). (d) ADSC-SCC-co-culture with pSCCs on the six-well plate and ADSCs in the transwell insert significantly decreased the growth of pSCCs ( P

    Techniques Used: Co-Culture Assay, Activity Assay, Cell Culture

    Migration assay. a) Migratory capacity of the ADSCs (black bar) and A431-SCCs (red bar) alone and in co-culture, measured as the level of optical density at 560 nm, with a standard deviation (SD). When co-cultured with A431-SCCs (black bar with red checkerboard pattern) ADSCs showed a significantly higher migration as in mono-culture ( P = 0.014). Co-culture of A431-SCCs with ADSCs (red bars with black diagonal slashes) does not lead to a significant change in the migratory properties of A431-SCCs ( P > 0.05). b) Migratory capacity of the ADSCs (black bar) and pSCCs (yellow bar) alone and in co-culture, measured as the level of optical density at 560 nm, with a SD. When co-cultured with pSCCs (black bar with yellow checkerboard pattern) ADSCs showed a higher migration as in mono-culture ( P > 0.05). Co-culture of pSCCs with ADSCs (yellow bars with black diagonal slashes) resulted in a significantly increased migration of pSCCs ( P = 0.009). ADSC, adipose tissue derived stem cells; pSCCs, primary squamous cell carcinoma cells; SCCs, squamous cell carcinoma cells.
    Figure Legend Snippet: Migration assay. a) Migratory capacity of the ADSCs (black bar) and A431-SCCs (red bar) alone and in co-culture, measured as the level of optical density at 560 nm, with a standard deviation (SD). When co-cultured with A431-SCCs (black bar with red checkerboard pattern) ADSCs showed a significantly higher migration as in mono-culture ( P = 0.014). Co-culture of A431-SCCs with ADSCs (red bars with black diagonal slashes) does not lead to a significant change in the migratory properties of A431-SCCs ( P > 0.05). b) Migratory capacity of the ADSCs (black bar) and pSCCs (yellow bar) alone and in co-culture, measured as the level of optical density at 560 nm, with a SD. When co-cultured with pSCCs (black bar with yellow checkerboard pattern) ADSCs showed a higher migration as in mono-culture ( P > 0.05). Co-culture of pSCCs with ADSCs (yellow bars with black diagonal slashes) resulted in a significantly increased migration of pSCCs ( P = 0.009). ADSC, adipose tissue derived stem cells; pSCCs, primary squamous cell carcinoma cells; SCCs, squamous cell carcinoma cells.

    Techniques Used: Migration, Co-Culture Assay, Standard Deviation, Cell Culture, Derivative Assay

    Induction of angiogenesis. Angiogenesis assay with incubation of HUVEC cells with conditioned media from mono-cultured ADSCs (a, e) , A431-SCCs (b) , pSCCs (f) , co-cultured ADSC-A431-SCCs (c) , and co-cultured ADSCs-pSCCs (g) . Controls (d, h) were induced with PMA. Tube formation could be detected with conditioned media from co-cultured ADSC-A431-SCCs, slightly from co-cultured ADSCs-pSCCs, and mono-cultured ADSCs or A431-SCCs. No significant angiogenesis could be detected when conditioned media from pSCC- or A431-SCC-mono-culture was added to the system. ADSC, adipose tissue derived stem cells; HUVEC, human umbilical vein endothelial cells; PMA, phorbol 12-myristate 13-acetate; pSCCs, primary squamous cell carcinoma cells; SCCs, squamous cell carcinoma cells.
    Figure Legend Snippet: Induction of angiogenesis. Angiogenesis assay with incubation of HUVEC cells with conditioned media from mono-cultured ADSCs (a, e) , A431-SCCs (b) , pSCCs (f) , co-cultured ADSC-A431-SCCs (c) , and co-cultured ADSCs-pSCCs (g) . Controls (d, h) were induced with PMA. Tube formation could be detected with conditioned media from co-cultured ADSC-A431-SCCs, slightly from co-cultured ADSCs-pSCCs, and mono-cultured ADSCs or A431-SCCs. No significant angiogenesis could be detected when conditioned media from pSCC- or A431-SCC-mono-culture was added to the system. ADSC, adipose tissue derived stem cells; HUVEC, human umbilical vein endothelial cells; PMA, phorbol 12-myristate 13-acetate; pSCCs, primary squamous cell carcinoma cells; SCCs, squamous cell carcinoma cells.

    Techniques Used: Angiogenesis Assay, Incubation, Cell Culture, Derivative Assay

    5) Product Images from "Prokaryotes in Subsoil—Evidence for a Strong Spatial Separation of Different Phyla by Analysing Co-occurrence Networks"

    Article Title: Prokaryotes in Subsoil—Evidence for a Strong Spatial Separation of Different Phyla by Analysing Co-occurrence Networks

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2015.01269

    Principal component analysis (PCA) of archaeal (A) and bacterial (B) OTUs based on 16S rRNA gene amplicons at 95% similarity level . The first three components from the PCA of relative, Hellinger-transformed data are shown.
    Figure Legend Snippet: Principal component analysis (PCA) of archaeal (A) and bacterial (B) OTUs based on 16S rRNA gene amplicons at 95% similarity level . The first three components from the PCA of relative, Hellinger-transformed data are shown.

    Techniques Used: Transformation Assay

    Relation of 16S rRNA copies between archaea and bacteria (A) and Shannon diversity index at 90% similarity level of archaea (B) and bacteria (C) in soil compartments of topsoil and subsoil . Different letters indicate significant differences ( P ≤ 0.05).
    Figure Legend Snippet: Relation of 16S rRNA copies between archaea and bacteria (A) and Shannon diversity index at 90% similarity level of archaea (B) and bacteria (C) in soil compartments of topsoil and subsoil . Different letters indicate significant differences ( P ≤ 0.05).

    Techniques Used:

    6) Product Images from "Transcriptome dynamics and molecular cross-talk between bovine oocyte and its companion cumulus cells"

    Article Title: Transcriptome dynamics and molecular cross-talk between bovine oocyte and its companion cumulus cells

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-12-57

    Diagrammatic illustration of the experimental design showing groups that were used for total RNA isolation, cDNA synthesis, array hybridization and quantitative and semi-quantitative RT-PCR validation of the array data . Numbers, 1 - 10, under each oocyte and CCs figure, represent the samples that were used for RNA isolation, cDNA synthesis and array hybridisation, as explained in materials and methods section. For all experimental groups, the samples were derived from GV stage COCs.
    Figure Legend Snippet: Diagrammatic illustration of the experimental design showing groups that were used for total RNA isolation, cDNA synthesis, array hybridization and quantitative and semi-quantitative RT-PCR validation of the array data . Numbers, 1 - 10, under each oocyte and CCs figure, represent the samples that were used for RNA isolation, cDNA synthesis and array hybridisation, as explained in materials and methods section. For all experimental groups, the samples were derived from GV stage COCs.

    Techniques Used: Isolation, Hybridization, Quantitative RT-PCR, Derivative Assay

    7) Product Images from "Investigation of HOXA9 promoter methylation as a biomarker to distinguish oral cancer patients at low risk of neck metastasis"

    Article Title: Investigation of HOXA9 promoter methylation as a biomarker to distinguish oral cancer patients at low risk of neck metastasis

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-353

    HOXA9 is methylated and expressed at low levels in oral cancer cell lines. a . Methylation levels (mean and standard deviation) as determined by pyrosequencing. b . Transcript levels determined by quantitative RT-PCR
    Figure Legend Snippet: HOXA9 is methylated and expressed at low levels in oral cancer cell lines. a . Methylation levels (mean and standard deviation) as determined by pyrosequencing. b . Transcript levels determined by quantitative RT-PCR

    Techniques Used: Methylation, Standard Deviation, Quantitative RT-PCR

    8) Product Images from "Alterations of gene expression and protein synthesis in co-cultured adipose tissue-derived stem cells and squamous cell-carcinoma cells: consequences for clinical applications"

    Article Title: Alterations of gene expression and protein synthesis in co-cultured adipose tissue-derived stem cells and squamous cell-carcinoma cells: consequences for clinical applications

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/scrt454

    Invasion assay. a) Invasive behavior of ADSCs (black bar) and A431-SCCs (red bar) alone and in co-culture, measured as the level of optical density at 560 nm with a standard deviation (SD). When co-cultured with A431-SCCs (black bar with red checkerboard pattern) ADSCs showed a significantly higher invasive capacity as in mono-culture ( P = 0.014). Co-culture of SCCs with ADSCs (red bars with black diagonal slashes) also leads to a significant increase in the invasive behavior of A431-SCCs ( P
    Figure Legend Snippet: Invasion assay. a) Invasive behavior of ADSCs (black bar) and A431-SCCs (red bar) alone and in co-culture, measured as the level of optical density at 560 nm with a standard deviation (SD). When co-cultured with A431-SCCs (black bar with red checkerboard pattern) ADSCs showed a significantly higher invasive capacity as in mono-culture ( P = 0.014). Co-culture of SCCs with ADSCs (red bars with black diagonal slashes) also leads to a significant increase in the invasive behavior of A431-SCCs ( P

    Techniques Used: Invasion Assay, Co-Culture Assay, Standard Deviation, Cell Culture

    Effect of ADSC-SCC-co-culture on cells’ proliferative activity. (a) The growth of ADSCs on the plate significantly increased ( P = 0.038) in co-culture with A431-SCCs in the transwell insert compared to mono-culture. The growth of co-cultured A431-SCCs in the transwell insert was not significantly affected ( P > 0.05). (b) ADSC-SCC-co-culture with A431-SCCs on the six-well plate and ADSCs in the transwell insert did not significantly affect cell growth of both cell types ( P > 0.05). (c) ADSC-SCC-co-culture with ADSCs on the six-well plate and pSCCs in the transwell insert significantly reduced ADSCs’ growth compared to mono-culture ( P = 0.03). The proliferative activity of pSCCs in co-culture was not affected ( P > 0.05). (d) ADSC-SCC-co-culture with pSCCs on the six-well plate and ADSCs in the transwell insert significantly decreased the growth of pSCCs ( P
    Figure Legend Snippet: Effect of ADSC-SCC-co-culture on cells’ proliferative activity. (a) The growth of ADSCs on the plate significantly increased ( P = 0.038) in co-culture with A431-SCCs in the transwell insert compared to mono-culture. The growth of co-cultured A431-SCCs in the transwell insert was not significantly affected ( P > 0.05). (b) ADSC-SCC-co-culture with A431-SCCs on the six-well plate and ADSCs in the transwell insert did not significantly affect cell growth of both cell types ( P > 0.05). (c) ADSC-SCC-co-culture with ADSCs on the six-well plate and pSCCs in the transwell insert significantly reduced ADSCs’ growth compared to mono-culture ( P = 0.03). The proliferative activity of pSCCs in co-culture was not affected ( P > 0.05). (d) ADSC-SCC-co-culture with pSCCs on the six-well plate and ADSCs in the transwell insert significantly decreased the growth of pSCCs ( P

    Techniques Used: Co-Culture Assay, Activity Assay, Cell Culture

    Migration assay. a) Migratory capacity of the ADSCs (black bar) and A431-SCCs (red bar) alone and in co-culture, measured as the level of optical density at 560 nm, with a standard deviation (SD). When co-cultured with A431-SCCs (black bar with red checkerboard pattern) ADSCs showed a significantly higher migration as in mono-culture ( P = 0.014). Co-culture of A431-SCCs with ADSCs (red bars with black diagonal slashes) does not lead to a significant change in the migratory properties of A431-SCCs ( P > 0.05). b) Migratory capacity of the ADSCs (black bar) and pSCCs (yellow bar) alone and in co-culture, measured as the level of optical density at 560 nm, with a SD. When co-cultured with pSCCs (black bar with yellow checkerboard pattern) ADSCs showed a higher migration as in mono-culture ( P > 0.05). Co-culture of pSCCs with ADSCs (yellow bars with black diagonal slashes) resulted in a significantly increased migration of pSCCs ( P = 0.009). ADSC, adipose tissue derived stem cells; pSCCs, primary squamous cell carcinoma cells; SCCs, squamous cell carcinoma cells.
    Figure Legend Snippet: Migration assay. a) Migratory capacity of the ADSCs (black bar) and A431-SCCs (red bar) alone and in co-culture, measured as the level of optical density at 560 nm, with a standard deviation (SD). When co-cultured with A431-SCCs (black bar with red checkerboard pattern) ADSCs showed a significantly higher migration as in mono-culture ( P = 0.014). Co-culture of A431-SCCs with ADSCs (red bars with black diagonal slashes) does not lead to a significant change in the migratory properties of A431-SCCs ( P > 0.05). b) Migratory capacity of the ADSCs (black bar) and pSCCs (yellow bar) alone and in co-culture, measured as the level of optical density at 560 nm, with a SD. When co-cultured with pSCCs (black bar with yellow checkerboard pattern) ADSCs showed a higher migration as in mono-culture ( P > 0.05). Co-culture of pSCCs with ADSCs (yellow bars with black diagonal slashes) resulted in a significantly increased migration of pSCCs ( P = 0.009). ADSC, adipose tissue derived stem cells; pSCCs, primary squamous cell carcinoma cells; SCCs, squamous cell carcinoma cells.

    Techniques Used: Migration, Co-Culture Assay, Standard Deviation, Cell Culture, Derivative Assay

    Induction of angiogenesis. Angiogenesis assay with incubation of HUVEC cells with conditioned media from mono-cultured ADSCs (a, e) , A431-SCCs (b) , pSCCs (f) , co-cultured ADSC-A431-SCCs (c) , and co-cultured ADSCs-pSCCs (g) . Controls (d, h) were induced with PMA. Tube formation could be detected with conditioned media from co-cultured ADSC-A431-SCCs, slightly from co-cultured ADSCs-pSCCs, and mono-cultured ADSCs or A431-SCCs. No significant angiogenesis could be detected when conditioned media from pSCC- or A431-SCC-mono-culture was added to the system. ADSC, adipose tissue derived stem cells; HUVEC, human umbilical vein endothelial cells; PMA, phorbol 12-myristate 13-acetate; pSCCs, primary squamous cell carcinoma cells; SCCs, squamous cell carcinoma cells.
    Figure Legend Snippet: Induction of angiogenesis. Angiogenesis assay with incubation of HUVEC cells with conditioned media from mono-cultured ADSCs (a, e) , A431-SCCs (b) , pSCCs (f) , co-cultured ADSC-A431-SCCs (c) , and co-cultured ADSCs-pSCCs (g) . Controls (d, h) were induced with PMA. Tube formation could be detected with conditioned media from co-cultured ADSC-A431-SCCs, slightly from co-cultured ADSCs-pSCCs, and mono-cultured ADSCs or A431-SCCs. No significant angiogenesis could be detected when conditioned media from pSCC- or A431-SCC-mono-culture was added to the system. ADSC, adipose tissue derived stem cells; HUVEC, human umbilical vein endothelial cells; PMA, phorbol 12-myristate 13-acetate; pSCCs, primary squamous cell carcinoma cells; SCCs, squamous cell carcinoma cells.

    Techniques Used: Angiogenesis Assay, Incubation, Cell Culture, Derivative Assay

    9) Product Images from "P2Y6 Receptors Regulate CXCL10 Expression and Secretion in Mouse Intestinal Epithelial Cells"

    Article Title: P2Y6 Receptors Regulate CXCL10 Expression and Secretion in Mouse Intestinal Epithelial Cells

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.00149

    Characterization of primary IEC cultures. IECs were prepared and cultured as mentioned in the section “Materials and Methods” to obtain a monolayer of differentiated cells. RNA was isolated and the expression of P2Y receptors (A) , P2X receptors (B) , the differentiated epithelial cell markers villin and ALPi (C) , ectonucleotidases (D) , and P1 receptors (E) were analyzed by qRT-PCR. Data are normalized to GAPDH mRNA level. Data presented are the mean ± SEM of three independent experiments each with cells pooled from three mice.
    Figure Legend Snippet: Characterization of primary IEC cultures. IECs were prepared and cultured as mentioned in the section “Materials and Methods” to obtain a monolayer of differentiated cells. RNA was isolated and the expression of P2Y receptors (A) , P2X receptors (B) , the differentiated epithelial cell markers villin and ALPi (C) , ectonucleotidases (D) , and P1 receptors (E) were analyzed by qRT-PCR. Data are normalized to GAPDH mRNA level. Data presented are the mean ± SEM of three independent experiments each with cells pooled from three mice.

    Techniques Used: Cell Culture, Isolation, Expressing, Quantitative RT-PCR, Mouse Assay

    10) Product Images from "Phosphoproteomic analysis reveals Smad protein family activation following Rift Valley fever virus infection"

    Article Title: Phosphoproteomic analysis reveals Smad protein family activation following Rift Valley fever virus infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0191983

    siRNA knockdown of Smad1, -2 and 4 does not impact RVFV replication. HSAECs transfected with media alone (Mock) or siRNAs (75nM) directed against a nontargeting control (NTC), Smad1, -2, or -4 were infected with MP12 virus (MOI 0.1). Both extracellular media supernatants (A) and protein lysates (B,C) were harvested at 9 and 24hpi. A) Infectious viral titers were determined by plaque assay. Data plotted as a bar graph of the mean with standard deviation of four replicates. Protein lysates from single (B) and double knockdowns (C) were analyzed by western blot for actin, total Smad1, -2, and -4 expression.
    Figure Legend Snippet: siRNA knockdown of Smad1, -2 and 4 does not impact RVFV replication. HSAECs transfected with media alone (Mock) or siRNAs (75nM) directed against a nontargeting control (NTC), Smad1, -2, or -4 were infected with MP12 virus (MOI 0.1). Both extracellular media supernatants (A) and protein lysates (B,C) were harvested at 9 and 24hpi. A) Infectious viral titers were determined by plaque assay. Data plotted as a bar graph of the mean with standard deviation of four replicates. Protein lysates from single (B) and double knockdowns (C) were analyzed by western blot for actin, total Smad1, -2, and -4 expression.

    Techniques Used: Transfection, Infection, Plaque Assay, Standard Deviation, Western Blot, Expressing

    Smad phosphorylation is dependent on RVFV replication and NSs expression. A) Analysis of HSAECs infected with MP12, ZH548 or their UV-inactivated (UVin) controls at 9 and 18hpi. Protein lysates were analyzed by western blot for pSmad1/5/9 MH2, pSmad1/5 MH2, pSmad2 MH2, pSmad2 L, RVFV NP, actin as well as total protein levels for Smad1 and Smad2. M: Mock; MP: MP12; ZH: ZH548. B) Smad phosphorylation in HSAECs infected with increasing MOIs (0.1, 0.5, 1, and 5) of MP12 virus at 9hpi was examined as described in panel A. C) Analysis of Vero cells infected at an MOI 3 with MP12 or MP12 ΔNSs. With the exception of pSmad1/5/9 MH2, protein lysates were analyzed by western blot as described in panel A. D) Vero cells transfected with media alone, pcDNA3.1 vector (2.5 μg), or pcDNA3.1-Flag-NSs (2.5 μg) were harvested at 24 hours post transfection. Protein lysates were analyzed by western blot for pSmad1/5 MH2, pSmad2 MH2, pSmad2 L, Flag-NSs as well as total protein levels for Smad1 and Smad2.
    Figure Legend Snippet: Smad phosphorylation is dependent on RVFV replication and NSs expression. A) Analysis of HSAECs infected with MP12, ZH548 or their UV-inactivated (UVin) controls at 9 and 18hpi. Protein lysates were analyzed by western blot for pSmad1/5/9 MH2, pSmad1/5 MH2, pSmad2 MH2, pSmad2 L, RVFV NP, actin as well as total protein levels for Smad1 and Smad2. M: Mock; MP: MP12; ZH: ZH548. B) Smad phosphorylation in HSAECs infected with increasing MOIs (0.1, 0.5, 1, and 5) of MP12 virus at 9hpi was examined as described in panel A. C) Analysis of Vero cells infected at an MOI 3 with MP12 or MP12 ΔNSs. With the exception of pSmad1/5/9 MH2, protein lysates were analyzed by western blot as described in panel A. D) Vero cells transfected with media alone, pcDNA3.1 vector (2.5 μg), or pcDNA3.1-Flag-NSs (2.5 μg) were harvested at 24 hours post transfection. Protein lysates were analyzed by western blot for pSmad1/5 MH2, pSmad2 MH2, pSmad2 L, Flag-NSs as well as total protein levels for Smad1 and Smad2.

    Techniques Used: Expressing, Infection, Western Blot, Transfection, Plasmid Preparation

    11) Product Images from "Sensitive, Efficient Quantitation of 13C-Enriched Nucleic Acids via Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry for Applications in Stable Isotope Probing"

    Article Title: Sensitive, Efficient Quantitation of 13C-Enriched Nucleic Acids via Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry for Applications in Stable Isotope Probing

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02223-14

    Principle coordinate analyses were conducted based on the Bray-Curtis dissimilarity of bacterial 16S rRNA gene pyrosequencing libraries from soil samples. Blue or pink symbols, respectively, correspond to DNA samples from high-density gradient fractions from incubations with labeled or unlabeled cellulose. Symbol shapes correspond to different sources of DNA template, namely, microcosm DNA (triangles) or unincubated soil DNA extract (cross-hairs [+]).
    Figure Legend Snippet: Principle coordinate analyses were conducted based on the Bray-Curtis dissimilarity of bacterial 16S rRNA gene pyrosequencing libraries from soil samples. Blue or pink symbols, respectively, correspond to DNA samples from high-density gradient fractions from incubations with labeled or unlabeled cellulose. Symbol shapes correspond to different sources of DNA template, namely, microcosm DNA (triangles) or unincubated soil DNA extract (cross-hairs [+]).

    Techniques Used: Labeling

    Quantitation of atom% 13 C in total DNA during soil incubations with [ 13 C]lignin or [ 13 C]cellulose. *, samples that yielded adequate quantities of [ 13 C]DNA (∼50 to 100 ng) for our downstream sequencing needs (i.e., preparation of 16S rRNA gene, ITS region, and metagenomic libraries). Note the difference in timescales along the x axis for the cellulose and lignin incubations.
    Figure Legend Snippet: Quantitation of atom% 13 C in total DNA during soil incubations with [ 13 C]lignin or [ 13 C]cellulose. *, samples that yielded adequate quantities of [ 13 C]DNA (∼50 to 100 ng) for our downstream sequencing needs (i.e., preparation of 16S rRNA gene, ITS region, and metagenomic libraries). Note the difference in timescales along the x axis for the cellulose and lignin incubations.

    Techniques Used: Quantitation Assay, Sequencing

    12) Product Images from "RAD51 interconnects between DNA replication, DNA repair and immunity"

    Article Title: RAD51 interconnects between DNA replication, DNA repair and immunity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx126

    Inhibition of MRE11 exonuclease activity blocks the expression of innate immune response genes in RAD51-depleted cells. (A and B ) The quantification of ssDNA and dsDNA DNA in the cytosol. RAD51-proficient and -depleted cells were pre-treated with mirin (25 μM), irradiated with 2 Gy and harvested 8 h after irradiation. Subsequently, cells were subjected to sub-cellular fractionation and the amount of cytosolic ssDNA ( A ) and dsDNA ( B ) were quantified using OliGreen and PicoGreen Quant-iT reagents, respectively. The bars represent the changes in the cytoplasmic DNA concentration relative to respective mock-treated samples. Error bars represent STDEV from four independent experiments; *** P
    Figure Legend Snippet: Inhibition of MRE11 exonuclease activity blocks the expression of innate immune response genes in RAD51-depleted cells. (A and B ) The quantification of ssDNA and dsDNA DNA in the cytosol. RAD51-proficient and -depleted cells were pre-treated with mirin (25 μM), irradiated with 2 Gy and harvested 8 h after irradiation. Subsequently, cells were subjected to sub-cellular fractionation and the amount of cytosolic ssDNA ( A ) and dsDNA ( B ) were quantified using OliGreen and PicoGreen Quant-iT reagents, respectively. The bars represent the changes in the cytoplasmic DNA concentration relative to respective mock-treated samples. Error bars represent STDEV from four independent experiments; *** P

    Techniques Used: Inhibition, Activity Assay, Expressing, Irradiation, Cell Fractionation, Concentration Assay

    Excessive DNA accumulates in the cytosol of RAD51-depleted cells. (A and B) Quantification of single-strand (ssDNA) and double-strand (dsDNA) DNA in the cytosol (cyto): RAD51-proficient and -depleted HT1080 cells were irradiated with 2 Gy and were harvested at the indicated times. Subsequently, cells underwent sub-cellular fractionation and the amount of cytosolic ssDNA ( A ) and dsDNA ( B ) were quantified using OliGreen and PicoGreen Quant-iT reagents, respectively. Bars represent fold changes in the cytoplasmic DNA concentration relative to RAD51-proficient mock-treated samples. Error bars represent the SEM from four independent experiments; ** P
    Figure Legend Snippet: Excessive DNA accumulates in the cytosol of RAD51-depleted cells. (A and B) Quantification of single-strand (ssDNA) and double-strand (dsDNA) DNA in the cytosol (cyto): RAD51-proficient and -depleted HT1080 cells were irradiated with 2 Gy and were harvested at the indicated times. Subsequently, cells underwent sub-cellular fractionation and the amount of cytosolic ssDNA ( A ) and dsDNA ( B ) were quantified using OliGreen and PicoGreen Quant-iT reagents, respectively. Bars represent fold changes in the cytoplasmic DNA concentration relative to RAD51-proficient mock-treated samples. Error bars represent the SEM from four independent experiments; ** P

    Techniques Used: Irradiation, Cell Fractionation, Concentration Assay

    13) Product Images from "Oxidized mitochondrial nucleoids released by neutrophils drive type I interferon production in human lupus"

    Article Title: Oxidized mitochondrial nucleoids released by neutrophils drive type I interferon production in human lupus

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20151876

    Live neutrophils extrude mtDNA–protein complexes. (A) Neutrophil supernatants from three healthy donors (HD) were run on agarose gels. High molecular weight complexes (mtC) yield a single DNA band of ∼16 Kb upon digestion with proteinase K (PK). (B) Amplification of the mitochondrial gene ND1 , but not the nuclear gene GAPDH , from DNA isolated from live neutrophil supernatants. Total neutrophil DNA was used as control. (C) Neutrophil supernatant (Orig Sup) was immunoprecipitated with anti-dsDNA antibody. The beads (IP Beads) and the resulting supernatants (IP Sup) were analyzed by agarose gel (left) or by Western blot with anti-TFAM or anti-H3 antibodies (right). (D) Abundance of mitochondrial and genomic DNA was assessed on 5 ng of isolated DNA from live, NETotic, or necrotic neutrophil supernatants by Real-Time PCR (mtDNA Copy Number; top) or by conventional PCR (bottom). (E) LDH activity was measured in cell-free supernatants from live, NETotic, or necrotic neutrophil cultures. (F) Quantification of extruded mtDNA by Real-Time PCR in the presence of DPI (NADPH Inhibitor) or MT (mtROS scavenger). (G, left) Apoptosis progression in untreated or GM-CSF–treated neutrophils was assessed by TUNEL assay. The percentage of TUNEL + cells is shown. (right) The amount of extruded mtDNA was assessed by Real-Time PCR (mtDNA Copy Number). (H, left) Extruded mtDNA was quantified by Picogreen. (right) mtDNA Copy Number in total cell DNA was quantified by Real-Time PCR. (I) The presence of TFAM, MnSOD, and TOMM20 was assessed in concentrated cell-free supernatants from live or necrotic neutrophils by Western blot. Coomassie staining of the BSA band was used as loading control. * indicates nonspecific band. (J) Surface expression of TOMM20, LAMP1, or Rab7 was assessed by flow cytometry on permeabilized (Total) or nonpermeabilized (Surface) neutrophils. Open gray histograms represent the isotype control. Δ MFI (Mean Fluorescence Intensity) = MFI antibody – MFI isotype control. (K–L) Extruded mtDNA (K) and TOMM20 plasma membrane expression (L) were measured in neutrophils treated with R837 (TLR7 agonist) in the presence or absence of IRS661 (TLR7 inhibitor) or with ODN2216 (TLR9 agonist). Data in B, C, and I are representative of three independent experiments with n = 3. Bars represent mean ± SD from at least three independent experiments, with n = 3–8. *, P
    Figure Legend Snippet: Live neutrophils extrude mtDNA–protein complexes. (A) Neutrophil supernatants from three healthy donors (HD) were run on agarose gels. High molecular weight complexes (mtC) yield a single DNA band of ∼16 Kb upon digestion with proteinase K (PK). (B) Amplification of the mitochondrial gene ND1 , but not the nuclear gene GAPDH , from DNA isolated from live neutrophil supernatants. Total neutrophil DNA was used as control. (C) Neutrophil supernatant (Orig Sup) was immunoprecipitated with anti-dsDNA antibody. The beads (IP Beads) and the resulting supernatants (IP Sup) were analyzed by agarose gel (left) or by Western blot with anti-TFAM or anti-H3 antibodies (right). (D) Abundance of mitochondrial and genomic DNA was assessed on 5 ng of isolated DNA from live, NETotic, or necrotic neutrophil supernatants by Real-Time PCR (mtDNA Copy Number; top) or by conventional PCR (bottom). (E) LDH activity was measured in cell-free supernatants from live, NETotic, or necrotic neutrophil cultures. (F) Quantification of extruded mtDNA by Real-Time PCR in the presence of DPI (NADPH Inhibitor) or MT (mtROS scavenger). (G, left) Apoptosis progression in untreated or GM-CSF–treated neutrophils was assessed by TUNEL assay. The percentage of TUNEL + cells is shown. (right) The amount of extruded mtDNA was assessed by Real-Time PCR (mtDNA Copy Number). (H, left) Extruded mtDNA was quantified by Picogreen. (right) mtDNA Copy Number in total cell DNA was quantified by Real-Time PCR. (I) The presence of TFAM, MnSOD, and TOMM20 was assessed in concentrated cell-free supernatants from live or necrotic neutrophils by Western blot. Coomassie staining of the BSA band was used as loading control. * indicates nonspecific band. (J) Surface expression of TOMM20, LAMP1, or Rab7 was assessed by flow cytometry on permeabilized (Total) or nonpermeabilized (Surface) neutrophils. Open gray histograms represent the isotype control. Δ MFI (Mean Fluorescence Intensity) = MFI antibody – MFI isotype control. (K–L) Extruded mtDNA (K) and TOMM20 plasma membrane expression (L) were measured in neutrophils treated with R837 (TLR7 agonist) in the presence or absence of IRS661 (TLR7 inhibitor) or with ODN2216 (TLR9 agonist). Data in B, C, and I are representative of three independent experiments with n = 3. Bars represent mean ± SD from at least three independent experiments, with n = 3–8. *, P

    Techniques Used: Molecular Weight, Amplification, Isolation, Immunoprecipitation, Agarose Gel Electrophoresis, Western Blot, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Activity Assay, TUNEL Assay, Staining, Expressing, Flow Cytometry, Cytometry, Fluorescence

    14) Product Images from "Growth factor and ultrasound-assisted bioreactor synergism for human mesenchymal stem cell chondrogenesis"

    Article Title: Growth factor and ultrasound-assisted bioreactor synergism for human mesenchymal stem cell chondrogenesis

    Journal: Journal of Tissue Engineering

    doi: 10.1177/2041731414566529

    (a) Enhancement of hMSC proliferation using US. MSCs were grown on glass coverslips for 4 days in M2 medium with and without US (14 kPa; 3 min; 1, 2, or 4 times/day). Cells were fixed and stained for Ki67 (mitosis marker) and Hoechst (nuclear marker). Five pictures were randomly taken on three coverslips per condition ( n = 15), and Ki67 and Hoechst positive cells were counted using ImageJ ™ . Data were expressed as percent Ki67 positive. (b) hMSC proliferation is increased with US application. hMSC-seeded scaffolds were cultured in CDM for 14 days and in M3 media for another 7 days in the US-assisted bioreactor according to the culture conditions outlined in Table 2 . dsDNA was assayed by Picogreen assay. Data were presented as average ± standard deviation ( n = 5–8 constructs). Statistically significant data were accounted with respect to respective control and shown as * p
    Figure Legend Snippet: (a) Enhancement of hMSC proliferation using US. MSCs were grown on glass coverslips for 4 days in M2 medium with and without US (14 kPa; 3 min; 1, 2, or 4 times/day). Cells were fixed and stained for Ki67 (mitosis marker) and Hoechst (nuclear marker). Five pictures were randomly taken on three coverslips per condition ( n = 15), and Ki67 and Hoechst positive cells were counted using ImageJ ™ . Data were expressed as percent Ki67 positive. (b) hMSC proliferation is increased with US application. hMSC-seeded scaffolds were cultured in CDM for 14 days and in M3 media for another 7 days in the US-assisted bioreactor according to the culture conditions outlined in Table 2 . dsDNA was assayed by Picogreen assay. Data were presented as average ± standard deviation ( n = 5–8 constructs). Statistically significant data were accounted with respect to respective control and shown as * p

    Techniques Used: Staining, Marker, Cell Culture, Picogreen Assay, Standard Deviation, Construct

    15) Product Images from "Periodic mild heat stimuli diminish extracellular matrix synthesis in pellet cultured human chondrocytes"

    Article Title: Periodic mild heat stimuli diminish extracellular matrix synthesis in pellet cultured human chondrocytes

    Journal: BMC Research Notes

    doi: 10.1186/s13104-019-4058-x

    Collagen and proteoglycan accumulation. a Macro images and wet weight. Synthesized pellets on day 21 were subjected to macro photography (i) and wet weight measurement (ii). b Collagen accumulation. Collagen synthesis in pellets on day 21 was evaluated by picrosirius red staining (i) and hydroxyproline assay (ii). The values of collagen were normalized to DNA content. c Proteoglycan accumulation. Proteoglycan synthesis in pellets on day 21 was evaluated by safranin-O staining (i) and DMMB colorimetric assay (ii). The values of proteoglycan were normalized to DNA content. d DNA content. DNA content of pellets on day 21 was evaluated using the Quanti-iT™ PicoGreen assay. Values represent the means and standard deviations. * and ** indicates P
    Figure Legend Snippet: Collagen and proteoglycan accumulation. a Macro images and wet weight. Synthesized pellets on day 21 were subjected to macro photography (i) and wet weight measurement (ii). b Collagen accumulation. Collagen synthesis in pellets on day 21 was evaluated by picrosirius red staining (i) and hydroxyproline assay (ii). The values of collagen were normalized to DNA content. c Proteoglycan accumulation. Proteoglycan synthesis in pellets on day 21 was evaluated by safranin-O staining (i) and DMMB colorimetric assay (ii). The values of proteoglycan were normalized to DNA content. d DNA content. DNA content of pellets on day 21 was evaluated using the Quanti-iT™ PicoGreen assay. Values represent the means and standard deviations. * and ** indicates P

    Techniques Used: Synthesized, Staining, Hydroxyproline Assay, Colorimetric Assay, Picogreen Assay

    16) Product Images from "Development of a Cytocompatible Scaffold from Pig Immature Testicular Tissue Allowing Human Sertoli Cell Attachment, Proliferation and Functionality"

    Article Title: Development of a Cytocompatible Scaffold from Pig Immature Testicular Tissue Allowing Human Sertoli Cell Attachment, Proliferation and Functionality

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19010227

    DNA quantification in DT. ( A ) Nuclei were counted on H and E-stained slides of native tissue and DT and represented as the number of nuclei per µm 2 ; ( B ) Following tissue digestion, DNA was extracted and quantified using the Quant-iT™ PicoGreen ® dsDNA assay. The results are presented as means ± SD. *** p
    Figure Legend Snippet: DNA quantification in DT. ( A ) Nuclei were counted on H and E-stained slides of native tissue and DT and represented as the number of nuclei per µm 2 ; ( B ) Following tissue digestion, DNA was extracted and quantified using the Quant-iT™ PicoGreen ® dsDNA assay. The results are presented as means ± SD. *** p

    Techniques Used: Staining, Picogreen Assay

    17) Product Images from "The Acellular Myocardial Flap: A Novel Extracellular Matrix Scaffold Enriched with Patent Microvascular Networks and Biocompatible Cell Niches"

    Article Title: The Acellular Myocardial Flap: A Novel Extracellular Matrix Scaffold Enriched with Patent Microvascular Networks and Biocompatible Cell Niches

    Journal: Tissue Engineering. Part C, Methods

    doi: 10.1089/ten.tec.2012.0536

    A pedicled flap of (A) native myocardium excised from the left ventricular wall of the porcine heart and a representative image of the decellularized (Decelled) scaffolds resulting from perfusion decellularization. The cannulae used to connect the vasculature to the perfusion system are shown secured with orange-zip ties. Red arrows indicate the open end of the coronary artery before cannulation (Native) and the inlet to the coronary artery after cannulation (Decelled). Similarly, blue arrows indicate the same features for the cardiac vein. Arrows also indicate the direction of anteriograde blood and decellularization solution flow. Areas designated by circles indicate origins of specimens of decellularized coronary arterial extracellular matrix (CA ECM) and decellularized myocardial extracellular matrix (MYO ECM) used in histological, immunohistochemical (IHC), DNA, biochemical, mechanical, and cell-seeding analyses. Time-course plot (B) of the mean recorded masses of scaffolds exposed to 4 different decellularization methods. Results of a (C) PicoGreen ® quantitative assay for DNA are shown, which compare the DNA content of both decellularized MYO ECM and CA ECM samples generated with various decellularization treatments to that of native myocardium and native coronary arteries, respectively. DNA content of (D) MYO ECM samples from scaffolds generated with the group 4 decellularization treatment and subsequent exposure to various reagents as measured by PicoGreen assay. DNA content of group 4 and native myocardium represented in (D) are carried over from C for comparison (*: indicates statistical significance from all other groups, p
    Figure Legend Snippet: A pedicled flap of (A) native myocardium excised from the left ventricular wall of the porcine heart and a representative image of the decellularized (Decelled) scaffolds resulting from perfusion decellularization. The cannulae used to connect the vasculature to the perfusion system are shown secured with orange-zip ties. Red arrows indicate the open end of the coronary artery before cannulation (Native) and the inlet to the coronary artery after cannulation (Decelled). Similarly, blue arrows indicate the same features for the cardiac vein. Arrows also indicate the direction of anteriograde blood and decellularization solution flow. Areas designated by circles indicate origins of specimens of decellularized coronary arterial extracellular matrix (CA ECM) and decellularized myocardial extracellular matrix (MYO ECM) used in histological, immunohistochemical (IHC), DNA, biochemical, mechanical, and cell-seeding analyses. Time-course plot (B) of the mean recorded masses of scaffolds exposed to 4 different decellularization methods. Results of a (C) PicoGreen ® quantitative assay for DNA are shown, which compare the DNA content of both decellularized MYO ECM and CA ECM samples generated with various decellularization treatments to that of native myocardium and native coronary arteries, respectively. DNA content of (D) MYO ECM samples from scaffolds generated with the group 4 decellularization treatment and subsequent exposure to various reagents as measured by PicoGreen assay. DNA content of group 4 and native myocardium represented in (D) are carried over from C for comparison (*: indicates statistical significance from all other groups, p

    Techniques Used: Flow Cytometry, Immunohistochemistry, Generated, Picogreen Assay

    18) Product Images from "High Magnesium Corrosion Rate has an Effect on Osteoclast and Mesenchymal Stem Cell Role During Bone Remodelling"

    Article Title: High Magnesium Corrosion Rate has an Effect on Osteoclast and Mesenchymal Stem Cell Role During Bone Remodelling

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-28476-w

    Effect of Mg conditioned media on hMSCs metabolic activity and DNA concentration was investigated using AlamarBlue assay and PicoGreen assay over a period of 7 days. hMSCs were cultured in the presence of various concentrations of Mg conditioned medium, ( A ) filtered medium and ( B ) non-filtered medium. A reduction in metabolic activity was observed when cells were cultured in Mg100 non-filtered medium on day 3 and 7 in comparison to the control. DNA concentration of hMSCs was also investigated following culture in ( C ) filtered medium and ( D ) non-filtered medium. A significant decrease ( # p
    Figure Legend Snippet: Effect of Mg conditioned media on hMSCs metabolic activity and DNA concentration was investigated using AlamarBlue assay and PicoGreen assay over a period of 7 days. hMSCs were cultured in the presence of various concentrations of Mg conditioned medium, ( A ) filtered medium and ( B ) non-filtered medium. A reduction in metabolic activity was observed when cells were cultured in Mg100 non-filtered medium on day 3 and 7 in comparison to the control. DNA concentration of hMSCs was also investigated following culture in ( C ) filtered medium and ( D ) non-filtered medium. A significant decrease ( # p

    Techniques Used: Activity Assay, Concentration Assay, Alamar Blue Assay, Picogreen Assay, Cell Culture

    19) Product Images from "Pullulan: a new cytoadhesive for cell-mediated cartilage repair"

    Article Title: Pullulan: a new cytoadhesive for cell-mediated cartilage repair

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-015-0011-7

    MSC viability and proliferation was maintained up to seven days in the presence of pullulan. (A) MSC metabolic activity was assessed by quantitative formazan production (A490). MSCs were cultured in 0%, 2% and 5% pullulan for one, three and seven days. An increase in metabolism at day 1 was observed in MSCs cultured in 5% pullulan as compared to 0% controls. Metabolic activity was maintained at days 3 and 7 in all cultures, but consistently increased over time with no significant change due to pullulan treatment. (B) An increase in MSC DNA concentration as assayed by PicoGreen incorporation was observed in all culture conditions between days 1 and 3, with a significant decrease in DNA concentration of MSCs cultured in 2% pullulan after seven days as compared to controls at the same time point. (C) The ratio of metabolic activity normalised to DNA content indicated a non-significant increase in MSC metabolism at day 1 in MSCs exposed to 5% pullulan; however, metabolic activity in all cultures decreased with time, returning to day 1 levels at day 7. Data are presented as the mean ± SD of n = 3 biological replicates generated using triplicate measurements, ** = P ≤ 0.01. MSC mesenchymal stem cell; SD, standard deviation.
    Figure Legend Snippet: MSC viability and proliferation was maintained up to seven days in the presence of pullulan. (A) MSC metabolic activity was assessed by quantitative formazan production (A490). MSCs were cultured in 0%, 2% and 5% pullulan for one, three and seven days. An increase in metabolism at day 1 was observed in MSCs cultured in 5% pullulan as compared to 0% controls. Metabolic activity was maintained at days 3 and 7 in all cultures, but consistently increased over time with no significant change due to pullulan treatment. (B) An increase in MSC DNA concentration as assayed by PicoGreen incorporation was observed in all culture conditions between days 1 and 3, with a significant decrease in DNA concentration of MSCs cultured in 2% pullulan after seven days as compared to controls at the same time point. (C) The ratio of metabolic activity normalised to DNA content indicated a non-significant increase in MSC metabolism at day 1 in MSCs exposed to 5% pullulan; however, metabolic activity in all cultures decreased with time, returning to day 1 levels at day 7. Data are presented as the mean ± SD of n = 3 biological replicates generated using triplicate measurements, ** = P ≤ 0.01. MSC mesenchymal stem cell; SD, standard deviation.

    Techniques Used: Activity Assay, Cell Culture, Concentration Assay, Generated, Standard Deviation

    20) Product Images from "A Non-woven Path: Electrospun Poly(lactic acid) Scaffolds for Kidney Tissue Engineering"

    Article Title: A Non-woven Path: Electrospun Poly(lactic acid) Scaffolds for Kidney Tissue Engineering

    Journal: Tissue Engineering and Regenerative Medicine

    doi: 10.1007/s13770-017-0107-5

    DNA quantity per scaffold at 3 and 7 days, assessed by PicoGreen assay. This confirms the ability of all scaffold architectures to support primary kidney cell life. Analysis using a one-way ANOVA showed significant differences F(7,23) = 4.79, p = 0.002, post hoc Tukey analysis shows that was in regards to cryogenic scaffolds. Data presented as mean ± 95% confidence intervals, circles denote individual data points
    Figure Legend Snippet: DNA quantity per scaffold at 3 and 7 days, assessed by PicoGreen assay. This confirms the ability of all scaffold architectures to support primary kidney cell life. Analysis using a one-way ANOVA showed significant differences F(7,23) = 4.79, p = 0.002, post hoc Tukey analysis shows that was in regards to cryogenic scaffolds. Data presented as mean ± 95% confidence intervals, circles denote individual data points

    Techniques Used: Picogreen Assay

    21) Product Images from "The Transcriptional Stress Response of Candida albicans to Weak Organic Acids"

    Article Title: The Transcriptional Stress Response of Candida albicans to Weak Organic Acids

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.114.015941

    Weak organic acids reduce total RNA and ribosomal RNA in Candida albicans : Total RNA content and relative rRNA abundance in cell pellets used for RNA deep-sequencing experiment. (A) Total RNA extraction yield quantified by RiboGreen assay. (B) rRNA vs. total RNA ratio estimated by areas under the peaks found on Bioanalyzer electropherograms. n = 4; * P
    Figure Legend Snippet: Weak organic acids reduce total RNA and ribosomal RNA in Candida albicans : Total RNA content and relative rRNA abundance in cell pellets used for RNA deep-sequencing experiment. (A) Total RNA extraction yield quantified by RiboGreen assay. (B) rRNA vs. total RNA ratio estimated by areas under the peaks found on Bioanalyzer electropherograms. n = 4; * P

    Techniques Used: Sequencing, RNA Extraction

    22) Product Images from "Zwitterionic Nanocarrier Surface Chemistry Improves siRNA Tumor Delivery and Silencing Activity Relative to Polyethylene Glycol"

    Article Title: Zwitterionic Nanocarrier Surface Chemistry Improves siRNA Tumor Delivery and Silencing Activity Relative to Polyethylene Glycol

    Journal: ACS nano

    doi: 10.1021/acsnano.7b01110

    Polyplexes with different corona chemistries have similar size, zeta potential, and cargo loading but varied stability against high salt concentrations. (A, B) Polyplex siRNA encapsulation efficiency and stability is highest at N + :P − 20. (A) Ribogreen assay reveals polyplex encapsulation plateaus by N + :P − ratio of 10. (B) Polyplexes retain higher stability after a 30 min incubation in 30% FBS at N + :P − 20 compared to N + :P − 10 ( p
    Figure Legend Snippet: Polyplexes with different corona chemistries have similar size, zeta potential, and cargo loading but varied stability against high salt concentrations. (A, B) Polyplex siRNA encapsulation efficiency and stability is highest at N + :P − 20. (A) Ribogreen assay reveals polyplex encapsulation plateaus by N + :P − ratio of 10. (B) Polyplexes retain higher stability after a 30 min incubation in 30% FBS at N + :P − 20 compared to N + :P − 10 ( p

    Techniques Used: Incubation

    23) Product Images from "Low abundance of telomerase in yeast: Implications for telomerase haploinsufficiency"

    Article Title: Low abundance of telomerase in yeast: Implications for telomerase haploinsufficiency

    Journal: RNA

    doi: 10.1261/rna.134706

    Quantitative Northern blot analysis of TLC1 molecules in total RNA. ( A ) The leftmost and rightmost sets of lanes, respectively, show twofold serial dilutions of in vitro transcribed TLC1 1167 RNA (beginning with 6.25 × 10 8 molecules) and TLC1 1261A55 RNA (beginning with 3.12 × 10 8 molecules). The center lanes show 10 μg, 7.5 μg, and 5 μg total RNA isolated from wild-type haploid cells (WT N), wild-type diploid cells (WT 2N), and TLC1/tlc1Δ heterozygous diploid cells (Het 2N). RNAs were hybridized with TLC1 and U1 snRNA probes. ( B ) A standard curve for TLC1 quantification was made by plotting the counts per peak area against the known amount of each standard in the TLC1 1167 RNA dilution series. Linear regression analysis of the seven-point curve yielded an r 2 value of 0.997, indicating that the Northern assay is linear from 9.76 × 10 6 to 6.25 × 10 8 TLC1 molecules; only the five lower points are shown in the graph because the interpolated TLC1 copy numbers in total RNA fall within this lower range. The TLC1 signal in each 10 μg total RNA sample is plotted on the line. ( C ) The number of TLC1 molecules in each total RNA sample was interpolated from either the TLC1 1167 standard curve or the TLC1 1261A55 standard curve (r 2 = 0.998). TLC1 molecules per nanogram total RNA were calculated by dividing the interpolated number by the amount of RNA loaded, i.e., either 10 μg, 7.5 μg, or 5 μg. To express these numbers relative to the A 260 quantification of the RNA standards, we then multiplied the RiboGreen-based values by the concentration ratio of A 260 /RiboGreen (see Materials and Methods). TLC1 molecules per cell were calculated by multiplying TLC1 molecules per nanogram RNA by 0.0012 ng RNA per haploid cell or 0.0018 ng RNA per diploid cell. Each number shown is the average of the 10 μg, 7.5 μg, and 5 μg samples, with the standard deviations shown in parentheses.
    Figure Legend Snippet: Quantitative Northern blot analysis of TLC1 molecules in total RNA. ( A ) The leftmost and rightmost sets of lanes, respectively, show twofold serial dilutions of in vitro transcribed TLC1 1167 RNA (beginning with 6.25 × 10 8 molecules) and TLC1 1261A55 RNA (beginning with 3.12 × 10 8 molecules). The center lanes show 10 μg, 7.5 μg, and 5 μg total RNA isolated from wild-type haploid cells (WT N), wild-type diploid cells (WT 2N), and TLC1/tlc1Δ heterozygous diploid cells (Het 2N). RNAs were hybridized with TLC1 and U1 snRNA probes. ( B ) A standard curve for TLC1 quantification was made by plotting the counts per peak area against the known amount of each standard in the TLC1 1167 RNA dilution series. Linear regression analysis of the seven-point curve yielded an r 2 value of 0.997, indicating that the Northern assay is linear from 9.76 × 10 6 to 6.25 × 10 8 TLC1 molecules; only the five lower points are shown in the graph because the interpolated TLC1 copy numbers in total RNA fall within this lower range. The TLC1 signal in each 10 μg total RNA sample is plotted on the line. ( C ) The number of TLC1 molecules in each total RNA sample was interpolated from either the TLC1 1167 standard curve or the TLC1 1261A55 standard curve (r 2 = 0.998). TLC1 molecules per nanogram total RNA were calculated by dividing the interpolated number by the amount of RNA loaded, i.e., either 10 μg, 7.5 μg, or 5 μg. To express these numbers relative to the A 260 quantification of the RNA standards, we then multiplied the RiboGreen-based values by the concentration ratio of A 260 /RiboGreen (see Materials and Methods). TLC1 molecules per cell were calculated by multiplying TLC1 molecules per nanogram RNA by 0.0012 ng RNA per haploid cell or 0.0018 ng RNA per diploid cell. Each number shown is the average of the 10 μg, 7.5 μg, and 5 μg samples, with the standard deviations shown in parentheses.

    Techniques Used: Northern Blot, In Vitro, Isolation, Concentration Assay

    24) Product Images from "Shep regulates Drosophila neuronal remodeling by controlling transcription of its chromatin targets"

    Article Title: Shep regulates Drosophila neuronal remodeling by controlling transcription of its chromatin targets

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.154047

    Loss of shep strongly affects transcriptome of P14 pupal neurons. (A) Genome-wide fold changes of gene expression in response to loss of shep at third instar larval and P14 pupal stages. Dashed line indicates where gene expression is equally affected between the two stages. Note that the expression of dawdle is upregulated to more than 10-fold at both stages, which is out of plot range in both A and B. Genes inhibited by shep in pupae (red), larvae (yellow) and both (aqua) or promoted by shep in pupae (sky blue), larvae (green) or both (pink) are indicated. (B) The shep gene specifically regulates stage-biased genes during metamorphosis. Genes with (gray) or without (black) stage-biased expression during normal metamorphosis are indicated. Stage-biased genes that are shep- inhibited (red) or shep -promoted (blue) in pupae are indicated. Non-stage biased shep -regulated genes are indicated in purple. (C) The binary heat maps apply the same color scheme and summarize overlap between shep- regulated genes and genes with expression changes during normal metamorphosis. (D) Top GO terms of gene expression changes as a result of normal metamorphosis that are either larval-biased (gray) or pupal-biased (black) overlaid with changes due to loss of shep in pupal neurons that are shep -inhibited (red) or shep -promoted (blue). (E,F) Screenshots of RNA-seq signals in loss-of- shep P14 pupal neurons, and Shep, Su(Hw) and Mod(mdg4)67.2 ChIP-seq signals in BG3 cells at the Dad (E) and Myc (F) loci. (G) Validation of RNA-seq results by RT-qPCR in sorted pupal neurons. Three biological replicates for each genotype were analyzed with Student's t -test (* P
    Figure Legend Snippet: Loss of shep strongly affects transcriptome of P14 pupal neurons. (A) Genome-wide fold changes of gene expression in response to loss of shep at third instar larval and P14 pupal stages. Dashed line indicates where gene expression is equally affected between the two stages. Note that the expression of dawdle is upregulated to more than 10-fold at both stages, which is out of plot range in both A and B. Genes inhibited by shep in pupae (red), larvae (yellow) and both (aqua) or promoted by shep in pupae (sky blue), larvae (green) or both (pink) are indicated. (B) The shep gene specifically regulates stage-biased genes during metamorphosis. Genes with (gray) or without (black) stage-biased expression during normal metamorphosis are indicated. Stage-biased genes that are shep- inhibited (red) or shep -promoted (blue) in pupae are indicated. Non-stage biased shep -regulated genes are indicated in purple. (C) The binary heat maps apply the same color scheme and summarize overlap between shep- regulated genes and genes with expression changes during normal metamorphosis. (D) Top GO terms of gene expression changes as a result of normal metamorphosis that are either larval-biased (gray) or pupal-biased (black) overlaid with changes due to loss of shep in pupal neurons that are shep -inhibited (red) or shep -promoted (blue). (E,F) Screenshots of RNA-seq signals in loss-of- shep P14 pupal neurons, and Shep, Su(Hw) and Mod(mdg4)67.2 ChIP-seq signals in BG3 cells at the Dad (E) and Myc (F) loci. (G) Validation of RNA-seq results by RT-qPCR in sorted pupal neurons. Three biological replicates for each genotype were analyzed with Student's t -test (* P

    Techniques Used: Genome Wide, Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR

    25) Product Images from "Cytokine therapy‐mediated neuroprotection in a Friedreich's ataxia mouse model"

    Article Title: Cytokine therapy‐mediated neuroprotection in a Friedreich's ataxia mouse model

    Journal: Annals of Neurology

    doi: 10.1002/ana.24846

    Both frataxin and regulatory factors implicated in controlling frataxin transcription are elevated in the cerebellum and spinal cord of YG8R mice treated with G‐CSF and/or SCF. (A) A schematic of the 5’ end of the human frataxin ( FXN ) gene showing approximate locations of the binding sites (yellow bars) for HIF‐2A, SRF, TFAP2A, and p53 in human or murine cells. The locations of the promotor (PR), Exon1, Exon2, and Intron1 regions are depicted. Different transcription start sites (TSS1 and TSS2) are shown upstream of Exon1, which holds the ATG translation start site. The directions of transcription for FXN (red arrows) and FXN antisense transcript ( FAST‐1 ; dashed black arrow) are shown. The red triangle indicates the site of the trinucleotide GAA repeat expansion within intron 1 of FXN gene of patients with Friedreich's ataxia. The relative (B) mRNA and protein expression levels of frataxin within the cerebellum and spinal cord of YG8R mice (normalized to NeuN or β actin); (C) mRNA expression levels of transcription factors implicated in controlling frataxin expression Epas1, Srf, Tfap2a , and Trp53 (normalized to NeuN). (D) Correlation and linear regression analysis of FXN and Epas1 mRNA levels (normalized to NeuN) in the spinal cord and cerebellum of treated YG8R mice (lines of best fit and 95% confidence interval [CI] are depicted); r = Spearman's correlation coefficient. All statistical comparisons are versus YG8R mice. Comparisons between control and untreated YG8R mice were analyzed using unpaired t tests or Mann–Whitney U tests. For all other analyses, either one‐way analysis of variance followed by Dunnett's multiple comparison test or Kruskal‐Wallis followed by Dunn's multiple comparison test was applied. * p
    Figure Legend Snippet: Both frataxin and regulatory factors implicated in controlling frataxin transcription are elevated in the cerebellum and spinal cord of YG8R mice treated with G‐CSF and/or SCF. (A) A schematic of the 5’ end of the human frataxin ( FXN ) gene showing approximate locations of the binding sites (yellow bars) for HIF‐2A, SRF, TFAP2A, and p53 in human or murine cells. The locations of the promotor (PR), Exon1, Exon2, and Intron1 regions are depicted. Different transcription start sites (TSS1 and TSS2) are shown upstream of Exon1, which holds the ATG translation start site. The directions of transcription for FXN (red arrows) and FXN antisense transcript ( FAST‐1 ; dashed black arrow) are shown. The red triangle indicates the site of the trinucleotide GAA repeat expansion within intron 1 of FXN gene of patients with Friedreich's ataxia. The relative (B) mRNA and protein expression levels of frataxin within the cerebellum and spinal cord of YG8R mice (normalized to NeuN or β actin); (C) mRNA expression levels of transcription factors implicated in controlling frataxin expression Epas1, Srf, Tfap2a , and Trp53 (normalized to NeuN). (D) Correlation and linear regression analysis of FXN and Epas1 mRNA levels (normalized to NeuN) in the spinal cord and cerebellum of treated YG8R mice (lines of best fit and 95% confidence interval [CI] are depicted); r = Spearman's correlation coefficient. All statistical comparisons are versus YG8R mice. Comparisons between control and untreated YG8R mice were analyzed using unpaired t tests or Mann–Whitney U tests. For all other analyses, either one‐way analysis of variance followed by Dunnett's multiple comparison test or Kruskal‐Wallis followed by Dunn's multiple comparison test was applied. * p

    Techniques Used: Mouse Assay, Binding Assay, Expressing, MANN-WHITNEY

    26) Product Images from "SNP detection using peptide nucleic acid probes and conjugated polymers: Applications in neurodegenerative disease identification"

    Article Title: SNP detection using peptide nucleic acid probes and conjugated polymers: Applications in neurodegenerative disease identification

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

    doi: 10.1073/pnas.0407578101

    Long ssDNA target sequences (black) are digested by S1 nuclease, leaving intact only those regions bound to the PNA probe (red). CPs (blue) added directly to the resulting solutions can only associate with the remaining PNA-FL/DNA duplex. Any PNA/DNA mismatches will result in complete DNA digestion; therefore, energy transfer from the CP occurs only for the perfect PNA-FL/DNA complement.
    Figure Legend Snippet: Long ssDNA target sequences (black) are digested by S1 nuclease, leaving intact only those regions bound to the PNA probe (red). CPs (blue) added directly to the resulting solutions can only associate with the remaining PNA-FL/DNA duplex. Any PNA/DNA mismatches will result in complete DNA digestion; therefore, energy transfer from the CP occurs only for the perfect PNA-FL/DNA complement.

    Techniques Used:

    Cationic CPs (blue) have the ability to associate nonspecifically along any region of a targeted ssDNA sequence (black). ( A ) Association of a short DNA target with the CP. ( B ) The probability of CP association along a longer DNA target. CPs complexed at distances greater than R o from the FL acceptor will be less efficient donors to the PNA-FL probe (red and green).
    Figure Legend Snippet: Cationic CPs (blue) have the ability to associate nonspecifically along any region of a targeted ssDNA sequence (black). ( A ) Association of a short DNA target with the CP. ( B ) The probability of CP association along a longer DNA target. CPs complexed at distances greater than R o from the FL acceptor will be less efficient donors to the PNA-FL probe (red and green).

    Techniques Used: Sequencing

    Fluorescence of PNA-FL annealed to complementary 28 (black, D0) and 249 (red, WT) base ssDNA targets ([PNA/ssDNA] = 1 × 10 -8 M) upon addition and excitation (λ ex = 380 nm) of the CP ([CP] = 2 × 10 -7 M) in 30 mM potassium phosphate buffer, pH 7.4. The noncomplementary (28 base, D3) DNA target is shown in blue for reference. The arrow indicates the drop in the relative FL-to-CP emission peak ratio between the two different DNA target lengths.
    Figure Legend Snippet: Fluorescence of PNA-FL annealed to complementary 28 (black, D0) and 249 (red, WT) base ssDNA targets ([PNA/ssDNA] = 1 × 10 -8 M) upon addition and excitation (λ ex = 380 nm) of the CP ([CP] = 2 × 10 -7 M) in 30 mM potassium phosphate buffer, pH 7.4. The noncomplementary (28 base, D3) DNA target is shown in blue for reference. The arrow indicates the drop in the relative FL-to-CP emission peak ratio between the two different DNA target lengths.

    Techniques Used: Fluorescence

    27) Product Images from "MicroRNA expression in preimplantation mouse embryos from Ped gene positive compared to Ped gene negative mice"

    Article Title: MicroRNA expression in preimplantation mouse embryos from Ped gene positive compared to Ped gene negative mice

    Journal: Journal of Assisted Reproduction and Genetics

    doi: 10.1007/s10815-008-9211-8

    Reverse transcription of ES cell and preimplantation embryo RNA for miR-125a and miR-125b detection
    Figure Legend Snippet: Reverse transcription of ES cell and preimplantation embryo RNA for miR-125a and miR-125b detection

    Techniques Used:

    28) Product Images from "A Novel Bvg-Repressed Promoter Causes vrg-Like Transcription of fim3 but Does Not Result in the Production of Serotype 3 Fimbriae in Bvg− Mode Bordetella pertussis"

    Article Title: A Novel Bvg-Repressed Promoter Causes vrg-Like Transcription of fim3 but Does Not Result in the Production of Serotype 3 Fimbriae in Bvg− Mode Bordetella pertussis

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00175-18

    Quantitative PCR of in vivo fim2 , fim3 , and vrgX expression. RNA prepared from B. pertussis strains BP536 (P fim3 -13C) and QC3980 (P fim3 -15C) grown on BG agar in the absence (white bar) or presence (black bar) of 50 mM MgSO 4 was reverse transcribed to cDNA, followed by qPCR analysis of genes fim2 , fim3 , and vrgX . Expression levels, after normalization to the rpoD gene, used as an internal control, are given as fold difference relative to fim3 expression in BP536 in the presence of MgSO 4 . The mean values and standard deviations, in error bars, were calculated from three biologically independent determinations and used to conduct statistical analysis using unpaired two-tailed t test between two samples. NS, P > 0.05; **** , P ≤ 0.0001.
    Figure Legend Snippet: Quantitative PCR of in vivo fim2 , fim3 , and vrgX expression. RNA prepared from B. pertussis strains BP536 (P fim3 -13C) and QC3980 (P fim3 -15C) grown on BG agar in the absence (white bar) or presence (black bar) of 50 mM MgSO 4 was reverse transcribed to cDNA, followed by qPCR analysis of genes fim2 , fim3 , and vrgX . Expression levels, after normalization to the rpoD gene, used as an internal control, are given as fold difference relative to fim3 expression in BP536 in the presence of MgSO 4 . The mean values and standard deviations, in error bars, were calculated from three biologically independent determinations and used to conduct statistical analysis using unpaired two-tailed t test between two samples. NS, P > 0.05; **** , P ≤ 0.0001.

    Techniques Used: Real-time Polymerase Chain Reaction, In Vivo, Expressing, Two Tailed Test

    29) Product Images from "P2Y6 Receptors Regulate CXCL10 Expression and Secretion in Mouse Intestinal Epithelial Cells"

    Article Title: P2Y6 Receptors Regulate CXCL10 Expression and Secretion in Mouse Intestinal Epithelial Cells

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.00149

    Characterization of primary IEC cultures. IECs were prepared and cultured as mentioned in the section “Materials and Methods” to obtain a monolayer of differentiated cells. RNA was isolated and the expression of P2Y receptors (A) , P2X receptors (B) , the differentiated epithelial cell markers villin and ALPi (C) , ectonucleotidases (D) , and P1 receptors (E) were analyzed by qRT-PCR. Data are normalized to GAPDH mRNA level. Data presented are the mean ± SEM of three independent experiments each with cells pooled from three mice.
    Figure Legend Snippet: Characterization of primary IEC cultures. IECs were prepared and cultured as mentioned in the section “Materials and Methods” to obtain a monolayer of differentiated cells. RNA was isolated and the expression of P2Y receptors (A) , P2X receptors (B) , the differentiated epithelial cell markers villin and ALPi (C) , ectonucleotidases (D) , and P1 receptors (E) were analyzed by qRT-PCR. Data are normalized to GAPDH mRNA level. Data presented are the mean ± SEM of three independent experiments each with cells pooled from three mice.

    Techniques Used: Cell Culture, Isolation, Expressing, Quantitative RT-PCR, Mouse Assay

    30) Product Images from "Inflammatory Cytokine Tumor Necrosis Factor ? Confers Precancerous Phenotype in an Organoid Model of Normal Human Ovarian Surface Epithelial Cells 1"

    Article Title: Inflammatory Cytokine Tumor Necrosis Factor ? Confers Precancerous Phenotype in an Organoid Model of Normal Human Ovarian Surface Epithelial Cells 1

    Journal:

    doi:

    Tumor necrosis factor α induced disintegration of basement membrane and expression of MMP-10 in ovarian epithelial cystic structures. (A) Immunofluorescent staining of COL4 in control HOSE spheroids and TNF-α-treated cystic structures
    Figure Legend Snippet: Tumor necrosis factor α induced disintegration of basement membrane and expression of MMP-10 in ovarian epithelial cystic structures. (A) Immunofluorescent staining of COL4 in control HOSE spheroids and TNF-α-treated cystic structures

    Techniques Used: Expressing, Staining

    Tumor necrosis factor α induced enlargement, disorganization, multilayer stratification, and disruption of cell polarity in ovarian epithelial cystic structures. (A) Representative phase-contrast images of normal HOSE spheroids and TNF-α-treated
    Figure Legend Snippet: Tumor necrosis factor α induced enlargement, disorganization, multilayer stratification, and disruption of cell polarity in ovarian epithelial cystic structures. (A) Representative phase-contrast images of normal HOSE spheroids and TNF-α-treated

    Techniques Used:

    Tumor necrosis factor α induced invasive phenotypes and overexpression of ovarian cancer markers in ovarian epithelial cystic structures. (A) Fluorescence labeling of F-actin (phalloidin) and nuclei (DAPI) in control HOSE spheroids and TNF-α-treated
    Figure Legend Snippet: Tumor necrosis factor α induced invasive phenotypes and overexpression of ovarian cancer markers in ovarian epithelial cystic structures. (A) Fluorescence labeling of F-actin (phalloidin) and nuclei (DAPI) in control HOSE spheroids and TNF-α-treated

    Techniques Used: Over Expression, Fluorescence, Labeling

    31) Product Images from "Identification of RNA-binding proteins in exosomes capable of interacting with different types of RNA: RBP-facilitated transport of RNAs into exosomes"

    Article Title: Identification of RNA-binding proteins in exosomes capable of interacting with different types of RNA: RBP-facilitated transport of RNAs into exosomes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0195969

    Validation of MVP. Transfected cells are expressing MVP-biotin and un-transfected cells were used as negative control. (A) Western blot analysis for the detection of MVP in HEK293F cell lysate and exosomal protein extracts shows that plasmid (MVP-biotin) was successfully expressed in HEK293F cells and was partitioned into exosomes isolated from these transfected cells. ( B ) Protein samples from different stages of MVP purification were separated by SDS-PAGE and the protein bands were detected by Comassie staining. After the pull down assay, the presence of MVP within exosomes was confirmed, i.e. MVP in exosomes from biotinylated MVP-transfected cells PD (pull-down lane), whereas it was absent in exosomes from untransfected cells. The position of the MVP-biotin band is indicated by squares. A representative experiment out of 3 is shown. ( C ) Total amount of RNAs coupled to MVP were extracted and quantified by Quant-iT™ RiboGreen® RNA Assay Kit (ThermoFisher Scientific). Captured RNA was expressed as a percentage of RNA eluted from the beads after the pull-down respect to the total RNA incubated with the beads. Exosomal-MVP was coupled with RNAs and the quantification of RNAs that had co-eluted with MVP showed that the amount of RNA present in the MVP elute (i.e. from MVP-RNA complex) was significantly higher than that from the untransfected control. The experiment was performed in 3-replicates and the graph shows the range of values and median (grey). Average and standard error of three independent experiments are shown. Samples: W = proteins from different wash steps during the MVP purification, and PD = pull-down proteins (MVP) after elution, I = input, and UB = unbound after RBP capture.
    Figure Legend Snippet: Validation of MVP. Transfected cells are expressing MVP-biotin and un-transfected cells were used as negative control. (A) Western blot analysis for the detection of MVP in HEK293F cell lysate and exosomal protein extracts shows that plasmid (MVP-biotin) was successfully expressed in HEK293F cells and was partitioned into exosomes isolated from these transfected cells. ( B ) Protein samples from different stages of MVP purification were separated by SDS-PAGE and the protein bands were detected by Comassie staining. After the pull down assay, the presence of MVP within exosomes was confirmed, i.e. MVP in exosomes from biotinylated MVP-transfected cells PD (pull-down lane), whereas it was absent in exosomes from untransfected cells. The position of the MVP-biotin band is indicated by squares. A representative experiment out of 3 is shown. ( C ) Total amount of RNAs coupled to MVP were extracted and quantified by Quant-iT™ RiboGreen® RNA Assay Kit (ThermoFisher Scientific). Captured RNA was expressed as a percentage of RNA eluted from the beads after the pull-down respect to the total RNA incubated with the beads. Exosomal-MVP was coupled with RNAs and the quantification of RNAs that had co-eluted with MVP showed that the amount of RNA present in the MVP elute (i.e. from MVP-RNA complex) was significantly higher than that from the untransfected control. The experiment was performed in 3-replicates and the graph shows the range of values and median (grey). Average and standard error of three independent experiments are shown. Samples: W = proteins from different wash steps during the MVP purification, and PD = pull-down proteins (MVP) after elution, I = input, and UB = unbound after RBP capture.

    Techniques Used: Transfection, Expressing, Negative Control, Western Blot, Plasmid Preparation, Isolation, Purification, SDS Page, Staining, Pull Down Assay, Incubation

    The gene transcripts encoding six RBPs identified in exosomes were silenced in the cytoplasm by siRNA and the subsequent effect on the amount of esRNA was assessed. A significant reduction of total RNA in exosomes (esRNA) was shown by post-transcriptional silencing of MVP. (A) Confirmation of gene silencing (silenced transcripts) in the cells after transfection with the siRNAs against the transcripts of HSP90AB1, XPO5, hnRNPH1, hnRNPM, hnRNPA2B1, and MVP with respect to the Negative Control siRNA (scrambled siRNA). Quantitative PCR was performed using cDNA, Fast SYBR Green Master Mix, and gene-specific primers. Gene expression was normalized to that of the housekeeping gene GAPDH . The error bars represent average of 4 biological replicates for hnRNPA2B1 and MVP, and 2 biological replicates for other four transcripts, and the average fold change values presented as RQ (relative quantification). (B) Quantification (the percentage-change, %) of total RNA present in exosomes (ng total esRNA / μg total exosomal proteins) after each gene silencing, i . e . silencing of HSP90AB1, XPO5, hnRNPH1, hnRNPM, and hnRNPA2B1. The silencing of hnRNPA2B1 caused a slight reduction of total RNA present in exosomes, but at non-significant level ≈13%. However, after silencing of hnRNPH1 the amount of RNA in exosomes was increased as compared to negative control. The experiment was performed in duplicates, except from hnRNPA2B1 that was performed in 4 replicates. The graph shows the range of values and median (grey). (C-D) Quantification of total RNA (ng total RNA / μg total protein) present in cells and exosomes after gene silencing of MVP. (C) A slight increase (but not significant, p-value 0.57) of the amount of total RNA in cytoplasm was observed. (D) On the contrary, down regulation of MVP (silencing) caused a significant reduction of the total RNA present in exosomes (esRNA), approximately by 50% (p-value 0.02). The experiment for MVP silencing was performed in four replicates; the graph shows the range of values and median (grey).
    Figure Legend Snippet: The gene transcripts encoding six RBPs identified in exosomes were silenced in the cytoplasm by siRNA and the subsequent effect on the amount of esRNA was assessed. A significant reduction of total RNA in exosomes (esRNA) was shown by post-transcriptional silencing of MVP. (A) Confirmation of gene silencing (silenced transcripts) in the cells after transfection with the siRNAs against the transcripts of HSP90AB1, XPO5, hnRNPH1, hnRNPM, hnRNPA2B1, and MVP with respect to the Negative Control siRNA (scrambled siRNA). Quantitative PCR was performed using cDNA, Fast SYBR Green Master Mix, and gene-specific primers. Gene expression was normalized to that of the housekeeping gene GAPDH . The error bars represent average of 4 biological replicates for hnRNPA2B1 and MVP, and 2 biological replicates for other four transcripts, and the average fold change values presented as RQ (relative quantification). (B) Quantification (the percentage-change, %) of total RNA present in exosomes (ng total esRNA / μg total exosomal proteins) after each gene silencing, i . e . silencing of HSP90AB1, XPO5, hnRNPH1, hnRNPM, and hnRNPA2B1. The silencing of hnRNPA2B1 caused a slight reduction of total RNA present in exosomes, but at non-significant level ≈13%. However, after silencing of hnRNPH1 the amount of RNA in exosomes was increased as compared to negative control. The experiment was performed in duplicates, except from hnRNPA2B1 that was performed in 4 replicates. The graph shows the range of values and median (grey). (C-D) Quantification of total RNA (ng total RNA / μg total protein) present in cells and exosomes after gene silencing of MVP. (C) A slight increase (but not significant, p-value 0.57) of the amount of total RNA in cytoplasm was observed. (D) On the contrary, down regulation of MVP (silencing) caused a significant reduction of the total RNA present in exosomes (esRNA), approximately by 50% (p-value 0.02). The experiment for MVP silencing was performed in four replicates; the graph shows the range of values and median (grey).

    Techniques Used: Transfection, Negative Control, Real-time Polymerase Chain Reaction, SYBR Green Assay, Expressing, Significance Assay

    Network analysis of all (30) identified RBPs in exosomes. ) to build a biological network of the RBPs in exosomes. The network included 35 nodes (gene products), 27 of which were among the identified exosomal-RBPs including MVP (indicated in grey). The network shows the involvement of exosomal-RBPs in ‘RNA posttranscriptional modifications’.
    Figure Legend Snippet: Network analysis of all (30) identified RBPs in exosomes. ) to build a biological network of the RBPs in exosomes. The network included 35 nodes (gene products), 27 of which were among the identified exosomal-RBPs including MVP (indicated in grey). The network shows the involvement of exosomal-RBPs in ‘RNA posttranscriptional modifications’.

    Techniques Used:

    Two hypothetical paths ( A and B) for the packaging of RNA–protein–complexes (RNPs) into exosomes during biosynthesis of these vesicles from the multivesicular bodies (MVBs): ( A ]. The intermediate vesicles are matured and transformed into intraluminal vesicles inside MVB. Upon fusion of MVB with the plasma membrane, the intraluminal vesicles (exosomes) will be released outside containing RNA-RNPs complexes into the extracellular environment. In the second model ( B ) RNA–protein complexes (RNPs)—transported via nuclear pore complex (NPC)—are attracted to the internalization site having different receptor-proteins that could mediate inward budding of MVBs, to form intraluminal vesicles. Upon fusion with the plasma membrane, the MVBs release the intraluminal vesicles (exosomes) containing RNA–RNPs complexes into the extracellular environment.
    Figure Legend Snippet: Two hypothetical paths ( A and B) for the packaging of RNA–protein–complexes (RNPs) into exosomes during biosynthesis of these vesicles from the multivesicular bodies (MVBs): ( A ]. The intermediate vesicles are matured and transformed into intraluminal vesicles inside MVB. Upon fusion of MVB with the plasma membrane, the intraluminal vesicles (exosomes) will be released outside containing RNA-RNPs complexes into the extracellular environment. In the second model ( B ) RNA–protein complexes (RNPs)—transported via nuclear pore complex (NPC)—are attracted to the internalization site having different receptor-proteins that could mediate inward budding of MVBs, to form intraluminal vesicles. Upon fusion with the plasma membrane, the MVBs release the intraluminal vesicles (exosomes) containing RNA–RNPs complexes into the extracellular environment.

    Techniques Used: Transformation Assay

    (A) RNA 3’-end labelling of cellular (miRNA, mRNA) and exosomal-RNAs (esRNA). The dot blot shows the efficiency of biotinylation of cellular miRNAs and mRNA, and exosomal total RNA (esRNA), relative to the biotinylated IRE control. A 43 nucleotide synthetic RNA template was used as a positive control for the reaction and as a general mass control. Serial dilutions of the labelled samples were loaded onto a nylon membrane, UV cross-linked, and detected by chemiluminescence. Dot blots show the efficiency of the RNA labelling reaction and the amount of the biotinylated RNA obtained. The biotinylation reaction for all RNA species esRNA, cell-miRNA and cell-mRNA was highly efficient, with biotinylation rates greater than 75%. (B) RNA electrophoretic mobility shift sssays (REMSAs) of cellular and exosomal RNA-RBP-complexes: Total exosomal-RNA (esRNA), cell-miRNA and cell-mRNA fractions extracted from HTB (lung) cells were incubated in binding reactions with exosomal proteins and with HTB cellular proteins. In all experiments, an electrophoretic shift was observed when the biotinylated RNA was incubated with the proteins from exosomes and cells. The specificities of the RNA-protein interactions were determined using competition assays. From left to right : the interaction of IRE RNA and cytosolic liver extract (positive control), the interaction of IRE RNA with cellular proteins, the interaction of cellular miRNAs with cellular proteins, the interaction of esRNA with exosomal proteins, and the interaction of cellular miRNA with exosomal proteins. The band-shifts are indicated by squares.
    Figure Legend Snippet: (A) RNA 3’-end labelling of cellular (miRNA, mRNA) and exosomal-RNAs (esRNA). The dot blot shows the efficiency of biotinylation of cellular miRNAs and mRNA, and exosomal total RNA (esRNA), relative to the biotinylated IRE control. A 43 nucleotide synthetic RNA template was used as a positive control for the reaction and as a general mass control. Serial dilutions of the labelled samples were loaded onto a nylon membrane, UV cross-linked, and detected by chemiluminescence. Dot blots show the efficiency of the RNA labelling reaction and the amount of the biotinylated RNA obtained. The biotinylation reaction for all RNA species esRNA, cell-miRNA and cell-mRNA was highly efficient, with biotinylation rates greater than 75%. (B) RNA electrophoretic mobility shift sssays (REMSAs) of cellular and exosomal RNA-RBP-complexes: Total exosomal-RNA (esRNA), cell-miRNA and cell-mRNA fractions extracted from HTB (lung) cells were incubated in binding reactions with exosomal proteins and with HTB cellular proteins. In all experiments, an electrophoretic shift was observed when the biotinylated RNA was incubated with the proteins from exosomes and cells. The specificities of the RNA-protein interactions were determined using competition assays. From left to right : the interaction of IRE RNA and cytosolic liver extract (positive control), the interaction of IRE RNA with cellular proteins, the interaction of cellular miRNAs with cellular proteins, the interaction of esRNA with exosomal proteins, and the interaction of cellular miRNA with exosomal proteins. The band-shifts are indicated by squares.

    Techniques Used: Dot Blot, Positive Control, Electrophoretic Mobility Shift Assay, Incubation, Binding Assay

    32) Product Images from "Establishment and characterization of a telomerase immortalized human gingival epithelial cell line"

    Article Title: Establishment and characterization of a telomerase immortalized human gingival epithelial cell line

    Journal: Journal of periodontal research

    doi: 10.1111/jre.12059

    Expression of transcripts for toll-like receptors (TLR) in TIGKs RT-PCR of TIGK mRNA from two separate experiments, superscript 1 and 2, using primers for the TLRs indicated. Genomic DNA templates serve as positive controls (PC), and negative controls are DNase I-treated total RNA (R). Each template cDNA was run in duplicate reactions for each experiment.
    Figure Legend Snippet: Expression of transcripts for toll-like receptors (TLR) in TIGKs RT-PCR of TIGK mRNA from two separate experiments, superscript 1 and 2, using primers for the TLRs indicated. Genomic DNA templates serve as positive controls (PC), and negative controls are DNase I-treated total RNA (R). Each template cDNA was run in duplicate reactions for each experiment.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    33) Product Images from "Stormwater influences phytoplankton assemblages within the diverse, but impacted Sydney Harbour estuary"

    Article Title: Stormwater influences phytoplankton assemblages within the diverse, but impacted Sydney Harbour estuary

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0209857

    Distance-based redundancy analysis plot of the eukaryotic community structure (using 18S rRNA gene V9 region) for the Sydney Harbour estuary, during high and low rainfall periods. Samples are colour-coded based on clusters (assigned using hierarchical cluster analysis with SIMPROF test based on Bray-Curtis similarity) for the high rainfall period: a (light green), b (dark blue), c (red), d (dark green); and for the low rainfall period: e (orange), f (light blue), g (blue), h (grey). Symbols represent geographic region sampled: Parramatta River (*), Lane Cove River (●), Western Central Harbour (▼), Eastern Central Harbour (▲), Middle Harbour (■) and Marine/Harbour Heads (♦). TN–Total nitrogen; Si–Silicate.
    Figure Legend Snippet: Distance-based redundancy analysis plot of the eukaryotic community structure (using 18S rRNA gene V9 region) for the Sydney Harbour estuary, during high and low rainfall periods. Samples are colour-coded based on clusters (assigned using hierarchical cluster analysis with SIMPROF test based on Bray-Curtis similarity) for the high rainfall period: a (light green), b (dark blue), c (red), d (dark green); and for the low rainfall period: e (orange), f (light blue), g (blue), h (grey). Symbols represent geographic region sampled: Parramatta River (*), Lane Cove River (●), Western Central Harbour (▼), Eastern Central Harbour (▲), Middle Harbour (■) and Marine/Harbour Heads (♦). TN–Total nitrogen; Si–Silicate.

    Techniques Used: Western Blot

    Eukaryotic community profile, based on 18S rRNA gene V9 region, in Sydney Harbour estuary during high and low rainfall periods. a) Multi-dimensional scaling plot of the community composition during the two periods. Samples are colour-coded based on clusters (assigned using hierarchical cluster analysis with SIMPROF test based on Bray-Curtis similarity) from the high rainfall period: a (light green), b (dark blue), c (red), d (dark green); and from the low rainfall period: e (orange), f (light blue), g (blue), h (grey). Symbols represent geographic region sampled: Parramatta River (*), Lane Cove River (●), Western Central Harbour (▼), Eastern Central Harbour (▲), Middle Harbour (■) and Marine/Harbour Heads (♦). b) Relative abundance profile of the eukaryotic community during the sampling periods, at taxonomic level of family or lower. Each bar represents the cluster of sites, as above. The rarefied number of reads assigned to each lineage was averaged across sites of each cluster.
    Figure Legend Snippet: Eukaryotic community profile, based on 18S rRNA gene V9 region, in Sydney Harbour estuary during high and low rainfall periods. a) Multi-dimensional scaling plot of the community composition during the two periods. Samples are colour-coded based on clusters (assigned using hierarchical cluster analysis with SIMPROF test based on Bray-Curtis similarity) from the high rainfall period: a (light green), b (dark blue), c (red), d (dark green); and from the low rainfall period: e (orange), f (light blue), g (blue), h (grey). Symbols represent geographic region sampled: Parramatta River (*), Lane Cove River (●), Western Central Harbour (▼), Eastern Central Harbour (▲), Middle Harbour (■) and Marine/Harbour Heads (♦). b) Relative abundance profile of the eukaryotic community during the sampling periods, at taxonomic level of family or lower. Each bar represents the cluster of sites, as above. The rarefied number of reads assigned to each lineage was averaged across sites of each cluster.

    Techniques Used: Western Blot, Sampling

    34) Product Images from "Citrullinated histone H3 as a novel prognostic blood marker in patients with advanced cancer"

    Article Title: Citrullinated histone H3 as a novel prognostic blood marker in patients with advanced cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0191231

    Inflammatory cytokines are elevated and correlate with H3Cit in patients with advanced cancer. ( A-E ) Plasma levels of IL-8, IL-6, TNFα, IL-1β, and G-CSF were all significantly higher in cancer patients compared to healthy individuals, as well as to severely ill patients without known cancer, with the exception of G-CSF which was similarly elevated in cancer patients and severely ill patients without known cancer. (F) Plasma levels of H3Cit correlated to plasma levels of IL-8 and IL-6, but weaker or no correlations were found to TNFα, IL-1β and G-CSF. ( G ) Multivariable regression confirmed the predictive influence of plasma cfDNA, NE, MPO, MPO-DNA complexes, IL-8, and IL-6 on plasma H3Cit levels (p
    Figure Legend Snippet: Inflammatory cytokines are elevated and correlate with H3Cit in patients with advanced cancer. ( A-E ) Plasma levels of IL-8, IL-6, TNFα, IL-1β, and G-CSF were all significantly higher in cancer patients compared to healthy individuals, as well as to severely ill patients without known cancer, with the exception of G-CSF which was similarly elevated in cancer patients and severely ill patients without known cancer. (F) Plasma levels of H3Cit correlated to plasma levels of IL-8 and IL-6, but weaker or no correlations were found to TNFα, IL-1β and G-CSF. ( G ) Multivariable regression confirmed the predictive influence of plasma cfDNA, NE, MPO, MPO-DNA complexes, IL-8, and IL-6 on plasma H3Cit levels (p

    Techniques Used:

    Cancer-associated neutrophil activation correlates with circulating H3Cit and cfDNA. ( A ) Peripheral neutrophil count was higher in cancer patients compared to severely ill patients without known cancer (dotted line represents upper reference interval). ( B ) Flow cytometry analysis showed a higher number of neutrophils positive for intracellular H3Cit in cancer patients compared to both healthy individuals and severely ill patients without known cancer. No significant difference was observed between severely ill patients without known cancer and healthy individuals. ( C-D ) Plasma NE ( C ) and MPO ( D ) were equally elevated in cancer patients and severely ill patients without known cancer compared to healthy individuals. (E) Plasma MPO-DNA complexes were significantly higher in cancer patients compared to both healthy individuals and severely ill patients without known cancer. The levels were also significantly higher in severely ill patients without known cancer compared to healthy individuals. (F) H3Cit and cfDNA correlated positively with NE, MPO and MPO-DNA complexes in cancer patients. ( G ) Similar correlations were seen in severely ill patients without known cancer, with the exception of correlations between H3Cit and NE and MPO. Lines represent medians with IQR. Groups were compared with the Mann-Whitney U test. Significance of correlation was analyzed with Pearson correlation coefficient after log transformed data to obtain a normal distribution. NS P > 0.05, * P
    Figure Legend Snippet: Cancer-associated neutrophil activation correlates with circulating H3Cit and cfDNA. ( A ) Peripheral neutrophil count was higher in cancer patients compared to severely ill patients without known cancer (dotted line represents upper reference interval). ( B ) Flow cytometry analysis showed a higher number of neutrophils positive for intracellular H3Cit in cancer patients compared to both healthy individuals and severely ill patients without known cancer. No significant difference was observed between severely ill patients without known cancer and healthy individuals. ( C-D ) Plasma NE ( C ) and MPO ( D ) were equally elevated in cancer patients and severely ill patients without known cancer compared to healthy individuals. (E) Plasma MPO-DNA complexes were significantly higher in cancer patients compared to both healthy individuals and severely ill patients without known cancer. The levels were also significantly higher in severely ill patients without known cancer compared to healthy individuals. (F) H3Cit and cfDNA correlated positively with NE, MPO and MPO-DNA complexes in cancer patients. ( G ) Similar correlations were seen in severely ill patients without known cancer, with the exception of correlations between H3Cit and NE and MPO. Lines represent medians with IQR. Groups were compared with the Mann-Whitney U test. Significance of correlation was analyzed with Pearson correlation coefficient after log transformed data to obtain a normal distribution. NS P > 0.05, * P

    Techniques Used: Activation Assay, Flow Cytometry, Cytometry, MANN-WHITNEY, Transformation Assay

    35) Product Images from "Identification of RNA-binding proteins in exosomes capable of interacting with different types of RNA: RBP-facilitated transport of RNAs into exosomes"

    Article Title: Identification of RNA-binding proteins in exosomes capable of interacting with different types of RNA: RBP-facilitated transport of RNAs into exosomes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0195969

    Validation of MVP. Transfected cells are expressing MVP-biotin and un-transfected cells were used as negative control. (A) Western blot analysis for the detection of MVP in HEK293F cell lysate and exosomal protein extracts shows that plasmid (MVP-biotin) was successfully expressed in HEK293F cells and was partitioned into exosomes isolated from these transfected cells. ( B ) Protein samples from different stages of MVP purification were separated by SDS-PAGE and the protein bands were detected by Comassie staining. After the pull down assay, the presence of MVP within exosomes was confirmed, i.e. MVP in exosomes from biotinylated MVP-transfected cells PD (pull-down lane), whereas it was absent in exosomes from untransfected cells. The position of the MVP-biotin band is indicated by squares. A representative experiment out of 3 is shown. ( C ) Total amount of RNAs coupled to MVP were extracted and quantified by Quant-iT™ RiboGreen® RNA Assay Kit (ThermoFisher Scientific). Captured RNA was expressed as a percentage of RNA eluted from the beads after the pull-down respect to the total RNA incubated with the beads. Exosomal-MVP was coupled with RNAs and the quantification of RNAs that had co-eluted with MVP showed that the amount of RNA present in the MVP elute (i.e. from MVP-RNA complex) was significantly higher than that from the untransfected control. The experiment was performed in 3-replicates and the graph shows the range of values and median (grey). Average and standard error of three independent experiments are shown. Samples: W = proteins from different wash steps during the MVP purification, and PD = pull-down proteins (MVP) after elution, I = input, and UB = unbound after RBP capture.
    Figure Legend Snippet: Validation of MVP. Transfected cells are expressing MVP-biotin and un-transfected cells were used as negative control. (A) Western blot analysis for the detection of MVP in HEK293F cell lysate and exosomal protein extracts shows that plasmid (MVP-biotin) was successfully expressed in HEK293F cells and was partitioned into exosomes isolated from these transfected cells. ( B ) Protein samples from different stages of MVP purification were separated by SDS-PAGE and the protein bands were detected by Comassie staining. After the pull down assay, the presence of MVP within exosomes was confirmed, i.e. MVP in exosomes from biotinylated MVP-transfected cells PD (pull-down lane), whereas it was absent in exosomes from untransfected cells. The position of the MVP-biotin band is indicated by squares. A representative experiment out of 3 is shown. ( C ) Total amount of RNAs coupled to MVP were extracted and quantified by Quant-iT™ RiboGreen® RNA Assay Kit (ThermoFisher Scientific). Captured RNA was expressed as a percentage of RNA eluted from the beads after the pull-down respect to the total RNA incubated with the beads. Exosomal-MVP was coupled with RNAs and the quantification of RNAs that had co-eluted with MVP showed that the amount of RNA present in the MVP elute (i.e. from MVP-RNA complex) was significantly higher than that from the untransfected control. The experiment was performed in 3-replicates and the graph shows the range of values and median (grey). Average and standard error of three independent experiments are shown. Samples: W = proteins from different wash steps during the MVP purification, and PD = pull-down proteins (MVP) after elution, I = input, and UB = unbound after RBP capture.

    Techniques Used: Transfection, Expressing, Negative Control, Western Blot, Plasmid Preparation, Isolation, Purification, SDS Page, Staining, Pull Down Assay, Incubation

    36) Product Images from "Convergent Sets of Data from In Vivo and In Vitro Methods Point to an Active Role of Hsp60 in Chronic Obstructive Pulmonary Disease Pathogenesis"

    Article Title: Convergent Sets of Data from In Vivo and In Vitro Methods Point to an Active Role of Hsp60 in Chronic Obstructive Pulmonary Disease Pathogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028200

    Impact of oxidative stress on the levels of Hsp60 in a human bronchial epithelial cell line. Panel A: Western blotting showing significantly increased levels of Hsp60 in 16HBE after treatment with 50 or 100 μM of H 2 O 2 compared to untreated (UT) cells. The difference between 100 and 50 μM was also significant. Histograms represent the mean (SD) of the Hsp60/actin ratio. Panel B: RT-PCR showing significantly increased levels of Hsp60 only after treatment with 100 μM of H 2 O 2 compared to UT treated cells. Panel C: Hsp60 increased levels in the extracellular medium (measured by ELISA tests) after treatment with 50 or 100 μM of H 2 O 2 compared to untreated (UT) cells. ^: p
    Figure Legend Snippet: Impact of oxidative stress on the levels of Hsp60 in a human bronchial epithelial cell line. Panel A: Western blotting showing significantly increased levels of Hsp60 in 16HBE after treatment with 50 or 100 μM of H 2 O 2 compared to untreated (UT) cells. The difference between 100 and 50 μM was also significant. Histograms represent the mean (SD) of the Hsp60/actin ratio. Panel B: RT-PCR showing significantly increased levels of Hsp60 only after treatment with 100 μM of H 2 O 2 compared to UT treated cells. Panel C: Hsp60 increased levels in the extracellular medium (measured by ELISA tests) after treatment with 50 or 100 μM of H 2 O 2 compared to untreated (UT) cells. ^: p

    Techniques Used: Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    37) Product Images from "Estimates of Soil Bacterial Ribosome Content and Diversity Are Significantly Affected by the Nucleic Acid Extraction Method Employed"

    Article Title: Estimates of Soil Bacterial Ribosome Content and Diversity Are Significantly Affected by the Nucleic Acid Extraction Method Employed

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00019-16

    Yield of total DNA and copies of 16S rRNA genes (A) and yield of total RNA and copies of 16S rRNA transcripts (B). The squares (total DNA or total RNA, white squares; Bacteria , black squares; Acidobacteria , gray squares) represent mean values for the
    Figure Legend Snippet: Yield of total DNA and copies of 16S rRNA genes (A) and yield of total RNA and copies of 16S rRNA transcripts (B). The squares (total DNA or total RNA, white squares; Bacteria , black squares; Acidobacteria , gray squares) represent mean values for the

    Techniques Used:

    z-transformed relative abundances of abundant phyla and proteobacterial classes detected in DNA and RNA extracts. Data were subjected to z-transformation ([x − mean] [standard deviation] −1 ). After z-transformation, values around 0 approximate
    Figure Legend Snippet: z-transformed relative abundances of abundant phyla and proteobacterial classes detected in DNA and RNA extracts. Data were subjected to z-transformation ([x − mean] [standard deviation] −1 ). After z-transformation, values around 0 approximate

    Techniques Used: Transformation Assay, Standard Deviation

    Unconstrained principal-component analysis of z-transformed concentration, copy number, and quality values of DNA extracts (A), of RNA extracts (B), and of relative abundances of 16 abundant phyla and proteobacterial classes detected in DNA extracts (C)
    Figure Legend Snippet: Unconstrained principal-component analysis of z-transformed concentration, copy number, and quality values of DNA extracts (A), of RNA extracts (B), and of relative abundances of 16 abundant phyla and proteobacterial classes detected in DNA extracts (C)

    Techniques Used: Transformation Assay, Concentration Assay

    38) Product Images from "Infectious Lassa Virus, but Not Filoviruses, Is Restricted by BST-2/Tetherin ▿"

    Article Title: Infectious Lassa Virus, but Not Filoviruses, Is Restricted by BST-2/Tetherin ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00103-10

    Human BST-2 overexpression inhibits infectious LASV, but not ZEBOV, release and spread. (A) Human 293 cells stably expressing human BST-2 (FLP-BST-2), CAT (FLP-CAT), or an empty plasmid (FLP) were infected with LASV at the indicated MOIs. After 72 h, viral RNA was extracted from the media, and the viral copy number was determined by qRT-PCR. (B to D) Alternatively, cells were infected with ZEBOV (B), CPXV-GFP (C), or RVFV (D), fixed in formalin and stained for high-content quantitative image-based analysis with virus-specific antibodies 24 or 48 h (C and D) or 72 h (B) after infection. Error bars indicate standard deviations.
    Figure Legend Snippet: Human BST-2 overexpression inhibits infectious LASV, but not ZEBOV, release and spread. (A) Human 293 cells stably expressing human BST-2 (FLP-BST-2), CAT (FLP-CAT), or an empty plasmid (FLP) were infected with LASV at the indicated MOIs. After 72 h, viral RNA was extracted from the media, and the viral copy number was determined by qRT-PCR. (B to D) Alternatively, cells were infected with ZEBOV (B), CPXV-GFP (C), or RVFV (D), fixed in formalin and stained for high-content quantitative image-based analysis with virus-specific antibodies 24 or 48 h (C and D) or 72 h (B) after infection. Error bars indicate standard deviations.

    Techniques Used: Over Expression, Stable Transfection, Expressing, Plasmid Preparation, Infection, Quantitative RT-PCR, Staining

    BST-2 localization in relation to filoviral GP 1,2 . (A) Human 293 cells stably expressing human BST-2 were infected with ZEBOV. Cells were fixed in formalin 24 h later and stained with antibodies against BST-2 and ZEBOV GP 1,2 . (B and C) Alternatively, cells were infected with MARV for 24 h (B) or 48 h (C) and stained with antibodies against BST-2 and MARV GP 1,2 . Immunofluorescence of anti-filoviral GP 1,2 (green) and anti-BST-2 (red) in a single confocal plain is shown as indicated. Right columns show the merged images for triple staining of BST-2 (red), filoviral GP 1,2 (green), and the nucleus (blue), as well as bright-field images.
    Figure Legend Snippet: BST-2 localization in relation to filoviral GP 1,2 . (A) Human 293 cells stably expressing human BST-2 were infected with ZEBOV. Cells were fixed in formalin 24 h later and stained with antibodies against BST-2 and ZEBOV GP 1,2 . (B and C) Alternatively, cells were infected with MARV for 24 h (B) or 48 h (C) and stained with antibodies against BST-2 and MARV GP 1,2 . Immunofluorescence of anti-filoviral GP 1,2 (green) and anti-BST-2 (red) in a single confocal plain is shown as indicated. Right columns show the merged images for triple staining of BST-2 (red), filoviral GP 1,2 (green), and the nucleus (blue), as well as bright-field images.

    Techniques Used: Stable Transfection, Expressing, Infection, Staining, Immunofluorescence

    BST-2 inhibits budding of arenaviral, filoviral, and paramyxoviral VLPs. (A) Human 293T cells were cotransfected with plasmids encoding hemagglutinin (HA)-tagged matrix proteins of LASV, MACV, ZEBOV, MARV, or NiV, together with an empty plasmid, or plasmid encoding VPS4A-E228Q, human BST-2 (hBST-2), or murine BST-2 (mBST-2). Cell lysates and supernatants were harvested 48 h after transfection. VLPs in clarified medium were pelleted through a sucrose cushion, and HA-tagged matrix proteins in VLPs were analyzed by Western blotting. Numbers below each lane indicate values obtained with densitometric scanning using the ImageJ program (NIH). (B) Expression of the HA-tagged matrix proteins, actin, VPS4A-E228Q, hBST-2, or mBST-2 in cell lysates was determined by Western blotting. Shown is a representative Western blot from three independent experiments.
    Figure Legend Snippet: BST-2 inhibits budding of arenaviral, filoviral, and paramyxoviral VLPs. (A) Human 293T cells were cotransfected with plasmids encoding hemagglutinin (HA)-tagged matrix proteins of LASV, MACV, ZEBOV, MARV, or NiV, together with an empty plasmid, or plasmid encoding VPS4A-E228Q, human BST-2 (hBST-2), or murine BST-2 (mBST-2). Cell lysates and supernatants were harvested 48 h after transfection. VLPs in clarified medium were pelleted through a sucrose cushion, and HA-tagged matrix proteins in VLPs were analyzed by Western blotting. Numbers below each lane indicate values obtained with densitometric scanning using the ImageJ program (NIH). (B) Expression of the HA-tagged matrix proteins, actin, VPS4A-E228Q, hBST-2, or mBST-2 in cell lysates was determined by Western blotting. Shown is a representative Western blot from three independent experiments.

    Techniques Used: Plasmid Preparation, Transfection, Western Blot, Expressing

    Knockdown of human BST-2 expression enhances infectious LASV release, but not ZEBOV or MARV spread. (A) HeLa cells were transfected with siRNA targeting BST-2 or with a control siRNA. After 24, 48, or 72 h, cellular RNA was extracted, and the relative BST-2 expression levels were determined by qRT-PCR. BST-2 protein expression levels in cell lysates were also determined by Western blotting. (B) Cells were infected with LASV (MOI = 0.1) 24 h after transfection. Viral RNA was extracted from the medium 72 h later, and the viral copy number was determined by qRT-PCR. (C) Alternatively, cells were infected with ZEBOV-GFP (MOI = 10) or MARV (MOI = 3). Cells were fixed in formalin 72 h later and stained for high-content quantitative image-based analysis with virus-specific antibodies. Error bars indicate standard deviations.
    Figure Legend Snippet: Knockdown of human BST-2 expression enhances infectious LASV release, but not ZEBOV or MARV spread. (A) HeLa cells were transfected with siRNA targeting BST-2 or with a control siRNA. After 24, 48, or 72 h, cellular RNA was extracted, and the relative BST-2 expression levels were determined by qRT-PCR. BST-2 protein expression levels in cell lysates were also determined by Western blotting. (B) Cells were infected with LASV (MOI = 0.1) 24 h after transfection. Viral RNA was extracted from the medium 72 h later, and the viral copy number was determined by qRT-PCR. (C) Alternatively, cells were infected with ZEBOV-GFP (MOI = 10) or MARV (MOI = 3). Cells were fixed in formalin 72 h later and stained for high-content quantitative image-based analysis with virus-specific antibodies. Error bars indicate standard deviations.

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Infection, Staining

    39) Product Images from "Stormwater influences phytoplankton assemblages within the diverse, but impacted Sydney Harbour estuary"

    Article Title: Stormwater influences phytoplankton assemblages within the diverse, but impacted Sydney Harbour estuary

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0209857

    Distance-based redundancy analysis plot of the eukaryotic community structure (using 18S rRNA gene V9 region) for the Sydney Harbour estuary, during high and low rainfall periods. Samples are colour-coded based on clusters (assigned using hierarchical cluster analysis with SIMPROF test based on Bray-Curtis similarity) for the high rainfall period: a (light green), b (dark blue), c (red), d (dark green); and for the low rainfall period: e (orange), f (light blue), g (blue), h (grey). Symbols represent geographic region sampled: Parramatta River (*), Lane Cove River (●), Western Central Harbour (▼), Eastern Central Harbour (▲), Middle Harbour (■) and Marine/Harbour Heads (♦). TN–Total nitrogen; Si–Silicate.
    Figure Legend Snippet: Distance-based redundancy analysis plot of the eukaryotic community structure (using 18S rRNA gene V9 region) for the Sydney Harbour estuary, during high and low rainfall periods. Samples are colour-coded based on clusters (assigned using hierarchical cluster analysis with SIMPROF test based on Bray-Curtis similarity) for the high rainfall period: a (light green), b (dark blue), c (red), d (dark green); and for the low rainfall period: e (orange), f (light blue), g (blue), h (grey). Symbols represent geographic region sampled: Parramatta River (*), Lane Cove River (●), Western Central Harbour (▼), Eastern Central Harbour (▲), Middle Harbour (■) and Marine/Harbour Heads (♦). TN–Total nitrogen; Si–Silicate.

    Techniques Used: Western Blot

    Eukaryotic community profile, based on 18S rRNA gene V9 region, in Sydney Harbour estuary during high and low rainfall periods. a) Multi-dimensional scaling plot of the community composition during the two periods. Samples are colour-coded based on clusters (assigned using hierarchical cluster analysis with SIMPROF test based on Bray-Curtis similarity) from the high rainfall period: a (light green), b (dark blue), c (red), d (dark green); and from the low rainfall period: e (orange), f (light blue), g (blue), h (grey). Symbols represent geographic region sampled: Parramatta River (*), Lane Cove River (●), Western Central Harbour (▼), Eastern Central Harbour (▲), Middle Harbour (■) and Marine/Harbour Heads (♦). b) Relative abundance profile of the eukaryotic community during the sampling periods, at taxonomic level of family or lower. Each bar represents the cluster of sites, as above. The rarefied number of reads assigned to each lineage was averaged across sites of each cluster.
    Figure Legend Snippet: Eukaryotic community profile, based on 18S rRNA gene V9 region, in Sydney Harbour estuary during high and low rainfall periods. a) Multi-dimensional scaling plot of the community composition during the two periods. Samples are colour-coded based on clusters (assigned using hierarchical cluster analysis with SIMPROF test based on Bray-Curtis similarity) from the high rainfall period: a (light green), b (dark blue), c (red), d (dark green); and from the low rainfall period: e (orange), f (light blue), g (blue), h (grey). Symbols represent geographic region sampled: Parramatta River (*), Lane Cove River (●), Western Central Harbour (▼), Eastern Central Harbour (▲), Middle Harbour (■) and Marine/Harbour Heads (♦). b) Relative abundance profile of the eukaryotic community during the sampling periods, at taxonomic level of family or lower. Each bar represents the cluster of sites, as above. The rarefied number of reads assigned to each lineage was averaged across sites of each cluster.

    Techniques Used: Western Blot, Sampling

    40) Product Images from "Influence of Activin A Supplementation During Human Embryonic Stem Cell Derivation on Germ Cell Differentiation Potential"

    Article Title: Influence of Activin A Supplementation During Human Embryonic Stem Cell Derivation on Germ Cell Differentiation Potential

    Journal: Stem Cells and Development

    doi: 10.1089/scd.2013.0024

    Gene expression profiles of all hESC lines upon germ cell-directed differentiation as EBs in the presence of bone morphogenic protein 4 (BMP4) for early primordial germ cell (PGC) markers STELLA, cKIT, FRAGILIS (a–c) , late PGC marker VASA (d) , pluripotency markers OCT4, SOX2, NANOG (e–g) , and the differentiation marker SSEA1 (h) . Activin A-derived hESC lines UGent11-4-ActA and UGent12-3-ActA show significantly higher expression for STELLA and cKIT at day 0 and significant upregulation of VASA at day 7, when FRAGILIS is downregulated. For all hESC lines, pluripotency markers OCT4, SOX2, NANOG were significantly downregulated, and SSEA1 expression increased as differentiation proceeded from day 0 to day 14. STELLA, cKIT: UGent11-4-ActA (a′) compared to standard hESC lines at day 0 (a) , P ≤0.001; UGent12-3-ActA (b′) compared to standard hESC lines at day 0 (b) , P ≤0.001; FRAGILIS: Day 7 UGent12-3-ActA EBs (a′) compared to day 0 and day 3 EBs (a) , P
    Figure Legend Snippet: Gene expression profiles of all hESC lines upon germ cell-directed differentiation as EBs in the presence of bone morphogenic protein 4 (BMP4) for early primordial germ cell (PGC) markers STELLA, cKIT, FRAGILIS (a–c) , late PGC marker VASA (d) , pluripotency markers OCT4, SOX2, NANOG (e–g) , and the differentiation marker SSEA1 (h) . Activin A-derived hESC lines UGent11-4-ActA and UGent12-3-ActA show significantly higher expression for STELLA and cKIT at day 0 and significant upregulation of VASA at day 7, when FRAGILIS is downregulated. For all hESC lines, pluripotency markers OCT4, SOX2, NANOG were significantly downregulated, and SSEA1 expression increased as differentiation proceeded from day 0 to day 14. STELLA, cKIT: UGent11-4-ActA (a′) compared to standard hESC lines at day 0 (a) , P ≤0.001; UGent12-3-ActA (b′) compared to standard hESC lines at day 0 (b) , P ≤0.001; FRAGILIS: Day 7 UGent12-3-ActA EBs (a′) compared to day 0 and day 3 EBs (a) , P

    Techniques Used: Expressing, Pyrolysis Gas Chromatography, Marker, Derivative Assay

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    Concentration Assay:

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  • 99
    Thermo Fisher quant it picogreen dsdna assay kit
    DNA quantification in DT. ( A ) Nuclei were counted on H and E-stained slides of native tissue and DT and represented as the number of nuclei per µm 2 ; ( B ) Following tissue digestion, DNA was extracted and quantified using the Quant-iT™ <t>PicoGreen</t> ® <t>dsDNA</t> assay. The results are presented as means ± SD. *** p
    Quant It Picogreen Dsdna Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher quant it ribogreen assay kit
    Polyplexes with different corona chemistries have similar size, zeta potential, and cargo loading but varied stability against high salt concentrations. (A, B) Polyplex siRNA encapsulation efficiency and stability is highest at N + :P − 20. (A) <t>Ribogreen</t> assay reveals polyplex encapsulation plateaus by N + :P − ratio of 10. (B) Polyplexes retain higher stability after a 30 min incubation in 30% FBS at N + :P − 20 compared to N + :P − 10 ( p
    Quant It Ribogreen Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quant it ribogreen assay kit/product/Thermo Fisher
    Average 99 stars, based on 81 article reviews
    Price from $9.99 to $1999.99
    quant it ribogreen assay kit - by Bioz Stars, 2020-05
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    DNA quantification in DT. ( A ) Nuclei were counted on H and E-stained slides of native tissue and DT and represented as the number of nuclei per µm 2 ; ( B ) Following tissue digestion, DNA was extracted and quantified using the Quant-iT™ PicoGreen ® dsDNA assay. The results are presented as means ± SD. *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Development of a Cytocompatible Scaffold from Pig Immature Testicular Tissue Allowing Human Sertoli Cell Attachment, Proliferation and Functionality

    doi: 10.3390/ijms19010227

    Figure Lengend Snippet: DNA quantification in DT. ( A ) Nuclei were counted on H and E-stained slides of native tissue and DT and represented as the number of nuclei per µm 2 ; ( B ) Following tissue digestion, DNA was extracted and quantified using the Quant-iT™ PicoGreen ® dsDNA assay. The results are presented as means ± SD. *** p

    Article Snippet: For quantification, DNA was extracted using the PureLink™ genomic DNA mini kit (Thermo Fisher, Ghent, Belgium, K1820-01) and quantified using the Quant-iT™ PicoGreen® dsDNA assay kit (Thermo Fisher, P7589) following the manufacturer’s instructions.

    Techniques: Staining, Picogreen Assay

    DNA quantity per scaffold at 3 and 7 days, assessed by PicoGreen assay. This confirms the ability of all scaffold architectures to support primary kidney cell life. Analysis using a one-way ANOVA showed significant differences F(7,23) = 4.79, p = 0.002, post hoc Tukey analysis shows that was in regards to cryogenic scaffolds. Data presented as mean ± 95% confidence intervals, circles denote individual data points

    Journal: Tissue Engineering and Regenerative Medicine

    Article Title: A Non-woven Path: Electrospun Poly(lactic acid) Scaffolds for Kidney Tissue Engineering

    doi: 10.1007/s13770-017-0107-5

    Figure Lengend Snippet: DNA quantity per scaffold at 3 and 7 days, assessed by PicoGreen assay. This confirms the ability of all scaffold architectures to support primary kidney cell life. Analysis using a one-way ANOVA showed significant differences F(7,23) = 4.79, p = 0.002, post hoc Tukey analysis shows that was in regards to cryogenic scaffolds. Data presented as mean ± 95% confidence intervals, circles denote individual data points

    Article Snippet: Total DNA content of the samples was calculated using a Quant-iT™ PicoGreen® assay kit (ThermoFisher, UK) as per the manufacturers’ instructions.

    Techniques: Picogreen Assay

    Polyplexes with different corona chemistries have similar size, zeta potential, and cargo loading but varied stability against high salt concentrations. (A, B) Polyplex siRNA encapsulation efficiency and stability is highest at N + :P − 20. (A) Ribogreen assay reveals polyplex encapsulation plateaus by N + :P − ratio of 10. (B) Polyplexes retain higher stability after a 30 min incubation in 30% FBS at N + :P − 20 compared to N + :P − 10 ( p

    Journal: ACS nano

    Article Title: Zwitterionic Nanocarrier Surface Chemistry Improves siRNA Tumor Delivery and Silencing Activity Relative to Polyethylene Glycol

    doi: 10.1021/acsnano.7b01110

    Figure Lengend Snippet: Polyplexes with different corona chemistries have similar size, zeta potential, and cargo loading but varied stability against high salt concentrations. (A, B) Polyplex siRNA encapsulation efficiency and stability is highest at N + :P − 20. (A) Ribogreen assay reveals polyplex encapsulation plateaus by N + :P − ratio of 10. (B) Polyplexes retain higher stability after a 30 min incubation in 30% FBS at N + :P − 20 compared to N + :P − 10 ( p

    Article Snippet: Polyplex encapsulation efficiency at various N+ :P− ratios was evaluated using a Quant-iT Ribogreen assay kit (ThermoFisher, USA).

    Techniques: Incubation

    Validation of MVP. Transfected cells are expressing MVP-biotin and un-transfected cells were used as negative control. (A) Western blot analysis for the detection of MVP in HEK293F cell lysate and exosomal protein extracts shows that plasmid (MVP-biotin) was successfully expressed in HEK293F cells and was partitioned into exosomes isolated from these transfected cells. ( B ) Protein samples from different stages of MVP purification were separated by SDS-PAGE and the protein bands were detected by Comassie staining. After the pull down assay, the presence of MVP within exosomes was confirmed, i.e. MVP in exosomes from biotinylated MVP-transfected cells PD (pull-down lane), whereas it was absent in exosomes from untransfected cells. The position of the MVP-biotin band is indicated by squares. A representative experiment out of 3 is shown. ( C ) Total amount of RNAs coupled to MVP were extracted and quantified by Quant-iT™ RiboGreen® RNA Assay Kit (ThermoFisher Scientific). Captured RNA was expressed as a percentage of RNA eluted from the beads after the pull-down respect to the total RNA incubated with the beads. Exosomal-MVP was coupled with RNAs and the quantification of RNAs that had co-eluted with MVP showed that the amount of RNA present in the MVP elute (i.e. from MVP-RNA complex) was significantly higher than that from the untransfected control. The experiment was performed in 3-replicates and the graph shows the range of values and median (grey). Average and standard error of three independent experiments are shown. Samples: W = proteins from different wash steps during the MVP purification, and PD = pull-down proteins (MVP) after elution, I = input, and UB = unbound after RBP capture.

    Journal: PLoS ONE

    Article Title: Identification of RNA-binding proteins in exosomes capable of interacting with different types of RNA: RBP-facilitated transport of RNAs into exosomes

    doi: 10.1371/journal.pone.0195969

    Figure Lengend Snippet: Validation of MVP. Transfected cells are expressing MVP-biotin and un-transfected cells were used as negative control. (A) Western blot analysis for the detection of MVP in HEK293F cell lysate and exosomal protein extracts shows that plasmid (MVP-biotin) was successfully expressed in HEK293F cells and was partitioned into exosomes isolated from these transfected cells. ( B ) Protein samples from different stages of MVP purification were separated by SDS-PAGE and the protein bands were detected by Comassie staining. After the pull down assay, the presence of MVP within exosomes was confirmed, i.e. MVP in exosomes from biotinylated MVP-transfected cells PD (pull-down lane), whereas it was absent in exosomes from untransfected cells. The position of the MVP-biotin band is indicated by squares. A representative experiment out of 3 is shown. ( C ) Total amount of RNAs coupled to MVP were extracted and quantified by Quant-iT™ RiboGreen® RNA Assay Kit (ThermoFisher Scientific). Captured RNA was expressed as a percentage of RNA eluted from the beads after the pull-down respect to the total RNA incubated with the beads. Exosomal-MVP was coupled with RNAs and the quantification of RNAs that had co-eluted with MVP showed that the amount of RNA present in the MVP elute (i.e. from MVP-RNA complex) was significantly higher than that from the untransfected control. The experiment was performed in 3-replicates and the graph shows the range of values and median (grey). Average and standard error of three independent experiments are shown. Samples: W = proteins from different wash steps during the MVP purification, and PD = pull-down proteins (MVP) after elution, I = input, and UB = unbound after RBP capture.

    Article Snippet: RNA was quantified by Quant-iT™ RiboGreen® RNA Assay Kit (ThermoFisher Scientific).

    Techniques: Transfection, Expressing, Negative Control, Western Blot, Plasmid Preparation, Isolation, Purification, SDS Page, Staining, Pull Down Assay, Incubation