rnase  (Thermo Fisher)


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    Name:
    Ribonuclease H
    Description:
    Ribonuclease H RNase H is an endoribonuclease that specifically degrades the RNA strand of an RNA DNA hybrid to produce 5 phosphate terminated oligoribonucleotides and single stranded DNA Applications Removal of mRNA during second strand cDNA synthesis Removal of poly A sequences from mRNA in the presence of oligo dT Oligodeoxyribonucleotide directed cleavage of RNA Source Purified from E coli expressing the E coli RNase H gene on a plasmid Performance and quality testing Ribonuclease nonspecific endodeoxyribonuclease 3 and 5 exodeoxyribonuclease Unit definition One unit hydrolyzes 1 nmol of RNA in 3H labeled poly A poly dT to acid soluble material in 20 min at 37°C Unit reaction conditions 20 mM Tris HCl pH 7 5 0 1 M KCl 10 mM MgCl2 0 1 mM DTT 5 w v sucrose 0 5 nmol 3H labeled poly A poly dT and enzyme in 50 µL for 20 min at 37°C
    Catalog Number:
    18021014
    Price:
    None
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher rnase
    <t>U937</t> cell-derived extracellular vesicles (EV) incorporate Poly(I:C) and may protect Poly(I:C) from degradation with <t>RNase</t> III. (A) The presence of Fluorescein Poly(I:C) in U937-derived EV. SSC-H/FSC-H and SSC-H/FL1 profiles of EV with percentage of gated events, as measured by flow cytometry. Shown are a representative sample of n = 3 biological replicates and quantification of flow cytometry data as percentage of Fluorescein Poly(I:C)-positive EV. Control EV (Con EV)-derived from untreated U937 cells. (B) Detection of soluble and vesicular Fluorescein Poly(I:C) digested or not with RNAse III. SSC-H/FSC-H profiles and FL1 histograms of EV and Poly(I:C), as measured by flow cytometry. Shown is one of n = 3–4 biological replicates for EV and one of n = 2 replicates for soluble Fluorescein Poly(I:C). Quantification of changes in median fluorescence intensities (MFI) of vesicular and soluble Fluorescein Poly(I:C) in the presence and absence of RNAse III. (C) Association of control EV (Con EV) with Poly(I:C) or Rhodamine Poly(I:C) in the presence or absence of RNase III, as measured by flow cytometry, shown is one from n = 3 biological replicates. Quantification of MFI changes in Rhodamine Poly(I:C) EV in the presence of RNAse III. Statistics: (A,B) one-way ANOVA with Tukey’s multiple comparisons test, (C) two-tailed paired t -test, ns, not significant.
    Ribonuclease H RNase H is an endoribonuclease that specifically degrades the RNA strand of an RNA DNA hybrid to produce 5 phosphate terminated oligoribonucleotides and single stranded DNA Applications Removal of mRNA during second strand cDNA synthesis Removal of poly A sequences from mRNA in the presence of oligo dT Oligodeoxyribonucleotide directed cleavage of RNA Source Purified from E coli expressing the E coli RNase H gene on a plasmid Performance and quality testing Ribonuclease nonspecific endodeoxyribonuclease 3 and 5 exodeoxyribonuclease Unit definition One unit hydrolyzes 1 nmol of RNA in 3H labeled poly A poly dT to acid soluble material in 20 min at 37°C Unit reaction conditions 20 mM Tris HCl pH 7 5 0 1 M KCl 10 mM MgCl2 0 1 mM DTT 5 w v sucrose 0 5 nmol 3H labeled poly A poly dT and enzyme in 50 µL for 20 min at 37°C
    https://www.bioz.com/result/rnase/product/Thermo Fisher
    Average 99 stars, based on 431 article reviews
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    rnase - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "TLR3 Ligand Poly(I:C) Exerts Distinct Actions in Synovial Fibroblasts When Delivered by Extracellular Vesicles"

    Article Title: TLR3 Ligand Poly(I:C) Exerts Distinct Actions in Synovial Fibroblasts When Delivered by Extracellular Vesicles

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00028

    U937 cell-derived extracellular vesicles (EV) incorporate Poly(I:C) and may protect Poly(I:C) from degradation with RNase III. (A) The presence of Fluorescein Poly(I:C) in U937-derived EV. SSC-H/FSC-H and SSC-H/FL1 profiles of EV with percentage of gated events, as measured by flow cytometry. Shown are a representative sample of n = 3 biological replicates and quantification of flow cytometry data as percentage of Fluorescein Poly(I:C)-positive EV. Control EV (Con EV)-derived from untreated U937 cells. (B) Detection of soluble and vesicular Fluorescein Poly(I:C) digested or not with RNAse III. SSC-H/FSC-H profiles and FL1 histograms of EV and Poly(I:C), as measured by flow cytometry. Shown is one of n = 3–4 biological replicates for EV and one of n = 2 replicates for soluble Fluorescein Poly(I:C). Quantification of changes in median fluorescence intensities (MFI) of vesicular and soluble Fluorescein Poly(I:C) in the presence and absence of RNAse III. (C) Association of control EV (Con EV) with Poly(I:C) or Rhodamine Poly(I:C) in the presence or absence of RNase III, as measured by flow cytometry, shown is one from n = 3 biological replicates. Quantification of MFI changes in Rhodamine Poly(I:C) EV in the presence of RNAse III. Statistics: (A,B) one-way ANOVA with Tukey’s multiple comparisons test, (C) two-tailed paired t -test, ns, not significant.
    Figure Legend Snippet: U937 cell-derived extracellular vesicles (EV) incorporate Poly(I:C) and may protect Poly(I:C) from degradation with RNase III. (A) The presence of Fluorescein Poly(I:C) in U937-derived EV. SSC-H/FSC-H and SSC-H/FL1 profiles of EV with percentage of gated events, as measured by flow cytometry. Shown are a representative sample of n = 3 biological replicates and quantification of flow cytometry data as percentage of Fluorescein Poly(I:C)-positive EV. Control EV (Con EV)-derived from untreated U937 cells. (B) Detection of soluble and vesicular Fluorescein Poly(I:C) digested or not with RNAse III. SSC-H/FSC-H profiles and FL1 histograms of EV and Poly(I:C), as measured by flow cytometry. Shown is one of n = 3–4 biological replicates for EV and one of n = 2 replicates for soluble Fluorescein Poly(I:C). Quantification of changes in median fluorescence intensities (MFI) of vesicular and soluble Fluorescein Poly(I:C) in the presence and absence of RNAse III. (C) Association of control EV (Con EV) with Poly(I:C) or Rhodamine Poly(I:C) in the presence or absence of RNase III, as measured by flow cytometry, shown is one from n = 3 biological replicates. Quantification of MFI changes in Rhodamine Poly(I:C) EV in the presence of RNAse III. Statistics: (A,B) one-way ANOVA with Tukey’s multiple comparisons test, (C) two-tailed paired t -test, ns, not significant.

    Techniques Used: Derivative Assay, Flow Cytometry, Cytometry, Fluorescence, Two Tailed Test

    2) Product Images from "Bone Marrow-Derived Mesenchymal Stem Cells Repaired but Did Not Prevent Gentamicin-Induced Acute Kidney Injury through Paracrine Effects in Rats"

    Article Title: Bone Marrow-Derived Mesenchymal Stem Cells Repaired but Did Not Prevent Gentamicin-Induced Acute Kidney Injury through Paracrine Effects in Rats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044092

    (A) Transmission Electron Microscopy of the exossomes-like microvesicles extracted from the BMSC conditioned medium. (B) RT-PCR for 18S RNA of exosome extracted from BMSC culture medium (EXO) untreated and treated with RNase (RNase+EXO). (C) The light micrographs of the kidney sections stained with hematoxylin eosin. (D) A graphic representation showing the quantitative analysis of the micrographs. The normal histology of the kidney tissue from control rats (CTL 15 ) and rats treated with G for 15 days (G 15 ). Some groups presented with massive tubular necrosis and unstained nuclei (G 15 , G 15 +RNAse+EXO 10 ), while other groups presented with intensely stained nuclei (G 15 +EXO 10 ). Data are expressed as the mean ± S.E.M. (* p
    Figure Legend Snippet: (A) Transmission Electron Microscopy of the exossomes-like microvesicles extracted from the BMSC conditioned medium. (B) RT-PCR for 18S RNA of exosome extracted from BMSC culture medium (EXO) untreated and treated with RNase (RNase+EXO). (C) The light micrographs of the kidney sections stained with hematoxylin eosin. (D) A graphic representation showing the quantitative analysis of the micrographs. The normal histology of the kidney tissue from control rats (CTL 15 ) and rats treated with G for 15 days (G 15 ). Some groups presented with massive tubular necrosis and unstained nuclei (G 15 , G 15 +RNAse+EXO 10 ), while other groups presented with intensely stained nuclei (G 15 +EXO 10 ). Data are expressed as the mean ± S.E.M. (* p

    Techniques Used: Transmission Assay, Electron Microscopy, Reverse Transcription Polymerase Chain Reaction, Staining, CTL Assay

    The schematic of the experiment protocol. In the control group (CTL), the rats were treated with daily i.p. injections of a vehicle (water), while the G groups received gentamicin (40 mg/Kg BW) continuously for 10, 11, 12, 15 or 20 days. For the prevention groups, the rats received BMSC 1×106 i.v. injections 24 hours before (G 10 +BMSC −1 ) or in the 5th day (G10d+BMSC5) of a 10-day treatment with G. For the treated groups, the rats received BMSC 1×106 i.v. injections on the 10th day of G treatment and continued receiving G for an additional 1 (G 11 +BMSC 10 ), 2 (G 12 +BMSC 10 ), 5 (G 15 +BMSC 10 ) or 10 (G 20 +BMSC 10 ) days. For the conditioned media protocol, the rats received G (40 mg/Kg/BW, i.p., daily) or water (CTL) for 15 or 20 days, and on the 10th day of the G treatment, the animals received 500 µl of a single dose of CM. In some experiments, the BMSCs were cultured for 12 h with trypsin and a concentration of 100 ug/ml (CM+TPS, 100 µg/ml) or RNase A at a concentration of 40 ug/ml, 280 Units (CM+RNAse) in DMEM without FBS.
    Figure Legend Snippet: The schematic of the experiment protocol. In the control group (CTL), the rats were treated with daily i.p. injections of a vehicle (water), while the G groups received gentamicin (40 mg/Kg BW) continuously for 10, 11, 12, 15 or 20 days. For the prevention groups, the rats received BMSC 1×106 i.v. injections 24 hours before (G 10 +BMSC −1 ) or in the 5th day (G10d+BMSC5) of a 10-day treatment with G. For the treated groups, the rats received BMSC 1×106 i.v. injections on the 10th day of G treatment and continued receiving G for an additional 1 (G 11 +BMSC 10 ), 2 (G 12 +BMSC 10 ), 5 (G 15 +BMSC 10 ) or 10 (G 20 +BMSC 10 ) days. For the conditioned media protocol, the rats received G (40 mg/Kg/BW, i.p., daily) or water (CTL) for 15 or 20 days, and on the 10th day of the G treatment, the animals received 500 µl of a single dose of CM. In some experiments, the BMSCs were cultured for 12 h with trypsin and a concentration of 100 ug/ml (CM+TPS, 100 µg/ml) or RNase A at a concentration of 40 ug/ml, 280 Units (CM+RNAse) in DMEM without FBS.

    Techniques Used: CTL Assay, Cell Culture, Concentration Assay

    (A) RT-PCR for 18S RNA in culture medium (CM) from BMSC controls and culture medium treated with RNase (CM+RNAse). (B) BMSC viability after treatment with Trypsin and RNase by Trypan blue extrusion. (C) The light micrographs of the kidney sections stained with hematoxylin eosin and with immunochemistry for caspase 3 and KI67. (D) A graphic representation showing the quantitative analysis of the control rat micrographs (CTL 15 ; CTL 15 +BMSC 10 ). The histology of the kidney tissue from rats treated with G for 15 days (G 15 ). Some groups presented with massive tubular necrosis and unstained nuclei (G 15 , G 15 +RNAse+CM 15 ), while other groups presented with intensely stained nuclei (G 15 +BMSC 10 , G 15 +CM 10 ). Data are expressed as the mean ± S.E.M. (* p
    Figure Legend Snippet: (A) RT-PCR for 18S RNA in culture medium (CM) from BMSC controls and culture medium treated with RNase (CM+RNAse). (B) BMSC viability after treatment with Trypsin and RNase by Trypan blue extrusion. (C) The light micrographs of the kidney sections stained with hematoxylin eosin and with immunochemistry for caspase 3 and KI67. (D) A graphic representation showing the quantitative analysis of the control rat micrographs (CTL 15 ; CTL 15 +BMSC 10 ). The histology of the kidney tissue from rats treated with G for 15 days (G 15 ). Some groups presented with massive tubular necrosis and unstained nuclei (G 15 , G 15 +RNAse+CM 15 ), while other groups presented with intensely stained nuclei (G 15 +BMSC 10 , G 15 +CM 10 ). Data are expressed as the mean ± S.E.M. (* p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Staining, CTL Assay

    3) Product Images from "Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury"

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033115

    RNase treatment does not modify MV size, but reduces RNA content of MVs. A) Representative MV size analyses by direct measurement with NTA, showing no difference among MVs treated or not with RNase. B) Representative Bioanalyzer profile, showing the size distribution of total RNA extracted from MVs treated or not with RNAse. The first peak (left side of each panel) represents an internal standard. The two peaks in Sample 1 (black arrows) represent 18 S (left) and 28 S (right) ribosomal RNA, only partially detectable in MVs. The red arrows showed the reduction of 18 and 28 S fragment inside RNAse-treated MVs. C) Histogram showing the expression level of SUMO-1 , POLR2 and Act B transcripts in MVs treated or not with RNase, express as 2 -δCt , as described in material and methods.
    Figure Legend Snippet: RNase treatment does not modify MV size, but reduces RNA content of MVs. A) Representative MV size analyses by direct measurement with NTA, showing no difference among MVs treated or not with RNase. B) Representative Bioanalyzer profile, showing the size distribution of total RNA extracted from MVs treated or not with RNAse. The first peak (left side of each panel) represents an internal standard. The two peaks in Sample 1 (black arrows) represent 18 S (left) and 28 S (right) ribosomal RNA, only partially detectable in MVs. The red arrows showed the reduction of 18 and 28 S fragment inside RNAse-treated MVs. C) Histogram showing the expression level of SUMO-1 , POLR2 and Act B transcripts in MVs treated or not with RNase, express as 2 -δCt , as described in material and methods.

    Techniques Used: Expressing, Activated Clotting Time Assay

    MV infusion protects SCID mice with cisplatin-induced AKI from tubular injury. Representative micrographs of renal histology of healthy SCID mice and of SCID mice treated with cisplatin and injected with vehicle alone or with MV pre-treated with RNase or with different regiments of MVs (single or multiple injections) and sacrificed at different time points (day 4, 14 and 21). Original Magnification: ×200. The typical aspect of intra-tubular casts, tubular necrosis and tubular atrophy are respectively shown by asterisks, arrows and head arrows.
    Figure Legend Snippet: MV infusion protects SCID mice with cisplatin-induced AKI from tubular injury. Representative micrographs of renal histology of healthy SCID mice and of SCID mice treated with cisplatin and injected with vehicle alone or with MV pre-treated with RNase or with different regiments of MVs (single or multiple injections) and sacrificed at different time points (day 4, 14 and 21). Original Magnification: ×200. The typical aspect of intra-tubular casts, tubular necrosis and tubular atrophy are respectively shown by asterisks, arrows and head arrows.

    Techniques Used: Mouse Assay, Injection

    4) Product Images from "The Human-Associated Archaeon Methanosphaera stadtmanae Is Recognized through Its RNA and Induces TLR8-Dependent NLRP3 Inflammasome Activation"

    Article Title: The Human-Associated Archaeon Methanosphaera stadtmanae Is Recognized through Its RNA and Induces TLR8-Dependent NLRP3 Inflammasome Activation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01535

    Methanosphaera stadtmanae and its RNA inducing an immune response with antiviral characteristics. (A,B) The expression of genes encoding for IFN-α14, IFN-β, and IFN-λ1 in moDCs (A) and PBMCs (B) after stimulation with M. stadtmanae for 3, 6, and 12 h was measured by qRT-PCR. The log2 ratios of all three genes to the reference gene HPRT are presented. The data from at least three different donors are shown as the mean ± SEM ( n = 3–4). ns, not significant; * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001 (all compared with unstimulated control group; repeated measures one-way ANOVA with Dunnett’s post hoc test). (C) Confocal microscopy of cellular location of NF-κB p65, IRF1, and IRF5 (green) in moDCs after stimulation with M. stadtmanae for 4 h by immunolabelling. Nuclei were counterstained with Hoechst 33342 (blue). Scale bars: 10 µM. The images shown are representative examples from one of three independent experiments ( n = 3). (D,E) ELISA of TNF-α and IL-1β in the supernatants of stimulated moDCs (D) or PBMCs (E) after 18 h. Cells were either untreated, treated with 10 7 cells of M. stadtmanae , or with 5 µg/mL of total RNA from M. stadtmanae . RNA was complexed to DOTAP and pre-treated for 30 min at 37°C with RNase A where indicated. (F,G) ELISA of TNF-α and IL-1β in the supernatants of moDCs (F) or PBMCs (G) stimulated for 18 h with 10 7 cells of M. stadtmanae or 2.5 µg/mL of purified rRNAs (complexed to DOTAP). In (D–G) , the data shown are the mean ± SEM of at least four different donors ( n = 4–7). ns, not significant; * P ≤ 0.05 and ** P ≤ 0.01 (one-way ANOVA with Tukey post hoc test; in (D,E) all compared with unstimulated control group and in (F,G) the rRNA fractions are compared with each other). moDCs, monocyte-derived dendritic cells; PBMCs, peripheral blood mononuclear cells; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; ANOVA, analysis of variance; TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1 beta.
    Figure Legend Snippet: Methanosphaera stadtmanae and its RNA inducing an immune response with antiviral characteristics. (A,B) The expression of genes encoding for IFN-α14, IFN-β, and IFN-λ1 in moDCs (A) and PBMCs (B) after stimulation with M. stadtmanae for 3, 6, and 12 h was measured by qRT-PCR. The log2 ratios of all three genes to the reference gene HPRT are presented. The data from at least three different donors are shown as the mean ± SEM ( n = 3–4). ns, not significant; * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001 (all compared with unstimulated control group; repeated measures one-way ANOVA with Dunnett’s post hoc test). (C) Confocal microscopy of cellular location of NF-κB p65, IRF1, and IRF5 (green) in moDCs after stimulation with M. stadtmanae for 4 h by immunolabelling. Nuclei were counterstained with Hoechst 33342 (blue). Scale bars: 10 µM. The images shown are representative examples from one of three independent experiments ( n = 3). (D,E) ELISA of TNF-α and IL-1β in the supernatants of stimulated moDCs (D) or PBMCs (E) after 18 h. Cells were either untreated, treated with 10 7 cells of M. stadtmanae , or with 5 µg/mL of total RNA from M. stadtmanae . RNA was complexed to DOTAP and pre-treated for 30 min at 37°C with RNase A where indicated. (F,G) ELISA of TNF-α and IL-1β in the supernatants of moDCs (F) or PBMCs (G) stimulated for 18 h with 10 7 cells of M. stadtmanae or 2.5 µg/mL of purified rRNAs (complexed to DOTAP). In (D–G) , the data shown are the mean ± SEM of at least four different donors ( n = 4–7). ns, not significant; * P ≤ 0.05 and ** P ≤ 0.01 (one-way ANOVA with Tukey post hoc test; in (D,E) all compared with unstimulated control group and in (F,G) the rRNA fractions are compared with each other). moDCs, monocyte-derived dendritic cells; PBMCs, peripheral blood mononuclear cells; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; ANOVA, analysis of variance; TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1 beta.

    Techniques Used: Expressing, Quantitative RT-PCR, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Purification, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

    5) Product Images from "Base Pairing Interaction between 5?- and 3?-UTRs Controls icaR mRNA Translation in Staphylococcus aureus"

    Article Title: Base Pairing Interaction between 5?- and 3?-UTRs Controls icaR mRNA Translation in Staphylococcus aureus

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004001

    Deletion of rnc gene, which encodes the double stranded endoribonuclease RNase III, affects icaR mRNA stability and IcaR protein levels. (A) Half-life measurement of icaR wild type and Δ3′-UTR mRNAs constitutively expressed in the wild type and Δ rnc mutant strains. These strains were grown in TSB-gluc at 37°C until exponential phase (OD 600 nm = 0.8) and then rifampicin (300 µg/ml) was added. Samples for RNA extraction were taken at the indicated time points (min). The experiment was repeated three times and representative images are shown. (B) Levels of icaR mRNA were quantified by densitometry of Northern blot autoradiographies using ImageJ ( http://rsbweb.nih.gov/ij/ ). Each of mRNA levels was relativized to mRNA levels at time 0. The logarithm values of relative mRNA levels were subjected to linear regression analysis and plotted as a function of time. Error bars indicate the standard deviation of mRNA levels from three independent experiments. Dashed lines indicate the time at which 50% of mRNA remained. The half-life of mRNAs is shown above of X-axis. (C) Representative Western blot showing IcaR protein levels in different mutant strains constitutively expressing the 3XFLAG tagged IcaR from the P blaZ promoter. Tagged IcaR protein was detected with commercial anti-3XFLAG antibodies. On the left, a Coomassie stained gel portion is shown as loading control. rnc , double-stranded endoribonuclease RNase III; pnp , polynucleotide phosphorylase PNPase; yqfR , (SAOUHSC_01659), ATP-dependent RNA helicase containing a DEAD box domain; hfq , RNA chaperone, host factor-1 protein.
    Figure Legend Snippet: Deletion of rnc gene, which encodes the double stranded endoribonuclease RNase III, affects icaR mRNA stability and IcaR protein levels. (A) Half-life measurement of icaR wild type and Δ3′-UTR mRNAs constitutively expressed in the wild type and Δ rnc mutant strains. These strains were grown in TSB-gluc at 37°C until exponential phase (OD 600 nm = 0.8) and then rifampicin (300 µg/ml) was added. Samples for RNA extraction were taken at the indicated time points (min). The experiment was repeated three times and representative images are shown. (B) Levels of icaR mRNA were quantified by densitometry of Northern blot autoradiographies using ImageJ ( http://rsbweb.nih.gov/ij/ ). Each of mRNA levels was relativized to mRNA levels at time 0. The logarithm values of relative mRNA levels were subjected to linear regression analysis and plotted as a function of time. Error bars indicate the standard deviation of mRNA levels from three independent experiments. Dashed lines indicate the time at which 50% of mRNA remained. The half-life of mRNAs is shown above of X-axis. (C) Representative Western blot showing IcaR protein levels in different mutant strains constitutively expressing the 3XFLAG tagged IcaR from the P blaZ promoter. Tagged IcaR protein was detected with commercial anti-3XFLAG antibodies. On the left, a Coomassie stained gel portion is shown as loading control. rnc , double-stranded endoribonuclease RNase III; pnp , polynucleotide phosphorylase PNPase; yqfR , (SAOUHSC_01659), ATP-dependent RNA helicase containing a DEAD box domain; hfq , RNA chaperone, host factor-1 protein.

    Techniques Used: Mutagenesis, RNA Extraction, Northern Blot, Standard Deviation, Western Blot, Expressing, Staining

    In vitro and in vivo RNase III-mediated processing of the double stranded region generated by icaR 5′-3′-UTRs interaction. (A) In vitro RNase III activity assay. A 32 P-labelled 5′-UTR fragment was incubated with purified recombinant S. aureus RNase III during different times in the absence or presence of either the 3′-UTR fragment or the substituted 3′-UTR fragment. The two RNA bands that are generated by the presence of the wild type 3′-UTR are indicated with arrows. (B) Schematic representation showing in vivo mRACE results. Mapping of icaR mRNA fragments naturally generated in vivo was carried out with circularized RNAs and two outward primers (RT and PCR) that pair next to the transcriptional terminator. Black and white triangles indicate in vivo processing sites identified in the icaR mRNA wild type and the icaR mRNA with the UCCCC substitution respectively.
    Figure Legend Snippet: In vitro and in vivo RNase III-mediated processing of the double stranded region generated by icaR 5′-3′-UTRs interaction. (A) In vitro RNase III activity assay. A 32 P-labelled 5′-UTR fragment was incubated with purified recombinant S. aureus RNase III during different times in the absence or presence of either the 3′-UTR fragment or the substituted 3′-UTR fragment. The two RNA bands that are generated by the presence of the wild type 3′-UTR are indicated with arrows. (B) Schematic representation showing in vivo mRACE results. Mapping of icaR mRNA fragments naturally generated in vivo was carried out with circularized RNAs and two outward primers (RT and PCR) that pair next to the transcriptional terminator. Black and white triangles indicate in vivo processing sites identified in the icaR mRNA wild type and the icaR mRNA with the UCCCC substitution respectively.

    Techniques Used: In Vitro, In Vivo, Generated, Activity Assay, Incubation, Purification, Recombinant, Polymerase Chain Reaction

    Modulation of IcaR expression by 5′-3′-UTRs interaction. A model of the potential post-transcriptional regulatory mechanism controlling IcaR expression mediated by the 3′-UTR interaction with the Shine-Dalgarno region is shown. Once icaR gene is transcribed, the 3′-UTR interacts either in trans or cis with the 5′-UTR through the anti-SD UCCCCUG motif. This interaction has two main consequences: i) it interferes with ribosome access to the SD region to inhibit the formation of the translational initiation complex and ii) it promotes RNase III-dependent mRNA decay. In consequence, IcaR repressor is less expressed and thus icaADBC transcription occurs, favouring PIA-PNAG biosynthesis and biofilm development. When the interaction between icaR 3′- and 5′-UTR regions does not happen, ribosome binds the SD and proceeds with IcaR protein translation. The resulting IcaR protein binds to icaADBC operon promoter inhibiting its transcription and consequently biofilm formation.
    Figure Legend Snippet: Modulation of IcaR expression by 5′-3′-UTRs interaction. A model of the potential post-transcriptional regulatory mechanism controlling IcaR expression mediated by the 3′-UTR interaction with the Shine-Dalgarno region is shown. Once icaR gene is transcribed, the 3′-UTR interacts either in trans or cis with the 5′-UTR through the anti-SD UCCCCUG motif. This interaction has two main consequences: i) it interferes with ribosome access to the SD region to inhibit the formation of the translational initiation complex and ii) it promotes RNase III-dependent mRNA decay. In consequence, IcaR repressor is less expressed and thus icaADBC transcription occurs, favouring PIA-PNAG biosynthesis and biofilm development. When the interaction between icaR 3′- and 5′-UTR regions does not happen, ribosome binds the SD and proceeds with IcaR protein translation. The resulting IcaR protein binds to icaADBC operon promoter inhibiting its transcription and consequently biofilm formation.

    Techniques Used: Expressing

    6) Product Images from "A Complex Small RNA Repertoire Is Generated by a Plant/Fungal-Like Machinery and Effected by a Metazoan-Like Argonaute in the Single-Cell Human Parasite Toxoplasma gondii"

    Article Title: A Complex Small RNA Repertoire Is Generated by a Plant/Fungal-Like Machinery and Effected by a Metazoan-Like Argonaute in the Single-Cell Human Parasite Toxoplasma gondii

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000920

    Domain organization and phylogenetic analysis of Argonaute and PIWI proteins. (A) Evolutionary relationships between Argonaute and Piwi proteins from the following species: AT: Arabidopsis thaliana , GL: Giardia lambdia , SP: Schizosaccharomyces pombe , DM: Drosophila melanogaster , HS: Homo sapiens , CE: Caenorhabditis elegans , CR: Chlamydomonas reinhardtii , NC: Neospora caninum , TT: Tetrahymena thermophila , TG: Toxoplasma gondii . Tg -AGO (accession number: GU046561) is pointed out by a pink arrow. Argonaute (in pink), Piwi (in blue) and group 3 (in red) branch family are represented as shaded boxes. Numbers above the nodes indicate neighbor-joining bootstrap percentages. (B) Schematic depiction of Toxoplasma Tg- AGO and Arabidopsis At -AGO1 (GeneID: 841262) domain according to archaeon Aquifex aeolicus X-ray crystal structure. The amino-terminal domain (grey or blue) is linked to the PAZ domain (red). The MID domain (green) connects the PAZ domain with the PIWI domain (pink) at the carboxy-terminal end of the protein. In the PAZ domain, residues important for binding of small RNA 3′ ends are indicated. In the MID domain, the residues required for 5′ end binding to small RNAs and binding to the 7-methylguanine (m7G) cap of target mRNAs are shown. The conserved residues for RNase H cleavage activity (DDH) in the PIWI domain are also shown. RGG indicates the domain of Tg- AGO containing arginine-glycine-glycine repeats. Poly Q, polyglutamine-containing domain in Arabidopsis Ago1. (C) Sequence alignment of conserved 5′–phosphate binding residues (MID domain) across various species of Argonaute proteins. Prefix Hs, Homo sapiens ; Dm, Drosophila melanogaster ; Aa, Aquifex aeolicus . Conserved residues are shaded in red.
    Figure Legend Snippet: Domain organization and phylogenetic analysis of Argonaute and PIWI proteins. (A) Evolutionary relationships between Argonaute and Piwi proteins from the following species: AT: Arabidopsis thaliana , GL: Giardia lambdia , SP: Schizosaccharomyces pombe , DM: Drosophila melanogaster , HS: Homo sapiens , CE: Caenorhabditis elegans , CR: Chlamydomonas reinhardtii , NC: Neospora caninum , TT: Tetrahymena thermophila , TG: Toxoplasma gondii . Tg -AGO (accession number: GU046561) is pointed out by a pink arrow. Argonaute (in pink), Piwi (in blue) and group 3 (in red) branch family are represented as shaded boxes. Numbers above the nodes indicate neighbor-joining bootstrap percentages. (B) Schematic depiction of Toxoplasma Tg- AGO and Arabidopsis At -AGO1 (GeneID: 841262) domain according to archaeon Aquifex aeolicus X-ray crystal structure. The amino-terminal domain (grey or blue) is linked to the PAZ domain (red). The MID domain (green) connects the PAZ domain with the PIWI domain (pink) at the carboxy-terminal end of the protein. In the PAZ domain, residues important for binding of small RNA 3′ ends are indicated. In the MID domain, the residues required for 5′ end binding to small RNAs and binding to the 7-methylguanine (m7G) cap of target mRNAs are shown. The conserved residues for RNase H cleavage activity (DDH) in the PIWI domain are also shown. RGG indicates the domain of Tg- AGO containing arginine-glycine-glycine repeats. Poly Q, polyglutamine-containing domain in Arabidopsis Ago1. (C) Sequence alignment of conserved 5′–phosphate binding residues (MID domain) across various species of Argonaute proteins. Prefix Hs, Homo sapiens ; Dm, Drosophila melanogaster ; Aa, Aquifex aeolicus . Conserved residues are shaded in red.

    Techniques Used: Binding Assay, Activity Assay, Sequencing

    Small RNA loading, sub-cellular localization and polysome association of Tg -AGO. (A) Northern blot analyses of Tg -miRNAs and Tg -rdsRNAs associated with Tg -AGO. Immuno-precipitation using anti-Flag antibody was performed from RH strains expressing ectopically HAFlag-tagged either full-length or delta-RGG truncated Tg -AGO. RNAs isolated from the immunoprecipitates were probed for Tg -miR-4, Tg -miR-43, Tg -rdsRNA-17 and Tg -rdsRNA-28. Mock: non-immune IgG (negative control). (B) Stably expressed recombinant protein HAFlag-TgAGO FL was detected by immunofluorescence assay using an HA antibody (in green) and compared to nuclear localization of acetylated histone H4 (in red). (C) To evaluate the sedimentation characteristics of Tg- AGO complexes, protein extracts containing HAFlag-TgAGO FL or HAFlag-TgAGO DRGG were subjected to sedimentation on 5%–45% sucrose gradients in the presence of cycloheximide (to preserve polyribosomes) or 30 mM EDTA (to disrupt polyribosomes). Aliquots of total extracts, either untreated (-RNase T1) or digested with RNase (+ RNase T1) were centrifuged through sucrose gradients and fractionated as described in Methods . Aliquots (equal volume) from indicated fractions were analyzed by Western blots with antibody against HA tag. (D) HAFlag-TgAGO FL and HAFlag-645.m00319 (negative control) were transiently co-expressed with Myc- Tg -p100 (Tudor/SN). Following Flag affinity purification, the bound proteins were analyzed by Western blot using the anti-HA and the anti-myc antibodies. Molecular weight markers are indicated on the left.
    Figure Legend Snippet: Small RNA loading, sub-cellular localization and polysome association of Tg -AGO. (A) Northern blot analyses of Tg -miRNAs and Tg -rdsRNAs associated with Tg -AGO. Immuno-precipitation using anti-Flag antibody was performed from RH strains expressing ectopically HAFlag-tagged either full-length or delta-RGG truncated Tg -AGO. RNAs isolated from the immunoprecipitates were probed for Tg -miR-4, Tg -miR-43, Tg -rdsRNA-17 and Tg -rdsRNA-28. Mock: non-immune IgG (negative control). (B) Stably expressed recombinant protein HAFlag-TgAGO FL was detected by immunofluorescence assay using an HA antibody (in green) and compared to nuclear localization of acetylated histone H4 (in red). (C) To evaluate the sedimentation characteristics of Tg- AGO complexes, protein extracts containing HAFlag-TgAGO FL or HAFlag-TgAGO DRGG were subjected to sedimentation on 5%–45% sucrose gradients in the presence of cycloheximide (to preserve polyribosomes) or 30 mM EDTA (to disrupt polyribosomes). Aliquots of total extracts, either untreated (-RNase T1) or digested with RNase (+ RNase T1) were centrifuged through sucrose gradients and fractionated as described in Methods . Aliquots (equal volume) from indicated fractions were analyzed by Western blots with antibody against HA tag. (D) HAFlag-TgAGO FL and HAFlag-645.m00319 (negative control) were transiently co-expressed with Myc- Tg -p100 (Tudor/SN). Following Flag affinity purification, the bound proteins were analyzed by Western blot using the anti-HA and the anti-myc antibodies. Molecular weight markers are indicated on the left.

    Techniques Used: Northern Blot, Immunoprecipitation, Expressing, Isolation, Negative Control, Stable Transfection, Recombinant, Immunofluorescence, Sedimentation, Western Blot, Affinity Purification, Molecular Weight

    7) Product Images from "Microparticles from Kidney-Derived Mesenchymal Stem Cells Act as Carriers of Proangiogenic Signals and Contribute to Recovery from Acute Kidney Injury"

    Article Title: Microparticles from Kidney-Derived Mesenchymal Stem Cells Act as Carriers of Proangiogenic Signals and Contribute to Recovery from Acute Kidney Injury

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0087853

    In vitro cell proliferation and anti-apoptotic effects of MPs. (A) Proliferation of HUVEC treated with vehicle control, with different doses of MPs or MP-treated with RNase (MP-RNase). (B) Apoptosis assay of HUVEC treated with vehicle control or with different doses of MPs. (C) Apoptosis assay of HMVEC treated with vehicle control or with different doses of MPs. Results are expressed as % compared to vehicle control and mean ± SE of three different experiments. Kruskal-Wallis test was performed; * P
    Figure Legend Snippet: In vitro cell proliferation and anti-apoptotic effects of MPs. (A) Proliferation of HUVEC treated with vehicle control, with different doses of MPs or MP-treated with RNase (MP-RNase). (B) Apoptosis assay of HUVEC treated with vehicle control or with different doses of MPs. (C) Apoptosis assay of HMVEC treated with vehicle control or with different doses of MPs. Results are expressed as % compared to vehicle control and mean ± SE of three different experiments. Kruskal-Wallis test was performed; * P

    Techniques Used: In Vitro, Apoptosis Assay

    In vitro angiogenic effects of MPs. (A) Representative images of angiogenic effects of HUVEC pretreated with vehicle control, with different doses of MPs, or MPs preincubated with RNase (MP-RNase). (B) Quantitative analysis of tube formation of HUVEC was performed. Results are expressed as % versus vehicle alone and mean ± SEM of six different experiments. Results are expressed as mean ± SE of three different experiments. Kruskal-Wallis test was performed; * P
    Figure Legend Snippet: In vitro angiogenic effects of MPs. (A) Representative images of angiogenic effects of HUVEC pretreated with vehicle control, with different doses of MPs, or MPs preincubated with RNase (MP-RNase). (B) Quantitative analysis of tube formation of HUVEC was performed. Results are expressed as % versus vehicle alone and mean ± SEM of six different experiments. Results are expressed as mean ± SE of three different experiments. Kruskal-Wallis test was performed; * P

    Techniques Used: In Vitro

    Anti-apoptotic effects of MPs on tubular epithelial and peritubular capillary endothelial cell in I/R injury kidney. (A) Representative confocal microscopic images of TUNEL (green) and TUNEL/CD 31 (red) double staining and (B, C) quantitative analysis of TUNEL-positive cells in the kidney 3 days after I/R injury. The total number of TUNEL-positive nuclei (B) and both TUNEL and CD 31-positive peritubular capillary nuclei (C) significantly decreased in kidneys injected with KMSCs and KMSC-derived MPs compared to those injected with vehicle control or MP-treated with RNase (MP-RNase) 3 days after IRI. Results are expressed as mean ± SE of six different experiments. Kruskal-Wallis test was performed; * P
    Figure Legend Snippet: Anti-apoptotic effects of MPs on tubular epithelial and peritubular capillary endothelial cell in I/R injury kidney. (A) Representative confocal microscopic images of TUNEL (green) and TUNEL/CD 31 (red) double staining and (B, C) quantitative analysis of TUNEL-positive cells in the kidney 3 days after I/R injury. The total number of TUNEL-positive nuclei (B) and both TUNEL and CD 31-positive peritubular capillary nuclei (C) significantly decreased in kidneys injected with KMSCs and KMSC-derived MPs compared to those injected with vehicle control or MP-treated with RNase (MP-RNase) 3 days after IRI. Results are expressed as mean ± SE of six different experiments. Kruskal-Wallis test was performed; * P

    Techniques Used: TUNEL Assay, Double Staining, Injection, Derivative Assay

    Effects of MPs on tubular epithelial and peritubular capillary endothelial cell proliferation in I/R injury kidney. (A) Representative images of PCNA and CD 31 staining in I/R injury kidney. (B), (C) Quantitative analysis of PCNA-positive cells in kidneys 3 days after I/R injury injury. PCNA-positive counting was used to assess the proliferation of tubular epithelial cells and peritubular capillaries. There were significantly more PCNA-positive tubules (B) and both PCNA and CD 31 positive peritubular capillaries (C) in I/R injury kidneys treated with KMSC and KMSC-derived MPs compared those treated with vehicle control or MP-treated with RNase (MP-RNase) 3 days after I/R injury. Results are expressed as mean ± SE of six different experiments. Kruskal-Wallis test was performed; * P
    Figure Legend Snippet: Effects of MPs on tubular epithelial and peritubular capillary endothelial cell proliferation in I/R injury kidney. (A) Representative images of PCNA and CD 31 staining in I/R injury kidney. (B), (C) Quantitative analysis of PCNA-positive cells in kidneys 3 days after I/R injury injury. PCNA-positive counting was used to assess the proliferation of tubular epithelial cells and peritubular capillaries. There were significantly more PCNA-positive tubules (B) and both PCNA and CD 31 positive peritubular capillaries (C) in I/R injury kidneys treated with KMSC and KMSC-derived MPs compared those treated with vehicle control or MP-treated with RNase (MP-RNase) 3 days after I/R injury. Results are expressed as mean ± SE of six different experiments. Kruskal-Wallis test was performed; * P

    Techniques Used: Staining, Derivative Assay

    Semi-quantitative injury scoring of I/R injury kidney. (A) Representative images of PAS staining in I/R injury kidney with vehicle control, KMSC, KMSC-derived MPs, and MP-treated with RNase (MP-RNase). (B) Quantitative analysis of I/R injury kidney was determined by semi-quantitative injury scoring (0–5). Injury score was significantly decreased in kidneys injected with KMSC and KMSC-derived MPs compared to those injected with vehicle control or MP-RNase 3 days after IRI. Results are expressed as % versus vehicle alone and mean ± SE of six different experiments. Kruskal-Wallis test was performed; * P
    Figure Legend Snippet: Semi-quantitative injury scoring of I/R injury kidney. (A) Representative images of PAS staining in I/R injury kidney with vehicle control, KMSC, KMSC-derived MPs, and MP-treated with RNase (MP-RNase). (B) Quantitative analysis of I/R injury kidney was determined by semi-quantitative injury scoring (0–5). Injury score was significantly decreased in kidneys injected with KMSC and KMSC-derived MPs compared to those injected with vehicle control or MP-RNase 3 days after IRI. Results are expressed as % versus vehicle alone and mean ± SE of six different experiments. Kruskal-Wallis test was performed; * P

    Techniques Used: Staining, Derivative Assay, Injection

    Effects of MPs on microvascular rarefaction of I/R injury kidney. (A) Representative images of CD 31 stainings in I/R injury kidney with vehicle control, KMSC, KMSC-derived MPs, and MP-treated with RNase (MP-RNase). (B) Quantitative analysis of peritubular capillary rarefaction index was determined by CD31 staining. The degree of microvascular rarefaction was significantly decreased in kidneys injected with KMSC and KMSC-derived MPs compared to those injected with vehicle control or MP-RNase 3 days after I/R injury. Results are expressed as % versus vehicle alone and mean ± SE of six different experiments. Kruskal-Wallis test was performed; * P
    Figure Legend Snippet: Effects of MPs on microvascular rarefaction of I/R injury kidney. (A) Representative images of CD 31 stainings in I/R injury kidney with vehicle control, KMSC, KMSC-derived MPs, and MP-treated with RNase (MP-RNase). (B) Quantitative analysis of peritubular capillary rarefaction index was determined by CD31 staining. The degree of microvascular rarefaction was significantly decreased in kidneys injected with KMSC and KMSC-derived MPs compared to those injected with vehicle control or MP-RNase 3 days after I/R injury. Results are expressed as % versus vehicle alone and mean ± SE of six different experiments. Kruskal-Wallis test was performed; * P

    Techniques Used: Derivative Assay, Staining, Injection

    8) Product Images from "Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins"

    Article Title: Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-20421-1

    Genetic cargo of mRPC derived extracellular vesicles. ( A ) A 1.5% denaturing agarose gel loaded with total RNA from mRPCs and EVs. Total RNA from EVs consisted primarily of species below 800 nucleotides (nt) lacking 28S and 18S rRNA. EVs were treated with RNase and no difference was detected when compared with non-treated EVs, indicating the RNA of EVs was enclosed within the vesicle membrane. ( B ) Transcription factors, a cell-cycle regulator and intermediate filaments were identified in both mRPCs and EVs included Pax6, Hes1, Sox2, Ki67, GFAP and Nestin. The transcription factors identified are collectively involved in facilitating mRPC multipotency, cell-cycle and fate specification during retinogenesis. GFP, GAPDH and β-actin mRNAs were also detected in mRPCs and EVs. Next, the presence of miRNAs with established expression and function during retinogensis were chosen for analysis. ( C ) Selected miRNA species analyzed included Let7d, miR-9, miR-182 and miR-204. U6 snRNA was used as control. Data presented were combined from four independent replicates.
    Figure Legend Snippet: Genetic cargo of mRPC derived extracellular vesicles. ( A ) A 1.5% denaturing agarose gel loaded with total RNA from mRPCs and EVs. Total RNA from EVs consisted primarily of species below 800 nucleotides (nt) lacking 28S and 18S rRNA. EVs were treated with RNase and no difference was detected when compared with non-treated EVs, indicating the RNA of EVs was enclosed within the vesicle membrane. ( B ) Transcription factors, a cell-cycle regulator and intermediate filaments were identified in both mRPCs and EVs included Pax6, Hes1, Sox2, Ki67, GFAP and Nestin. The transcription factors identified are collectively involved in facilitating mRPC multipotency, cell-cycle and fate specification during retinogenesis. GFP, GAPDH and β-actin mRNAs were also detected in mRPCs and EVs. Next, the presence of miRNAs with established expression and function during retinogensis were chosen for analysis. ( C ) Selected miRNA species analyzed included Let7d, miR-9, miR-182 and miR-204. U6 snRNA was used as control. Data presented were combined from four independent replicates.

    Techniques Used: Derivative Assay, Agarose Gel Electrophoresis, Expressing

    Extracellular vesicle internalization and transfer of GFP mRNA ( A ) Super resolution 3D reconstruction of GFP+ mRPC following 24 h incubation with PKH26 labeled extracellular vesicles. Red vesicles are visibly localized near the cell surface and within cytoplasm. In the XZ axis, GFP (green), EVs (red) and nuclei (blue, DAPI). ( B ) same as ( A ) with GFP (FITC) channel removed to increase visibility of PKH26 (TRITC) labeled EVs. Each panel contains three cross-sectional views (xy, xz, and yz). Scale: 5 µm. ( C ) RT-PCR analysis of GFP mRNA transfer between GFP+ mRPCs and non-GFP hRPCs. Non-GFP hRPCs served as negative control; GFP+ mRPCs served as postive control. GAPDH served as the internal control gene. EVs were treated using an RNase-Free DNase Set to remove DNA comtamination before cDNA synthesis. ( D ) Intensities of RT-PCR images were measured with ImageJ software and normalized to GAPDH. Relative levels of hRPC GFP after transfer of EVs is significantly higher than negative control.
    Figure Legend Snippet: Extracellular vesicle internalization and transfer of GFP mRNA ( A ) Super resolution 3D reconstruction of GFP+ mRPC following 24 h incubation with PKH26 labeled extracellular vesicles. Red vesicles are visibly localized near the cell surface and within cytoplasm. In the XZ axis, GFP (green), EVs (red) and nuclei (blue, DAPI). ( B ) same as ( A ) with GFP (FITC) channel removed to increase visibility of PKH26 (TRITC) labeled EVs. Each panel contains three cross-sectional views (xy, xz, and yz). Scale: 5 µm. ( C ) RT-PCR analysis of GFP mRNA transfer between GFP+ mRPCs and non-GFP hRPCs. Non-GFP hRPCs served as negative control; GFP+ mRPCs served as postive control. GAPDH served as the internal control gene. EVs were treated using an RNase-Free DNase Set to remove DNA comtamination before cDNA synthesis. ( D ) Intensities of RT-PCR images were measured with ImageJ software and normalized to GAPDH. Relative levels of hRPC GFP after transfer of EVs is significantly higher than negative control.

    Techniques Used: Incubation, Labeling, Reverse Transcription Polymerase Chain Reaction, Negative Control, Software

    9) Product Images from "Nanoparticle core stability and surface functionalization drive the mTOR signaling pathway in hepatocellular cell lines"

    Article Title: Nanoparticle core stability and surface functionalization drive the mTOR signaling pathway in hepatocellular cell lines

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-16447-6

    Functionalized nanoparticles bearing hard protein corona colocalize with lysosomes in Huh7 cells. Cells were incubated with BSA or RNase (both 50 µM) labelled with Atto633 (red dye) for 1 h. Additionally, cells were incubated with either Si-OH or Si-NH 2 or PS-NH2 NPs (all 50 µg/ml) bearing either BSA or RNase as hard protein corona (both 50 µM). Acidic organelles were stained with LysoTracker probe (Invitrogen, green dye), and the cells were analyzed using confocal microscopy. Colocalization is yellow.
    Figure Legend Snippet: Functionalized nanoparticles bearing hard protein corona colocalize with lysosomes in Huh7 cells. Cells were incubated with BSA or RNase (both 50 µM) labelled with Atto633 (red dye) for 1 h. Additionally, cells were incubated with either Si-OH or Si-NH 2 or PS-NH2 NPs (all 50 µg/ml) bearing either BSA or RNase as hard protein corona (both 50 µM). Acidic organelles were stained with LysoTracker probe (Invitrogen, green dye), and the cells were analyzed using confocal microscopy. Colocalization is yellow.

    Techniques Used: Incubation, Staining, Confocal Microscopy

    Inhibition of proliferation in Huh7 cell line by PS-NH 2 nanoparticles. ( A ) Cells were cultured in medium for 24 h in the presence or absence of hydroxyl- (Si-OH), amino-functionalized (Si-NH 2 ) silica, or amino-functionalized (PS-NH 2 ) PS NPs. Cell viability was assessed by the WST-1 assay. The data were normalized to control values (no particle exposure) and expressed as mean ± SEM, n = 3 each. ( B ) Analysis of cytotoxicity in Huh7 cultured with Si-OH, Si-NH 2 , or PS-NH 2 NPs bearing RNase as hard protein corona. Cells were cultured in the presence or absence of Si-OH or Si-NH 2 NPs (all 100 µg/ml) pre-incubated with increasing concentrations of RNase for 1 h. Cell viability was assessed by the WST-1 assay. The data were normalized to control values (no particle exposure) and expressed as mean ± SEM, n = 3 each. ( C ) Analysis of cytotoxicity in Huh7 cultured with Si-OH or Si-NH 2 , NPs bearing BSA as hard protein corona. Cells were cultured in the presence or absence of Si-OH or Si-NH 2 NPs (all 100 µg/ml) pre-incubated with increasing concentrations of BSA for 1 h. Cell viability was assessed by the WST-1 assay. The data were normalized to control values (no particle exposure) and expressed as mean ± SEM, n = 3 each. ( D ) Comparison of proliferative activity of Huh7 cultured with Si-OH, Si-NH 2 , or PS-NH 2 NPs bearing BSA or RNase (100 µM both) as hard protein corona or bare NPs. Cell were treated as in ( A – C ). The data were normalized to control values (no particle exposure) and expressed as mean ± SEM, n = 3 each.
    Figure Legend Snippet: Inhibition of proliferation in Huh7 cell line by PS-NH 2 nanoparticles. ( A ) Cells were cultured in medium for 24 h in the presence or absence of hydroxyl- (Si-OH), amino-functionalized (Si-NH 2 ) silica, or amino-functionalized (PS-NH 2 ) PS NPs. Cell viability was assessed by the WST-1 assay. The data were normalized to control values (no particle exposure) and expressed as mean ± SEM, n = 3 each. ( B ) Analysis of cytotoxicity in Huh7 cultured with Si-OH, Si-NH 2 , or PS-NH 2 NPs bearing RNase as hard protein corona. Cells were cultured in the presence or absence of Si-OH or Si-NH 2 NPs (all 100 µg/ml) pre-incubated with increasing concentrations of RNase for 1 h. Cell viability was assessed by the WST-1 assay. The data were normalized to control values (no particle exposure) and expressed as mean ± SEM, n = 3 each. ( C ) Analysis of cytotoxicity in Huh7 cultured with Si-OH or Si-NH 2 , NPs bearing BSA as hard protein corona. Cells were cultured in the presence or absence of Si-OH or Si-NH 2 NPs (all 100 µg/ml) pre-incubated with increasing concentrations of BSA for 1 h. Cell viability was assessed by the WST-1 assay. The data were normalized to control values (no particle exposure) and expressed as mean ± SEM, n = 3 each. ( D ) Comparison of proliferative activity of Huh7 cultured with Si-OH, Si-NH 2 , or PS-NH 2 NPs bearing BSA or RNase (100 µM both) as hard protein corona or bare NPs. Cell were treated as in ( A – C ). The data were normalized to control values (no particle exposure) and expressed as mean ± SEM, n = 3 each.

    Techniques Used: Inhibition, Cell Culture, WST-1 Assay, Incubation, Activity Assay

    Inhibition of proliferation in HepG2 cell line by PS-NH2 nanoparticles. ( A ) Comparison of proliferative activity of HepG2 cultured with Si-OH, Si-NH 2 , or PS-NH 2 NPs bearing BSA or RNase (100 µM both) as hard protein corona or bare NPs. Cells were cultured in medium for 24 h in the presence or absence of hydroxyl- (Si-OH), amino-functionalized (Si-NH 2 ) silica, or amino-functionalized (PS-NH 2 ) PS NPs. Cell viability was assessed by the WST-1 assay. The data were normalized to control values (no particle exposure) and expressed as mean ± SEM, n = 3 each. * p
    Figure Legend Snippet: Inhibition of proliferation in HepG2 cell line by PS-NH2 nanoparticles. ( A ) Comparison of proliferative activity of HepG2 cultured with Si-OH, Si-NH 2 , or PS-NH 2 NPs bearing BSA or RNase (100 µM both) as hard protein corona or bare NPs. Cells were cultured in medium for 24 h in the presence or absence of hydroxyl- (Si-OH), amino-functionalized (Si-NH 2 ) silica, or amino-functionalized (PS-NH 2 ) PS NPs. Cell viability was assessed by the WST-1 assay. The data were normalized to control values (no particle exposure) and expressed as mean ± SEM, n = 3 each. * p

    Techniques Used: Inhibition, Activity Assay, Cell Culture, WST-1 Assay

    RagC complexes isolated by immunoprecipitation from Huh7 cells. ( A ) Huh7 cultured with Si-OH, Si-NH2, or PS-NH2 NPs (all 50 µg/ml) bearing BSA or RNase (both 50 µM) as hard protein corona or bare NPs for 4 h. After 4 h post NP treatment, cells were lysed with lysis buffer from immunoprecipitation kit (Abcam). RagC-mTOR complexes were co-immunoprecipitated from the precleared cell lysates with appropriate Ab as described in the manufacturer’s instruction. The resulting protein complex was eluted from the beads with Laemmli protein sample buffer for SDS-PAGE (Bio-Rad) and resolved on SDS-PAGE with specific antibody against mTOR (Cell Signaling) (full blots are presented in Supporting Information). ( B ) Scheme of district biochemical signaling activation in cells after stimulation with amino-functionalized non-biodegradable PS and biodegradable silica NPs. ∆mΦ – mitochondrial membrane potential; LMP – lysosomal membrane permeabilization.
    Figure Legend Snippet: RagC complexes isolated by immunoprecipitation from Huh7 cells. ( A ) Huh7 cultured with Si-OH, Si-NH2, or PS-NH2 NPs (all 50 µg/ml) bearing BSA or RNase (both 50 µM) as hard protein corona or bare NPs for 4 h. After 4 h post NP treatment, cells were lysed with lysis buffer from immunoprecipitation kit (Abcam). RagC-mTOR complexes were co-immunoprecipitated from the precleared cell lysates with appropriate Ab as described in the manufacturer’s instruction. The resulting protein complex was eluted from the beads with Laemmli protein sample buffer for SDS-PAGE (Bio-Rad) and resolved on SDS-PAGE with specific antibody against mTOR (Cell Signaling) (full blots are presented in Supporting Information). ( B ) Scheme of district biochemical signaling activation in cells after stimulation with amino-functionalized non-biodegradable PS and biodegradable silica NPs. ∆mΦ – mitochondrial membrane potential; LMP – lysosomal membrane permeabilization.

    Techniques Used: Isolation, Immunoprecipitation, Cell Culture, Lysis, SDS Page, Activation Assay

    EPR spectra of ( A ) BSA and ( B ) RNase labelled with bromoacetamido-methylproxyl spin label (black traces). Labeled proteins adsorbed on silica Si-OH (blue traces) and Si NH 2 (red traces) NPs. All spectra have been recorded under the same instrumental conditions: microwave frequency, 9.3 GHz; modulation amplitude, 1 G; modulation frequency, 100 kHz; microwave power, 1 mW; time constant, 163 ms; 30 scans; and T = 25 °C. RNase activity in presence of functionalized silica NPs as function of protein concentration ( C ) or NP concentration ( D ). ( C ) 100 µg/ml of either Si-OH or Si-NH 2 NPs were incubated with increasing RNase concentrations in PBS pH 7.4 for 1 h. RNase activity was assessed using Ambion® RNaseAlert® Lab Test kit (Thermo Fisher Scientific), according to the manufacturer’s instruction. The kit contains a fluorescent substrate that emits a green fluorescence if it is cleaved by RNase. The data expressed as mean ± SEM, n = 3 each. * p
    Figure Legend Snippet: EPR spectra of ( A ) BSA and ( B ) RNase labelled with bromoacetamido-methylproxyl spin label (black traces). Labeled proteins adsorbed on silica Si-OH (blue traces) and Si NH 2 (red traces) NPs. All spectra have been recorded under the same instrumental conditions: microwave frequency, 9.3 GHz; modulation amplitude, 1 G; modulation frequency, 100 kHz; microwave power, 1 mW; time constant, 163 ms; 30 scans; and T = 25 °C. RNase activity in presence of functionalized silica NPs as function of protein concentration ( C ) or NP concentration ( D ). ( C ) 100 µg/ml of either Si-OH or Si-NH 2 NPs were incubated with increasing RNase concentrations in PBS pH 7.4 for 1 h. RNase activity was assessed using Ambion® RNaseAlert® Lab Test kit (Thermo Fisher Scientific), according to the manufacturer’s instruction. The kit contains a fluorescent substrate that emits a green fluorescence if it is cleaved by RNase. The data expressed as mean ± SEM, n = 3 each. * p

    Techniques Used: Electron Paramagnetic Resonance, Labeling, Mass Spectrometry, Activity Assay, Protein Concentration, Concentration Assay, Incubation, Fluorescence

    Physicochemical characterization of the chemically distinct nanoparticles. ( A ) Characterization of hydroxyl-, amino-functionalized silica and amino-functionalized PS NPs. Physical parameters of NPs were characterized by dynamic light scattering (DLS) using a Zetasizer Nano. PDI, polydispersity index. ( B ) Scheme of the experimental setup. Protein adsorption on silica NPs characterized using fluorescence correlation spectroscopy. Positively (Si-NH 2 ) and negatively (Si-OH) charged silica NPs were titrated with fluorescently labeled BSA and RNase. ( C ) Exemplary autocorrelation curves normalized to 0.8 or 1.2. ( D ) Number of particles, ( E ) their brightness, and ( F ) mean diffusion time. Curves in logarithmic plots ( D , F ) were fitted with straight lines according to Freundlich adsorption model. Error bars show SEM for n = 3.
    Figure Legend Snippet: Physicochemical characterization of the chemically distinct nanoparticles. ( A ) Characterization of hydroxyl-, amino-functionalized silica and amino-functionalized PS NPs. Physical parameters of NPs were characterized by dynamic light scattering (DLS) using a Zetasizer Nano. PDI, polydispersity index. ( B ) Scheme of the experimental setup. Protein adsorption on silica NPs characterized using fluorescence correlation spectroscopy. Positively (Si-NH 2 ) and negatively (Si-OH) charged silica NPs were titrated with fluorescently labeled BSA and RNase. ( C ) Exemplary autocorrelation curves normalized to 0.8 or 1.2. ( D ) Number of particles, ( E ) their brightness, and ( F ) mean diffusion time. Curves in logarithmic plots ( D , F ) were fitted with straight lines according to Freundlich adsorption model. Error bars show SEM for n = 3.

    Techniques Used: Adsorption, Fluorescence, Spectroscopy, Labeling, Diffusion-based Assay

    mTOR signalling in Huh7 cells exposed to different nanoparticles. ( A ) Huh7 cultured with Si-OH, Si-NH2, or PS-NH2 NPs (all 50 µg/ml) bearing BSA or RNase (both 50 µM) as hard protein corona or bare NPs for 4 h. Activation of mTOR was analyzed by Western immunoblotting (full blots are presented in Supporting Information). Actin served as a loading control. Representative blots out of 3. ( B ) Scheme illustrating the mTOR involvement in NP signalling. ( C ) Activation of mTOR on lysosomal membranes was analyzed in Huh7 stimulated as in ( A ) and analyzed by confocal microscopy using LAMP1 antibody as marker of lysosomes (red) and pmTOR (green). Colocalization is shown in yellow. Increased cell fluorescence due to mTOR phosphorylation is presented ( D ) as corrected total cell fluorescence (CTCF). Quantifications performed using ImageJ are presented as means of n = 30 cells. * p
    Figure Legend Snippet: mTOR signalling in Huh7 cells exposed to different nanoparticles. ( A ) Huh7 cultured with Si-OH, Si-NH2, or PS-NH2 NPs (all 50 µg/ml) bearing BSA or RNase (both 50 µM) as hard protein corona or bare NPs for 4 h. Activation of mTOR was analyzed by Western immunoblotting (full blots are presented in Supporting Information). Actin served as a loading control. Representative blots out of 3. ( B ) Scheme illustrating the mTOR involvement in NP signalling. ( C ) Activation of mTOR on lysosomal membranes was analyzed in Huh7 stimulated as in ( A ) and analyzed by confocal microscopy using LAMP1 antibody as marker of lysosomes (red) and pmTOR (green). Colocalization is shown in yellow. Increased cell fluorescence due to mTOR phosphorylation is presented ( D ) as corrected total cell fluorescence (CTCF). Quantifications performed using ImageJ are presented as means of n = 30 cells. * p

    Techniques Used: Cell Culture, Activation Assay, Western Blot, Confocal Microscopy, Marker, Fluorescence

    10) Product Images from "Human Cytomegalovirus Elicits a Coordinated Cellular Antiviral Response via Envelope Glycoprotein B"

    Article Title: Human Cytomegalovirus Elicits a Coordinated Cellular Antiviral Response via Envelope Glycoprotein B

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.3.1202-1211.2004

    Effects of CMV in endothelial cells. (A) CMV induces ISG expression in endothelial cells. Human fibroblasts or endothelial cells were mock infected (M) or infected with CMV (strain AD169 or VHL/E) at an MOI of 2 PFU/cell. At the indicated times postinfection, total cellular RNA was harvested and subjected to RNase protection analysis with OAS- and actin-specific probes. (B) CMV triggers an antiviral state in endothelial cells. HUVEC were stimulated as indicated (interferon, 100 U/ml). At 6 h poststimulation, the treatments were removed and the monolayers were challenged with approximately 100 PFU of VSV per well. Plaque formation was visualized by crystal violet staining at 48 h postinfection.
    Figure Legend Snippet: Effects of CMV in endothelial cells. (A) CMV induces ISG expression in endothelial cells. Human fibroblasts or endothelial cells were mock infected (M) or infected with CMV (strain AD169 or VHL/E) at an MOI of 2 PFU/cell. At the indicated times postinfection, total cellular RNA was harvested and subjected to RNase protection analysis with OAS- and actin-specific probes. (B) CMV triggers an antiviral state in endothelial cells. HUVEC were stimulated as indicated (interferon, 100 U/ml). At 6 h poststimulation, the treatments were removed and the monolayers were challenged with approximately 100 PFU of VSV per well. Plaque formation was visualized by crystal violet staining at 48 h postinfection.

    Techniques Used: Expressing, Infection, Staining

    Cell response to treatment with CMV, gB, and gB 1-460 . (A and B) CMV and gB trigger a functional antiviral state in cells. Human fibroblasts were stimulated as indicated (interferon, 100 U/ml). At 6 h poststimulation, the treatments were removed and the monolayers were challenged with approximately 100 PFU of VSV per well. The mock-treated cells were not infected with VSV. Plaque formation was visualized by crystal violet staining at 48 h postinfection. CMV replicates with much slower kinetics than VSV; thus, any visible plaque formation is the result of VSV, not CMV, growth. (C) gB 1-460 minimally induces ISG activation. Human fibroblasts were mock treated or treated with gB 1-750 or gB 1-460 (1 μg/ml). At 8 h posttreatment, total RNA was harvested from cells and subjected to RNase protection analysis with ISG54- and actin-specific probes.
    Figure Legend Snippet: Cell response to treatment with CMV, gB, and gB 1-460 . (A and B) CMV and gB trigger a functional antiviral state in cells. Human fibroblasts were stimulated as indicated (interferon, 100 U/ml). At 6 h poststimulation, the treatments were removed and the monolayers were challenged with approximately 100 PFU of VSV per well. The mock-treated cells were not infected with VSV. Plaque formation was visualized by crystal violet staining at 48 h postinfection. CMV replicates with much slower kinetics than VSV; thus, any visible plaque formation is the result of VSV, not CMV, growth. (C) gB 1-460 minimally induces ISG activation. Human fibroblasts were mock treated or treated with gB 1-750 or gB 1-460 (1 μg/ml). At 8 h posttreatment, total RNA was harvested from cells and subjected to RNase protection analysis with ISG54- and actin-specific probes.

    Techniques Used: Functional Assay, Infection, Staining, Activation Assay

    Soluble gB induces ISG expression in endothelial cells. Endothelial cells from three different sources were mock treated (M) or stimulated with soluble gB. At the indicated times posttreatment, total cellular RNA was harvested and subjected to RNase protection analysis with OAS-, ISG54-, and actin-specific probes.
    Figure Legend Snippet: Soluble gB induces ISG expression in endothelial cells. Endothelial cells from three different sources were mock treated (M) or stimulated with soluble gB. At the indicated times posttreatment, total cellular RNA was harvested and subjected to RNase protection analysis with OAS-, ISG54-, and actin-specific probes.

    Techniques Used: Expressing

    11) Product Images from "Targeted gene silencing in vivo by platelet membrane–coated metal-organic framework nanoparticles"

    Article Title: Targeted gene silencing in vivo by platelet membrane–coated metal-organic framework nanoparticles

    Journal: Science Advances

    doi: 10.1126/sciadv.aaz6108

    Formulation and characterization. ( A ) Diameter of pristine MOF, MOF-siRNA, P-MOF, P-MOF-siRNA, and platelet (PLT) membrane vesicles after formulation ( n = 3, mean + SD). ( B ) Zeta potential of pristine MOF, MOF-siRNA, P-MOF, P-MOF-siRNA, and platelet membrane vesicles after formulation ( n = 3, mean + SD). ( C ) Transmission electron microscopy image of P-MOF-siRNA negatively stained with uranyl acetate (scale bar, 200 nm). ( D ) Encapsulation efficiency of siRNA inside P-MOF-siRNA at various siRNA inputs ( n = 3, mean + SD). ( E ) Stability of MOF-siRNA and P-MOF-siRNA over time in PBS or serum-containing medium ( n = 3, mean ± SD). ( F ) siRNA release from P-MOF-siRNA at pH 5.0 or pH 7.4 over time ( n = 3, mean ± SD). ( G and H ) Degradation of siRNA, either in free form or in P-MOF-siRNA, when exposed to purified RNase (G) or serum-containing medium (H) for increasing amounts of time. ( I ) Western blots for three characteristic platelet surface markers (CD41, CD61, and P-selectin) in MOF-siRNA, platelet membrane vesicles, and P-MOF-siRNA. ( J ) Dot blot intensity of P-MOF-siRNA probed with antibodies against the intracellular or extracellular domains of CD47 ( n = 3, mean + SD).
    Figure Legend Snippet: Formulation and characterization. ( A ) Diameter of pristine MOF, MOF-siRNA, P-MOF, P-MOF-siRNA, and platelet (PLT) membrane vesicles after formulation ( n = 3, mean + SD). ( B ) Zeta potential of pristine MOF, MOF-siRNA, P-MOF, P-MOF-siRNA, and platelet membrane vesicles after formulation ( n = 3, mean + SD). ( C ) Transmission electron microscopy image of P-MOF-siRNA negatively stained with uranyl acetate (scale bar, 200 nm). ( D ) Encapsulation efficiency of siRNA inside P-MOF-siRNA at various siRNA inputs ( n = 3, mean + SD). ( E ) Stability of MOF-siRNA and P-MOF-siRNA over time in PBS or serum-containing medium ( n = 3, mean ± SD). ( F ) siRNA release from P-MOF-siRNA at pH 5.0 or pH 7.4 over time ( n = 3, mean ± SD). ( G and H ) Degradation of siRNA, either in free form or in P-MOF-siRNA, when exposed to purified RNase (G) or serum-containing medium (H) for increasing amounts of time. ( I ) Western blots for three characteristic platelet surface markers (CD41, CD61, and P-selectin) in MOF-siRNA, platelet membrane vesicles, and P-MOF-siRNA. ( J ) Dot blot intensity of P-MOF-siRNA probed with antibodies against the intracellular or extracellular domains of CD47 ( n = 3, mean + SD).

    Techniques Used: Transmission Assay, Electron Microscopy, Staining, Purification, Western Blot, Dot Blot

    12) Product Images from "Encapsidation of Host RNAs by Cucumber Necrosis Virus Coat Protein during both Agroinfiltration and Infection"

    Article Title: Encapsidation of Host RNAs by Cucumber Necrosis Virus Coat Protein during both Agroinfiltration and Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.01466-15

    Agarose gel electrophoresis and Northern blot analysis of host RNA species present in CNVCPpBin(+) VLPs and CNV particles. (A) Agarose gel electrophoresis of RNA extracted from RNase-treated pCNVCPpBin(+) particles. Lane 1, RNA extracted from pCNVCPpBin(+) VLPs; lane 2, CNV virion RNA (vRNA); lane 3, total RNA from uninfected leaves (mock infected). The sizes of cytoplasmic 26S rRNA (3.3 kb), 18S rRNA (1.8 kb), and 16S rRNA (1.5 kb) are indicated on the right. Asterisks, the major RNAs of a discrete size extracted from pCNVCPpBin(+) VLPs; brackets, a series of smaller RNAs ranging from ∼0.5 to ∼1.3 kb. (B) Northern blot analysis. Fifty nanograms of RNA extracted from RNase-treated CNVCPpBin(+) VLPs (lane 1) or 2.5 μg of RNA extracted from RNase-treated CNV particles was electrophoresed through a 1% denaturing agarose gel and blotted onto a Zeta-probe membrane (Bio-Rad). The probe was a 32 P-labeled randomly primed cDNA to total leaf RNA extracted from N. benthamiana . Lane 3, 50 ng of total leaf RNA used as a control. Lanes 1 and 3 were exposed for approximately 1/12 the time that lane 2 was exposed.
    Figure Legend Snippet: Agarose gel electrophoresis and Northern blot analysis of host RNA species present in CNVCPpBin(+) VLPs and CNV particles. (A) Agarose gel electrophoresis of RNA extracted from RNase-treated pCNVCPpBin(+) particles. Lane 1, RNA extracted from pCNVCPpBin(+) VLPs; lane 2, CNV virion RNA (vRNA); lane 3, total RNA from uninfected leaves (mock infected). The sizes of cytoplasmic 26S rRNA (3.3 kb), 18S rRNA (1.8 kb), and 16S rRNA (1.5 kb) are indicated on the right. Asterisks, the major RNAs of a discrete size extracted from pCNVCPpBin(+) VLPs; brackets, a series of smaller RNAs ranging from ∼0.5 to ∼1.3 kb. (B) Northern blot analysis. Fifty nanograms of RNA extracted from RNase-treated CNVCPpBin(+) VLPs (lane 1) or 2.5 μg of RNA extracted from RNase-treated CNV particles was electrophoresed through a 1% denaturing agarose gel and blotted onto a Zeta-probe membrane (Bio-Rad). The probe was a 32 P-labeled randomly primed cDNA to total leaf RNA extracted from N. benthamiana . Lane 3, 50 ng of total leaf RNA used as a control. Lanes 1 and 3 were exposed for approximately 1/12 the time that lane 2 was exposed.

    Techniques Used: Agarose Gel Electrophoresis, Northern Blot, Infection, Labeling

    13) Product Images from "Packaging of viral RNAs in virions of adenoviruses"

    Article Title: Packaging of viral RNAs in virions of adenoviruses

    Journal: Virology Journal

    doi: 10.1186/1743-422X-6-16

    Analysis of RNA in HAV5.EGFP capsids with or without RNase/DNase treatment . (A) Total yields of RNAs isolated from mature and empty/intermediate capsids with or without RNase/DNase treatment. (B) The [ 32 P]-labeled cDNAs were made by reverse transcription of 2 μg, RNase-free DNase treated RNAs from mature and empty/intermediate capsids with or without RNase/DNase treatment and hybridized to Hind III-digested pFHAV5, which contains E1A-deleted HAdV-5 genome. RT was primed by oligo-dT/hexamers. (C) Viral RNAs detected in RNAs from mature (Ma) and empty/intermediate (E-I) capsids after RNase/DNase treatment in Southern hybridization in panel B were quantitated by using PhosphorImager software.
    Figure Legend Snippet: Analysis of RNA in HAV5.EGFP capsids with or without RNase/DNase treatment . (A) Total yields of RNAs isolated from mature and empty/intermediate capsids with or without RNase/DNase treatment. (B) The [ 32 P]-labeled cDNAs were made by reverse transcription of 2 μg, RNase-free DNase treated RNAs from mature and empty/intermediate capsids with or without RNase/DNase treatment and hybridized to Hind III-digested pFHAV5, which contains E1A-deleted HAdV-5 genome. RT was primed by oligo-dT/hexamers. (C) Viral RNAs detected in RNAs from mature (Ma) and empty/intermediate (E-I) capsids after RNase/DNase treatment in Southern hybridization in panel B were quantitated by using PhosphorImager software.

    Techniques Used: Isolation, Labeling, Hybridization, Software

    14) Product Images from "Genetic editing and interrogation with Cpf1 and caged truncated pre-tRNA-like crRNA in mammalian cells"

    Article Title: Genetic editing and interrogation with Cpf1 and caged truncated pre-tRNA-like crRNA in mammalian cells

    Journal: Cell Discovery

    doi: 10.1038/s41421-018-0035-0

    Design of RNase-resistant caged truncated pre-tRNA-like crRNA (catRNA). a Structures of conventional crRNA, caged crRNA (caRNA), and caged truncated pre-tRNA-like crRNA (catRNA) transcribed by Pol III and Pol II polymerases. Red, crRNA spacer sequence. Oligo-T (Pol III) or oligo-T-Poly A (Pol II) are incorporated into crRNA but not caRNA or catRNA. b Structures of pre-tRNA and truncated pre-tRNA. Pre-tRNA is matured by RNase P by removal of the leader sequence and subsequently modified and processed by RNase Z. RNase P processing is blocked by disrupting the 7-base pair stem structure of pre-tRNA, which is critical for RNase P recognition and cleavage. Subsequent RNase Z cleavage is also abolished due to RNase P processing failure. c Illustration of the RNase-resistant property of catRNA
    Figure Legend Snippet: Design of RNase-resistant caged truncated pre-tRNA-like crRNA (catRNA). a Structures of conventional crRNA, caged crRNA (caRNA), and caged truncated pre-tRNA-like crRNA (catRNA) transcribed by Pol III and Pol II polymerases. Red, crRNA spacer sequence. Oligo-T (Pol III) or oligo-T-Poly A (Pol II) are incorporated into crRNA but not caRNA or catRNA. b Structures of pre-tRNA and truncated pre-tRNA. Pre-tRNA is matured by RNase P by removal of the leader sequence and subsequently modified and processed by RNase Z. RNase P processing is blocked by disrupting the 7-base pair stem structure of pre-tRNA, which is critical for RNase P recognition and cleavage. Subsequent RNase Z cleavage is also abolished due to RNase P processing failure. c Illustration of the RNase-resistant property of catRNA

    Techniques Used: Sequencing, Modification

    15) Product Images from "Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury"

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033115

    RNase treatment does not modify MV size, but reduces RNA content of MVs. A) Representative MV size analyses by direct measurement with NTA, showing no difference among MVs treated or not with RNase. B) Representative Bioanalyzer profile, showing the size distribution of total RNA extracted from MVs treated or not with RNAse. The first peak (left side of each panel) represents an internal standard. The two peaks in Sample 1 (black arrows) represent 18 S (left) and 28 S (right) ribosomal RNA, only partially detectable in MVs. The red arrows showed the reduction of 18 and 28 S fragment inside RNAse-treated MVs. C) Histogram showing the expression level of SUMO-1 , POLR2 and Act B transcripts in MVs treated or not with RNase, express as 2 -δCt , as described in material and methods.
    Figure Legend Snippet: RNase treatment does not modify MV size, but reduces RNA content of MVs. A) Representative MV size analyses by direct measurement with NTA, showing no difference among MVs treated or not with RNase. B) Representative Bioanalyzer profile, showing the size distribution of total RNA extracted from MVs treated or not with RNAse. The first peak (left side of each panel) represents an internal standard. The two peaks in Sample 1 (black arrows) represent 18 S (left) and 28 S (right) ribosomal RNA, only partially detectable in MVs. The red arrows showed the reduction of 18 and 28 S fragment inside RNAse-treated MVs. C) Histogram showing the expression level of SUMO-1 , POLR2 and Act B transcripts in MVs treated or not with RNase, express as 2 -δCt , as described in material and methods.

    Techniques Used: Expressing, Activated Clotting Time Assay

    MV infusion protects SCID mice with cisplatin-induced AKI from tubular injury. Representative micrographs of renal histology of healthy SCID mice and of SCID mice treated with cisplatin and injected with vehicle alone or with MV pre-treated with RNase or with different regiments of MVs (single or multiple injections) and sacrificed at different time points (day 4, 14 and 21). Original Magnification: ×200. The typical aspect of intra-tubular casts, tubular necrosis and tubular atrophy are respectively shown by asterisks, arrows and head arrows.
    Figure Legend Snippet: MV infusion protects SCID mice with cisplatin-induced AKI from tubular injury. Representative micrographs of renal histology of healthy SCID mice and of SCID mice treated with cisplatin and injected with vehicle alone or with MV pre-treated with RNase or with different regiments of MVs (single or multiple injections) and sacrificed at different time points (day 4, 14 and 21). Original Magnification: ×200. The typical aspect of intra-tubular casts, tubular necrosis and tubular atrophy are respectively shown by asterisks, arrows and head arrows.

    Techniques Used: Mouse Assay, Injection

    16) Product Images from "Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes"

    Article Title: Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0107351

    Full-length enrichment for library cloning and next generation sequencing (NGS). Full-length (blue line with 5′ cap) or truncated (short blue line without 5′ cap) mRNAs were reverse transcribed into first-strand cDNA using oligo-dT primers (red arrow). The mRNA:cDNA hybrid was treated with RNase I (scissor) to remove the single-stranded RNA that was not fully extended by the first-strand cDNA, followed by selection for full-length transcripts using Cap-antibody magnetic beads to enrich the full-length mRNA:cDNA. The full-length single-stranded DNA (FLssDNA) was eluted from beads and used for both cDNA library cloning (lower left) and NGS (lower right). For full-length library cloning, a double-stranded adaptor (green) was linked to the 5′ end of ssDNA. Second-strand cDNA synthesis was then carried out, followed by cloning into a vector. For NGS, the full-length enriched ssDNA was fragmented by sonication to target fragments in the range of 200–400 bp, followed by ligation of the double-stranded DNA sequencing adaptor mixture (purple) to 3′ and 5′ ends of ssDNA. To maintain the complexity of the library while enriching the full-length cDNA for NGS, the original polyA mRNA was also fragmented using RNAse III, followed by ligation of the double-stranded RNA sequencing adaptor mixture (brown) to 3′ and 5′ ends of mRNA. After first- and second-strand synthesis, the polyA and capped mRNA and polyA and non-capped mRNA samples were mixed in a 3∶1 ratio and applied to the downstream NGS procedure.
    Figure Legend Snippet: Full-length enrichment for library cloning and next generation sequencing (NGS). Full-length (blue line with 5′ cap) or truncated (short blue line without 5′ cap) mRNAs were reverse transcribed into first-strand cDNA using oligo-dT primers (red arrow). The mRNA:cDNA hybrid was treated with RNase I (scissor) to remove the single-stranded RNA that was not fully extended by the first-strand cDNA, followed by selection for full-length transcripts using Cap-antibody magnetic beads to enrich the full-length mRNA:cDNA. The full-length single-stranded DNA (FLssDNA) was eluted from beads and used for both cDNA library cloning (lower left) and NGS (lower right). For full-length library cloning, a double-stranded adaptor (green) was linked to the 5′ end of ssDNA. Second-strand cDNA synthesis was then carried out, followed by cloning into a vector. For NGS, the full-length enriched ssDNA was fragmented by sonication to target fragments in the range of 200–400 bp, followed by ligation of the double-stranded DNA sequencing adaptor mixture (purple) to 3′ and 5′ ends of ssDNA. To maintain the complexity of the library while enriching the full-length cDNA for NGS, the original polyA mRNA was also fragmented using RNAse III, followed by ligation of the double-stranded RNA sequencing adaptor mixture (brown) to 3′ and 5′ ends of mRNA. After first- and second-strand synthesis, the polyA and capped mRNA and polyA and non-capped mRNA samples were mixed in a 3∶1 ratio and applied to the downstream NGS procedure.

    Techniques Used: Clone Assay, Next-Generation Sequencing, Selection, Magnetic Beads, cDNA Library Assay, Plasmid Preparation, Sonication, Ligation, DNA Sequencing, RNA Sequencing Assay

    17) Product Images from "Differential amplicons (ΔAmp)—a new molecular method to assess RNA integrity"

    Article Title: Differential amplicons (ΔAmp)—a new molecular method to assess RNA integrity

    Journal: Biomolecular Detection and Quantification

    doi: 10.1016/j.bdq.2015.09.002

    RNase I treated HeLa cells. Cq and RQI plotted against [RNase I] (A). ΔΔAmp ERR (RNase) = ΔAmp ERR ([RNase]) − Δ ERR ([RNase] = 0) and RQI plotted against [RNase I] (B).
    Figure Legend Snippet: RNase I treated HeLa cells. Cq and RQI plotted against [RNase I] (A). ΔΔAmp ERR (RNase) = ΔAmp ERR ([RNase]) − Δ ERR ([RNase] = 0) and RQI plotted against [RNase I] (B).

    Techniques Used:

    18) Product Images from "Microvesicles Derived from Inflammation-Challenged Endothelial Cells Modulate Vascular Smooth Muscle Cell Functions"

    Article Title: Microvesicles Derived from Inflammation-Challenged Endothelial Cells Modulate Vascular Smooth Muscle Cell Functions

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2016.00692

    RNase digestion of EMV and effects of EMV and RNase- EMV on viability of HBVSMC. (A) Summarized data showing effective digestion of EMV total RNAs by RNase treatment. (B) Summary data showing that EMV promoted HBVSMC proliferation, and RNase-EMV was less effective. ** p
    Figure Legend Snippet: RNase digestion of EMV and effects of EMV and RNase- EMV on viability of HBVSMC. (A) Summarized data showing effective digestion of EMV total RNAs by RNase treatment. (B) Summary data showing that EMV promoted HBVSMC proliferation, and RNase-EMV was less effective. ** p

    Techniques Used:

    MiR-146a-5p expression in EMV and HBVSMC . Summary data showing effective digestion of miR-146a-5p in EMV by RNase treatment. ∧∧ p
    Figure Legend Snippet: MiR-146a-5p expression in EMV and HBVSMC . Summary data showing effective digestion of miR-146a-5p in EMV by RNase treatment. ∧∧ p

    Techniques Used: Expressing

    Effects of EMV and RNase-EMV on migration and expression of Mek1/2 and p-Erk1/2/Erk1/2 in HBVSMC. (A) Migration of HBVSMC treated with EMV, RNase-EMV or EMV+Mek1/2 inhibitor (PD0325901). (B) Expression of Mek1/2 and p-Erk1/2/Erk1/2. ** p
    Figure Legend Snippet: Effects of EMV and RNase-EMV on migration and expression of Mek1/2 and p-Erk1/2/Erk1/2 in HBVSMC. (A) Migration of HBVSMC treated with EMV, RNase-EMV or EMV+Mek1/2 inhibitor (PD0325901). (B) Expression of Mek1/2 and p-Erk1/2/Erk1/2. ** p

    Techniques Used: Migration, Expressing

    Effects of EMV and RNase-EMV on apoptosis and expression of cleaved caspase-3 and Bcl-2 expression in HBVSMC. (A) Apoptosis analysis by flow cytometry. (B) Apoptosis determined by Hoechst 33258 staining (Red arrows represent apoptotic cells, white arrows represent normal cells). Scale bar: 50 μm. (C) Protein levels of cleaved caspase-3 and Bcl-2 in HBVSMC. * p
    Figure Legend Snippet: Effects of EMV and RNase-EMV on apoptosis and expression of cleaved caspase-3 and Bcl-2 expression in HBVSMC. (A) Apoptosis analysis by flow cytometry. (B) Apoptosis determined by Hoechst 33258 staining (Red arrows represent apoptotic cells, white arrows represent normal cells). Scale bar: 50 μm. (C) Protein levels of cleaved caspase-3 and Bcl-2 in HBVSMC. * p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining

    19) Product Images from "RNA from a simple-tandem repeat is required for sperm maturation and male fertility in Drosophila melanogaster"

    Article Title: RNA from a simple-tandem repeat is required for sperm maturation and male fertility in Drosophila melanogaster

    Journal: eLife

    doi: 10.7554/eLife.48940

    AAGAG RNA foci contain single-stranded RNA and are not associated with R-loops. Confocal sections of embryonic nuclei in cycle 14 (with exception of left panel in ‘b’), nuclear periphery outlined in dotted circles. ( a ) No RNase control. ( b ) Treated with RNaseIII (left nucleus is cycle 12) ( c ) RNaseH ( d ) RNase1 and ( e ) RNaseA.
    Figure Legend Snippet: AAGAG RNA foci contain single-stranded RNA and are not associated with R-loops. Confocal sections of embryonic nuclei in cycle 14 (with exception of left panel in ‘b’), nuclear periphery outlined in dotted circles. ( a ) No RNase control. ( b ) Treated with RNaseIII (left nucleus is cycle 12) ( c ) RNaseH ( d ) RNase1 and ( e ) RNaseA.

    Techniques Used:

    20) Product Images from "Minute Virus of Canines NP1 Protein Interacts with the Cellular Factor CPSF6 To Regulate Viral Alternative RNA Processing"

    Article Title: Minute Virus of Canines NP1 Protein Interacts with the Cellular Factor CPSF6 To Regulate Viral Alternative RNA Processing

    Journal: Journal of Virology

    doi: 10.1128/JVI.01530-18

    CPSF6 affects MVC alternative RNA processing. (A) Transcription profile of MVC showing the P6 promoter, transcription starting site (TSS), splice donors (D) and acceptors (A), and proximal [(pA)p], and distal [(pA)d] polyadenylation sites. The annotated nucleotides delineate the boundaries of the transcription landmarks indicated within the MVC genome. The position of the RNase protection probes, 2A/3D (nt 2344 to 2550), 3A (nt 2910 to 3110), and (pA)p (nt 3107 to 3333), are indicated. NP1 and NS-66 mRNA species polyadenylated at (pA)p or (pA)d are shown. (B) RPAs of total RNA extracted 48 h following transfection of 293FT or CPSF6(-)293T cells with MVC Rep-Cap (lanes 1 and 2), IMVC-WT (lanes 3 and 4), and IMVC-5X (lanes 5 and 6) using the (pA)p probe. The sizes of the probe and various protected bands are shown on the left. The protected bands representing RNA species extending through (pA)p to (pA)d or cleaved at the various cleavage sites as described for (pA)d and (pA)p are indicated to the right. Quantifications below show the ratio of (pA)p/(pA)d RNAs from transfected CPSF6 knockout cells compared to the level in 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (C) Immunoprecipitation analysis reveals the association of CPSF6 and MVC NP1 in 293FT cells. Equal amounts of cell lysates were immunoprecipitated (IP) using protein-G magnetic beads (lane 2) or anti-HA magnetic beads (lane 3), as described in Materials and Methods, followed by immunoblotting with antibodies against CPSF6 and HA-tagged NP1. (D) RPAs of total RNA as described for panel B using the 3A probe. The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species (3Aunspl, unspliced at the third intron acceptor [3A]; 3Aspl, third intron acceptor spliced) are indicated to the right. Quantifications below show the ratio of spliced/unspliced RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (E) RPAs of total RNA as described for panels B and D using the 2A/3D probe (nt 2910 to 3100). The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species [(pA)d, read-through of the second intron acceptor (2A) and third intron donor (3D); 2Aspl/3Dun, the second intron acceptor spliced but third intron unspliced; 2Aspl/3Dspl, both second intron acceptor and third intron spliced] are indicated to the right. Quantifications below show the ratio of 2Aspl/3Dspl to read-through RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (F) 293FT or CPSF6(-)293T cells transfected with constructs described for panels B, D, and E were harvested and analyzed by immunoblotting using antibodies directed against CPSF6, NS, and NP1 (the individual epitopes are described in Materials and Methods). Immunoblotting for β-actin was used as loading control.
    Figure Legend Snippet: CPSF6 affects MVC alternative RNA processing. (A) Transcription profile of MVC showing the P6 promoter, transcription starting site (TSS), splice donors (D) and acceptors (A), and proximal [(pA)p], and distal [(pA)d] polyadenylation sites. The annotated nucleotides delineate the boundaries of the transcription landmarks indicated within the MVC genome. The position of the RNase protection probes, 2A/3D (nt 2344 to 2550), 3A (nt 2910 to 3110), and (pA)p (nt 3107 to 3333), are indicated. NP1 and NS-66 mRNA species polyadenylated at (pA)p or (pA)d are shown. (B) RPAs of total RNA extracted 48 h following transfection of 293FT or CPSF6(-)293T cells with MVC Rep-Cap (lanes 1 and 2), IMVC-WT (lanes 3 and 4), and IMVC-5X (lanes 5 and 6) using the (pA)p probe. The sizes of the probe and various protected bands are shown on the left. The protected bands representing RNA species extending through (pA)p to (pA)d or cleaved at the various cleavage sites as described for (pA)d and (pA)p are indicated to the right. Quantifications below show the ratio of (pA)p/(pA)d RNAs from transfected CPSF6 knockout cells compared to the level in 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (C) Immunoprecipitation analysis reveals the association of CPSF6 and MVC NP1 in 293FT cells. Equal amounts of cell lysates were immunoprecipitated (IP) using protein-G magnetic beads (lane 2) or anti-HA magnetic beads (lane 3), as described in Materials and Methods, followed by immunoblotting with antibodies against CPSF6 and HA-tagged NP1. (D) RPAs of total RNA as described for panel B using the 3A probe. The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species (3Aunspl, unspliced at the third intron acceptor [3A]; 3Aspl, third intron acceptor spliced) are indicated to the right. Quantifications below show the ratio of spliced/unspliced RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (E) RPAs of total RNA as described for panels B and D using the 2A/3D probe (nt 2910 to 3100). The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species [(pA)d, read-through of the second intron acceptor (2A) and third intron donor (3D); 2Aspl/3Dun, the second intron acceptor spliced but third intron unspliced; 2Aspl/3Dspl, both second intron acceptor and third intron spliced] are indicated to the right. Quantifications below show the ratio of 2Aspl/3Dspl to read-through RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (F) 293FT or CPSF6(-)293T cells transfected with constructs described for panels B, D, and E were harvested and analyzed by immunoblotting using antibodies directed against CPSF6, NS, and NP1 (the individual epitopes are described in Materials and Methods). Immunoblotting for β-actin was used as loading control.

    Techniques Used: Transfection, Knock-Out, Derivative Assay, Immunoprecipitation, Magnetic Beads, Construct

    21) Product Images from "Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins"

    Article Title: Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-20421-1

    Genetic cargo of mRPC derived extracellular vesicles. ( A ) A 1.5% denaturing agarose gel loaded with total RNA from mRPCs and EVs. Total RNA from EVs consisted primarily of species below 800 nucleotides (nt) lacking 28S and 18S rRNA. EVs were treated with RNase and no difference was detected when compared with non-treated EVs, indicating the RNA of EVs was enclosed within the vesicle membrane. ( B ) Transcription factors, a cell-cycle regulator and intermediate filaments were identified in both mRPCs and EVs included Pax6, Hes1, Sox2, Ki67, GFAP and Nestin. The transcription factors identified are collectively involved in facilitating mRPC multipotency, cell-cycle and fate specification during retinogenesis. GFP, GAPDH and β-actin mRNAs were also detected in mRPCs and EVs. Next, the presence of miRNAs with established expression and function during retinogensis were chosen for analysis. ( C ) Selected miRNA species analyzed included Let7d, miR-9, miR-182 and miR-204. U6 snRNA was used as control. Data presented were combined from four independent replicates.
    Figure Legend Snippet: Genetic cargo of mRPC derived extracellular vesicles. ( A ) A 1.5% denaturing agarose gel loaded with total RNA from mRPCs and EVs. Total RNA from EVs consisted primarily of species below 800 nucleotides (nt) lacking 28S and 18S rRNA. EVs were treated with RNase and no difference was detected when compared with non-treated EVs, indicating the RNA of EVs was enclosed within the vesicle membrane. ( B ) Transcription factors, a cell-cycle regulator and intermediate filaments were identified in both mRPCs and EVs included Pax6, Hes1, Sox2, Ki67, GFAP and Nestin. The transcription factors identified are collectively involved in facilitating mRPC multipotency, cell-cycle and fate specification during retinogenesis. GFP, GAPDH and β-actin mRNAs were also detected in mRPCs and EVs. Next, the presence of miRNAs with established expression and function during retinogensis were chosen for analysis. ( C ) Selected miRNA species analyzed included Let7d, miR-9, miR-182 and miR-204. U6 snRNA was used as control. Data presented were combined from four independent replicates.

    Techniques Used: Derivative Assay, Agarose Gel Electrophoresis, Expressing

    Extracellular vesicle internalization and transfer of GFP mRNA ( A ) Super resolution 3D reconstruction of GFP+ mRPC following 24 h incubation with PKH26 labeled extracellular vesicles. Red vesicles are visibly localized near the cell surface and within cytoplasm. In the XZ axis, GFP (green), EVs (red) and nuclei (blue, DAPI). ( B ) same as ( A ) with GFP (FITC) channel removed to increase visibility of PKH26 (TRITC) labeled EVs. Each panel contains three cross-sectional views (xy, xz, and yz). Scale: 5 µm. ( C ) RT-PCR analysis of GFP mRNA transfer between GFP+ mRPCs and non-GFP hRPCs. Non-GFP hRPCs served as negative control; GFP+ mRPCs served as postive control. GAPDH served as the internal control gene. EVs were treated using an RNase-Free DNase Set to remove DNA comtamination before cDNA synthesis. ( D ) Intensities of RT-PCR images were measured with ImageJ software and normalized to GAPDH. Relative levels of hRPC GFP after transfer of EVs is significantly higher than negative control.
    Figure Legend Snippet: Extracellular vesicle internalization and transfer of GFP mRNA ( A ) Super resolution 3D reconstruction of GFP+ mRPC following 24 h incubation with PKH26 labeled extracellular vesicles. Red vesicles are visibly localized near the cell surface and within cytoplasm. In the XZ axis, GFP (green), EVs (red) and nuclei (blue, DAPI). ( B ) same as ( A ) with GFP (FITC) channel removed to increase visibility of PKH26 (TRITC) labeled EVs. Each panel contains three cross-sectional views (xy, xz, and yz). Scale: 5 µm. ( C ) RT-PCR analysis of GFP mRNA transfer between GFP+ mRPCs and non-GFP hRPCs. Non-GFP hRPCs served as negative control; GFP+ mRPCs served as postive control. GAPDH served as the internal control gene. EVs were treated using an RNase-Free DNase Set to remove DNA comtamination before cDNA synthesis. ( D ) Intensities of RT-PCR images were measured with ImageJ software and normalized to GAPDH. Relative levels of hRPC GFP after transfer of EVs is significantly higher than negative control.

    Techniques Used: Incubation, Labeling, Reverse Transcription Polymerase Chain Reaction, Negative Control, Software

    22) Product Images from "Selective inhibition of carbonic anhydrase IX over carbonic anhydrase XII in breast cancer cells using benzene sulfonamides: Disconnect between activity and growth inhibition"

    Article Title: Selective inhibition of carbonic anhydrase IX over carbonic anhydrase XII in breast cancer cells using benzene sulfonamides: Disconnect between activity and growth inhibition

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0207417

    Effects of USBs on cell cycle transition in breast cancer cells. Cell cycle analysis was performed in UFH-001 (Panel A) and T47D (Panel B) cells treated with varying concentrations of USB compounds for 48 h, under normoxic conditions. Post treatment, cells were stained with propidium iodine containing RNase A. Cell cycle distribution data (gated for live, diploid cells only) was analyzed using the FCS Express Version 5 from De Novo Software from data obtained using FACS caliber. The images in A and B are representative of there repetitions and were generated by ModFit LT software. The percentage of breast cancer cells in G-phase (Panel C), all phases of the cell cycle in UFH-001 (Panel D) and T47D cells (Panel E), were quantified using Prism. Data are represented as mean of at least three independent experiments ± SEM and NC = negative control.
    Figure Legend Snippet: Effects of USBs on cell cycle transition in breast cancer cells. Cell cycle analysis was performed in UFH-001 (Panel A) and T47D (Panel B) cells treated with varying concentrations of USB compounds for 48 h, under normoxic conditions. Post treatment, cells were stained with propidium iodine containing RNase A. Cell cycle distribution data (gated for live, diploid cells only) was analyzed using the FCS Express Version 5 from De Novo Software from data obtained using FACS caliber. The images in A and B are representative of there repetitions and were generated by ModFit LT software. The percentage of breast cancer cells in G-phase (Panel C), all phases of the cell cycle in UFH-001 (Panel D) and T47D cells (Panel E), were quantified using Prism. Data are represented as mean of at least three independent experiments ± SEM and NC = negative control.

    Techniques Used: Cell Cycle Assay, Staining, Software, FACS, Generated, Negative Control

    23) Product Images from "Strain-Specific Role of RNAs in Prion Replication"

    Article Title: Strain-Specific Role of RNAs in Prion Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.01286-12

    Addition of RNA enhances prion in vitro amplification. (A) Total RNA enhances ME7 conversion efficiency. Normal brain homogenates (NBHs; 10%) were treated with RNase A or RNase Out. After treatment, RNase A was inhibited with RNase Out (parts 2 and 3)
    Figure Legend Snippet: Addition of RNA enhances prion in vitro amplification. (A) Total RNA enhances ME7 conversion efficiency. Normal brain homogenates (NBHs; 10%) were treated with RNase A or RNase Out. After treatment, RNase A was inhibited with RNase Out (parts 2 and 3)

    Techniques Used: In Vitro, Amplification

    24) Product Images from "The Transcriptional Repressor HBP1 Is a Target of the p38 Mitogen-Activated Protein Kinase Pathway in Cell Cycle Regulation"

    Article Title: The Transcriptional Repressor HBP1 Is a Target of the p38 Mitogen-Activated Protein Kinase Pathway in Cell Cycle Regulation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.23.8890-8901.2003

    HBP1 protein half-life, but not mRNA, is affected by p38 MAP kinase activity. (A) HBP1 mRNA expression level was not significantly affected by p38 MAP kinase activity . The level of HBP1 mRNA during fasting and refeeding (F/R) treatment was scored by RNase protection assay with total RNA that was isolated from liver tissue. 18S RNA was used as an RNA loading control. The results were quantified with a PhosphorImager and normalized for 18S RNA. These data are an average of two experiments that did not vary more than 10%. F, fasted mice. (B) p38 MAP kinase inhibition decreases HBP1 protein stability. The HA-HBP1 expression construct was transfected into a single 150-mm 2 plate of 293T cells, which was then distributed to multiple smaller dishes to ensure uniform expression of HA-HBP1 across all plates. Cells were placed in methionine-free media, treated with 10 μM SB203580 or with vehicle, pulsed with [ 35 S]methionine for 15 min and then chased with excess unlabeled methionine-supplemented media. Cells were harvested at the indicated timepoints. 35 S-labeled HBP1 was immunoprecipitated with anti-HA antibody. The labeled HBP1 protein was quantitated by phosphorimager analysis after SDS-10% PAGE (bottom panel). The decay of HBP1 followed the expected first order kinetics. The slope of the decay line was calculated by standard linear regression, and the protein half-life was determined accordingly (top panel) (see Results). In the presence of SB203580, the HBP1 half-life was 0.61 ± 0.13 h, compared to 1.47 ± 0.14 h for untreated cells ( P
    Figure Legend Snippet: HBP1 protein half-life, but not mRNA, is affected by p38 MAP kinase activity. (A) HBP1 mRNA expression level was not significantly affected by p38 MAP kinase activity . The level of HBP1 mRNA during fasting and refeeding (F/R) treatment was scored by RNase protection assay with total RNA that was isolated from liver tissue. 18S RNA was used as an RNA loading control. The results were quantified with a PhosphorImager and normalized for 18S RNA. These data are an average of two experiments that did not vary more than 10%. F, fasted mice. (B) p38 MAP kinase inhibition decreases HBP1 protein stability. The HA-HBP1 expression construct was transfected into a single 150-mm 2 plate of 293T cells, which was then distributed to multiple smaller dishes to ensure uniform expression of HA-HBP1 across all plates. Cells were placed in methionine-free media, treated with 10 μM SB203580 or with vehicle, pulsed with [ 35 S]methionine for 15 min and then chased with excess unlabeled methionine-supplemented media. Cells were harvested at the indicated timepoints. 35 S-labeled HBP1 was immunoprecipitated with anti-HA antibody. The labeled HBP1 protein was quantitated by phosphorimager analysis after SDS-10% PAGE (bottom panel). The decay of HBP1 followed the expected first order kinetics. The slope of the decay line was calculated by standard linear regression, and the protein half-life was determined accordingly (top panel) (see Results). In the presence of SB203580, the HBP1 half-life was 0.61 ± 0.13 h, compared to 1.47 ± 0.14 h for untreated cells ( P

    Techniques Used: Activity Assay, Expressing, Rnase Protection Assay, Isolation, Mouse Assay, Inhibition, Construct, Transfection, Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis

    25) Product Images from "Selective inhibition of carbonic anhydrase IX over carbonic anhydrase XII in breast cancer cells using benzene sulfonamides: Disconnect between activity and growth inhibition"

    Article Title: Selective inhibition of carbonic anhydrase IX over carbonic anhydrase XII in breast cancer cells using benzene sulfonamides: Disconnect between activity and growth inhibition

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0207417

    Effects of USBs on cell cycle transition in breast cancer cells. Cell cycle analysis was performed in UFH-001 (Panel A) and T47D (Panel B) cells treated with varying concentrations of USB compounds for 48 h, under normoxic conditions. Post treatment, cells were stained with propidium iodine containing RNase A. Cell cycle distribution data (gated for live, diploid cells only) was analyzed using the FCS Express Version 5 from De Novo Software from data obtained using FACS caliber. The images in A and B are representative of there repetitions and were generated by ModFit LT software. The percentage of breast cancer cells in G-phase (Panel C), all phases of the cell cycle in UFH-001 (Panel D) and T47D cells (Panel E), were quantified using Prism. Data are represented as mean of at least three independent experiments ± SEM and NC = negative control.
    Figure Legend Snippet: Effects of USBs on cell cycle transition in breast cancer cells. Cell cycle analysis was performed in UFH-001 (Panel A) and T47D (Panel B) cells treated with varying concentrations of USB compounds for 48 h, under normoxic conditions. Post treatment, cells were stained with propidium iodine containing RNase A. Cell cycle distribution data (gated for live, diploid cells only) was analyzed using the FCS Express Version 5 from De Novo Software from data obtained using FACS caliber. The images in A and B are representative of there repetitions and were generated by ModFit LT software. The percentage of breast cancer cells in G-phase (Panel C), all phases of the cell cycle in UFH-001 (Panel D) and T47D cells (Panel E), were quantified using Prism. Data are represented as mean of at least three independent experiments ± SEM and NC = negative control.

    Techniques Used: Cell Cycle Assay, Staining, Software, FACS, Generated, Negative Control

    26) Product Images from "Human mesenchymal stromal cell-derived extracellular vesicles alleviate renal ischemic reperfusion injury and enhance angiogenesis in rats"

    Article Title: Human mesenchymal stromal cell-derived extracellular vesicles alleviate renal ischemic reperfusion injury and enhance angiogenesis in rats

    Journal: American Journal of Translational Research

    doi:

    MSCs-EVs entered ischemia-reperfusion injured renal tissues and restored tubular injury. A. Representative renal sections from Sham, Vehicle, EVs and EVs-RNase groups (PAS Original magnification ×200); B. Renal injury scoring and quantitative
    Figure Legend Snippet: MSCs-EVs entered ischemia-reperfusion injured renal tissues and restored tubular injury. A. Representative renal sections from Sham, Vehicle, EVs and EVs-RNase groups (PAS Original magnification ×200); B. Renal injury scoring and quantitative

    Techniques Used:

    VEGF was up-regulated by MSC-EVs and HIF-1α was downregulated. A. Representative micrograph of HIF-1α staining (IHC, ×200) from Sham, Vehicle, EVs and EVs-RNase groups. B. Protein levels of HIF-1α and VEGF in renal tissues
    Figure Legend Snippet: VEGF was up-regulated by MSC-EVs and HIF-1α was downregulated. A. Representative micrograph of HIF-1α staining (IHC, ×200) from Sham, Vehicle, EVs and EVs-RNase groups. B. Protein levels of HIF-1α and VEGF in renal tissues

    Techniques Used: Staining, Immunohistochemistry

    MSCs-EVs enhanced cell proliferation and reduced apoptosis at 24 h after reperfusion. A. Representative micrograph of TUNEL staining (×200) and Ki67 staining (IHC, ×200) from Sham, Vehicle, EVs and EVs-RNase groups. B, C. Quantitative
    Figure Legend Snippet: MSCs-EVs enhanced cell proliferation and reduced apoptosis at 24 h after reperfusion. A. Representative micrograph of TUNEL staining (×200) and Ki67 staining (IHC, ×200) from Sham, Vehicle, EVs and EVs-RNase groups. B, C. Quantitative

    Techniques Used: TUNEL Assay, Staining, Immunohistochemistry

    27) Product Images from "Microvesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Facilitate Tubular Epithelial Cell Dedifferentiation and Growth via Hepatocyte Growth Factor Induction"

    Article Title: Microvesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Facilitate Tubular Epithelial Cell Dedifferentiation and Growth via Hepatocyte Growth Factor Induction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0121534

    Either in vivo or in vitro, human HGF mRNA present in MVs enters injured rat tubular cells and is translated into the corresponding protein. (A) Human HGF gene expression in cultured rat tubular cells. The human HGF gene transcript was detectable in MVs and in the cells of origin. After 24 h of incubation with MVs, the human HGF mRNA was present in injured rat tubular cells, whereas human HGF mRNA was absent in cells incubated with vehicle or with RNase-MVs. The Ct for human HGF and β-actin (rat or human) was determined for each sample. The data were collected from 5 independent experiments. TECs: tubular epithelial cells. (B) Human HGF gene expression in rat kidney tissues. The human HGF mRNA was not detected in the affected kidney tissues of MV-treated AKI animals at any given points in time. The Ct for human HGF and rat β-actin was determined for each kidney sample. The data were collected from 3 samples for each group. (C) Human HGF in vitro staining. After MV exposure for 24 or 48 h, a few injured rat tubular cells displayed human HGF-positive expression in the cytoplasm. As a control, no positive staining was observed in cells exposed to vehicle or RNase-MVs. Magnification, ×20. (D) Human HGF in vivo staining. At 24 or 48 h following MV administration, the human HGF protein was detected in a few tubular cells. No positive-stained kidney cells were identifiable in animals treated with vehicle or with RNase-MVs. Magnification, ×40. AKI: acute kidney injury.
    Figure Legend Snippet: Either in vivo or in vitro, human HGF mRNA present in MVs enters injured rat tubular cells and is translated into the corresponding protein. (A) Human HGF gene expression in cultured rat tubular cells. The human HGF gene transcript was detectable in MVs and in the cells of origin. After 24 h of incubation with MVs, the human HGF mRNA was present in injured rat tubular cells, whereas human HGF mRNA was absent in cells incubated with vehicle or with RNase-MVs. The Ct for human HGF and β-actin (rat or human) was determined for each sample. The data were collected from 5 independent experiments. TECs: tubular epithelial cells. (B) Human HGF gene expression in rat kidney tissues. The human HGF mRNA was not detected in the affected kidney tissues of MV-treated AKI animals at any given points in time. The Ct for human HGF and rat β-actin was determined for each kidney sample. The data were collected from 3 samples for each group. (C) Human HGF in vitro staining. After MV exposure for 24 or 48 h, a few injured rat tubular cells displayed human HGF-positive expression in the cytoplasm. As a control, no positive staining was observed in cells exposed to vehicle or RNase-MVs. Magnification, ×20. (D) Human HGF in vivo staining. At 24 or 48 h following MV administration, the human HGF protein was detected in a few tubular cells. No positive-stained kidney cells were identifiable in animals treated with vehicle or with RNase-MVs. Magnification, ×40. AKI: acute kidney injury.

    Techniques Used: In Vivo, In Vitro, Expressing, Cell Culture, Incubation, Staining

    At 48 h post-injury, kidney HGF gene and protein expression is substantially enhanced by MV administration. (A)-(D) HGF gene expression in injured kidney tissues. MV administration led to a significant up-regulation of kidney HGF gene expression. The examination of rat HGF expression in kidney tissues using species-specific primers also indicated a similar result. As negative controls, no rat HGF mRNA was identified in MVs or in the cells of origin (hUC-MSCs). RNase pretreatment abolished the effect of MVs. By contrast, EGF, IGF-1 or TGFβ1 gene expression was not altered by MV administration. Gene expression levels in sham-treated samples were regarded as the baseline levels (dotted line). The relative expression levels of each gene were calculated using the 2−ΔΔCt method. The data were collected from 6 rats for each experimental condition. *P
    Figure Legend Snippet: At 48 h post-injury, kidney HGF gene and protein expression is substantially enhanced by MV administration. (A)-(D) HGF gene expression in injured kidney tissues. MV administration led to a significant up-regulation of kidney HGF gene expression. The examination of rat HGF expression in kidney tissues using species-specific primers also indicated a similar result. As negative controls, no rat HGF mRNA was identified in MVs or in the cells of origin (hUC-MSCs). RNase pretreatment abolished the effect of MVs. By contrast, EGF, IGF-1 or TGFβ1 gene expression was not altered by MV administration. Gene expression levels in sham-treated samples were regarded as the baseline levels (dotted line). The relative expression levels of each gene were calculated using the 2−ΔΔCt method. The data were collected from 6 rats for each experimental condition. *P

    Techniques Used: Expressing

    MV administration reverses the abnormal kidney structure and function elicited by AKI at 2 wk post-injury. (A) Representative micrographs of injured kidneys. In AKI animals treated with RNase-MVs or vehicle, the damaged kidneys display a mottled color, in contrast to a uniform color on the surfaces of the kidneys from MV-treated animals. (B) Representative micrographs illustrating α-SMA staining and Masson’s tri-chrome staining. Weaker positive staining was observed for α-SMA and for collagen on kidney sections from AKI animals receiving MV treatment compared with animals treated with RNase-MVs or vehicle. Magnification, ×40. (C) Serum creatinine value at 2 wk post-injury. Ischemic injury led to a significant increase in the serum creatinine level at 2 wk post-injury, which was inhibited by MV treatment. All quantitative data were obtained from 6 different animals for each experimental condition. *P
    Figure Legend Snippet: MV administration reverses the abnormal kidney structure and function elicited by AKI at 2 wk post-injury. (A) Representative micrographs of injured kidneys. In AKI animals treated with RNase-MVs or vehicle, the damaged kidneys display a mottled color, in contrast to a uniform color on the surfaces of the kidneys from MV-treated animals. (B) Representative micrographs illustrating α-SMA staining and Masson’s tri-chrome staining. Weaker positive staining was observed for α-SMA and for collagen on kidney sections from AKI animals receiving MV treatment compared with animals treated with RNase-MVs or vehicle. Magnification, ×40. (C) Serum creatinine value at 2 wk post-injury. Ischemic injury led to a significant increase in the serum creatinine level at 2 wk post-injury, which was inhibited by MV treatment. All quantitative data were obtained from 6 different animals for each experimental condition. *P

    Techniques Used: Staining

    MV administration promotes tubular cell dedifferentiation and proliferation at 48 h post-injury, whereas cell apoptosis is inhibited. Representative micrographs showing vimentin, PCNA and TUNEL staining of tubular cells. Immuno-staining for vimentin and PCNA proteins, which are indictors for tubular cell dedifferentiation and cell proliferation, respectively, was employed. TUNEL staining was used to detect cell apoptosis. In contrast to the rats treated with vehicle or with RNase-MVs, the rats receiving MV treatment displayed more PCNA- and vimentin-positive stained tubular cells and fewer TUNEL-positive cells on kidney tissue sections. Magnification, ×40.
    Figure Legend Snippet: MV administration promotes tubular cell dedifferentiation and proliferation at 48 h post-injury, whereas cell apoptosis is inhibited. Representative micrographs showing vimentin, PCNA and TUNEL staining of tubular cells. Immuno-staining for vimentin and PCNA proteins, which are indictors for tubular cell dedifferentiation and cell proliferation, respectively, was employed. TUNEL staining was used to detect cell apoptosis. In contrast to the rats treated with vehicle or with RNase-MVs, the rats receiving MV treatment displayed more PCNA- and vimentin-positive stained tubular cells and fewer TUNEL-positive cells on kidney tissue sections. Magnification, ×40.

    Techniques Used: TUNEL Assay, Staining, Immunostaining

    28) Product Images from "Minimal-length Synthetic shRNAs Formulated with Lipid Nanoparticles are Potent Inhibitors of Hepatitis C Virus IRES-linked Gene Expression in Mice"

    Article Title: Minimal-length Synthetic shRNAs Formulated with Lipid Nanoparticles are Potent Inhibitors of Hepatitis C Virus IRES-linked Gene Expression in Mice

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1038/mtna.2013.50

    Liver uptake of LNP-SG220 in CD1 mice. ( a ) Denaturing 10% PAGE analysis of RNase protection assay to monitor SG220 present in livers of CD1 mice at the indicated doses and time points. A mouse injected with PBS only was used as a control (PBS). Bands corresponding to undigested probe and probe protected after RNase A/T1 digestion are labeled. For quantification, the amounts of SG220 indicated in the last four lanes were spiked into 50 µg of total liver RNA from untreated mice to generate a calibration curve for the quantification of the liver uptake of SG220. PP, protected probe; UP, undigested full-length probe. ( b ) Plots of background corrected counts ( 32 P) for individual mice for dosing groups 0.5 and 2.5 mg/kg. LNP, lipid nanoparticles.
    Figure Legend Snippet: Liver uptake of LNP-SG220 in CD1 mice. ( a ) Denaturing 10% PAGE analysis of RNase protection assay to monitor SG220 present in livers of CD1 mice at the indicated doses and time points. A mouse injected with PBS only was used as a control (PBS). Bands corresponding to undigested probe and probe protected after RNase A/T1 digestion are labeled. For quantification, the amounts of SG220 indicated in the last four lanes were spiked into 50 µg of total liver RNA from untreated mice to generate a calibration curve for the quantification of the liver uptake of SG220. PP, protected probe; UP, undigested full-length probe. ( b ) Plots of background corrected counts ( 32 P) for individual mice for dosing groups 0.5 and 2.5 mg/kg. LNP, lipid nanoparticles.

    Techniques Used: Mouse Assay, Polyacrylamide Gel Electrophoresis, Rnase Protection Assay, Injection, Labeling

    29) Product Images from "Mesenchymal Stem Cell-Derived Microvesicles Protect Against Acute Tubular Injury"

    Article Title: Mesenchymal Stem Cell-Derived Microvesicles Protect Against Acute Tubular Injury

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2008070798

    Cytofluorimetric characterization of mesenchymal stem cell (MSC)-derived microvesicles (MVs). Representative FACS analyses of MVs (A) and MVs treated with RNase (B) showing the size (with 1-, 2- and −4-μm beads used as internal size standards) and the expression of CD44, CD29, α-4 integrin, α-5 integrin, CD73, α-6 integrin, and HLA-class I (thick lines) surface molecules. Dot lines indicate the isotypic controls. Ten different MV preparations were analyzed with similar results. In the CD44, CD29, α-4 integrin, α-5 integrin, and CD73 experiments, the Kolmogrov-Smirnov statistical analyses between relevant antibodies and the isotypic control was significant ( P
    Figure Legend Snippet: Cytofluorimetric characterization of mesenchymal stem cell (MSC)-derived microvesicles (MVs). Representative FACS analyses of MVs (A) and MVs treated with RNase (B) showing the size (with 1-, 2- and −4-μm beads used as internal size standards) and the expression of CD44, CD29, α-4 integrin, α-5 integrin, CD73, α-6 integrin, and HLA-class I (thick lines) surface molecules. Dot lines indicate the isotypic controls. Ten different MV preparations were analyzed with similar results. In the CD44, CD29, α-4 integrin, α-5 integrin, and CD73 experiments, the Kolmogrov-Smirnov statistical analyses between relevant antibodies and the isotypic control was significant ( P

    Techniques Used: Derivative Assay, FACS, Expressing

    Proliferative and anti-apoptotic effects of mesenchymal stem cell (MSC)-derived microvesicles (MVs). (A) 10 μM BrdU was added to 4000 cells/well (TECs) into 96-well plates incubated for 48 h in DMEM deprived of FCS in the presence of vehicle alone or of different doses of MVs (black bars) or of RNase-treated MVs (white bars) or MVs pretreated with trypsin or with 100 μg/ml of sHA. EGF-induced (10 ng/ml) proliferation was also evaluated in TECs incubated or not with RNase-pretreated MVs (30 μg/ml). Results are expressed as mean ± SD of six different experiments. ANOVA with Newmann-Keuls multicomparison test was performed; * P
    Figure Legend Snippet: Proliferative and anti-apoptotic effects of mesenchymal stem cell (MSC)-derived microvesicles (MVs). (A) 10 μM BrdU was added to 4000 cells/well (TECs) into 96-well plates incubated for 48 h in DMEM deprived of FCS in the presence of vehicle alone or of different doses of MVs (black bars) or of RNase-treated MVs (white bars) or MVs pretreated with trypsin or with 100 μg/ml of sHA. EGF-induced (10 ng/ml) proliferation was also evaluated in TECs incubated or not with RNase-pretreated MVs (30 μg/ml). Results are expressed as mean ± SD of six different experiments. ANOVA with Newmann-Keuls multicomparison test was performed; * P

    Techniques Used: Derivative Assay, Incubation

    Incorporation of MVs in tubular epithelial cells (TECs). (A) Representative micrographs of internalization by TECs (30 min at 37 °C) of microvesicles (MVs) labeled with PKH26 preincubated or not with trypsin (0.5 mM) (Try); or with 100 μg/ml of sHA; or with 1 μg/ml blocking monoclonal antibody against CD44, CD29, and α4 integrin. Three experiments were performed with similar results. (B) Representative FACS analyses of internalization, after 30 min of incubation at 37 °C, by TECs of MVs labeled with PKH26 (black curves) preincubated or not with trypsin (Try) or with 100 μg/ml of sHA or with 1 μg/ml blocking monoclonal antibodies against CD44, CD29, and α4 integrin. Black curves indicate the internalization of untreated MVs. In the first panel, dot curve indicates the negative control (cells not incubated with MVs); red curve indicates the MVs treated with RNase and labeled with PKH26. In the other panels, dot curves indicate internalization of MVs after pretreatment with trypsin or incubation with blocking antibodies or sHA. Three experiments were performed with similar results.
    Figure Legend Snippet: Incorporation of MVs in tubular epithelial cells (TECs). (A) Representative micrographs of internalization by TECs (30 min at 37 °C) of microvesicles (MVs) labeled with PKH26 preincubated or not with trypsin (0.5 mM) (Try); or with 100 μg/ml of sHA; or with 1 μg/ml blocking monoclonal antibody against CD44, CD29, and α4 integrin. Three experiments were performed with similar results. (B) Representative FACS analyses of internalization, after 30 min of incubation at 37 °C, by TECs of MVs labeled with PKH26 (black curves) preincubated or not with trypsin (Try) or with 100 μg/ml of sHA or with 1 μg/ml blocking monoclonal antibodies against CD44, CD29, and α4 integrin. Black curves indicate the internalization of untreated MVs. In the first panel, dot curve indicates the negative control (cells not incubated with MVs); red curve indicates the MVs treated with RNase and labeled with PKH26. In the other panels, dot curves indicate internalization of MVs after pretreatment with trypsin or incubation with blocking antibodies or sHA. Three experiments were performed with similar results.

    Techniques Used: Labeling, Blocking Assay, FACS, Incubation, Negative Control

    Effects of intravenous injection of microvesicles (MVs) or mesenchymal stem cells (MSCs) into acute kidney injury (AKI) mice. Mice were given intramuscular injection of 8 ml/kg of 50% glycerol on day 0, followed by intravenous injection of MVs or RNase-treated MVs or MSCs or vehicle as control on day 3. (A and B) Creatinine and blood urea nitrogen values at the beginning of the experiments and on day 3, 5, 8, and 15 after glycerol administration. ANOVA with Dunnet's multicomparison test: * P
    Figure Legend Snippet: Effects of intravenous injection of microvesicles (MVs) or mesenchymal stem cells (MSCs) into acute kidney injury (AKI) mice. Mice were given intramuscular injection of 8 ml/kg of 50% glycerol on day 0, followed by intravenous injection of MVs or RNase-treated MVs or MSCs or vehicle as control on day 3. (A and B) Creatinine and blood urea nitrogen values at the beginning of the experiments and on day 3, 5, 8, and 15 after glycerol administration. ANOVA with Dunnet's multicomparison test: * P

    Techniques Used: Injection, Mouse Assay

    Schematic representation of the protocol of glycerol induced acute kidney injury (AKI) and treatment with mesenchymal stem cells (MSCs) or MSC-derived microvesicles (MVs). Glycerol was injected intramuscularly at time 0; the arrow at day 3 indicate the administration of 75,000 MSCs; or 15 μg of MSC-derived MVs; or MSC-derived MVs treated with RNase, trypsin, or sHA; or fibroblast-derived MVs; or vehicle alone; the subsequent arrows indicate the time of sacrifice.
    Figure Legend Snippet: Schematic representation of the protocol of glycerol induced acute kidney injury (AKI) and treatment with mesenchymal stem cells (MSCs) or MSC-derived microvesicles (MVs). Glycerol was injected intramuscularly at time 0; the arrow at day 3 indicate the administration of 75,000 MSCs; or 15 μg of MSC-derived MVs; or MSC-derived MVs treated with RNase, trypsin, or sHA; or fibroblast-derived MVs; or vehicle alone; the subsequent arrows indicate the time of sacrifice.

    Techniques Used: Derivative Assay, Injection

    30) Product Images from "Tumor-infiltrating DCs suppress nucleic acid\u2013mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1"

    Article Title: Tumor-infiltrating DCs suppress nucleic acid\u2013mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1

    Journal: Nature immunology

    doi: 10.1038/ni.2376

    TIM-3 impedes the antitumor effects of chemotherapy. ( a ) Quantification of IFN-β1 and IL-12 mRNA in TIM-3 + BMDCs cultured alone (UT) or together with apoptotic CDDP-treated MC38 cells with (MC38 DNase + RNase) or without (MC38) pretreatment with
    Figure Legend Snippet: TIM-3 impedes the antitumor effects of chemotherapy. ( a ) Quantification of IFN-β1 and IL-12 mRNA in TIM-3 + BMDCs cultured alone (UT) or together with apoptotic CDDP-treated MC38 cells with (MC38 DNase + RNase) or without (MC38) pretreatment with

    Techniques Used: Cell Culture

    31) Product Images from "Exosomes from Drug-Resistant Breast Cancer Cells Transmit Chemoresistance by a Horizontal Transfer of MicroRNAs"

    Article Title: Exosomes from Drug-Resistant Breast Cancer Cells Transmit Chemoresistance by a Horizontal Transfer of MicroRNAs

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095240

    Effects of different exosomes. (A) Flow cytometry of cell cycle distribution was analyzed after MCF-7/S was incubated with vehicle, S/exo, D/exo, RNase S/exo, and RNase D/exo for 72 h. Data are expressed as the mean ± SD, n = 3: * P
    Figure Legend Snippet: Effects of different exosomes. (A) Flow cytometry of cell cycle distribution was analyzed after MCF-7/S was incubated with vehicle, S/exo, D/exo, RNase S/exo, and RNase D/exo for 72 h. Data are expressed as the mean ± SD, n = 3: * P

    Techniques Used: Flow Cytometry, Cytometry, Incubation

    32) Product Images from "Inflammation potentiates miR-939 expression and packaging into small extracellular vesicles"

    Article Title: Inflammation potentiates miR-939 expression and packaging into small extracellular vesicles

    Journal: Journal of Extracellular Vesicles

    doi: 10.1080/20013078.2019.1650595

    Electroporation efficiently packages miRNA within THP-1 cell-derived sEVs. (a). Ten micrograms of THP-1 cell-derived sEVs were electroporated with 30 picomoles of miR-939 at different voltages and the amount of miR-939 incorporated within sEVs was estimated by Taqman-based qRT-PCR. (b). The sEVs subjected to passive incubation or 2 kV electroporation with miR-939 were subsequently treated with a combination of RNase A and detergents to evaluate the surface versus internal incorporation of miRNA. miR-939 within electroporated sEVs were resistant to RNase A digestion, while miR-939 incubated passively with sEVs were significantly degraded. Taqman-based qRT-PCR was used to determine the miR-939 content (normalized to miR-223) and significance was determined by one-way ANOVA with Sidak’s multiple comparison test ***, ### p
    Figure Legend Snippet: Electroporation efficiently packages miRNA within THP-1 cell-derived sEVs. (a). Ten micrograms of THP-1 cell-derived sEVs were electroporated with 30 picomoles of miR-939 at different voltages and the amount of miR-939 incorporated within sEVs was estimated by Taqman-based qRT-PCR. (b). The sEVs subjected to passive incubation or 2 kV electroporation with miR-939 were subsequently treated with a combination of RNase A and detergents to evaluate the surface versus internal incorporation of miRNA. miR-939 within electroporated sEVs were resistant to RNase A digestion, while miR-939 incubated passively with sEVs were significantly degraded. Taqman-based qRT-PCR was used to determine the miR-939 content (normalized to miR-223) and significance was determined by one-way ANOVA with Sidak’s multiple comparison test ***, ### p

    Techniques Used: Electroporation, Derivative Assay, Quantitative RT-PCR, Incubation

    33) Product Images from "Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria"

    Article Title: Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria

    Journal: bioRxiv

    doi: 10.1101/2020.10.25.354225

    Statistical and in vitro biochemical analysis of G1 pauses. a, Information content (Ri) for −10LR (−10-like region) encoded by all σ 70 -Δ greAB G1 pauses as a function of its distance from TSS. The second base in the −10-like hexamer marked the location of the −10LR. The highest Ri of the hexamers ranging from −1 to +2 was adopted and assigned to −10LR (n = 3099). b, Boxplot compares the Ri of −10LR for proximal G1p and distal G1d pauses. All σ 70 promoters from RegulonDB with a labeled −10 element were used as a control (n = 950). c, d, Read length distribution at G1p and G1d pauses, respectively. Ratio of reads, number of reads with specific length(es)/number of total reads. The cartoon on the top depict the backtracked translocation states of G1d complexes based on a significant difference of their read lengths. Note, that the short ≤15-nt RNAs detected at most G1p pauses were due to the close proximity of G1p pauses to TSS that precluded determination of translocation state of G1p complexes by treatment with RNase I. e, f, RNET-seq and RNA-seq profiles of two representative genomic regions containing G1p and G1d pauses identified by RNET-seq at mraZ and yieE promoters. The first 20 nt of mraZ and yieE transcripts are shown. The red capital letters and arrows indicate the TSS and the pause peaks from RNET-seq data. g, h, In vitro validation of the σ 70 -dependent G1p/G1d pauses at mraZ and yieE promoters. The left panel shows nascent RNA in the paused complexes obtained in the presence and absence of GreA or GreB. Immobilization on streptavidin beads through 5’-biotin DNA was used to confirm integrity of the RNA-labeled paused complexes (right panel). Eσ 70 with His-tagged σ 70 was used for the assay confirming presence of σ 70 in the paused complexes. RO, run-off transcripts; St, streptavidin; Ni, Ni 2+ -NTA agarose; S, supernatant; P, pellet. i, Sequence logo for σ 70 -ΔgreAB G1p and G1d promoters and for σ 70 promoters from RegulonDB. The DNA sequences were aligned relative to the TSS. Only the strongest pause was used for analysis of the TSSs following multiple pause sites. Coordinate “0” represents TSS (commonly marked as the +1 site) in the sequence logo, otherwise the standard “+1” TSS nomenclature was used. −10R, −10 promoter element; tssR, region surrounding TSS; −10LR, −10-like region; spacer, spacing region between − 10R and TSS. j, Boxplot comparing Ri of the −10 elements for G1p (top, n = 1069) and G1d (bottom, n = 407) promoters; −10R of the same numbers of randomly chosen promoters were used as a control. k, Heatmap showing correlation between distribution of spacer length and information content (Ri) of the promoter −10 element for all σ 70 promoters (top), promoters containing G1p (middle) and G1d (bottom) pauses. The two-tailed Mann-Whitney U -test was used for the statistical analysis shown above.
    Figure Legend Snippet: Statistical and in vitro biochemical analysis of G1 pauses. a, Information content (Ri) for −10LR (−10-like region) encoded by all σ 70 -Δ greAB G1 pauses as a function of its distance from TSS. The second base in the −10-like hexamer marked the location of the −10LR. The highest Ri of the hexamers ranging from −1 to +2 was adopted and assigned to −10LR (n = 3099). b, Boxplot compares the Ri of −10LR for proximal G1p and distal G1d pauses. All σ 70 promoters from RegulonDB with a labeled −10 element were used as a control (n = 950). c, d, Read length distribution at G1p and G1d pauses, respectively. Ratio of reads, number of reads with specific length(es)/number of total reads. The cartoon on the top depict the backtracked translocation states of G1d complexes based on a significant difference of their read lengths. Note, that the short ≤15-nt RNAs detected at most G1p pauses were due to the close proximity of G1p pauses to TSS that precluded determination of translocation state of G1p complexes by treatment with RNase I. e, f, RNET-seq and RNA-seq profiles of two representative genomic regions containing G1p and G1d pauses identified by RNET-seq at mraZ and yieE promoters. The first 20 nt of mraZ and yieE transcripts are shown. The red capital letters and arrows indicate the TSS and the pause peaks from RNET-seq data. g, h, In vitro validation of the σ 70 -dependent G1p/G1d pauses at mraZ and yieE promoters. The left panel shows nascent RNA in the paused complexes obtained in the presence and absence of GreA or GreB. Immobilization on streptavidin beads through 5’-biotin DNA was used to confirm integrity of the RNA-labeled paused complexes (right panel). Eσ 70 with His-tagged σ 70 was used for the assay confirming presence of σ 70 in the paused complexes. RO, run-off transcripts; St, streptavidin; Ni, Ni 2+ -NTA agarose; S, supernatant; P, pellet. i, Sequence logo for σ 70 -ΔgreAB G1p and G1d promoters and for σ 70 promoters from RegulonDB. The DNA sequences were aligned relative to the TSS. Only the strongest pause was used for analysis of the TSSs following multiple pause sites. Coordinate “0” represents TSS (commonly marked as the +1 site) in the sequence logo, otherwise the standard “+1” TSS nomenclature was used. −10R, −10 promoter element; tssR, region surrounding TSS; −10LR, −10-like region; spacer, spacing region between − 10R and TSS. j, Boxplot comparing Ri of the −10 elements for G1p (top, n = 1069) and G1d (bottom, n = 407) promoters; −10R of the same numbers of randomly chosen promoters were used as a control. k, Heatmap showing correlation between distribution of spacer length and information content (Ri) of the promoter −10 element for all σ 70 promoters (top), promoters containing G1p (middle) and G1d (bottom) pauses. The two-tailed Mann-Whitney U -test was used for the statistical analysis shown above.

    Techniques Used: In Vitro, Labeling, Translocation Assay, RNA Sequencing Assay, Sequencing, Two Tailed Test, MANN-WHITNEY

    34) Product Images from "Effects of Endothelial Progenitor Cell-Derived Microvesicles on Hypoxia/Reoxygenation-Induced Endothelial Dysfunction and Apoptosis"

    Article Title: Effects of Endothelial Progenitor Cell-Derived Microvesicles on Hypoxia/Reoxygenation-Induced Endothelial Dysfunction and Apoptosis

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2013/572729

    EPC-MV characterization, modification, caspase 3 and miR126 expression. (a) Flow cytometric plots showing Annexin V, CD34 and VEGFR2 expressions (isotype controls: left curves; antibodies: right curves) in EPC-MVs. (b) TEM image showing similar spherical morphology of sEPC-MVs and aEPC-MVs. Scale bar: 500 nm. (c) Summarized data showing effective digestion of EPC-MVs total RNAs by RNase treatment. (d) Caspase 3 and miR126 expression in control MVs (generated from basal condition), sEPC-MVs, and aEPC-MVs. * P
    Figure Legend Snippet: EPC-MV characterization, modification, caspase 3 and miR126 expression. (a) Flow cytometric plots showing Annexin V, CD34 and VEGFR2 expressions (isotype controls: left curves; antibodies: right curves) in EPC-MVs. (b) TEM image showing similar spherical morphology of sEPC-MVs and aEPC-MVs. Scale bar: 500 nm. (c) Summarized data showing effective digestion of EPC-MVs total RNAs by RNase treatment. (d) Caspase 3 and miR126 expression in control MVs (generated from basal condition), sEPC-MVs, and aEPC-MVs. * P

    Techniques Used: Modification, Expressing, Flow Cytometry, Transmission Electron Microscopy, Generated

    35) Product Images from "Minute Virus of Canines NP1 Protein Interacts with the Cellular Factor CPSF6 To Regulate Viral Alternative RNA Processing"

    Article Title: Minute Virus of Canines NP1 Protein Interacts with the Cellular Factor CPSF6 To Regulate Viral Alternative RNA Processing

    Journal: Journal of Virology

    doi: 10.1128/JVI.01530-18

    CPSF6 affects MVC alternative RNA processing. (A) Transcription profile of MVC showing the P6 promoter, transcription starting site (TSS), splice donors (D) and acceptors (A), and proximal [(pA)p], and distal [(pA)d] polyadenylation sites. The annotated nucleotides delineate the boundaries of the transcription landmarks indicated within the MVC genome. The position of the RNase protection probes, 2A/3D (nt 2344 to 2550), 3A (nt 2910 to 3110), and (pA)p (nt 3107 to 3333), are indicated. NP1 and NS-66 mRNA species polyadenylated at (pA)p or (pA)d are shown. (B) RPAs of total RNA extracted 48 h following transfection of 293FT or CPSF6(-)293T cells with MVC Rep-Cap (lanes 1 and 2), IMVC-WT (lanes 3 and 4), and IMVC-5X (lanes 5 and 6) using the (pA)p probe. The sizes of the probe and various protected bands are shown on the left. The protected bands representing RNA species extending through (pA)p to (pA)d or cleaved at the various cleavage sites as described for (pA)d and (pA)p are indicated to the right. Quantifications below show the ratio of (pA)p/(pA)d RNAs from transfected CPSF6 knockout cells compared to the level in 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (C) Immunoprecipitation analysis reveals the association of CPSF6 and MVC NP1 in 293FT cells. Equal amounts of cell lysates were immunoprecipitated (IP) using protein-G magnetic beads (lane 2) or anti-HA magnetic beads (lane 3), as described in Materials and Methods, followed by immunoblotting with antibodies against CPSF6 and HA-tagged NP1. (D) RPAs of total RNA as described for panel B using the 3A probe. The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species (3Aunspl, unspliced at the third intron acceptor [3A]; 3Aspl, third intron acceptor spliced) are indicated to the right. Quantifications below show the ratio of spliced/unspliced RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (E) RPAs of total RNA as described for panels B and D using the 2A/3D probe (nt 2910 to 3100). The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species [(pA)d, read-through of the second intron acceptor (2A) and third intron donor (3D); 2Aspl/3Dun, the second intron acceptor spliced but third intron unspliced; 2Aspl/3Dspl, both second intron acceptor and third intron spliced] are indicated to the right. Quantifications below show the ratio of 2Aspl/3Dspl to read-through RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (F) 293FT or CPSF6(-)293T cells transfected with constructs described for panels B, D, and E were harvested and analyzed by immunoblotting using antibodies directed against CPSF6, NS, and NP1 (the individual epitopes are described in Materials and Methods). Immunoblotting for β-actin was used as loading control.
    Figure Legend Snippet: CPSF6 affects MVC alternative RNA processing. (A) Transcription profile of MVC showing the P6 promoter, transcription starting site (TSS), splice donors (D) and acceptors (A), and proximal [(pA)p], and distal [(pA)d] polyadenylation sites. The annotated nucleotides delineate the boundaries of the transcription landmarks indicated within the MVC genome. The position of the RNase protection probes, 2A/3D (nt 2344 to 2550), 3A (nt 2910 to 3110), and (pA)p (nt 3107 to 3333), are indicated. NP1 and NS-66 mRNA species polyadenylated at (pA)p or (pA)d are shown. (B) RPAs of total RNA extracted 48 h following transfection of 293FT or CPSF6(-)293T cells with MVC Rep-Cap (lanes 1 and 2), IMVC-WT (lanes 3 and 4), and IMVC-5X (lanes 5 and 6) using the (pA)p probe. The sizes of the probe and various protected bands are shown on the left. The protected bands representing RNA species extending through (pA)p to (pA)d or cleaved at the various cleavage sites as described for (pA)d and (pA)p are indicated to the right. Quantifications below show the ratio of (pA)p/(pA)d RNAs from transfected CPSF6 knockout cells compared to the level in 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (C) Immunoprecipitation analysis reveals the association of CPSF6 and MVC NP1 in 293FT cells. Equal amounts of cell lysates were immunoprecipitated (IP) using protein-G magnetic beads (lane 2) or anti-HA magnetic beads (lane 3), as described in Materials and Methods, followed by immunoblotting with antibodies against CPSF6 and HA-tagged NP1. (D) RPAs of total RNA as described for panel B using the 3A probe. The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species (3Aunspl, unspliced at the third intron acceptor [3A]; 3Aspl, third intron acceptor spliced) are indicated to the right. Quantifications below show the ratio of spliced/unspliced RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (E) RPAs of total RNA as described for panels B and D using the 2A/3D probe (nt 2910 to 3100). The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species [(pA)d, read-through of the second intron acceptor (2A) and third intron donor (3D); 2Aspl/3Dun, the second intron acceptor spliced but third intron unspliced; 2Aspl/3Dspl, both second intron acceptor and third intron spliced] are indicated to the right. Quantifications below show the ratio of 2Aspl/3Dspl to read-through RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (F) 293FT or CPSF6(-)293T cells transfected with constructs described for panels B, D, and E were harvested and analyzed by immunoblotting using antibodies directed against CPSF6, NS, and NP1 (the individual epitopes are described in Materials and Methods). Immunoblotting for β-actin was used as loading control.

    Techniques Used: Transfection, Knock-Out, Derivative Assay, Immunoprecipitation, Magnetic Beads, Construct

    36) Product Images from "TLR3 Ligand Poly(I:C) Exerts Distinct Actions in Synovial Fibroblasts When Delivered by Extracellular Vesicles"

    Article Title: TLR3 Ligand Poly(I:C) Exerts Distinct Actions in Synovial Fibroblasts When Delivered by Extracellular Vesicles

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00028

    U937 cell-derived extracellular vesicles (EV) incorporate Poly(I:C) and may protect Poly(I:C) from degradation with RNase III. (A) The presence of Fluorescein Poly(I:C) in U937-derived EV. SSC-H/FSC-H and SSC-H/FL1 profiles of EV with percentage of gated events, as measured by flow cytometry. Shown are a representative sample of n = 3 biological replicates and quantification of flow cytometry data as percentage of Fluorescein Poly(I:C)-positive EV. Control EV (Con EV)-derived from untreated U937 cells. (B) Detection of soluble and vesicular Fluorescein Poly(I:C) digested or not with RNAse III. SSC-H/FSC-H profiles and FL1 histograms of EV and Poly(I:C), as measured by flow cytometry. Shown is one of n = 3–4 biological replicates for EV and one of n = 2 replicates for soluble Fluorescein Poly(I:C). Quantification of changes in median fluorescence intensities (MFI) of vesicular and soluble Fluorescein Poly(I:C) in the presence and absence of RNAse III. (C) Association of control EV (Con EV) with Poly(I:C) or Rhodamine Poly(I:C) in the presence or absence of RNase III, as measured by flow cytometry, shown is one from n = 3 biological replicates. Quantification of MFI changes in Rhodamine Poly(I:C) EV in the presence of RNAse III. Statistics: (A,B) one-way ANOVA with Tukey’s multiple comparisons test, (C) two-tailed paired t -test, ns, not significant.
    Figure Legend Snippet: U937 cell-derived extracellular vesicles (EV) incorporate Poly(I:C) and may protect Poly(I:C) from degradation with RNase III. (A) The presence of Fluorescein Poly(I:C) in U937-derived EV. SSC-H/FSC-H and SSC-H/FL1 profiles of EV with percentage of gated events, as measured by flow cytometry. Shown are a representative sample of n = 3 biological replicates and quantification of flow cytometry data as percentage of Fluorescein Poly(I:C)-positive EV. Control EV (Con EV)-derived from untreated U937 cells. (B) Detection of soluble and vesicular Fluorescein Poly(I:C) digested or not with RNAse III. SSC-H/FSC-H profiles and FL1 histograms of EV and Poly(I:C), as measured by flow cytometry. Shown is one of n = 3–4 biological replicates for EV and one of n = 2 replicates for soluble Fluorescein Poly(I:C). Quantification of changes in median fluorescence intensities (MFI) of vesicular and soluble Fluorescein Poly(I:C) in the presence and absence of RNAse III. (C) Association of control EV (Con EV) with Poly(I:C) or Rhodamine Poly(I:C) in the presence or absence of RNase III, as measured by flow cytometry, shown is one from n = 3 biological replicates. Quantification of MFI changes in Rhodamine Poly(I:C) EV in the presence of RNAse III. Statistics: (A,B) one-way ANOVA with Tukey’s multiple comparisons test, (C) two-tailed paired t -test, ns, not significant.

    Techniques Used: Derivative Assay, Flow Cytometry, Cytometry, Fluorescence, Two Tailed Test

    37) Product Images from "Dicer-2-Dependent Generation of Viral DNA from Defective Genomes of RNA Viruses Modulates Antiviral Immunity in Insects"

    Article Title: Dicer-2-Dependent Generation of Viral DNA from Defective Genomes of RNA Viruses Modulates Antiviral Immunity in Insects

    Journal: Cell Host & Microbe

    doi: 10.1016/j.chom.2018.02.001

    Circular vDNA Produced during RNA Virus Infection of Insects Confers Virus-Specific Protective Immunity (A) Wild-type flies were infected with FHV or mock infected (Tris). DNA was extracted from individual flies and RNase treated, and linear DNA was digested with Plasmid-Safe DNase (DNase-PS). Amplification of Rp49 by PCR shows linear DNA, while amplification of mitochondrial DNA (mtDNA) is a control for circular DNA. Linear and circular DNA from FHV RNA1 and RNA2, the two FHV genomic segments, is detected by PCR. (B) Flies were injected with DCV and processed as in (A). (C) Flies were injected with Sindbis virus and processed as in (A). (D) Mosquitoes were fed chikungunya virus (CHIKV) through a blood meal and processed as in (A). CHIKV circular vDNA presence was assessed by PCR, and actin was used as a control for linear DNA. (E) Wild-type flies were infected with Sindbis replicon, and linear and circular forms of vDNA were detected as described in (A). (F) Wild-type flies were immunized with 10 ng of circular vDNA isolated from FHV-infected S2 cells (cvDNA FHV), mitochondrial DNA isolated from uninfected S2 cells (mtDNA), or mock infected (Tris). Three days later, flies were challenged with 80 TCID 50 FHV or with Tris. “cvDNA FHV-FHV” indicates flies immunized with FHV cvDNA and challenged with FHV. (G) Wild-type flies were immunized as described in (F) and challenged with 100 TCID 50 DCV or with 80 TCID 50 FHV. Survival was monitored daily. Molecular weights (MW) are indicated. (A–G) Data are representative of three independent experiments. (F and G) Dots represent mean and SEM (n = 75 flies per condition). ns, not significant; ∗∗∗∗ p
    Figure Legend Snippet: Circular vDNA Produced during RNA Virus Infection of Insects Confers Virus-Specific Protective Immunity (A) Wild-type flies were infected with FHV or mock infected (Tris). DNA was extracted from individual flies and RNase treated, and linear DNA was digested with Plasmid-Safe DNase (DNase-PS). Amplification of Rp49 by PCR shows linear DNA, while amplification of mitochondrial DNA (mtDNA) is a control for circular DNA. Linear and circular DNA from FHV RNA1 and RNA2, the two FHV genomic segments, is detected by PCR. (B) Flies were injected with DCV and processed as in (A). (C) Flies were injected with Sindbis virus and processed as in (A). (D) Mosquitoes were fed chikungunya virus (CHIKV) through a blood meal and processed as in (A). CHIKV circular vDNA presence was assessed by PCR, and actin was used as a control for linear DNA. (E) Wild-type flies were infected with Sindbis replicon, and linear and circular forms of vDNA were detected as described in (A). (F) Wild-type flies were immunized with 10 ng of circular vDNA isolated from FHV-infected S2 cells (cvDNA FHV), mitochondrial DNA isolated from uninfected S2 cells (mtDNA), or mock infected (Tris). Three days later, flies were challenged with 80 TCID 50 FHV or with Tris. “cvDNA FHV-FHV” indicates flies immunized with FHV cvDNA and challenged with FHV. (G) Wild-type flies were immunized as described in (F) and challenged with 100 TCID 50 DCV or with 80 TCID 50 FHV. Survival was monitored daily. Molecular weights (MW) are indicated. (A–G) Data are representative of three independent experiments. (F and G) Dots represent mean and SEM (n = 75 flies per condition). ns, not significant; ∗∗∗∗ p

    Techniques Used: Produced, Infection, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Injection, Isolation

    38) Product Images from "Obesity reduces the pro-angiogenic potential of adipose tissue stem cell-derived extracellular vesicles (EVs) by impairing miR-126 content: impact on clinical applications"

    Article Title: Obesity reduces the pro-angiogenic potential of adipose tissue stem cell-derived extracellular vesicles (EVs) by impairing miR-126 content: impact on clinical applications

    Journal: International Journal of Obesity (2005)

    doi: 10.1038/ijo.2015.123

    EV-mediated miR-126 transfer is required for EV functional activities. ( a ) Migration assays performed on ECs treated with EVs recovered from nASCs that had either been treated with anti-miR-126 antagomir or nEVs+5U RNAse or not at all. The results are representative of five different experiments performed in triplicate ( n =5) (*** P
    Figure Legend Snippet: EV-mediated miR-126 transfer is required for EV functional activities. ( a ) Migration assays performed on ECs treated with EVs recovered from nASCs that had either been treated with anti-miR-126 antagomir or nEVs+5U RNAse or not at all. The results are representative of five different experiments performed in triplicate ( n =5) (*** P

    Techniques Used: Functional Assay, Migration

    39) Product Images from "Chronic lung infection by Pseudomonas aeruginosa biofilm is cured by L-Methionine in combination with antibiotic therapy"

    Article Title: Chronic lung infection by Pseudomonas aeruginosa biofilm is cured by L-Methionine in combination with antibiotic therapy

    Journal: Scientific Reports

    doi: 10.1038/srep16043

    L-Methionine (L-Met) degrades extracellular DNA. 72 h old PA biofilm culture media was centrifuged at 2000 g for 5 min and the supernatant of (a) Untreated culture (U-Sup) (b) L-Met treated culture (L-Met-Sup) were loaded in the agarose gel and visualized. (b) U-Sup was treated with DNase and RNase (Fermentas) for 1 h at 37 °C and then loaded in the agarose gel and visualized. Extracellular DNA was precipitated and DNA concentration was determined by spectrofluorimetrically from U-Sup and L-Met-Sup (c) with different duration of incubation with L-Met (0.5 μM) or (d) with different concentrations of L-Met and incubated for 72 h. Viscosity of U-Sup and L-Met-Sup (e) with different duration of incubation with L-Met (0.5 μM) or (f) with different concentrations of L-Met and incubated for 72 h was determined using Rheometer. (D F - 0.5 μM was compared with control). Statistical significance was calculated using One-way ANOVA. Asterisks indicate statistical significance as follows: **(p
    Figure Legend Snippet: L-Methionine (L-Met) degrades extracellular DNA. 72 h old PA biofilm culture media was centrifuged at 2000 g for 5 min and the supernatant of (a) Untreated culture (U-Sup) (b) L-Met treated culture (L-Met-Sup) were loaded in the agarose gel and visualized. (b) U-Sup was treated with DNase and RNase (Fermentas) for 1 h at 37 °C and then loaded in the agarose gel and visualized. Extracellular DNA was precipitated and DNA concentration was determined by spectrofluorimetrically from U-Sup and L-Met-Sup (c) with different duration of incubation with L-Met (0.5 μM) or (d) with different concentrations of L-Met and incubated for 72 h. Viscosity of U-Sup and L-Met-Sup (e) with different duration of incubation with L-Met (0.5 μM) or (f) with different concentrations of L-Met and incubated for 72 h was determined using Rheometer. (D F - 0.5 μM was compared with control). Statistical significance was calculated using One-way ANOVA. Asterisks indicate statistical significance as follows: **(p

    Techniques Used: Agarose Gel Electrophoresis, Concentration Assay, Incubation

    40) Product Images from "Mesenchymal stem cell-derived microparticles ameliorate peritubular capillary rarefaction via inhibition of endothelial-mesenchymal transition and decrease tubulointerstitial fibrosis in unilateral ureteral obstruction"

    Article Title: Mesenchymal stem cell-derived microparticles ameliorate peritubular capillary rarefaction via inhibition of endothelial-mesenchymal transition and decrease tubulointerstitial fibrosis in unilateral ureteral obstruction

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-015-0012-6

    Anti-fibrotic effects of KMSC or KMSC-derived MPs in UUO kidneys. Light-field microscopic analysis of Masson’s trichrome–stained sections of UUO kidneys treated with vehicle control, KMSC, KMSC-derived MPs, or MP-RNase (A) . Quantitative analysis of tubulointerstitial fibrosis in Masson’s trichrome–stained sections using the Image J program ( B , upper panel). Immunohistochemical study for α-SMA expression in UUO kidneys treated with vehicle control, KMSC, KMSC-derived MPs, or MP-RNase (A) . Quantitative analysis of tubulointerstitial α-SMA–positive area sections using the Image J program ( B , middle panel). Immunohistochemical study for F4/80 positive cells in UUO kidneys treated with vehicle control, KMSC, KMSC-derived MPs, or MP-RNase (A) . Quantification analysis of F4/80 positive cells per field ( B , lower panel). Ten randomly selected high-power fields were quantified and averaged to obtain the value for each mouse ( n = 6 for each experimental group). Results are expressed as the mean ± SE of three different experiments. The Kruskal-Wallis test was performed; * P
    Figure Legend Snippet: Anti-fibrotic effects of KMSC or KMSC-derived MPs in UUO kidneys. Light-field microscopic analysis of Masson’s trichrome–stained sections of UUO kidneys treated with vehicle control, KMSC, KMSC-derived MPs, or MP-RNase (A) . Quantitative analysis of tubulointerstitial fibrosis in Masson’s trichrome–stained sections using the Image J program ( B , upper panel). Immunohistochemical study for α-SMA expression in UUO kidneys treated with vehicle control, KMSC, KMSC-derived MPs, or MP-RNase (A) . Quantitative analysis of tubulointerstitial α-SMA–positive area sections using the Image J program ( B , middle panel). Immunohistochemical study for F4/80 positive cells in UUO kidneys treated with vehicle control, KMSC, KMSC-derived MPs, or MP-RNase (A) . Quantification analysis of F4/80 positive cells per field ( B , lower panel). Ten randomly selected high-power fields were quantified and averaged to obtain the value for each mouse ( n = 6 for each experimental group). Results are expressed as the mean ± SE of three different experiments. The Kruskal-Wallis test was performed; * P

    Techniques Used: Derivative Assay, Staining, Immunohistochemistry, Expressing

    In vitro cell proliferation effects of MPs. Cell proliferation of HUVEC non-pretreated control (‘Control’), pretreated with TGF-β1 alone, with TGF-β1 treatment simultaneously with KMSC-derived MP, and MPs preincubated with RNase. Non-treated control was expressed as ‘Control.’ Results are expressed as the mean ± SE of three different experiments. The Kruskal-Wallis test was performed; * P
    Figure Legend Snippet: In vitro cell proliferation effects of MPs. Cell proliferation of HUVEC non-pretreated control (‘Control’), pretreated with TGF-β1 alone, with TGF-β1 treatment simultaneously with KMSC-derived MP, and MPs preincubated with RNase. Non-treated control was expressed as ‘Control.’ Results are expressed as the mean ± SE of three different experiments. The Kruskal-Wallis test was performed; * P

    Techniques Used: In Vitro, Derivative Assay

    Proliferation, microvascular rarefaction, and apoptosis in UUO kidneys. Immunohistochemistry analysis showed representative images of CD31 and PCNA staining in UUO kidneys treated with vehicle control, KMSC, KMSC-derived MP, and MP-RNase ( A , upper panel, CD31; brown color, PCNA; red color). Quantitative analysis of PCNA-positive cells (black arrows) per field in UUO kidneys. The PCNA-positive counting was used to assess the proliferation of tubular epithelial cells and peritubular capillaries (B) . Quantitative analysis of PTC rarefaction index was determined by CD31 staining (B) . Representative images of TUNEL and CD31 staining in UUO kidneys by confocal laser microscope analysis ( A , lower panel, CD31; red color, TUNEL; green color). Quantitative analysis demonstrates the total number of TUNEL-positive nuclei and both TUNEL and CD31-positive PTC nuclei (white arrows) in UUO kidneys injected with KMSCs and KMSC-derived MPs, compared to those injected with vehicle control or MP-RNase. Results are expressed as the mean ± SE of six different experiments. The Kruskal-Wallis test was performed; * P
    Figure Legend Snippet: Proliferation, microvascular rarefaction, and apoptosis in UUO kidneys. Immunohistochemistry analysis showed representative images of CD31 and PCNA staining in UUO kidneys treated with vehicle control, KMSC, KMSC-derived MP, and MP-RNase ( A , upper panel, CD31; brown color, PCNA; red color). Quantitative analysis of PCNA-positive cells (black arrows) per field in UUO kidneys. The PCNA-positive counting was used to assess the proliferation of tubular epithelial cells and peritubular capillaries (B) . Quantitative analysis of PTC rarefaction index was determined by CD31 staining (B) . Representative images of TUNEL and CD31 staining in UUO kidneys by confocal laser microscope analysis ( A , lower panel, CD31; red color, TUNEL; green color). Quantitative analysis demonstrates the total number of TUNEL-positive nuclei and both TUNEL and CD31-positive PTC nuclei (white arrows) in UUO kidneys injected with KMSCs and KMSC-derived MPs, compared to those injected with vehicle control or MP-RNase. Results are expressed as the mean ± SE of six different experiments. The Kruskal-Wallis test was performed; * P

    Techniques Used: Immunohistochemistry, Staining, Derivative Assay, TUNEL Assay, Microscopy, Injection

    Related Articles

    Isolation:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: .. Total RNA was isolated from MVs, treated or not with RNase, using the mirVana RNA isolation kit (Ambion) according to the manufacturer’s protocol. .. RNA integrity and structure and the efficacy of RNase treatment were evaluated by Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA), using the eukaryotic total RNA 6000 Pico Kit (Agilent Tech.).

    Article Title: Bone Marrow-Derived Mesenchymal Stem Cells Repaired but Did Not Prevent Gentamicin-Induced Acute Kidney Injury through Paracrine Effects in Rats
    Article Snippet: .. RNA isolation, Reverse transcription and Quantitative Real Time PCR Total RNA from the BMSC culture medium treated with RNase (40 µg/ml) was extracted using a Trizol technique (Invitrogen Life Technologies) according to the manufacturer's instructions and published protocol . .. Reverse transcription was performed using a High Capacity cDNA Reverse Transcription kit for real-time PCR (Applied Biosystems).

    Labeling:

    Article Title: Human Cytomegalovirus Elicits a Coordinated Cellular Antiviral Response via Envelope Glycoprotein B
    Article Snippet: .. Gel-purified radioactively labeled probes and sample RNA were hybridized, digested with RNase, and separated by denaturing polyacrylamide gel electrophoresis (PAGE) according to the manufacturer's instructions (RPA III kit; Ambion). ..

    Purification:

    Article Title: Packaging of viral RNAs in virions of adenoviruses
    Article Snippet: .. Equal amounts (based on protein concentrations) of CsCl purified HAdV-5 capsids were treated with RNase and then extracted with Trizol reagent (Invitrogen) to isolate the virion RNAs. .. Two micrograms of virion RNAs isolated from mature or empty/intermediate capsids were reversely transcribed by reverse transcriptase II in the presence of [32 P]-dCTP.

    Article Title: Targeted gene silencing in vivo by platelet membrane–coated metal-organic framework nanoparticles
    Article Snippet: .. For the siRNA degradation study in purified RNase, a working solution of an RNase cocktail enzyme mix (Invitrogen) was prepared by diluting 1:1000 with distilled water. .. Then, 10 μl of the diluted enzyme mix was added into 100-μl aliquots containing 50 pmol of siRNA in free form or in P-MOF-siRNA, and the mixtures were incubated at 37°C for increasing amounts of time.

    Real-time Polymerase Chain Reaction:

    Article Title: Bone Marrow-Derived Mesenchymal Stem Cells Repaired but Did Not Prevent Gentamicin-Induced Acute Kidney Injury through Paracrine Effects in Rats
    Article Snippet: .. RNA isolation, Reverse transcription and Quantitative Real Time PCR Total RNA from the BMSC culture medium treated with RNase (40 µg/ml) was extracted using a Trizol technique (Invitrogen Life Technologies) according to the manufacturer's instructions and published protocol . .. Reverse transcription was performed using a High Capacity cDNA Reverse Transcription kit for real-time PCR (Applied Biosystems).

    Incubation:

    Article Title: Hypermethylation‐mediated down‐regulation of lncRNA TBX5‐AS1:2 in Tetralogy of Fallot inhibits cell proliferation by reducing TBX5 expression, et al. Hypermethylation‐mediated down‐regulation of lncRNA TBX5‐AS1:2 in Tetralogy of Fallot inhibits cell proliferation by reducing TBX5 expression
    Article Snippet: .. 2.12 Ribonuclease protection assay Each RNA sample from HEK293T cells was incubated for 1 hour at 37°C and then treated with RNAse A+T cocktail (Ambion) to digest single‐stranded but not duplex RNAs. .. After incubation for 30 minutes at 37°C, samples were dealt with proteinase K (Yeasen).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Human Cytomegalovirus Elicits a Coordinated Cellular Antiviral Response via Envelope Glycoprotein B
    Article Snippet: .. Gel-purified radioactively labeled probes and sample RNA were hybridized, digested with RNase, and separated by denaturing polyacrylamide gel electrophoresis (PAGE) according to the manufacturer's instructions (RPA III kit; Ambion). ..

    Recombinase Polymerase Amplification:

    Article Title: Human Cytomegalovirus Elicits a Coordinated Cellular Antiviral Response via Envelope Glycoprotein B
    Article Snippet: .. Gel-purified radioactively labeled probes and sample RNA were hybridized, digested with RNase, and separated by denaturing polyacrylamide gel electrophoresis (PAGE) according to the manufacturer's instructions (RPA III kit; Ambion). ..

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  • 99
    Thermo Fisher rnase iii
    Comparison of gradients from different rotors. The use of different rotors (SW40 Ti and SW41 Ti) in similar conditions led to the same protein distribution through the 25 fractions of 5% to 50% sucrose density gradients. a , top panel: western blot analysis for RNA-dependent protein CTCF and RNA-independent protein ASNS in 25 fractions of representative control and <t>RNase-treated</t> samples after ultracentrifugation in a SW40 Ti rotor (30,000 rpm/114,000 g for 18 h at 4° C). Bottom panel: graph of the quantitative analysis of <t>three</t> replicates (N = 3) depicting the mean of three experiments with standard error of the mean (SEM). Adapted from Caudron-Herger et al., Mol Cell (2019) (ref. 24). b , Same as in a , with a SW41 Ti rotor (N = 1) (30,000 rpm/111,000 g for 18 h at 4° C). For all western blots, fractions 1 to 25 were loaded onto two membranes (1: fractions 1 to 13 and 2: fractions 14 to 25).
    Rnase Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnase s1
    Enzymatic probing analysis of hmtRNA Thr , -G30A and -G30A/C40U. ( A ) Probing was performed using various <t>RNase</t> S1 concentrations. Lane C, control; lane G, ladder digested by RNase T1 under denaturing conditions; lane OH − , alkaline digestion. ( B ) Structure analysis by enzymatic probing. The red stars indicate the main RNase S1 cleavage sites.
    Rnase S1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher e coli rnase e
    In vitro screening of candidate small molecule inhibitors against E. coli <t>RNase</t> E NTD. (A) Representative 20% denaturing PAGE analysis of the cleavage of 1 μM 5′-p-RNA13-FAM-3′ by 5 nM E . coli RNase E NTD after incubation at 28 °C for 45 min in the absence of small molecule (-) or in the presence of 10 mM AS1, AS2, AS3, AS4, AS6, AS7, AS8, AS9, or 5′S1. This is a composite image assembled from multiple gels (complete gels are presented in Supplementary Fig. S2 ). The expected position of the bands representing the full-length FAM-labelled 5′-p-RNA13-FAM-3′ RNA substrate and the FAM-labelled pentamer 5′-p-AUUUG-FAM-3′ cleavage product are indicated on the right-hand-side of the gels. (B) The chemical structures of inhibitory small molecules AS2, AS4 and 5′S1 (further details are presented in Supplementary Table S1 ).
    E Coli Rnase E, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Comparison of gradients from different rotors. The use of different rotors (SW40 Ti and SW41 Ti) in similar conditions led to the same protein distribution through the 25 fractions of 5% to 50% sucrose density gradients. a , top panel: western blot analysis for RNA-dependent protein CTCF and RNA-independent protein ASNS in 25 fractions of representative control and RNase-treated samples after ultracentrifugation in a SW40 Ti rotor (30,000 rpm/114,000 g for 18 h at 4° C). Bottom panel: graph of the quantitative analysis of three replicates (N = 3) depicting the mean of three experiments with standard error of the mean (SEM). Adapted from Caudron-Herger et al., Mol Cell (2019) (ref. 24). b , Same as in a , with a SW41 Ti rotor (N = 1) (30,000 rpm/111,000 g for 18 h at 4° C). For all western blots, fractions 1 to 25 were loaded onto two membranes (1: fractions 1 to 13 and 2: fractions 14 to 25).

    Journal: Nature protocols

    Article Title: Identification, quantification and bioinformatic analysis of RNA-dependent proteins by RNase treatment and density gradient ultracentrifugation using R-DeeP

    doi: 10.1038/s41596-019-0261-4

    Figure Lengend Snippet: Comparison of gradients from different rotors. The use of different rotors (SW40 Ti and SW41 Ti) in similar conditions led to the same protein distribution through the 25 fractions of 5% to 50% sucrose density gradients. a , top panel: western blot analysis for RNA-dependent protein CTCF and RNA-independent protein ASNS in 25 fractions of representative control and RNase-treated samples after ultracentrifugation in a SW40 Ti rotor (30,000 rpm/114,000 g for 18 h at 4° C). Bottom panel: graph of the quantitative analysis of three replicates (N = 3) depicting the mean of three experiments with standard error of the mean (SEM). Adapted from Caudron-Herger et al., Mol Cell (2019) (ref. 24). b , Same as in a , with a SW41 Ti rotor (N = 1) (30,000 rpm/111,000 g for 18 h at 4° C). For all western blots, fractions 1 to 25 were loaded onto two membranes (1: fractions 1 to 13 and 2: fractions 14 to 25).

    Article Snippet: RNase III, 250 U (Thermo Fisher Scientific, cat. no. AM2290).

    Techniques: Western Blot

    TMT workflow for sucrose gradient fractions. Proteins in the 25 sucrose density gradient fractions from control or RNase-treated samples were TCA precipitated and digested into peptides using trypsin. For each fraction, three biological replicates of the control sample and three biological replicates of the RNase-treated sample were labeled with individual tandem mass reporter tags (TMT, here TMT-6 plex), combined on a per-fraction basis and analyzed by LC-MS/MS. Fraction 1 is shown as an example here. The procedure is repeated for fractions 2 to 25.

    Journal: Nature protocols

    Article Title: Identification, quantification and bioinformatic analysis of RNA-dependent proteins by RNase treatment and density gradient ultracentrifugation using R-DeeP

    doi: 10.1038/s41596-019-0261-4

    Figure Lengend Snippet: TMT workflow for sucrose gradient fractions. Proteins in the 25 sucrose density gradient fractions from control or RNase-treated samples were TCA precipitated and digested into peptides using trypsin. For each fraction, three biological replicates of the control sample and three biological replicates of the RNase-treated sample were labeled with individual tandem mass reporter tags (TMT, here TMT-6 plex), combined on a per-fraction basis and analyzed by LC-MS/MS. Fraction 1 is shown as an example here. The procedure is repeated for fractions 2 to 25.

    Article Snippet: RNase III, 250 U (Thermo Fisher Scientific, cat. no. AM2290).

    Techniques: Labeling, Liquid Chromatography with Mass Spectroscopy

    Enzymatic probing analysis of hmtRNA Thr , -G30A and -G30A/C40U. ( A ) Probing was performed using various RNase S1 concentrations. Lane C, control; lane G, ladder digested by RNase T1 under denaturing conditions; lane OH − , alkaline digestion. ( B ) Structure analysis by enzymatic probing. The red stars indicate the main RNase S1 cleavage sites.

    Journal: Nucleic Acids Research

    Article Title: A natural non-Watson–Crick base pair in human mitochondrial tRNAThr causes structural and functional susceptibility to local mutations

    doi: 10.1093/nar/gky243

    Figure Lengend Snippet: Enzymatic probing analysis of hmtRNA Thr , -G30A and -G30A/C40U. ( A ) Probing was performed using various RNase S1 concentrations. Lane C, control; lane G, ladder digested by RNase T1 under denaturing conditions; lane OH − , alkaline digestion. ( B ) Structure analysis by enzymatic probing. The red stars indicate the main RNase S1 cleavage sites.

    Article Snippet: T4 DNA ligase, T4 PNK (polynucleotide kinase), RNase T1, RNase S1, and restriction endonucleases were obtained from Thermo Scientific (Pittsburgh, PA, USA).

    Techniques:

    In vitro screening of candidate small molecule inhibitors against E. coli RNase E NTD. (A) Representative 20% denaturing PAGE analysis of the cleavage of 1 μM 5′-p-RNA13-FAM-3′ by 5 nM E . coli RNase E NTD after incubation at 28 °C for 45 min in the absence of small molecule (-) or in the presence of 10 mM AS1, AS2, AS3, AS4, AS6, AS7, AS8, AS9, or 5′S1. This is a composite image assembled from multiple gels (complete gels are presented in Supplementary Fig. S2 ). The expected position of the bands representing the full-length FAM-labelled 5′-p-RNA13-FAM-3′ RNA substrate and the FAM-labelled pentamer 5′-p-AUUUG-FAM-3′ cleavage product are indicated on the right-hand-side of the gels. (B) The chemical structures of inhibitory small molecules AS2, AS4 and 5′S1 (further details are presented in Supplementary Table S1 ).

    Journal: Biochemistry and Biophysics Reports

    Article Title: Identification and analysis of novel small molecule inhibitors of RNase E: Implications for antibacterial targeting and regulation of RNase E

    doi: 10.1016/j.bbrep.2020.100773

    Figure Lengend Snippet: In vitro screening of candidate small molecule inhibitors against E. coli RNase E NTD. (A) Representative 20% denaturing PAGE analysis of the cleavage of 1 μM 5′-p-RNA13-FAM-3′ by 5 nM E . coli RNase E NTD after incubation at 28 °C for 45 min in the absence of small molecule (-) or in the presence of 10 mM AS1, AS2, AS3, AS4, AS6, AS7, AS8, AS9, or 5′S1. This is a composite image assembled from multiple gels (complete gels are presented in Supplementary Fig. S2 ). The expected position of the bands representing the full-length FAM-labelled 5′-p-RNA13-FAM-3′ RNA substrate and the FAM-labelled pentamer 5′-p-AUUUG-FAM-3′ cleavage product are indicated on the right-hand-side of the gels. (B) The chemical structures of inhibitory small molecules AS2, AS4 and 5′S1 (further details are presented in Supplementary Table S1 ).

    Article Snippet: A putative small molecule-binding site at the active site of E. coli RNase E was selected in the apo-2BX2 structure based on the presence of the catalytic residues D303, N305 and D346 and a putative small molecule-binding site at the 5′ sensor region was selected in the 2VMK structure based on the presence of the key amino acids G124, V128, R169 and T170.

    Techniques: In Vitro, Polyacrylamide Gel Electrophoresis, Incubation

    Inhibition of F. tularensis and A. baumannii RNase E NTDs by AS2, AS4 and 5′S1. The relative rates of cleavage of 1 μM modified target-guide substrate by 5 nM E. coli (blue), F. tularensis (orange) or A. baumannii (grey) RNase E NTD in the absence of inhibitor and in the presence of either 2 mM AS2, AS4 or 5′S1. For each RNase E NTD, the data have been normalised to the cleavage rate when there was no inhibitor present. Data are the average from duplicate experiments and the error bars represent the standard error of the mean. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Journal: Biochemistry and Biophysics Reports

    Article Title: Identification and analysis of novel small molecule inhibitors of RNase E: Implications for antibacterial targeting and regulation of RNase E

    doi: 10.1016/j.bbrep.2020.100773

    Figure Lengend Snippet: Inhibition of F. tularensis and A. baumannii RNase E NTDs by AS2, AS4 and 5′S1. The relative rates of cleavage of 1 μM modified target-guide substrate by 5 nM E. coli (blue), F. tularensis (orange) or A. baumannii (grey) RNase E NTD in the absence of inhibitor and in the presence of either 2 mM AS2, AS4 or 5′S1. For each RNase E NTD, the data have been normalised to the cleavage rate when there was no inhibitor present. Data are the average from duplicate experiments and the error bars represent the standard error of the mean. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Article Snippet: A putative small molecule-binding site at the active site of E. coli RNase E was selected in the apo-2BX2 structure based on the presence of the catalytic residues D303, N305 and D346 and a putative small molecule-binding site at the 5′ sensor region was selected in the 2VMK structure based on the presence of the key amino acids G124, V128, R169 and T170.

    Techniques: Inhibition, Modification

    Potency of AS2, AS4 and 5′S1 as small molecule inhibitors of RNase E. (A) Schematic of the FRET-based real-time RNase E assay. The substrate is a partially double-stranded RNA (modified target-guide) consisting of a 5′ hydroxylated, 3′ FAM-labelled 18-mer “target” RNA and a 5′ monophosphorylated, 3′ TAMRA-labelled 13-mer “guide” RNA, which anneals to the target RNA with partial complementarity. In the uncleaved modified target-guide substrate, the fluorescence of the 3′ FAM group of the 18-mer target RNA is quenched by the 3′ TAMRA group of the 13-mer guide RNA. Endoribonucleolytic cleavage of the single-stranded A/U-rich region of the 18-mer target RNA by RNase E NTD, at the position indicated by the arrow, results in the release of a 3′ FAM-labelled pentamer and the unquenching of the FAM fluorescence. The increase in fluorescence can be monitored in real-time. (B) Plots of the rate of cleavage of 1 μM modified target-guide RNA substrate by 5 nM E . coli RNase E NTD in the presence of 0.25, 0.5, 1, 2, 3, 4, 5 and 10 mM AS2, AS4 or 5′S1. Data are the average from three experiments and the error bars represent the standard error of the mean. Data were fitted as described in Materials and Methods to determine the IC 50 , which is indicated for the respective small molecule inhibitor in the top right-hand corner of the plot.

    Journal: Biochemistry and Biophysics Reports

    Article Title: Identification and analysis of novel small molecule inhibitors of RNase E: Implications for antibacterial targeting and regulation of RNase E

    doi: 10.1016/j.bbrep.2020.100773

    Figure Lengend Snippet: Potency of AS2, AS4 and 5′S1 as small molecule inhibitors of RNase E. (A) Schematic of the FRET-based real-time RNase E assay. The substrate is a partially double-stranded RNA (modified target-guide) consisting of a 5′ hydroxylated, 3′ FAM-labelled 18-mer “target” RNA and a 5′ monophosphorylated, 3′ TAMRA-labelled 13-mer “guide” RNA, which anneals to the target RNA with partial complementarity. In the uncleaved modified target-guide substrate, the fluorescence of the 3′ FAM group of the 18-mer target RNA is quenched by the 3′ TAMRA group of the 13-mer guide RNA. Endoribonucleolytic cleavage of the single-stranded A/U-rich region of the 18-mer target RNA by RNase E NTD, at the position indicated by the arrow, results in the release of a 3′ FAM-labelled pentamer and the unquenching of the FAM fluorescence. The increase in fluorescence can be monitored in real-time. (B) Plots of the rate of cleavage of 1 μM modified target-guide RNA substrate by 5 nM E . coli RNase E NTD in the presence of 0.25, 0.5, 1, 2, 3, 4, 5 and 10 mM AS2, AS4 or 5′S1. Data are the average from three experiments and the error bars represent the standard error of the mean. Data were fitted as described in Materials and Methods to determine the IC 50 , which is indicated for the respective small molecule inhibitor in the top right-hand corner of the plot.

    Article Snippet: A putative small molecule-binding site at the active site of E. coli RNase E was selected in the apo-2BX2 structure based on the presence of the catalytic residues D303, N305 and D346 and a putative small molecule-binding site at the 5′ sensor region was selected in the 2VMK structure based on the presence of the key amino acids G124, V128, R169 and T170.

    Techniques: Modification, Fluorescence

    Molecular docking of potential small molecule inhibitors into E. coli RNase E NTD. The lowest-energy RNase E-small molecule complex conformations obtained from the molecular docking of potential small molecule inhibitors AS1-9 into the active site (A) and 5′S1 and 5′S2 into the 5′ sensor region (B) of E. coli RNase E NTD using 100 starting placement poses. The corresponding docking score is shown above each panel. The docked small molecule is shown as sticks and labelled in each panel. E. coli RNase E NTD is shown as a ribbon representation (S1 subdomain, blue; DNase I subdomain, red; 5′ sensor subdomain, gold; small subdomain, grey). Key amino acids, required for catalytic activity and/or substrate binding, are shown as sticks and labelled. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Journal: Biochemistry and Biophysics Reports

    Article Title: Identification and analysis of novel small molecule inhibitors of RNase E: Implications for antibacterial targeting and regulation of RNase E

    doi: 10.1016/j.bbrep.2020.100773

    Figure Lengend Snippet: Molecular docking of potential small molecule inhibitors into E. coli RNase E NTD. The lowest-energy RNase E-small molecule complex conformations obtained from the molecular docking of potential small molecule inhibitors AS1-9 into the active site (A) and 5′S1 and 5′S2 into the 5′ sensor region (B) of E. coli RNase E NTD using 100 starting placement poses. The corresponding docking score is shown above each panel. The docked small molecule is shown as sticks and labelled in each panel. E. coli RNase E NTD is shown as a ribbon representation (S1 subdomain, blue; DNase I subdomain, red; 5′ sensor subdomain, gold; small subdomain, grey). Key amino acids, required for catalytic activity and/or substrate binding, are shown as sticks and labelled. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Article Snippet: A putative small molecule-binding site at the active site of E. coli RNase E was selected in the apo-2BX2 structure based on the presence of the catalytic residues D303, N305 and D346 and a putative small molecule-binding site at the 5′ sensor region was selected in the 2VMK structure based on the presence of the key amino acids G124, V128, R169 and T170.

    Techniques: Activity Assay, Binding Assay