media purified raav  (Millipore)


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    Structured Review

    Millipore media purified raav
    <t>rAAV</t> capsids manufactured with the human and baculovirus- Sf9 platforms are post-translationally modified and exhibit differential PTM profiles. (a) PTM identities and residue positions along the length of the rAAV8 polypeptide from N to C-terminus in baculo- Sf9 vector. PTMs colored by type (acetylation = green, methylation = blue, phosphorylation = cyan, deamidation = orange, O-GlcNAcylation = magenta). Residues above the sequence are externally facing on the capsid. Residues below are lumenal or buried. Residues within the grey box from 1-220 represent the disordered region of AAV8 yet to be crystallized. The two regions for LamR binding (491-547 and 593-623) are highlighted in yellow boxes. (b) Cumulative capsid PTMs observed from all baculo- Sf9 rAAV8 lots, purified from both cell lysates and media. Same color code as in (a). (c) Same as (a) but with human-produced rAAV8. (d) Same as (b) but with human rAAV8. (e) Shared and unique capsid PTMs for rAAV8 produced in the baculo- Sf9 (yellow) and human platforms (purple). Same color code as in (a). Excluded are deamidation degradation marks which are universal. (f) Negative staining and TEM imaging of baculo- Sf9 rAAV8 cell-purified vector. White arrow = full capsid, red arrow = empty capsid, for reference for panels f-i (percent full capsids noted on left). (g) Same as (f) but media purified vector. (h) Same as (f) but with human rAAV8 cell-purified vector. (i) Same as (h) but with media-purified vector. (j) Silver stain of capsid VP species present in vector lots from panels f-i. (k) 2D gel images from human-produced rAAV8 from pH 3-10. VP1 (87 kDa), VP2 (72 kDa), and VP3 (62 kDa) bands = black arrowheads. (l) 2D gel images from baculo- Sf9 produced rAAV8. (m) Thermal capsid melt curves for rAAV8 vectors shown from 50-90°C, full melt curves from 30-95°C are in Fig S7a. Tm initiation = dashed black line; final Tm = dashed grey line.
    Media Purified Raav, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 3412 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Methods Matter -- Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors"

    Article Title: Methods Matter -- Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors

    Journal: bioRxiv

    doi: 10.1101/640169

    rAAV capsids manufactured with the human and baculovirus- Sf9 platforms are post-translationally modified and exhibit differential PTM profiles. (a) PTM identities and residue positions along the length of the rAAV8 polypeptide from N to C-terminus in baculo- Sf9 vector. PTMs colored by type (acetylation = green, methylation = blue, phosphorylation = cyan, deamidation = orange, O-GlcNAcylation = magenta). Residues above the sequence are externally facing on the capsid. Residues below are lumenal or buried. Residues within the grey box from 1-220 represent the disordered region of AAV8 yet to be crystallized. The two regions for LamR binding (491-547 and 593-623) are highlighted in yellow boxes. (b) Cumulative capsid PTMs observed from all baculo- Sf9 rAAV8 lots, purified from both cell lysates and media. Same color code as in (a). (c) Same as (a) but with human-produced rAAV8. (d) Same as (b) but with human rAAV8. (e) Shared and unique capsid PTMs for rAAV8 produced in the baculo- Sf9 (yellow) and human platforms (purple). Same color code as in (a). Excluded are deamidation degradation marks which are universal. (f) Negative staining and TEM imaging of baculo- Sf9 rAAV8 cell-purified vector. White arrow = full capsid, red arrow = empty capsid, for reference for panels f-i (percent full capsids noted on left). (g) Same as (f) but media purified vector. (h) Same as (f) but with human rAAV8 cell-purified vector. (i) Same as (h) but with media-purified vector. (j) Silver stain of capsid VP species present in vector lots from panels f-i. (k) 2D gel images from human-produced rAAV8 from pH 3-10. VP1 (87 kDa), VP2 (72 kDa), and VP3 (62 kDa) bands = black arrowheads. (l) 2D gel images from baculo- Sf9 produced rAAV8. (m) Thermal capsid melt curves for rAAV8 vectors shown from 50-90°C, full melt curves from 30-95°C are in Fig S7a. Tm initiation = dashed black line; final Tm = dashed grey line.
    Figure Legend Snippet: rAAV capsids manufactured with the human and baculovirus- Sf9 platforms are post-translationally modified and exhibit differential PTM profiles. (a) PTM identities and residue positions along the length of the rAAV8 polypeptide from N to C-terminus in baculo- Sf9 vector. PTMs colored by type (acetylation = green, methylation = blue, phosphorylation = cyan, deamidation = orange, O-GlcNAcylation = magenta). Residues above the sequence are externally facing on the capsid. Residues below are lumenal or buried. Residues within the grey box from 1-220 represent the disordered region of AAV8 yet to be crystallized. The two regions for LamR binding (491-547 and 593-623) are highlighted in yellow boxes. (b) Cumulative capsid PTMs observed from all baculo- Sf9 rAAV8 lots, purified from both cell lysates and media. Same color code as in (a). (c) Same as (a) but with human-produced rAAV8. (d) Same as (b) but with human rAAV8. (e) Shared and unique capsid PTMs for rAAV8 produced in the baculo- Sf9 (yellow) and human platforms (purple). Same color code as in (a). Excluded are deamidation degradation marks which are universal. (f) Negative staining and TEM imaging of baculo- Sf9 rAAV8 cell-purified vector. White arrow = full capsid, red arrow = empty capsid, for reference for panels f-i (percent full capsids noted on left). (g) Same as (f) but media purified vector. (h) Same as (f) but with human rAAV8 cell-purified vector. (i) Same as (h) but with media-purified vector. (j) Silver stain of capsid VP species present in vector lots from panels f-i. (k) 2D gel images from human-produced rAAV8 from pH 3-10. VP1 (87 kDa), VP2 (72 kDa), and VP3 (62 kDa) bands = black arrowheads. (l) 2D gel images from baculo- Sf9 produced rAAV8. (m) Thermal capsid melt curves for rAAV8 vectors shown from 50-90°C, full melt curves from 30-95°C are in Fig S7a. Tm initiation = dashed black line; final Tm = dashed grey line.

    Techniques Used: Modification, Plasmid Preparation, Methylation, Sequencing, Binding Assay, Purification, Produced, Negative Staining, Transmission Electron Microscopy, Imaging, Silver Staining, Two-Dimensional Gel Electrophoresis

    2) Product Images from "Human endogenous retrovirus (HERV) expression is not induced by treatment with the histone deacetylase (HDAC) inhibitors in cellular models of HIV-1 latency"

    Article Title: Human endogenous retrovirus (HERV) expression is not induced by treatment with the histone deacetylase (HDAC) inhibitors in cellular models of HIV-1 latency

    Journal: Retrovirology

    doi: 10.1186/s12977-016-0242-4

    Romidepsin and Prostratin do not increase HERV expression in U1s. The HERVs analysed were: HK2 env , HK2 pol , HERV-W env (syncytin-1) and HERV-FRD env (syncytin-2). The fold change in HERV expression following drug treatment was compared to the untreated control ( lines show 95 % CI) and was calculated relative to GAPDH expression. Romidepsin and prostratin were used at final concentrations of 0.2 and 1 μM, respectively. The data points represent the relative fold change in expression normalised with GAPDH ( lines show 95 % CI) in two independent experiments. A significant change of expression (i.e. higher than the untreated cells) would show the 95 % CI to be higher than and not overlap the dashed horizontal line which indicates 1× relative fold change (two-sided test)
    Figure Legend Snippet: Romidepsin and Prostratin do not increase HERV expression in U1s. The HERVs analysed were: HK2 env , HK2 pol , HERV-W env (syncytin-1) and HERV-FRD env (syncytin-2). The fold change in HERV expression following drug treatment was compared to the untreated control ( lines show 95 % CI) and was calculated relative to GAPDH expression. Romidepsin and prostratin were used at final concentrations of 0.2 and 1 μM, respectively. The data points represent the relative fold change in expression normalised with GAPDH ( lines show 95 % CI) in two independent experiments. A significant change of expression (i.e. higher than the untreated cells) would show the 95 % CI to be higher than and not overlap the dashed horizontal line which indicates 1× relative fold change (two-sided test)

    Techniques Used: Expressing

    The HDAC inhibitors panobinostat and vorinostat do not increase HERV expression in J-LAT-8.4 cells. The HERVs analysed were: HK2 env , HK2 pol , HERV-W env (syncytin-1) and HERV-FRD env (syncytin-2). The fold change in HERV expression following drug treatment was compared to the untreated control ( lines show 95 % CI) and was calculated relative to GAPDH expression. The doses of the drugs used were vorinostat (1 μM/well), panobinostat (0.1 μM/well), PMA (0.1 μg/μL). The data points ( empty circles ) represent the relative fold change in expression normalised with GAPDH ( black squares show the median and orange lines show 95 % CI) for up to three replicates in four independent experiments. A significant change of expression (i.e. higher than the untreated cells) would show the 95 % CI to be higher than and not overlap the dashed horizontal line which indicates 1× relative fold change (two-sided test)
    Figure Legend Snippet: The HDAC inhibitors panobinostat and vorinostat do not increase HERV expression in J-LAT-8.4 cells. The HERVs analysed were: HK2 env , HK2 pol , HERV-W env (syncytin-1) and HERV-FRD env (syncytin-2). The fold change in HERV expression following drug treatment was compared to the untreated control ( lines show 95 % CI) and was calculated relative to GAPDH expression. The doses of the drugs used were vorinostat (1 μM/well), panobinostat (0.1 μM/well), PMA (0.1 μg/μL). The data points ( empty circles ) represent the relative fold change in expression normalised with GAPDH ( black squares show the median and orange lines show 95 % CI) for up to three replicates in four independent experiments. A significant change of expression (i.e. higher than the untreated cells) would show the 95 % CI to be higher than and not overlap the dashed horizontal line which indicates 1× relative fold change (two-sided test)

    Techniques Used: Expressing

    The HDAC inhibitors panobinostat and vorinostat do not increase HERV expression in U1 cells following treatment with higher doses of the drugs for 5 h. The doses of the drugs used in this experiments were: vorinostat 1 μM/well, panobinostat 0.1 μM/well and PMA 0.1 μg/μL. The HERVs analysed were: HK2 env , HK2 pol , HERV-W env (syncytin-1) and HERV-FRD env (syncytin-2). The fold change in HERV expression following drug treatment was compared to the untreated control ( lines show 95 % CI) and was calculated relative to GAPDH expression. The data points represent the relative fold change in expression normalised with GAPDH ( lines show 95 % CI) for up to three replicates in two independent experiments. A significant change of expression (i.e. higher than the untreated cells) would show the 95 % CI to be higher than and not overlap the dashed horizontal line which indicates 1× relative fold change (two-sided test)
    Figure Legend Snippet: The HDAC inhibitors panobinostat and vorinostat do not increase HERV expression in U1 cells following treatment with higher doses of the drugs for 5 h. The doses of the drugs used in this experiments were: vorinostat 1 μM/well, panobinostat 0.1 μM/well and PMA 0.1 μg/μL. The HERVs analysed were: HK2 env , HK2 pol , HERV-W env (syncytin-1) and HERV-FRD env (syncytin-2). The fold change in HERV expression following drug treatment was compared to the untreated control ( lines show 95 % CI) and was calculated relative to GAPDH expression. The data points represent the relative fold change in expression normalised with GAPDH ( lines show 95 % CI) for up to three replicates in two independent experiments. A significant change of expression (i.e. higher than the untreated cells) would show the 95 % CI to be higher than and not overlap the dashed horizontal line which indicates 1× relative fold change (two-sided test)

    Techniques Used: Expressing

    The HDAC inhibitors panobinostat and vorinostat do not increase HERV expression in U1 cells. The HERVs analysed were: HK2 env , HK2 pol , HERV-W env (syncytin-1) and HERV-FRD env (syncytin-2). The fold change in HERV expression following drug treatment was compared to the untreated control ( lines show 95 % CI) and was calculated relative to GAPDH expression. The doses of the drugs used were vorinostat (1 μM/well), panobinostat (0.1 μM/well), PMA (0.1 μg/μL) and IL-1β (10 pg/mL). The data points represent the relative fold change in expression normalised with GAPDH ( lines show 95 % CI) for up to three replicates ( lines show 95 % CI) in four independent experiments. A significant change of expression (i.e. higher than the untreated cells) would show the 95 % CI to be higher than and not overlap the dashed horizontal line which indicates 1× relative fold change (two-sided test)
    Figure Legend Snippet: The HDAC inhibitors panobinostat and vorinostat do not increase HERV expression in U1 cells. The HERVs analysed were: HK2 env , HK2 pol , HERV-W env (syncytin-1) and HERV-FRD env (syncytin-2). The fold change in HERV expression following drug treatment was compared to the untreated control ( lines show 95 % CI) and was calculated relative to GAPDH expression. The doses of the drugs used were vorinostat (1 μM/well), panobinostat (0.1 μM/well), PMA (0.1 μg/μL) and IL-1β (10 pg/mL). The data points represent the relative fold change in expression normalised with GAPDH ( lines show 95 % CI) for up to three replicates ( lines show 95 % CI) in four independent experiments. A significant change of expression (i.e. higher than the untreated cells) would show the 95 % CI to be higher than and not overlap the dashed horizontal line which indicates 1× relative fold change (two-sided test)

    Techniques Used: Expressing

    3) Product Images from "Meta-Analysis of Multiple Sclerosis Microarray Data Reveals Dysregulation in RNA Splicing Regulatory Genes"

    Article Title: Meta-Analysis of Multiple Sclerosis Microarray Data Reveals Dysregulation in RNA Splicing Regulatory Genes

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms161023463

    Splicing pattern analysis of the NFAT5 gene. ( A ) Schematic representation of the NFAT5 gene between exons 1 and 5. Exons and introns are represented by boxes and lines, and are approximately drawn to scale. Exons indicated in black are those undergoing alternatively skipping (alone or in combination). The positions of the primer couple used for competitive fluorescent RT-PCR experiments are shown by arrows (the forward primer was labeled with the FAM fluorophore). The C > T transition (rs12599391) abolishing the intron-2 ISE element is also indicated; ( B ) Fluorescent RT-PCR products, obtained from the RNA extracted from PBMCs of a control individual (heterozygous CT for the rs12599391 polymorphism), were separated by using capillary electrophoresis. The panel on the left is a close-up view of the GeneMapper window encompassing all peaks corresponding to different isoforms. A schematic representation of the obtained RT-PCR products is also depicted on the right; ( C ) Boxplot diagram showing the percentage of the exon-2-including transcripts among all NFAT5 mRNAs, according to disease status and by stratifying individuals on the basis of their rs12599391 genotype. Expression levels were measured by fluorescent competitive RT-PCRs. Significance levels of t -tests and of the one-way ANOVA analyses, are shown above and below the boxplots, respectively. * p
    Figure Legend Snippet: Splicing pattern analysis of the NFAT5 gene. ( A ) Schematic representation of the NFAT5 gene between exons 1 and 5. Exons and introns are represented by boxes and lines, and are approximately drawn to scale. Exons indicated in black are those undergoing alternatively skipping (alone or in combination). The positions of the primer couple used for competitive fluorescent RT-PCR experiments are shown by arrows (the forward primer was labeled with the FAM fluorophore). The C > T transition (rs12599391) abolishing the intron-2 ISE element is also indicated; ( B ) Fluorescent RT-PCR products, obtained from the RNA extracted from PBMCs of a control individual (heterozygous CT for the rs12599391 polymorphism), were separated by using capillary electrophoresis. The panel on the left is a close-up view of the GeneMapper window encompassing all peaks corresponding to different isoforms. A schematic representation of the obtained RT-PCR products is also depicted on the right; ( C ) Boxplot diagram showing the percentage of the exon-2-including transcripts among all NFAT5 mRNAs, according to disease status and by stratifying individuals on the basis of their rs12599391 genotype. Expression levels were measured by fluorescent competitive RT-PCRs. Significance levels of t -tests and of the one-way ANOVA analyses, are shown above and below the boxplots, respectively. * p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Labeling, Electrophoresis, Expressing

    4) Product Images from "Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods"

    Article Title: Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.03661-15

    Determination of data acceptance criteria for HF183/BacR287 and HumM2 qPCR method amplification inhibition and sample processing controls.
    Figure Legend Snippet: Determination of data acceptance criteria for HF183/BacR287 and HumM2 qPCR method amplification inhibition and sample processing controls.

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

    Scatter plot showing estimated log 10 copies per reaction for 100-ml impaired water samples tested across all participating laboratories using the HF183/BacR287 and HumM2 qPCR methods. ✖, samples that failed the amplification efficiency ( E ) data
    Figure Legend Snippet: Scatter plot showing estimated log 10 copies per reaction for 100-ml impaired water samples tested across all participating laboratories using the HF183/BacR287 and HumM2 qPCR methods. ✖, samples that failed the amplification efficiency ( E ) data

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    5) Product Images from "Inhibition of Herpes Simplex Virus Type 1 and Type 2 Infections by Peptide-Derivatized Dendrimers ▿Inhibition of Herpes Simplex Virus Type 1 and Type 2 Infections by Peptide-Derivatized Dendrimers ▿ †"

    Article Title: Inhibition of Herpes Simplex Virus Type 1 and Type 2 Infections by Peptide-Derivatized Dendrimers ▿Inhibition of Herpes Simplex Virus Type 1 and Type 2 Infections by Peptide-Derivatized Dendrimers ▿ †

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00149-11

    SB105_A10 binds to cell surface heparan sulfate. Vero cell monolayers were left untreated or treated with heparinase I or heparitinase I (2.5 U/ml) for 1 h at 37°C, washed with medium, and chilled at 4°C. Next, SB105_A10-FAM (5 μM)
    Figure Legend Snippet: SB105_A10 binds to cell surface heparan sulfate. Vero cell monolayers were left untreated or treated with heparinase I or heparitinase I (2.5 U/ml) for 1 h at 37°C, washed with medium, and chilled at 4°C. Next, SB105_A10-FAM (5 μM)

    Techniques Used:

    6) Product Images from "Max-E47, a Designed Minimalist Protein that Targets the E-Box DNA Site In Vivo and In Vitro"

    Article Title: Max-E47, a Designed Minimalist Protein that Targets the E-Box DNA Site In Vivo and In Vitro

    Journal: Journal of the American Chemical Society

    doi: 10.1021/ja901306q

    Max-E47 hybrids inhibit native MaxbHLHZ activation from the E-box (MY1H). ( A ) The HIS3 assay of the inhibition of native MaxbHLHZ by the Max-E47 hybrids. Plates a–d are transformations plated on SD/-H/-L + 10 mM 3-AT plates, which were incubated
    Figure Legend Snippet: Max-E47 hybrids inhibit native MaxbHLHZ activation from the E-box (MY1H). ( A ) The HIS3 assay of the inhibition of native MaxbHLHZ by the Max-E47 hybrids. Plates a–d are transformations plated on SD/-H/-L + 10 mM 3-AT plates, which were incubated

    Techniques Used: Activation Assay, Inhibition, Incubation

    Max-E47 hybrids activate transcription from the E-box (Y1H). ( A ) The HIS3 assay of Max-E47 hybrids expressed in pGAD424. SD/-H/-L + 10 mM 3-AT plates were incubated at 30 °C for 6 days. a . pGAD424 (negative control); clean. b . pGAD424/native MaxbHLHZ
    Figure Legend Snippet: Max-E47 hybrids activate transcription from the E-box (Y1H). ( A ) The HIS3 assay of Max-E47 hybrids expressed in pGAD424. SD/-H/-L + 10 mM 3-AT plates were incubated at 30 °C for 6 days. a . pGAD424 (negative control); clean. b . pGAD424/native MaxbHLHZ

    Techniques Used: Incubation, Negative Control

    7) Product Images from "Interplay between the catabolite repression control protein Crc, Hfq and RNA in Hfq-dependent translational regulation in Pseudomonas aeruginosa"

    Article Title: Interplay between the catabolite repression control protein Crc, Hfq and RNA in Hfq-dependent translational regulation in Pseudomonas aeruginosa

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1245

    The presence of Crc stabilizes the Hfq/RNA complex. ( A ) Electrophoretic mobility shift assays (EMSA) with 32 P- amiE 6ARN RNA and increasing amounts of Hfq 6 in the absence and presence of Crc. Lane 1, electrophoretic mobility of 32 P- amiE 6ARN RNA (A*) in the absence of proteins. Lanes 2–5, 32 P- amiE 6ARN RNA/Hfq 6 complex formation (A*H) with increasing concentrations of Hfq 6 . Lanes 6–10, 32 P- amiE 6ARN RNA/Crc/Hfq 6 complex formation (A*HC) with increasing concentrations of Hfq 6 . ( B and C ) MST analysis with 30 nM labelled Hfq 6 and increasing concentrations of amiE 6ARN RNA in the absence (B) and presence of 360 nM Crc (C). The results represent data from two independent experiments and are shown as mean ± standard deviation. Thermophoresis/T-jump analysis is shown. LED power of 90% and MST power of 60% were used. ( D ) Hfq-RNA dissociation in the absence and presence of Crc. 32 P- amiE 6ARN RNA was pre-incubated with Hfq (A*H complex; lanes 2–5) or with Hfq and Crc (A*HC complex; lanes 7–10). Then, unlabelled amiE 6ARN competitor RNA was added for the times given in seconds followed by electrophoresis on a native polyacrylamide gel. Lane 1, electrophoretic mobility of 32 P- amiE 6ARN RNA (A*) in the presence of unlabelled 32 P- amiE 6ARN RNA. Lane 6, electrophoretic mobility of 32 P- amiE 6ARN RNA (A*) in the presence of unlabelled 32 P- amiE 6ARN RNA and Crc. The concentrations of the ligands are given at the right.
    Figure Legend Snippet: The presence of Crc stabilizes the Hfq/RNA complex. ( A ) Electrophoretic mobility shift assays (EMSA) with 32 P- amiE 6ARN RNA and increasing amounts of Hfq 6 in the absence and presence of Crc. Lane 1, electrophoretic mobility of 32 P- amiE 6ARN RNA (A*) in the absence of proteins. Lanes 2–5, 32 P- amiE 6ARN RNA/Hfq 6 complex formation (A*H) with increasing concentrations of Hfq 6 . Lanes 6–10, 32 P- amiE 6ARN RNA/Crc/Hfq 6 complex formation (A*HC) with increasing concentrations of Hfq 6 . ( B and C ) MST analysis with 30 nM labelled Hfq 6 and increasing concentrations of amiE 6ARN RNA in the absence (B) and presence of 360 nM Crc (C). The results represent data from two independent experiments and are shown as mean ± standard deviation. Thermophoresis/T-jump analysis is shown. LED power of 90% and MST power of 60% were used. ( D ) Hfq-RNA dissociation in the absence and presence of Crc. 32 P- amiE 6ARN RNA was pre-incubated with Hfq (A*H complex; lanes 2–5) or with Hfq and Crc (A*HC complex; lanes 7–10). Then, unlabelled amiE 6ARN competitor RNA was added for the times given in seconds followed by electrophoresis on a native polyacrylamide gel. Lane 1, electrophoretic mobility of 32 P- amiE 6ARN RNA (A*) in the presence of unlabelled 32 P- amiE 6ARN RNA. Lane 6, electrophoretic mobility of 32 P- amiE 6ARN RNA (A*) in the presence of unlabelled 32 P- amiE 6ARN RNA and Crc. The concentrations of the ligands are given at the right.

    Techniques Used: Electrophoretic Mobility Shift Assay, Microscale Thermophoresis, Standard Deviation, Incubation, Electrophoresis

    Amino acid residues in Crc implicated in Hfq and RNA interactions. The C-side and N-side of Crc are termed according to the localization of the N- and C-terminus, respectively. (A and B) Ribbon diagram (top), surface representation (middle) and electrostatic surface potential (bottom) of the Crc C-side ( 24 ) containing the basic patch ( A ) and the N-side opposed to it ( B ). The positions of the N- and C-termini are depicted in the ribbon diagrams (top) and are colored in dark blue (N-terminus) and green (C-terminus) in the surface structure (middle), respectively. K residues that were found to be cross-linked in the Hfq/Crc/ amiE 6ARN complex are depicted in light blue. Residues in Crc that were found to interact with amiE 6ARN RNA are highlighted in yellow. Amino acid residues that were found to be altered in the PAO1Δ crcZ sup mutants are highlighted in red. α-helices are colored in black, β-strands in light grey, and coils in dark grey, respectively. Image visualization was performed with Chimera ( 38 ). The electrostatic surface potential was calculated by Coulomb's law and visualized by Chimera ( 38 ). The electrostatic potential ranges from -10 (red) to +10 (blue) kcal/(mol* e ) at 298K.
    Figure Legend Snippet: Amino acid residues in Crc implicated in Hfq and RNA interactions. The C-side and N-side of Crc are termed according to the localization of the N- and C-terminus, respectively. (A and B) Ribbon diagram (top), surface representation (middle) and electrostatic surface potential (bottom) of the Crc C-side ( 24 ) containing the basic patch ( A ) and the N-side opposed to it ( B ). The positions of the N- and C-termini are depicted in the ribbon diagrams (top) and are colored in dark blue (N-terminus) and green (C-terminus) in the surface structure (middle), respectively. K residues that were found to be cross-linked in the Hfq/Crc/ amiE 6ARN complex are depicted in light blue. Residues in Crc that were found to interact with amiE 6ARN RNA are highlighted in yellow. Amino acid residues that were found to be altered in the PAO1Δ crcZ sup mutants are highlighted in red. α-helices are colored in black, β-strands in light grey, and coils in dark grey, respectively. Image visualization was performed with Chimera ( 38 ). The electrostatic surface potential was calculated by Coulomb's law and visualized by Chimera ( 38 ). The electrostatic potential ranges from -10 (red) to +10 (blue) kcal/(mol* e ) at 298K.

    Techniques Used:

    In vitro association between Hfq and Crc in the presence of RNA. ( A ) Overlay of the 2D 15 N– 1 H BEST-TROSY HSQC recorded before and after addition of unlabelled Hfq 6 . The resulting spectra are colored according to the molar ratio of Crc: Hfq 6 (black 1:0; red 1:1; blue 1:2; magenta 1:3). NMR signals that experienced chemical shift changes are boxed. ( B ) Overlay of the 2D 13 C– 1 H HMQC spectra of 13 C-methyl-labelled Crc recorded before (black spectra) and after addition of equimolar amounts of the unlabelled Hfq 6 / amiE 6ARN complex (red spectra). ( C ) MST analysis with 30 nM labelled Hfq 6 , 30 nM amiE 6ARN and increasing amounts of Crc. ( D ) MST analysis with 30 nM labelled Hfq 6 and increasing amounts of Crc. Data from two independent experiments are shown as mean ± standard deviation. Thermophoresis/T-jump analysis is shown. LED power of 90% and MST power of 60% were used.
    Figure Legend Snippet: In vitro association between Hfq and Crc in the presence of RNA. ( A ) Overlay of the 2D 15 N– 1 H BEST-TROSY HSQC recorded before and after addition of unlabelled Hfq 6 . The resulting spectra are colored according to the molar ratio of Crc: Hfq 6 (black 1:0; red 1:1; blue 1:2; magenta 1:3). NMR signals that experienced chemical shift changes are boxed. ( B ) Overlay of the 2D 13 C– 1 H HMQC spectra of 13 C-methyl-labelled Crc recorded before (black spectra) and after addition of equimolar amounts of the unlabelled Hfq 6 / amiE 6ARN complex (red spectra). ( C ) MST analysis with 30 nM labelled Hfq 6 , 30 nM amiE 6ARN and increasing amounts of Crc. ( D ) MST analysis with 30 nM labelled Hfq 6 and increasing amounts of Crc. Data from two independent experiments are shown as mean ± standard deviation. Thermophoresis/T-jump analysis is shown. LED power of 90% and MST power of 60% were used.

    Techniques Used: In Vitro, Nuclear Magnetic Resonance, Microscale Thermophoresis, Standard Deviation

    The association between Hfq and Crc requires RNA binding to the distal side of Hfq. ( A ) In vitro co-IP experiments were performed with purified components as indicated on top of the Figures, anti-Hfq specific antibodies and magnetic protein G beads. The in vitro association of Hfq with Crc was visualized by western-blot analysis using anti-Crc or anti-Hfq specific antibodies. Lanes 1 and 2, 4 pmol Hfq and 12 pmol Crc were loaded, respectively. Lanes 3–5, control experiments in the absence of both proteins (lane 3) or presence of either only Hfq (lane 4) or Crc (lane 5). Lanes 6–16, co-IP with anti-Hfq antibodies in the presence of Hfq and Crc (lanes 7, 9, 11, 13, and 15) and in the absence of Crc lanes 8, 10, 12, 14, and 16), respectively, with no RNA added (lane 6) and in the presence of amiE 6ARN (lane 7), poly-(U) 14 (lane 9), CrcZ (lane 11), poly-(A) 27 (lane 13) and PrrF2 sRNA (lane 15), respectively. ( B ) BACTH analysis of the Crc-Hfq Y25D interaction in E. coli strains BTH101(pUT18, pKT25) (white bar), BTH101(pHfq-T18, pKT25-Crc) (black bar) and BTH101(pHfq Y25D -T18, pKT25-Crc) (yellow bar), respectively. The results of three independent experiments were averaged and are shown as mean ± standard deviation.
    Figure Legend Snippet: The association between Hfq and Crc requires RNA binding to the distal side of Hfq. ( A ) In vitro co-IP experiments were performed with purified components as indicated on top of the Figures, anti-Hfq specific antibodies and magnetic protein G beads. The in vitro association of Hfq with Crc was visualized by western-blot analysis using anti-Crc or anti-Hfq specific antibodies. Lanes 1 and 2, 4 pmol Hfq and 12 pmol Crc were loaded, respectively. Lanes 3–5, control experiments in the absence of both proteins (lane 3) or presence of either only Hfq (lane 4) or Crc (lane 5). Lanes 6–16, co-IP with anti-Hfq antibodies in the presence of Hfq and Crc (lanes 7, 9, 11, 13, and 15) and in the absence of Crc lanes 8, 10, 12, 14, and 16), respectively, with no RNA added (lane 6) and in the presence of amiE 6ARN (lane 7), poly-(U) 14 (lane 9), CrcZ (lane 11), poly-(A) 27 (lane 13) and PrrF2 sRNA (lane 15), respectively. ( B ) BACTH analysis of the Crc-Hfq Y25D interaction in E. coli strains BTH101(pUT18, pKT25) (white bar), BTH101(pHfq-T18, pKT25-Crc) (black bar) and BTH101(pHfq Y25D -T18, pKT25-Crc) (yellow bar), respectively. The results of three independent experiments were averaged and are shown as mean ± standard deviation.

    Techniques Used: RNA Binding Assay, In Vitro, Co-Immunoprecipitation Assay, Purification, Western Blot, Standard Deviation

    Crc affects binding of the sRNA PrrF2 to Hfq. MST analyses with 40 nM PrrF2-Cy5 RNA and ( A ) increasing concentrations of Hfq, ( B ), increasing concentrations of Hfq in the presence of 500 nM amiE 6ARN, and ( C ) increasing concentrations of Hfq in the presence of 500 nM amiE 6ARN and 1 μM Crc. The results represent data from two independent experiments and are shown as mean ± standard deviation. Thermophoresis/T-jump analysis is shown. LED power of 90% and MST power of 60% were used. ( D ) PrrF2 sRNA does not bind to the Hfq/Crc/ amiE 6ARN RNA complex. EMSA with 10 nM Cy5-labelled PrrF2 RNA (red bands) and 100 nM 6-FAM-labelled amiE 6ARN RNA (green bands) in the absence or presence of Hfq 6 and Crc. Lane 1, electrophoretic mobility of 6-FAM- amiE 6ARN RNA (A*). Lane 2, electrophoretic mobility of PrrF2-Cy5 RNA (P*). Lanes 3 and 4, electrophoretic mobility of 6-FAM- amiE 6ARN RNA (A*H; middle panel) and PrrF2-Cy5 RNA (P*H; upper panel), respectively, in the presence of 120 nM Hfq. Lane 5, electrophoretic mobility of PrrF2-Cy5 RNA and 6-FAM- amiE 6ARN RNA in the presence of 120 nM Hfq. As shown in the superimposition (bottom panel) both RNAs are in complex with Hfq (P*HA*). Lane 6, electrophoretic mobility of 6-FAM- amiE 6ARN RNA in the presence of 120 nM Hfq and 960 nM of Crc (A*HC). Lane 7, electrophoretic mobility of PrrF2-Cy5 RNA and 6-FAM- amiE 6ARN RNA in the presence of both, 120 nM Hfq and 960 nM Crc. As shown in the superimposition PrrF2-Cy5 RNA is not part of the Hfq/Crc/6-FAM- amiE 6ARN (A*HC) RNA complex. Only the Hfq bound state (P*H) is observed. ( E ) The strains PAO1Δ crc Δ crcZ (pTL antR , pME4510) (orange bar) and PAO1Δ crcΔcrcZ (pTL antR , pME4510 crc Flag ) (blue bar) were grown in BSM-succinate medium. Samples were withdrawn at an OD 600 of 2.0. The bars represent the β-galactosidase values conferred by the plasmid pTLantR encoded translational antR::lacZ fusion in the presence or absence of ectopic crc Flag expression, respectively. The error bars represent standard deviations from three independent experiments. Top panel, Crc Flag , S1, PrrF2 and 5S rRNA levels in strains PAO1Δ crc Δ crcZ (pTL antR , pME4510) (lane 1) and PAO1Δ crcΔcrcZ (pTL antR , pME4510 crc Flag ) (lane 2). The Crc Flag levels were determined by western-blot analysis using anti-Crc antibodies. Immuno-detection of ribosomal protein S1 served as a loading control. The PrrF2 and 5S rRNA (control) levels were determined by Northern blotting.
    Figure Legend Snippet: Crc affects binding of the sRNA PrrF2 to Hfq. MST analyses with 40 nM PrrF2-Cy5 RNA and ( A ) increasing concentrations of Hfq, ( B ), increasing concentrations of Hfq in the presence of 500 nM amiE 6ARN, and ( C ) increasing concentrations of Hfq in the presence of 500 nM amiE 6ARN and 1 μM Crc. The results represent data from two independent experiments and are shown as mean ± standard deviation. Thermophoresis/T-jump analysis is shown. LED power of 90% and MST power of 60% were used. ( D ) PrrF2 sRNA does not bind to the Hfq/Crc/ amiE 6ARN RNA complex. EMSA with 10 nM Cy5-labelled PrrF2 RNA (red bands) and 100 nM 6-FAM-labelled amiE 6ARN RNA (green bands) in the absence or presence of Hfq 6 and Crc. Lane 1, electrophoretic mobility of 6-FAM- amiE 6ARN RNA (A*). Lane 2, electrophoretic mobility of PrrF2-Cy5 RNA (P*). Lanes 3 and 4, electrophoretic mobility of 6-FAM- amiE 6ARN RNA (A*H; middle panel) and PrrF2-Cy5 RNA (P*H; upper panel), respectively, in the presence of 120 nM Hfq. Lane 5, electrophoretic mobility of PrrF2-Cy5 RNA and 6-FAM- amiE 6ARN RNA in the presence of 120 nM Hfq. As shown in the superimposition (bottom panel) both RNAs are in complex with Hfq (P*HA*). Lane 6, electrophoretic mobility of 6-FAM- amiE 6ARN RNA in the presence of 120 nM Hfq and 960 nM of Crc (A*HC). Lane 7, electrophoretic mobility of PrrF2-Cy5 RNA and 6-FAM- amiE 6ARN RNA in the presence of both, 120 nM Hfq and 960 nM Crc. As shown in the superimposition PrrF2-Cy5 RNA is not part of the Hfq/Crc/6-FAM- amiE 6ARN (A*HC) RNA complex. Only the Hfq bound state (P*H) is observed. ( E ) The strains PAO1Δ crc Δ crcZ (pTL antR , pME4510) (orange bar) and PAO1Δ crcΔcrcZ (pTL antR , pME4510 crc Flag ) (blue bar) were grown in BSM-succinate medium. Samples were withdrawn at an OD 600 of 2.0. The bars represent the β-galactosidase values conferred by the plasmid pTLantR encoded translational antR::lacZ fusion in the presence or absence of ectopic crc Flag expression, respectively. The error bars represent standard deviations from three independent experiments. Top panel, Crc Flag , S1, PrrF2 and 5S rRNA levels in strains PAO1Δ crc Δ crcZ (pTL antR , pME4510) (lane 1) and PAO1Δ crcΔcrcZ (pTL antR , pME4510 crc Flag ) (lane 2). The Crc Flag levels were determined by western-blot analysis using anti-Crc antibodies. Immuno-detection of ribosomal protein S1 served as a loading control. The PrrF2 and 5S rRNA (control) levels were determined by Northern blotting.

    Techniques Used: Binding Assay, Microscale Thermophoresis, Standard Deviation, Plasmid Preparation, Expressing, Western Blot, Northern Blot

    8) Product Images from "5-Hydroxytryptamine 5HT2C Receptors Form a Protein Complex with N-Methyl-d-aspartate GluN2A Subunits and Activate Phosphorylation of Src Protein to Modulate Motoneuronal Depolarization"

    Article Title: 5-Hydroxytryptamine 5HT2C Receptors Form a Protein Complex with N-Methyl-d-aspartate GluN2A Subunits and Activate Phosphorylation of Src Protein to Modulate Motoneuronal Depolarization

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.277806

    5HT2C agonist MK 212 increases NMDA-induced motorneuronal depolarization in the frog spinal cord. A, 5-HT 2C agonist MK 212 enhances NMDA-induced motoneuron depolarizations in a dose-dependent manner. Application of NMDA (100 μ m , 10 s) is indicated
    Figure Legend Snippet: 5HT2C agonist MK 212 increases NMDA-induced motorneuronal depolarization in the frog spinal cord. A, 5-HT 2C agonist MK 212 enhances NMDA-induced motoneuron depolarizations in a dose-dependent manner. Application of NMDA (100 μ m , 10 s) is indicated

    Techniques Used:

    5HT2C agonist MK 212 results in phosphorylation of Src 416 in rat spinal neuronal culture. Immunoblot analysis of primary rat spinal neuronal culture treated with MK 212 (3–30 μ m ) for 20 min resulted in significant increases in Src 416 phosphorylation
    Figure Legend Snippet: 5HT2C agonist MK 212 results in phosphorylation of Src 416 in rat spinal neuronal culture. Immunoblot analysis of primary rat spinal neuronal culture treated with MK 212 (3–30 μ m ) for 20 min resulted in significant increases in Src 416 phosphorylation

    Techniques Used:

    Model of 5HT2C modulation of the NMDA channel. The 5HT2C receptor and NMDA channel are shown in close proximity. We hypothesize activation of 5HT2C receptors results in phosphorylation of Src Tyr-416 , which then modifies the GluN2A subunit of the NMDA
    Figure Legend Snippet: Model of 5HT2C modulation of the NMDA channel. The 5HT2C receptor and NMDA channel are shown in close proximity. We hypothesize activation of 5HT2C receptors results in phosphorylation of Src Tyr-416 , which then modifies the GluN2A subunit of the NMDA

    Techniques Used: Activation Assay

    GluN2A, 5HT2C, and Src tyrosine kinase multiprotein association in synaptosomes and GluN2A/5HT2C localization in rat spinal cord tissue and spinal neuronal culture. A, synaptosomal membranes were isolated from rat spinal cord tissue as described. Synaptosomal
    Figure Legend Snippet: GluN2A, 5HT2C, and Src tyrosine kinase multiprotein association in synaptosomes and GluN2A/5HT2C localization in rat spinal cord tissue and spinal neuronal culture. A, synaptosomal membranes were isolated from rat spinal cord tissue as described. Synaptosomal

    Techniques Used: Isolation

    5HT2C mRNA isoforms in the rat spinal cord. Top , edited regions of four major isoforms of the 5HT2C receptor pre-mRNA. Bottom , quantification of mRNA for VNV, VSV, and VGV 5HT2C isoforms in rat thoracic spinal cord. Significantly less of the unedited
    Figure Legend Snippet: 5HT2C mRNA isoforms in the rat spinal cord. Top , edited regions of four major isoforms of the 5HT2C receptor pre-mRNA. Bottom , quantification of mRNA for VNV, VSV, and VGV 5HT2C isoforms in rat thoracic spinal cord. Significantly less of the unedited

    Techniques Used:

    9) Product Images from "Haploinsufficiency of A20 impairs protein–protein interactome and leads into caspase-8-dependent enhancement of NLRP3 inflammasome activation"

    Article Title: Haploinsufficiency of A20 impairs protein–protein interactome and leads into caspase-8-dependent enhancement of NLRP3 inflammasome activation

    Journal: RMD Open

    doi: 10.1136/rmdopen-2018-000740

    Expression and activation of the NLRP3 inflammasome is altered in peripheral blood mononuclear cells (PBMCs) of TNFAIP3 p.(Lys91*) carriers. Samples from TNFAIP3 p.(Lys91*) mutation carriers, patients 1 (II-1, woman, 30 years) and 2 (III-1, female, 8 years), were compared with sex-matched and age-matched controls 1 and 2, respectively. Relative expression of (A) NLRP3 inflammasome components and target cytokines and (B) inflammasome-independent proinflammatory cytokines was analysed in PBMCs by quantitative PCR and normalised against housekeeping gene expression (arbitrary units). (C) Whole blood was stimulated with LPS and ATP, followed by the detection of NLRP3 inflammasome-triggered caspase-1 activity in monocytes using a fluorescent FLICA probe; the data are presented as median fluorescence intensity (MFI).
    Figure Legend Snippet: Expression and activation of the NLRP3 inflammasome is altered in peripheral blood mononuclear cells (PBMCs) of TNFAIP3 p.(Lys91*) carriers. Samples from TNFAIP3 p.(Lys91*) mutation carriers, patients 1 (II-1, woman, 30 years) and 2 (III-1, female, 8 years), were compared with sex-matched and age-matched controls 1 and 2, respectively. Relative expression of (A) NLRP3 inflammasome components and target cytokines and (B) inflammasome-independent proinflammatory cytokines was analysed in PBMCs by quantitative PCR and normalised against housekeeping gene expression (arbitrary units). (C) Whole blood was stimulated with LPS and ATP, followed by the detection of NLRP3 inflammasome-triggered caspase-1 activity in monocytes using a fluorescent FLICA probe; the data are presented as median fluorescence intensity (MFI).

    Techniques Used: Expressing, Activation Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Activity Assay, Fluorescence

    10) Product Images from "A novel denaturing heteroduplex tracking assay for genotypic prediction of HIV-1 tropism"

    Article Title: A novel denaturing heteroduplex tracking assay for genotypic prediction of HIV-1 tropism

    Journal: Journal of virological methods

    doi: 10.1016/j.jviromet.2012.06.013

    Analysis of DNA heteroduplexes with multiple mismatches using HTA with denaturing MDE® gel under different formamide concentrations. (A) HTA gel pictures. Formamide concentration is indicated in each gel. Samples 2 to 19 were heteroduplexes with six to fifteen mismatches, whose sequence alignment is shown as (B). (B) Alignment of target HIV-1 V3 DNA sequences. The sequence starts after primer V3F and ends before primer V3R, corresponding to nucleotide position of HIV-1 V3 11 to 87.
    Figure Legend Snippet: Analysis of DNA heteroduplexes with multiple mismatches using HTA with denaturing MDE® gel under different formamide concentrations. (A) HTA gel pictures. Formamide concentration is indicated in each gel. Samples 2 to 19 were heteroduplexes with six to fifteen mismatches, whose sequence alignment is shown as (B). (B) Alignment of target HIV-1 V3 DNA sequences. The sequence starts after primer V3F and ends before primer V3R, corresponding to nucleotide position of HIV-1 V3 11 to 87.

    Techniques Used: Concentration Assay, Sequencing

    Tropism determination of HIV-1 from patient plasma samples by the HIV-1 V3 denaturing HTA. (A) HIV-1 V3 denaturing HTA result using plasma sample of patient WC202. 1. 100ng of purified HIV-1 V3 RT PCR product from plasma sample of patient WC202. 2. 50ng V3 DNA from clone WC202PR5b. 3. 50ng V3 DNA from clone WC202PR5f. 4. 50ng V3 DNA from clone WC202PX4. p. probe only. (B) HIV-1 V3 denaturing HTA result using plasma sample of patient WC28. 1. 100ng of purified HIV-1 V3 RT PCR product from plasma sample of patient WC28. 2. 50ng V3 DNA from clone WC28PR5. 3. 50ng V3 DNA from clone WC28PX4a. 4. 50ng V3 DNA from clone WC28PX4c. 5. 50ng V3 DNA from clone WC28PX4b. p. probe only. (C) Amino acid sequence alignment of V3 clones from samples WC202 and WC28.
    Figure Legend Snippet: Tropism determination of HIV-1 from patient plasma samples by the HIV-1 V3 denaturing HTA. (A) HIV-1 V3 denaturing HTA result using plasma sample of patient WC202. 1. 100ng of purified HIV-1 V3 RT PCR product from plasma sample of patient WC202. 2. 50ng V3 DNA from clone WC202PR5b. 3. 50ng V3 DNA from clone WC202PR5f. 4. 50ng V3 DNA from clone WC202PX4. p. probe only. (B) HIV-1 V3 denaturing HTA result using plasma sample of patient WC28. 1. 100ng of purified HIV-1 V3 RT PCR product from plasma sample of patient WC28. 2. 50ng V3 DNA from clone WC28PR5. 3. 50ng V3 DNA from clone WC28PX4a. 4. 50ng V3 DNA from clone WC28PX4c. 5. 50ng V3 DNA from clone WC28PX4b. p. probe only. (C) Amino acid sequence alignment of V3 clones from samples WC202 and WC28.

    Techniques Used: Purification, Reverse Transcription Polymerase Chain Reaction, Sequencing, Clone Assay

    11) Product Images from "Real-Time PCR for Diagnosing and Quantifying Co-infection by Two Globally Distributed Fungal Pathogens of Wheat"

    Article Title: Real-Time PCR for Diagnosing and Quantifying Co-infection by Two Globally Distributed Fungal Pathogens of Wheat

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.01086

    (A) The relationship between quantification cycle and logarithm of the concentration of fungal DNA in duplexed qPCR settings. Triplicate dilution series corresponding to gDNA concentrations of 5000, 500, 50, 5, and 0.5 pg μl -1 were prepared. No-template samples were included in every reaction as negative controls ( n = 10). The quantification cycle at which fluorescent signals were observed is plotted against the logarithm of DNA concentrations of P. tritici-repentis (A) and Pa. nodorum (B) . The corresponding regression equations and coefficient of determinations ( R 2 ) are shown on the plot. Data are means ± standard deviation where visible ( n = 3).
    Figure Legend Snippet: (A) The relationship between quantification cycle and logarithm of the concentration of fungal DNA in duplexed qPCR settings. Triplicate dilution series corresponding to gDNA concentrations of 5000, 500, 50, 5, and 0.5 pg μl -1 were prepared. No-template samples were included in every reaction as negative controls ( n = 10). The quantification cycle at which fluorescent signals were observed is plotted against the logarithm of DNA concentrations of P. tritici-repentis (A) and Pa. nodorum (B) . The corresponding regression equations and coefficient of determinations ( R 2 ) are shown on the plot. Data are means ± standard deviation where visible ( n = 3).

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

    Specificity testing of primers and their matching probes in singleplex real-time qPCR settings. Amplification curve for P. tritici-repentis is plotted in blue (A) and amplification curve of Pa. nodorum is plotted in orange (B) . Primers and probes ( Table 1 ) were tested against DNA from the five fungal species, a negative wheat control, and a no template control and reactions were run separately. Data represent means ± standard deviation ( n = 3).
    Figure Legend Snippet: Specificity testing of primers and their matching probes in singleplex real-time qPCR settings. Amplification curve for P. tritici-repentis is plotted in blue (A) and amplification curve of Pa. nodorum is plotted in orange (B) . Primers and probes ( Table 1 ) were tested against DNA from the five fungal species, a negative wheat control, and a no template control and reactions were run separately. Data represent means ± standard deviation ( n = 3).

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Standard Deviation

    Detection and quantification of P. tritici-repentis and Pa. nodorum in a simulated DNA matrix of various ratios. (A) P. tritici-repentis DNA was spiked with Pa. nodorum DNA at ratios of 1:1, 1:10, 1:100, 1:1000, and 1:10000. (B) Pa. nodorum was spiked with P. tritici-repentis DNA at same ratios. The starting concentration was 5 ng μl -1 of each pathogen DNA. The subsequent ratios were sequential 10-fold dilutions (0.5, 0.05, 0.005, and 0.0005 ng μl -1 ) of one pathogen DNA, with the other held constant at 5 ng μl -1 . Data represent means ± standard deviation ( n = 6).
    Figure Legend Snippet: Detection and quantification of P. tritici-repentis and Pa. nodorum in a simulated DNA matrix of various ratios. (A) P. tritici-repentis DNA was spiked with Pa. nodorum DNA at ratios of 1:1, 1:10, 1:100, 1:1000, and 1:10000. (B) Pa. nodorum was spiked with P. tritici-repentis DNA at same ratios. The starting concentration was 5 ng μl -1 of each pathogen DNA. The subsequent ratios were sequential 10-fold dilutions (0.5, 0.05, 0.005, and 0.0005 ng μl -1 ) of one pathogen DNA, with the other held constant at 5 ng μl -1 . Data represent means ± standard deviation ( n = 6).

    Techniques Used: Concentration Assay, Standard Deviation

    Specificity testing of primers by agarose gel electrophoresis. (A) Primers of P. tritici-repentis, Ptr -Forward and Ptr -Reverse, were tested against Pa. nodorum DNA, and (B) primers of Pa. nodorum , Pn -Forward and Pn -Reverse, were tested against P. tritici-repentis DNA in two separate PCR reactions. Reactions included negative controls of DNA samples from five cereal fungal pathogens. Reactions including positive and negative controls were electrophoresed on 2% agarose with two technical replicates.
    Figure Legend Snippet: Specificity testing of primers by agarose gel electrophoresis. (A) Primers of P. tritici-repentis, Ptr -Forward and Ptr -Reverse, were tested against Pa. nodorum DNA, and (B) primers of Pa. nodorum , Pn -Forward and Pn -Reverse, were tested against P. tritici-repentis DNA in two separate PCR reactions. Reactions included negative controls of DNA samples from five cereal fungal pathogens. Reactions including positive and negative controls were electrophoresed on 2% agarose with two technical replicates.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction

    12) Product Images from "Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide"

    Article Title: Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004853

    Br- LPS induces activation of caspase 8 and 9 in PMNs. (A) Heparinized blood was incubated with 0.3 pmol/mL of Br- LPS or PBS for 30 minutes and stained with anti-active caspase 8 or anti-active caspase 9. PMNs population was analyzed by each caspase marker (B) Heparinized blood samples were treated with Z-VAD-FMK or PBS for 1 hour and then incubated with Br -LPS (1.5 pmol/mL) for 2 hours. PMNs population was analyzed by Annexin V. Geometric means of histograms are displayed as relative units. Experiments were repeated at least three times.
    Figure Legend Snippet: Br- LPS induces activation of caspase 8 and 9 in PMNs. (A) Heparinized blood was incubated with 0.3 pmol/mL of Br- LPS or PBS for 30 minutes and stained with anti-active caspase 8 or anti-active caspase 9. PMNs population was analyzed by each caspase marker (B) Heparinized blood samples were treated with Z-VAD-FMK or PBS for 1 hour and then incubated with Br -LPS (1.5 pmol/mL) for 2 hours. PMNs population was analyzed by Annexin V. Geometric means of histograms are displayed as relative units. Experiments were repeated at least three times.

    Techniques Used: Activation Assay, Incubation, Staining, Marker

    13) Product Images from "Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability"

    Article Title: Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw170

    Incorporation of 8-oxodGMP and 2-OH-dAMP in TNR sequences. ( A and B ) Incorporation of dGMP and 8-oxodGMP opposite cytosine. ( A ) The primer/template sequence and representative gels are shown. Primer/template substrate (160 nM) (S1/T1) was incubated with POL β and increasing concentration of 8-oxodGTP or dGTP at 37°C for 1 h (0–30 μM and 0–3 μM, respectively). M is the DNA substrate without enzyme. Reaction products were separated by 20% denaturing PAGE at 500 V for 2.5 h. Bands were visualized by fluorescence emission by Typhoon scanner and the analysis of the band intensities was performed by ImageJ software. ( B ) Percentage of incorporated dNMP plotted as a function of added dNTPs. Data were fitted by Kaleidagraph software to evaluate kinetics parameters. ( C and D ) Incorporation of 8-oxodGMP and dTMP opposite adenine. Primer/template sequences is S2/T1 (Supplementary Table S1). Experimental conditions and analyses were as described above. The concentration range of 8-oxodGTP and dTTP was 0–2 μM and 0–1 μM, respectively. ( E – F ) Incorporation of 2-OH-dAMP and dAMP in CAG/CTG repeat sequence. ( E ) Primer/template sequence is S3/T2 (Supplementary Table S1); ( F ) Primer/template duplex (160 nM) was incubated with POL β (0.1U) in 10 μl reaction buffer in the absence of dNTP (lane 1), after addition of 2-OH-dATP (lane 2), dGTP and 2-OH-dATP (lane 3); 2-OH-dATP, dGTP and dCTP (lane 4), dATP (lane 5); dATP and dGTP (lane 6), dATP, dGTP and dCTP (lane 7). All nucleotide triphosphates were at 10 μM final concentration. Reaction products were separated by 15% denaturing PAGE and image acquisition and analysis was performed as described before.
    Figure Legend Snippet: Incorporation of 8-oxodGMP and 2-OH-dAMP in TNR sequences. ( A and B ) Incorporation of dGMP and 8-oxodGMP opposite cytosine. ( A ) The primer/template sequence and representative gels are shown. Primer/template substrate (160 nM) (S1/T1) was incubated with POL β and increasing concentration of 8-oxodGTP or dGTP at 37°C for 1 h (0–30 μM and 0–3 μM, respectively). M is the DNA substrate without enzyme. Reaction products were separated by 20% denaturing PAGE at 500 V for 2.5 h. Bands were visualized by fluorescence emission by Typhoon scanner and the analysis of the band intensities was performed by ImageJ software. ( B ) Percentage of incorporated dNMP plotted as a function of added dNTPs. Data were fitted by Kaleidagraph software to evaluate kinetics parameters. ( C and D ) Incorporation of 8-oxodGMP and dTMP opposite adenine. Primer/template sequences is S2/T1 (Supplementary Table S1). Experimental conditions and analyses were as described above. The concentration range of 8-oxodGTP and dTTP was 0–2 μM and 0–1 μM, respectively. ( E – F ) Incorporation of 2-OH-dAMP and dAMP in CAG/CTG repeat sequence. ( E ) Primer/template sequence is S3/T2 (Supplementary Table S1); ( F ) Primer/template duplex (160 nM) was incubated with POL β (0.1U) in 10 μl reaction buffer in the absence of dNTP (lane 1), after addition of 2-OH-dATP (lane 2), dGTP and 2-OH-dATP (lane 3); 2-OH-dATP, dGTP and dCTP (lane 4), dATP (lane 5); dATP and dGTP (lane 6), dATP, dGTP and dCTP (lane 7). All nucleotide triphosphates were at 10 μM final concentration. Reaction products were separated by 15% denaturing PAGE and image acquisition and analysis was performed as described before.

    Techniques Used: Sequencing, Incubation, Concentration Assay, Polyacrylamide Gel Electrophoresis, Fluorescence, Software, CTG Assay

    Incorporation and extension of 8-oxodGMP by POL β. Primer/template (160 nM) (Supplementary Table S1, S3/T2 panel A; S1/T1 panel B) were used. Both substrates were incubated with POL β (0.1 U) and 8-oxodGTP or dGTP and others dNTPs (50 μM final concentration each). ( A ) Lane 1, primer; lane 2, 8-oxodGTP; lane 3, as lane 2 plus dCTP; lane 4, as lane 3 plus dATP; lane 5, as lane 4 plus dTTP; lane 6, primer plus dGTP; lane 7, as lane 6 plus dCTP; lane 8 as lane 7 plus dATP; lane 9, as lane 8 plus dTTP. ( B ) Lane 1, primer; lane 2, primer plus 8- oxodGTP; lane 3, as lane 2 plus dCTP; lane 4 as lane 3 plus dTTP; lane 5, as lane 4 plus dATP; lane 6, primer plus dGTP; lane 7, as lane 6 plus dCTP; lane 8 as lane 6 plus dTTP and dATP. ( C ) The DNA substrate (160 nM) was built by annealing three oligomers of 22, 77 and 100 bases respectively indicated as S1, S4 and T1 in Supplementary Table S1, in order to produce a preformed nicked duplex containing CTG/CAG repeats. Incorporation of dGTP and 8-oxodGTP (lanes 1 and 4) and elongation (lanes 2 and 5) was obtained by incubating the substrate (lane 3) with POL β (0.1 U) at 37°C for 1 h.
    Figure Legend Snippet: Incorporation and extension of 8-oxodGMP by POL β. Primer/template (160 nM) (Supplementary Table S1, S3/T2 panel A; S1/T1 panel B) were used. Both substrates were incubated with POL β (0.1 U) and 8-oxodGTP or dGTP and others dNTPs (50 μM final concentration each). ( A ) Lane 1, primer; lane 2, 8-oxodGTP; lane 3, as lane 2 plus dCTP; lane 4, as lane 3 plus dATP; lane 5, as lane 4 plus dTTP; lane 6, primer plus dGTP; lane 7, as lane 6 plus dCTP; lane 8 as lane 7 plus dATP; lane 9, as lane 8 plus dTTP. ( B ) Lane 1, primer; lane 2, primer plus 8- oxodGTP; lane 3, as lane 2 plus dCTP; lane 4 as lane 3 plus dTTP; lane 5, as lane 4 plus dATP; lane 6, primer plus dGTP; lane 7, as lane 6 plus dCTP; lane 8 as lane 6 plus dTTP and dATP. ( C ) The DNA substrate (160 nM) was built by annealing three oligomers of 22, 77 and 100 bases respectively indicated as S1, S4 and T1 in Supplementary Table S1, in order to produce a preformed nicked duplex containing CTG/CAG repeats. Incorporation of dGTP and 8-oxodGTP (lanes 1 and 4) and elongation (lanes 2 and 5) was obtained by incubating the substrate (lane 3) with POL β (0.1 U) at 37°C for 1 h.

    Techniques Used: Incubation, Concentration Assay, CTG Assay

    Novel contributors in TNR expansion process. Following an initial incision event mediated by OGG1 and APE1 at an 8-oxodG site in the top strand (red, step 1), POL drives repair synthesis by LP BER. Long flaps might eventually fold in stable secondary structures (step 2). A faulty removal by FEN1 depending on flap conformations might leave hairpins with unligatable dRP ends (step 3). Removal of dRP by POL β allows ligation by LIG1 (step 4). If 8-oxodGTP is present in the dNTPs pool, 8-oxodGMP can be incorporated opposite A in the complementary strand creating a substrate for MUTYH (step 5). MUTYH activity on the bottom strand (blue) allows the initiation of a new repair event, as well as an elongation process on this side (step 6). Realignements of the strands will result in TNR expansion (step 7). Newly synthesized tracts are represented by full rectangles. The proposed model has been modified from refs. ( 12 , 16 , 40 ).
    Figure Legend Snippet: Novel contributors in TNR expansion process. Following an initial incision event mediated by OGG1 and APE1 at an 8-oxodG site in the top strand (red, step 1), POL drives repair synthesis by LP BER. Long flaps might eventually fold in stable secondary structures (step 2). A faulty removal by FEN1 depending on flap conformations might leave hairpins with unligatable dRP ends (step 3). Removal of dRP by POL β allows ligation by LIG1 (step 4). If 8-oxodGTP is present in the dNTPs pool, 8-oxodGMP can be incorporated opposite A in the complementary strand creating a substrate for MUTYH (step 5). MUTYH activity on the bottom strand (blue) allows the initiation of a new repair event, as well as an elongation process on this side (step 6). Realignements of the strands will result in TNR expansion (step 7). Newly synthesized tracts are represented by full rectangles. The proposed model has been modified from refs. ( 12 , 16 , 40 ).

    Techniques Used: Ligation, Activity Assay, Synthesized, Modification

    14) Product Images from "Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples"

    Article Title: Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.04137-13

    Gel electrophoresis of TaqMan qPCR products showing PD products present in HF183/BFDrev products (A) and absent in HF183/BacR287 amplification products (B). Lanes 1 to 11, amplicons generated from 5 to 1 × 10 5 ng per reaction mixture of human composite DNA template; lanes 12, no-template control (the DNA target product was visible due to amplification of the IAC spike); lanes L, molecular size ladder. The lowest band on the molecular ladder indicates 50 bp.
    Figure Legend Snippet: Gel electrophoresis of TaqMan qPCR products showing PD products present in HF183/BFDrev products (A) and absent in HF183/BacR287 amplification products (B). Lanes 1 to 11, amplicons generated from 5 to 1 × 10 5 ng per reaction mixture of human composite DNA template; lanes 12, no-template control (the DNA target product was visible due to amplification of the IAC spike); lanes L, molecular size ladder. The lowest band on the molecular ladder indicates 50 bp.

    Techniques Used: Nucleic Acid Electrophoresis, Real-time Polymerase Chain Reaction, Amplification, Generated

    15) Product Images from "Exoribonuclease-Resistant RNAs Exist within both Coding and Noncoding Subgenomic RNAs"

    Article Title: Exoribonuclease-Resistant RNAs Exist within both Coding and Noncoding Subgenomic RNAs

    Journal: mBio

    doi: 10.1128/mBio.02461-18

    Widespread occurrence of Xrn1-resistant RNAs among plant viruses. (A) Consensus sequence and secondary structure of xrRNA D based on a comparative sequence alignment of 47 sequences of viruses belonging to the Tombusviridae and Luteoviridae families (shown in Fig. S1 in the supplemental material). Y = pyrimidine; R = purine. Non-Watson-Crick base pairs are shown using the Leontis-Westhof nomenclature ( 49 ). The numbering is that of the crystal structure of the SCNMV xrRNA ( 26 ). (B) Phylogenetic relationship between various plant viruses, based on the RNA-dependent RNA polymerase amino acid sequence ( 31 ). The viruses and corresponding genera in which we identified xrRNA D structures are marked by a star. Numbers at the nodes refer to bootstrap values as percentages obtained from 2,000 replications, shown only for branches supported by more than 40% of the data. Branch lengths are proportional to the number of changes. Further analysis will likely reveal xrRNA D elements in more of these viruses with additional sequence and structural variation.
    Figure Legend Snippet: Widespread occurrence of Xrn1-resistant RNAs among plant viruses. (A) Consensus sequence and secondary structure of xrRNA D based on a comparative sequence alignment of 47 sequences of viruses belonging to the Tombusviridae and Luteoviridae families (shown in Fig. S1 in the supplemental material). Y = pyrimidine; R = purine. Non-Watson-Crick base pairs are shown using the Leontis-Westhof nomenclature ( 49 ). The numbering is that of the crystal structure of the SCNMV xrRNA ( 26 ). (B) Phylogenetic relationship between various plant viruses, based on the RNA-dependent RNA polymerase amino acid sequence ( 31 ). The viruses and corresponding genera in which we identified xrRNA D structures are marked by a star. Numbers at the nodes refer to bootstrap values as percentages obtained from 2,000 replications, shown only for branches supported by more than 40% of the data. Branch lengths are proportional to the number of changes. Further analysis will likely reveal xrRNA D elements in more of these viruses with additional sequence and structural variation.

    Techniques Used: Sequencing

    Biochemical characterization of representative plant virus xrRNA D elements. (A) In vitro Xrn1 resistance assay of xrRNA D from various plant RNA viruses ( Table 1 ). The xrRNA from RCNMV was included as a positive control. Arrows indicate the size of full-length RNAs and Xrn1-resistant degradation products. (B) Classification of viruses used in the experiments represented in panel A ( Table 1 ). (C to E) In vitro Xrn1 resistance assay of wild-type (WT) and pseudoknot (PK) mutant versions of MCMV (C), PLRV (D), and HuPLV1 (E) xrRNAs. (F to H) Reverse transcription (RT) mapping of the Xrn1 stop site. Data represent distributions of RT products of Xrn1-resistant fragments of MCMV (F), PLRV (G), and HuPLV1 (H) degradation fragments. Experimentally validated stop sites are indicated on the secondary structure diagrams for all tested xrRNA D shown in Fig. S2 .
    Figure Legend Snippet: Biochemical characterization of representative plant virus xrRNA D elements. (A) In vitro Xrn1 resistance assay of xrRNA D from various plant RNA viruses ( Table 1 ). The xrRNA from RCNMV was included as a positive control. Arrows indicate the size of full-length RNAs and Xrn1-resistant degradation products. (B) Classification of viruses used in the experiments represented in panel A ( Table 1 ). (C to E) In vitro Xrn1 resistance assay of wild-type (WT) and pseudoknot (PK) mutant versions of MCMV (C), PLRV (D), and HuPLV1 (E) xrRNAs. (F to H) Reverse transcription (RT) mapping of the Xrn1 stop site. Data represent distributions of RT products of Xrn1-resistant fragments of MCMV (F), PLRV (G), and HuPLV1 (H) degradation fragments. Experimentally validated stop sites are indicated on the secondary structure diagrams for all tested xrRNA D shown in Fig. S2 .

    Techniques Used: In Vitro, Positive Control, Mutagenesis

    An expanding repertoire of structured RNAs for blocking exoribonuclease degradation. (Top) xrRNAs adopt a three-dimensional structure that blocks the progression of 5′-to-3′ exoribonucleases such as Xrn1 (gray). In the case of flaviviruses and dianthoviruses, xrRNAs are in the 3′UTR, resulting in accumulating noncoding sgRNAs. (Middle) Secondary structure diagrams of the two classes of xrRNAs from flaviviruses (xrRNA F1 and xrRNA F2 ) ( 15 , 22 , 23 ) and of xrRNA D from dianthoviruses ( 26 ). Secondary structure features are labeled, and nucleotides involved in tertiary interactions are shown in colors connected by dashed lines (pseudoknot shown in blue). Experimentally determined Xrn1 stop sites are indicated. (Bottom) The boxes below each secondary structure contain diagrams reflecting the currently available three-dimensional structures ( 24 – 26 ). The A8-G33 pair is highlighted in the open state of the Sweet clover necrotic mosaic virus (SCNMV) xrRNA (far left).
    Figure Legend Snippet: An expanding repertoire of structured RNAs for blocking exoribonuclease degradation. (Top) xrRNAs adopt a three-dimensional structure that blocks the progression of 5′-to-3′ exoribonucleases such as Xrn1 (gray). In the case of flaviviruses and dianthoviruses, xrRNAs are in the 3′UTR, resulting in accumulating noncoding sgRNAs. (Middle) Secondary structure diagrams of the two classes of xrRNAs from flaviviruses (xrRNA F1 and xrRNA F2 ) ( 15 , 22 , 23 ) and of xrRNA D from dianthoviruses ( 26 ). Secondary structure features are labeled, and nucleotides involved in tertiary interactions are shown in colors connected by dashed lines (pseudoknot shown in blue). Experimentally determined Xrn1 stop sites are indicated. (Bottom) The boxes below each secondary structure contain diagrams reflecting the currently available three-dimensional structures ( 24 – 26 ). The A8-G33 pair is highlighted in the open state of the Sweet clover necrotic mosaic virus (SCNMV) xrRNA (far left).

    Techniques Used: Blocking Assay, Labeling

    16) Product Images from "Novel monodisperse PEGtide dendrons: design, fabrication and evaluation of mannose receptor-mediated macrophage targeting"

    Article Title: Novel monodisperse PEGtide dendrons: design, fabrication and evaluation of mannose receptor-mediated macrophage targeting

    Journal: Bioconjugate chemistry

    doi: 10.1021/bc400011v

    Synthesis of PEGtide dendrons: (A) G1.0; and (B) G2.0-5.0. The dendrons were synthesized using Fmoc SPPS using following components: Fmoc-Lys(5-FAM)-OH; Fmoc-Lys(Fmoc)-OH; Fmoc-β-Ala-OH; and Fmoc-dPEG 6 -OH.
    Figure Legend Snippet: Synthesis of PEGtide dendrons: (A) G1.0; and (B) G2.0-5.0. The dendrons were synthesized using Fmoc SPPS using following components: Fmoc-Lys(5-FAM)-OH; Fmoc-Lys(Fmoc)-OH; Fmoc-β-Ala-OH; and Fmoc-dPEG 6 -OH.

    Techniques Used: Synthesized

    17) Product Images from "Stable reduction of CCR5 by RNAi through hematopoietic stem cell transplant in non-human primates"

    Article Title: Stable reduction of CCR5 by RNAi through hematopoietic stem cell transplant in non-human primates

    Journal:

    doi: 10.1073/pnas.0705474104

    Reduction of rhCCR5 by an shRNA(1005). An shRNA against rhCCR5(1005) was tested in rhCCR5-expressing 293T cells. Cells were transduced with an SIV-based lentiviral vector bearing shRNA(1005) against rhCCR5 and analyzed for CCR5 and EGFP expression by
    Figure Legend Snippet: Reduction of rhCCR5 by an shRNA(1005). An shRNA against rhCCR5(1005) was tested in rhCCR5-expressing 293T cells. Cells were transduced with an SIV-based lentiviral vector bearing shRNA(1005) against rhCCR5 and analyzed for CCR5 and EGFP expression by

    Techniques Used: shRNA, Expressing, Transduction, Plasmid Preparation

    18) Product Images from "Effects of hepatitis C virus core protein and nonstructural protein 4B on the Wnt/β-catenin pathway"

    Article Title: Effects of hepatitis C virus core protein and nonstructural protein 4B on the Wnt/β-catenin pathway

    Journal: BMC Microbiology

    doi: 10.1186/s12866-017-1032-4

    HCV core protein and NS4B promote, directly or under Wnt3a induction, the nuclear translocation of β-catenin in Huh7 cells and LO2 cells. a Subcellular localization of β-catenin in Huh7 cells (magnification, ×1000). β-catenin protein was detected by using an anti-β-catenin antibody which was visualized by an anti-rabbit secondary antibody conjugated with CF 488A dye ( green ). Nuclei were counterstained with DAPI ( blue ). b The histogram indicating the percentage of positive cells for β-catenin nuclear localization in Huh7 cells. HCV core protein and NS4B promote the nuclear translocation of β-catenin in Huh7 cells. Compared with Huh7-mkate2, ** P
    Figure Legend Snippet: HCV core protein and NS4B promote, directly or under Wnt3a induction, the nuclear translocation of β-catenin in Huh7 cells and LO2 cells. a Subcellular localization of β-catenin in Huh7 cells (magnification, ×1000). β-catenin protein was detected by using an anti-β-catenin antibody which was visualized by an anti-rabbit secondary antibody conjugated with CF 488A dye ( green ). Nuclei were counterstained with DAPI ( blue ). b The histogram indicating the percentage of positive cells for β-catenin nuclear localization in Huh7 cells. HCV core protein and NS4B promote the nuclear translocation of β-catenin in Huh7 cells. Compared with Huh7-mkate2, ** P

    Techniques Used: Translocation Assay

    19) Product Images from "Metformin Induces Cell Cycle Arrest and Apoptosis in Drug-Resistant Leukemia Cells"

    Article Title: Metformin Induces Cell Cycle Arrest and Apoptosis in Drug-Resistant Leukemia Cells

    Journal: Leukemia Research and Treatment

    doi: 10.1155/2015/516460

    Effects of metformin on apoptosis. CEM and 10E 1 -CEM cells were exposed to 10 mM metformin for 72 h and the induction of apoptosis was evaluated by (a, and Table 1 ) flow cytometry of annexin V-PI labeled cells and (b) by fluorescence microscopy of DAPI labeled cells. (c) 10E 1 -CEM cells were treated with 10 mM metformin for 96 h before caspase-3 and -7 activation was evaluated by flow cytometry. The numbers in the quadrants indicate the percentages of cells. A representative experiment of three performed. (a, and Table 1 ).
    Figure Legend Snippet: Effects of metformin on apoptosis. CEM and 10E 1 -CEM cells were exposed to 10 mM metformin for 72 h and the induction of apoptosis was evaluated by (a, and Table 1 ) flow cytometry of annexin V-PI labeled cells and (b) by fluorescence microscopy of DAPI labeled cells. (c) 10E 1 -CEM cells were treated with 10 mM metformin for 96 h before caspase-3 and -7 activation was evaluated by flow cytometry. The numbers in the quadrants indicate the percentages of cells. A representative experiment of three performed. (a, and Table 1 ).

    Techniques Used: Flow Cytometry, Cytometry, Labeling, Fluorescence, Microscopy, Activation Assay

    20) Product Images from "Polyarginine as a multifunctional fusion tag"

    Article Title: Polyarginine as a multifunctional fusion tag

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1110/ps.051393805

    Effect of an R 9 tag on the purification of a protein by cation-exchange chromatography. ( A ) RNase A-R 9 was purified by cation-exchange chromatography before (−CPB) and after (+CPB) the addition of carboxypeptidase B. ( B ) SDS-PAGE gel of RNase A-R 9 before (−CPB) and after (+CPB) the addition of carboxypeptidase B. Purified RNase A is a standard.
    Figure Legend Snippet: Effect of an R 9 tag on the purification of a protein by cation-exchange chromatography. ( A ) RNase A-R 9 was purified by cation-exchange chromatography before (−CPB) and after (+CPB) the addition of carboxypeptidase B. ( B ) SDS-PAGE gel of RNase A-R 9 before (−CPB) and after (+CPB) the addition of carboxypeptidase B. Purified RNase A is a standard.

    Techniques Used: Purification, Chromatography, SDS Page

    Effect of an R 9 tag on the adsorption of a protein to a glass slide and silica resin. ( A ) Fluorescent images of fluorescein-labeled RNase A-R 9 and RNase A (10–0.01 μM) adsorbed on to a glass slide. ( B ) Ribonucleolytic activity in a solution containing silica resin with adsorbed RNase A-R 9 or RNase A, and in the supernatant upon removal of the silica resin with adsorbed RNase A-R 9 .
    Figure Legend Snippet: Effect of an R 9 tag on the adsorption of a protein to a glass slide and silica resin. ( A ) Fluorescent images of fluorescein-labeled RNase A-R 9 and RNase A (10–0.01 μM) adsorbed on to a glass slide. ( B ) Ribonucleolytic activity in a solution containing silica resin with adsorbed RNase A-R 9 or RNase A, and in the supernatant upon removal of the silica resin with adsorbed RNase A-R 9 .

    Techniques Used: Adsorption, Labeling, Activity Assay

    Effect of an R 9 tag on the uptake of a protein by living mammalian cells. CHO-K1 cells were incubated with fluorescein-labeled RNase A-R 9 (10 μM, A ) or fluorescein-labeled RNase A (10 μM, B ) for 15 min at 37°C before visualization by fluorescence microscopy. Scale bar: 10 μm.
    Figure Legend Snippet: Effect of an R 9 tag on the uptake of a protein by living mammalian cells. CHO-K1 cells were incubated with fluorescein-labeled RNase A-R 9 (10 μM, A ) or fluorescein-labeled RNase A (10 μM, B ) for 15 min at 37°C before visualization by fluorescence microscopy. Scale bar: 10 μm.

    Techniques Used: Incubation, Labeling, Fluorescence, Microscopy

    21) Product Images from "Improved Identification of Rapidly Growing Mycobacteria by a 16S-23S Internal Transcribed Spacer Region PCR and Capillary Gel Electrophoresis"

    Article Title: Improved Identification of Rapidly Growing Mycobacteria by a 16S-23S Internal Transcribed Spacer Region PCR and Capillary Gel Electrophoresis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0102290

    Representative CGE electropherograms following PCR amplification of the 16S–23S rRNA internal transcribed spacer (ITS) for A) M. chelonae ; B) M. abscessus ; C) M. massiliense ; D) M. fortuitum ; and E) M. mucogenicum . Peaks correlate with the ITS fragment length(s) which is shown above each peak. Panel A and D highlight the phenomenon of spurious double peaks which were less than 1 bp apart. Panel F shows the ITS-CGE electropherogram following the pre-extraction mix of M. abscessus and M. fortuitum demonstrating the typical peaks for each isolate.
    Figure Legend Snippet: Representative CGE electropherograms following PCR amplification of the 16S–23S rRNA internal transcribed spacer (ITS) for A) M. chelonae ; B) M. abscessus ; C) M. massiliense ; D) M. fortuitum ; and E) M. mucogenicum . Peaks correlate with the ITS fragment length(s) which is shown above each peak. Panel A and D highlight the phenomenon of spurious double peaks which were less than 1 bp apart. Panel F shows the ITS-CGE electropherogram following the pre-extraction mix of M. abscessus and M. fortuitum demonstrating the typical peaks for each isolate.

    Techniques Used: Polymerase Chain Reaction, Amplification

    22) Product Images from "Most Anti-BrdU Antibodies React with 2?-Deoxy-5-Ethynyluridine -- The Method for the Effective Suppression of This Cross-Reactivity"

    Article Title: Most Anti-BrdU Antibodies React with 2?-Deoxy-5-Ethynyluridine -- The Method for the Effective Suppression of This Cross-Reactivity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051679

    The simultaneous localisation of BrdU and EdU. The results of simultaneous localisation of BrdU and EdU using two different protocols for BrdU revelation in cells labelled for 5 minutes with BrdU and then after 13 hours for 20 minutes with EdU. A. The revelation of BrdU by 4N HCl is shown in the picture. The upper set of images represents the detection of both signals without the application of the blocking step by means of 2 mM azidomethylphenylsulfide. The bottom set of images represents the detection of both signals with the application of the blocking step by means of 2 mM azidomethylphenylsulfide. Barr: 20 µm. B. The revelation of BrdU by copper(I) ions and exonuclease III is shown in the picture. The upper set of images represents the detection of both signals without the application of the blocking step by means of 2 mM azidomethylphenylsulfide. The bottom set of images represents the detection of both signals with the application of the blocking step by means of 2 mM azidomethylphenylsulfide. Barr: 20 µm.
    Figure Legend Snippet: The simultaneous localisation of BrdU and EdU. The results of simultaneous localisation of BrdU and EdU using two different protocols for BrdU revelation in cells labelled for 5 minutes with BrdU and then after 13 hours for 20 minutes with EdU. A. The revelation of BrdU by 4N HCl is shown in the picture. The upper set of images represents the detection of both signals without the application of the blocking step by means of 2 mM azidomethylphenylsulfide. The bottom set of images represents the detection of both signals with the application of the blocking step by means of 2 mM azidomethylphenylsulfide. Barr: 20 µm. B. The revelation of BrdU by copper(I) ions and exonuclease III is shown in the picture. The upper set of images represents the detection of both signals without the application of the blocking step by means of 2 mM azidomethylphenylsulfide. The bottom set of images represents the detection of both signals with the application of the blocking step by means of 2 mM azidomethylphenylsulfide. Barr: 20 µm.

    Techniques Used: Blocking Assay

    The suppression of the EdU signal using non-fluorescent azido molecules. The suppression of the signal provided by BU1/75 antibody after a click reaction with 2 or 20 mM 2-azidoethanol, 1-azido-2,3-dihydroxypropane or azidomethylphenylsulfide in cells labelled with EdU for 20 minutes. The images were acquired for 8 ms. Barr: 20 µm.
    Figure Legend Snippet: The suppression of the EdU signal using non-fluorescent azido molecules. The suppression of the signal provided by BU1/75 antibody after a click reaction with 2 or 20 mM 2-azidoethanol, 1-azido-2,3-dihydroxypropane or azidomethylphenylsulfide in cells labelled with EdU for 20 minutes. The images were acquired for 8 ms. Barr: 20 µm.

    Techniques Used: Mass Spectrometry

    23) Product Images from "Regional Assessment of Human Fecal Contamination in Southern California Coastal Drainages"

    Article Title: Regional Assessment of Human Fecal Contamination in Southern California Coastal Drainages

    Journal: International Journal of Environmental Research and Public Health

    doi: 10.3390/ijerph14080874

    Frequency of HF183 detection by site in wet (light grey filled bars) versus dry (dark-filled) weather conditions. Frequency of HF183 detection is defined as % samples that are positive for HF183 and a sample is deemed positive for HF183 if the HF183 marker amplified in any of the three qPCR replicates. Sites are sorted from left to right by frequency of detection under dry weather conditions.
    Figure Legend Snippet: Frequency of HF183 detection by site in wet (light grey filled bars) versus dry (dark-filled) weather conditions. Frequency of HF183 detection is defined as % samples that are positive for HF183 and a sample is deemed positive for HF183 if the HF183 marker amplified in any of the three qPCR replicates. Sites are sorted from left to right by frequency of detection under dry weather conditions.

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

    Site average HF183 concentration versus frequency of HF183 detection in summer dry conditions. Frequency of HF183 detection is defined as % samples that are positive for HF183, and a sample is considered positive for HF183 if the marker is amplified in any of the three qPCR replicates. The site average concentration is calculated as the geomean of sample concentrations with non-detected (ND) and DBLOD values substituted by the Poisson approach, as described in Appendix A .
    Figure Legend Snippet: Site average HF183 concentration versus frequency of HF183 detection in summer dry conditions. Frequency of HF183 detection is defined as % samples that are positive for HF183, and a sample is considered positive for HF183 if the marker is amplified in any of the three qPCR replicates. The site average concentration is calculated as the geomean of sample concentrations with non-detected (ND) and DBLOD values substituted by the Poisson approach, as described in Appendix A .

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

    Frequency of HF183 detection at the 22 sites in summer dry weather. Frequency of HF183 detection is defined as % samples that are positive for HF183, and a sample is deemed HF183 positive if HF183 amplified in any of the three qPCR replicates.
    Figure Legend Snippet: Frequency of HF183 detection at the 22 sites in summer dry weather. Frequency of HF183 detection is defined as % samples that are positive for HF183, and a sample is deemed HF183 positive if HF183 amplified in any of the three qPCR replicates.

    Techniques Used: Amplification, Real-time Polymerase Chain Reaction

    24) Product Images from "Development of TaqMan Probe-Based Insulated Isothermal PCR (iiPCR) for Sensitive and Specific On-Site Pathogen Detection"

    Article Title: Development of TaqMan Probe-Based Insulated Isothermal PCR (iiPCR) for Sensitive and Specific On-Site Pathogen Detection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045278

    Time course of WSSV TaqMan probe-iiPCR. WSSV TaqMan probe-iiPCR reactions containing 10 1 and 10 3 copies of pWSSV1 were allowed to be carried out for 5, 10, 15, 20, 25, or 30 min. Amplicons were detected by 12% polyacrylamide gel analysis (A), and the intensity of the bands was estimated by densitometry (ImageJ program, NCBI) (B). In addition, fluorescent signals of three independent experiments were collected, and S/N ratios and SDs were calculated (C). Arrows, iiPCR amplicons; M, DNA size markers; S/N ratio, fluorescent intensity after /fluorescent intensity before .
    Figure Legend Snippet: Time course of WSSV TaqMan probe-iiPCR. WSSV TaqMan probe-iiPCR reactions containing 10 1 and 10 3 copies of pWSSV1 were allowed to be carried out for 5, 10, 15, 20, 25, or 30 min. Amplicons were detected by 12% polyacrylamide gel analysis (A), and the intensity of the bands was estimated by densitometry (ImageJ program, NCBI) (B). In addition, fluorescent signals of three independent experiments were collected, and S/N ratios and SDs were calculated (C). Arrows, iiPCR amplicons; M, DNA size markers; S/N ratio, fluorescent intensity after /fluorescent intensity before .

    Techniques Used:

    Generation and detection of TaqMan probe hydrolysis in iiPCR. Target pWSSV1 and pIMNV plasmids (10 3 copies) were subjected to WSSV (A) and IMNV (C) iiPCR, respectively, for 30 min in the presence of WSSVprobe, IMNVprobe, or no probe. Fluorescent signals (520 nm) of individual reactions were collected before and after iiPCR by the modified iiPCR and shown below the gel image. After the reactions were completed, amplicons were also analyzed on a 12% polyacrylamide gel in 1X TAE buffer. WSSV (B) and IMNV (D) real time PCR assays containing the same components as in iiPCR were carried out as described in Materials and Methods . The result shown here is a representative of at least three experiments with similar results. Arrows, iiPCR amplicons; M, DNA size markers; NTC, no template control (ddH 2 O); “before”, fluorescent signals detected before reaction; “after”, fluorescent signals detected after reaction; S/N ratio, signal intensity after /signal intensity before for iiPCR; signal intensity cycle 40 /signal intensity cycle 1 for real time PCR.
    Figure Legend Snippet: Generation and detection of TaqMan probe hydrolysis in iiPCR. Target pWSSV1 and pIMNV plasmids (10 3 copies) were subjected to WSSV (A) and IMNV (C) iiPCR, respectively, for 30 min in the presence of WSSVprobe, IMNVprobe, or no probe. Fluorescent signals (520 nm) of individual reactions were collected before and after iiPCR by the modified iiPCR and shown below the gel image. After the reactions were completed, amplicons were also analyzed on a 12% polyacrylamide gel in 1X TAE buffer. WSSV (B) and IMNV (D) real time PCR assays containing the same components as in iiPCR were carried out as described in Materials and Methods . The result shown here is a representative of at least three experiments with similar results. Arrows, iiPCR amplicons; M, DNA size markers; NTC, no template control (ddH 2 O); “before”, fluorescent signals detected before reaction; “after”, fluorescent signals detected after reaction; S/N ratio, signal intensity after /signal intensity before for iiPCR; signal intensity cycle 40 /signal intensity cycle 1 for real time PCR.

    Techniques Used: Modification, Real-time Polymerase Chain Reaction

    25) Product Images from "Metformin Induces Cell Cycle Arrest and Apoptosis in Drug-Resistant Leukemia Cells"

    Article Title: Metformin Induces Cell Cycle Arrest and Apoptosis in Drug-Resistant Leukemia Cells

    Journal: Leukemia Research and Treatment

    doi: 10.1155/2015/516460

    The effect of metformin on leukemia cell proliferation. CEM and 10E 1 -CEM cells were treated with metformin (10 mM) for the times indicated and the viable cell number was determined as an arbitrary unit of absorbance after XTT tetrazolium salt incorporation. Values are represented as the cell number (±SD) relative to the controls of at least three independent experiments performed in quadruplicate ( n > 12): ∗∗ P
    Figure Legend Snippet: The effect of metformin on leukemia cell proliferation. CEM and 10E 1 -CEM cells were treated with metformin (10 mM) for the times indicated and the viable cell number was determined as an arbitrary unit of absorbance after XTT tetrazolium salt incorporation. Values are represented as the cell number (±SD) relative to the controls of at least three independent experiments performed in quadruplicate ( n > 12): ∗∗ P

    Techniques Used:

    Metformin induces cell cycle arrest. CCRF-CEM and 10E 1 -CEM cells were treated with 4 and 10 mM metformin for 72 h and cell cycle distribution was determined by flow cytometry. Representative histograms showing the distribution of cells on the basis of DNA content.
    Figure Legend Snippet: Metformin induces cell cycle arrest. CCRF-CEM and 10E 1 -CEM cells were treated with 4 and 10 mM metformin for 72 h and cell cycle distribution was determined by flow cytometry. Representative histograms showing the distribution of cells on the basis of DNA content.

    Techniques Used: Flow Cytometry, Cytometry

    Effects of metformin on apoptosis. CEM and 10E 1 -CEM cells were exposed to 10 mM metformin for 72 h and the induction of apoptosis was evaluated by (a, and Table 1 ) flow cytometry of annexin V-PI labeled cells and (b) by fluorescence microscopy of DAPI labeled cells. (c) 10E 1 -CEM cells were treated with 10 mM metformin for 96 h before caspase-3 and -7 activation was evaluated by flow cytometry. The numbers in the quadrants indicate the percentages of cells. A representative experiment of three performed. (a, and Table 1 ).
    Figure Legend Snippet: Effects of metformin on apoptosis. CEM and 10E 1 -CEM cells were exposed to 10 mM metformin for 72 h and the induction of apoptosis was evaluated by (a, and Table 1 ) flow cytometry of annexin V-PI labeled cells and (b) by fluorescence microscopy of DAPI labeled cells. (c) 10E 1 -CEM cells were treated with 10 mM metformin for 96 h before caspase-3 and -7 activation was evaluated by flow cytometry. The numbers in the quadrants indicate the percentages of cells. A representative experiment of three performed. (a, and Table 1 ).

    Techniques Used: Flow Cytometry, Cytometry, Labeling, Fluorescence, Microscopy, Activation Assay

    Metformin induces mitochondrial perturbations. CEM and 10E 1 -CEM cells were exposed to metformin (10 mM) for 48 h and then stained with DiOC 6 (3)/PI. The Δ ψ m was determined as DiOC 3 (6) emitted fluorescence (black: control cells, red: metformin-treated cells). Fluorescence by CEM cells treated with 4-HPR has been shown as positive control (CEM CTRL+). In all cases, differences of mean fluorescence intensity of metformin versus control ( n = 10.000 cells analyzed/assay) are statistically significant ( P
    Figure Legend Snippet: Metformin induces mitochondrial perturbations. CEM and 10E 1 -CEM cells were exposed to metformin (10 mM) for 48 h and then stained with DiOC 6 (3)/PI. The Δ ψ m was determined as DiOC 3 (6) emitted fluorescence (black: control cells, red: metformin-treated cells). Fluorescence by CEM cells treated with 4-HPR has been shown as positive control (CEM CTRL+). In all cases, differences of mean fluorescence intensity of metformin versus control ( n = 10.000 cells analyzed/assay) are statistically significant ( P

    Techniques Used: Staining, Fluorescence, Positive Control

    Metformin induces changes in enzymes controlling energy metabolism. Western blots of PKC δ and PKC ε in CEM, 10E 1 -CEM, and R5-CEM cells exposed to 10 mM metformin for the times indicated. Representative results of three independent assays performed are shown.
    Figure Legend Snippet: Metformin induces changes in enzymes controlling energy metabolism. Western blots of PKC δ and PKC ε in CEM, 10E 1 -CEM, and R5-CEM cells exposed to 10 mM metformin for the times indicated. Representative results of three independent assays performed are shown.

    Techniques Used: Western Blot

    The effect of metformin on the induction of autophagy. Western blots probed for LC3B in CEM and 10E 1 -CEM cells exposed to metformin (10 mM) for the times indicated. Mel-Ho cells treated for 30 h with terfenadine (10 μ M) were used as a positive control where conversion of LC3B-I isoform to the LC3B-II is shown.
    Figure Legend Snippet: The effect of metformin on the induction of autophagy. Western blots probed for LC3B in CEM and 10E 1 -CEM cells exposed to metformin (10 mM) for the times indicated. Mel-Ho cells treated for 30 h with terfenadine (10 μ M) were used as a positive control where conversion of LC3B-I isoform to the LC3B-II is shown.

    Techniques Used: Western Blot, Positive Control

    Effect of metformin on leukemia cell viability. The leukemia cell lines CEM, 10E 1 -CEM, and R5-CEM were exposed to metformin for the times and at the concentrations indicated, and cell viability was determined with the XTT assay. The values are shown relative to the untreated control cells (±SD) from at least three independent experiments performed in quadruplicate ( n > 12): ∗∗ P
    Figure Legend Snippet: Effect of metformin on leukemia cell viability. The leukemia cell lines CEM, 10E 1 -CEM, and R5-CEM were exposed to metformin for the times and at the concentrations indicated, and cell viability was determined with the XTT assay. The values are shown relative to the untreated control cells (±SD) from at least three independent experiments performed in quadruplicate ( n > 12): ∗∗ P

    Techniques Used: XTT Assay

    The effect of metformin on cell cycle regulatory proteins. Western blot analysis of cdc2 p34, cyclin A, cyclin B1, and cyclin E in CEM, 10E 1 -CEM, and R5-CEM cells treated with metformin (10 mM) for the times indicated. Representative results of three independent assays performed are shown.
    Figure Legend Snippet: The effect of metformin on cell cycle regulatory proteins. Western blot analysis of cdc2 p34, cyclin A, cyclin B1, and cyclin E in CEM, 10E 1 -CEM, and R5-CEM cells treated with metformin (10 mM) for the times indicated. Representative results of three independent assays performed are shown.

    Techniques Used: Western Blot

    26) Product Images from "GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells"

    Article Title: GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells

    Journal: Scientific Reports

    doi: 10.1038/srep37721

    Effect of endocytosis inhibitors on GapmeR cellular internalization in human T-cells. Primary human T-cells were untreated ( A , control ) or pre-treated with amiloride [0.5 mM ( B ), 1.0 mM ( C ), 2.0 mM ( D ), 3.0 mM ( E ), 4.0 mM ( F ) or 5.0 mM ( G )], 1 μg/ml filipin ( H ), 10 μM chlorpromazine ( I ), 10 mM cytochalasin D ( J ) for 30 min. Cells were then incubated with 500 nM FAM-GapmeR for 24 h to allow gymnosis. Cellular internalization of GapmeR was analysed by flow cytometry. Results show dose-dependent inhibition of FAM-GapmeR cellular uptake in cells treated with amiloride ( K ) but not with other three inhibitors. Data represent at least three independent experiments using T-cells purified from at least 3 different donors, *p
    Figure Legend Snippet: Effect of endocytosis inhibitors on GapmeR cellular internalization in human T-cells. Primary human T-cells were untreated ( A , control ) or pre-treated with amiloride [0.5 mM ( B ), 1.0 mM ( C ), 2.0 mM ( D ), 3.0 mM ( E ), 4.0 mM ( F ) or 5.0 mM ( G )], 1 μg/ml filipin ( H ), 10 μM chlorpromazine ( I ), 10 mM cytochalasin D ( J ) for 30 min. Cells were then incubated with 500 nM FAM-GapmeR for 24 h to allow gymnosis. Cellular internalization of GapmeR was analysed by flow cytometry. Results show dose-dependent inhibition of FAM-GapmeR cellular uptake in cells treated with amiloride ( K ) but not with other three inhibitors. Data represent at least three independent experiments using T-cells purified from at least 3 different donors, *p

    Techniques Used: Incubation, Flow Cytometry, Cytometry, Inhibition, Purification

    27) Product Images from "Simultaneous single-molecule epigenetic imaging of DNA methylation and hydroxymethylation"

    Article Title: Simultaneous single-molecule epigenetic imaging of DNA methylation and hydroxymethylation

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

    doi: 10.1073/pnas.1600223113

    Reduced binding to MBD proteins is shown in 5hmC/5mCpGs compared with 5mC/5mCpGs. ( A ) Binding of 5mC/5mCpGs and 5hmC/5mCpGs containing DNA to varying concentrations of the methyl-CpG binding domain of human MBD2 protein assayed via EMSA. ( B ) Model for
    Figure Legend Snippet: Reduced binding to MBD proteins is shown in 5hmC/5mCpGs compared with 5mC/5mCpGs. ( A ) Binding of 5mC/5mCpGs and 5hmC/5mCpGs containing DNA to varying concentrations of the methyl-CpG binding domain of human MBD2 protein assayed via EMSA. ( B ) Model for

    Techniques Used: Binding Assay

    28) Product Images from "Nanovector-based prolyl hydroxylase domain 2 silencing system enhances the efficiency of stem cell transplantation for infarcted myocardium repair"

    Article Title: Nanovector-based prolyl hydroxylase domain 2 silencing system enhances the efficiency of stem cell transplantation for infarcted myocardium repair

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S71586

    Assessment of the optimized gene silencing system. Notes: (A) Real-time MSCs bio-behaviors during transfection. ( B ) Cell proliferation of non-transfected and transfected MSCs during 48 hours after transfection. ( C ) Cell apoptosis of non-transfected and transfected MSCs at 48 hours after transfection. ( D ) FAM-tagged siRNA transfected into MSCs. Scale bar =50 μm. ( E ) PHD2 mRNA expression after siRNA transfection. † P > 0.05 versus non-transfected MSCs; * P
    Figure Legend Snippet: Assessment of the optimized gene silencing system. Notes: (A) Real-time MSCs bio-behaviors during transfection. ( B ) Cell proliferation of non-transfected and transfected MSCs during 48 hours after transfection. ( C ) Cell apoptosis of non-transfected and transfected MSCs at 48 hours after transfection. ( D ) FAM-tagged siRNA transfected into MSCs. Scale bar =50 μm. ( E ) PHD2 mRNA expression after siRNA transfection. † P > 0.05 versus non-transfected MSCs; * P

    Techniques Used: Transfection, Expressing

    Schematic representation of Arg-G4-based PHD2 silencing system combined with MSC transplantation for infarcted myocardium repair. Abbreviations: Arg-G4, arginine-terminated G4; G4, generation 4 poly(amidoamine); MI, myocardial infarction; MSC, mesenchymal stem cell; PHD2, prolyl hydroxylase domain protein 2; siRNA, small interfering RNA.
    Figure Legend Snippet: Schematic representation of Arg-G4-based PHD2 silencing system combined with MSC transplantation for infarcted myocardium repair. Abbreviations: Arg-G4, arginine-terminated G4; G4, generation 4 poly(amidoamine); MI, myocardial infarction; MSC, mesenchymal stem cell; PHD2, prolyl hydroxylase domain protein 2; siRNA, small interfering RNA.

    Techniques Used: Transplantation Assay, Small Interfering RNA

    Optimization of Arg-G4-based gene silencing. Notes: ( A ) Arg-G4-siRNA transfection efficiency in MSCs at various N/P ratios using Lipofectamine 2000 as a control. ( B ) Cell viability in MSCs during transfection at various N/P ratios using Lipofectamine 2000 as a control. ( C ) Arg-G4-siRNA transfection efficiency in MSCs using various siRNA concentrations. ( D ) Cell viability in MSCs during transfection using various siRNA concentrations. * P
    Figure Legend Snippet: Optimization of Arg-G4-based gene silencing. Notes: ( A ) Arg-G4-siRNA transfection efficiency in MSCs at various N/P ratios using Lipofectamine 2000 as a control. ( B ) Cell viability in MSCs during transfection at various N/P ratios using Lipofectamine 2000 as a control. ( C ) Arg-G4-siRNA transfection efficiency in MSCs using various siRNA concentrations. ( D ) Cell viability in MSCs during transfection using various siRNA concentrations. * P

    Techniques Used: Transfection

    29) Product Images from "Bcl-2 inhibits apoptosis by increasing the time-to-death and intrinsic cell-to-cell variations in the mitochondrial pathway of cell death"

    Article Title: Bcl-2 inhibits apoptosis by increasing the time-to-death and intrinsic cell-to-cell variations in the mitochondrial pathway of cell death

    Journal: Apoptosis

    doi: 10.1007/s10495-010-0515-7

    Probabilistic computational model of the intrinsic pathway of apoptosis predicts the kinetics and bi-modal distribution of caspase activation. a Probability distribution of caspase 9 activation calculated from single cell activation data of caspase 9. Insets indicate the number of Bcl-2 molecules, corresponding to ~0.075 μM (45 molecules), ~0.75 μM (450 molecules), and ~3 μM (1800 molecules). Time is measured in Monte-Carlo (MC) steps. 1 MC step = 10 −4 s. b Analysis of early caspase 9 activation in cells treated with HA14-1 confirms rapid caspase 9 activation without intermediate events. Jurkat T cells were treated with HA14-1 (15 μM) for 6 h, labeled with FAM-LEHD-FMK (caspase 9 FLICA), TMRM and 7-AAD, and analyzed by flow cytometry. Late apoptotic events were excluded based on changes in 7-AAD versus forward scatter plots. Region R2, cells with energized mitochondria and lack of caspase 9 activation; region R3, cells with loss of Δψ m and no or initial caspase 9 activation; region R4, cells with loss of Δψ m and full caspase 9 activation. Note that there are nearly no intermediate events between the regions R3 and R4. c Jurkat T cells were treated for 6 h with HA14-1 at concentrations indicated, and analyzed as above. Note a characteristic shoulder on FLICA fluorescence plots (marked below the plots with ), probably indicative of early caspase 9 activation, as well as small number of cells having intermediate caspase 9 activation. Cells with no caspase activation are marked with , and cells with full caspase activation are marked with . d Upper panel the kinetics of caspase activation, analyzed using caspase 9 FLICA in 7-AAD negative Jurkat T cells treated with DMSO or 10 μM HA14-1; lower panel probability distribution of caspase 9 activation calculated from single cell activation data of caspase 9, for 45 molecules of Bcl-2 (~0.075 μM)
    Figure Legend Snippet: Probabilistic computational model of the intrinsic pathway of apoptosis predicts the kinetics and bi-modal distribution of caspase activation. a Probability distribution of caspase 9 activation calculated from single cell activation data of caspase 9. Insets indicate the number of Bcl-2 molecules, corresponding to ~0.075 μM (45 molecules), ~0.75 μM (450 molecules), and ~3 μM (1800 molecules). Time is measured in Monte-Carlo (MC) steps. 1 MC step = 10 −4 s. b Analysis of early caspase 9 activation in cells treated with HA14-1 confirms rapid caspase 9 activation without intermediate events. Jurkat T cells were treated with HA14-1 (15 μM) for 6 h, labeled with FAM-LEHD-FMK (caspase 9 FLICA), TMRM and 7-AAD, and analyzed by flow cytometry. Late apoptotic events were excluded based on changes in 7-AAD versus forward scatter plots. Region R2, cells with energized mitochondria and lack of caspase 9 activation; region R3, cells with loss of Δψ m and no or initial caspase 9 activation; region R4, cells with loss of Δψ m and full caspase 9 activation. Note that there are nearly no intermediate events between the regions R3 and R4. c Jurkat T cells were treated for 6 h with HA14-1 at concentrations indicated, and analyzed as above. Note a characteristic shoulder on FLICA fluorescence plots (marked below the plots with ), probably indicative of early caspase 9 activation, as well as small number of cells having intermediate caspase 9 activation. Cells with no caspase activation are marked with , and cells with full caspase activation are marked with . d Upper panel the kinetics of caspase activation, analyzed using caspase 9 FLICA in 7-AAD negative Jurkat T cells treated with DMSO or 10 μM HA14-1; lower panel probability distribution of caspase 9 activation calculated from single cell activation data of caspase 9, for 45 molecules of Bcl-2 (~0.075 μM)

    Techniques Used: Activation Assay, Labeling, Flow Cytometry, Cytometry, Fluorescence

    Intrinsic stochastic variability in apoptotic signaling is sufficient for cell-to-cell variability in time-to-death. Simulated timing of caspase 9 activation in HA14-1-treated cells. Mean protein concentration of Bcl-2 was set to a 4500 molecules (~7.5 μM), or b 2250 molecules (~3.75 μM). Concentrations of all proteins in the model vary with coefficient of variation (CV) of 0.25 ( upper panels ) or 0 ( lower panels ). Each line represents a single simulated cell. Insets indicate % cell death within the time of analysis. Total of 16 cells were analyzed. Time is measured in Monte-Carlo (MC) steps. 1 MC step = 10 −4 s
    Figure Legend Snippet: Intrinsic stochastic variability in apoptotic signaling is sufficient for cell-to-cell variability in time-to-death. Simulated timing of caspase 9 activation in HA14-1-treated cells. Mean protein concentration of Bcl-2 was set to a 4500 molecules (~7.5 μM), or b 2250 molecules (~3.75 μM). Concentrations of all proteins in the model vary with coefficient of variation (CV) of 0.25 ( upper panels ) or 0 ( lower panels ). Each line represents a single simulated cell. Insets indicate % cell death within the time of analysis. Total of 16 cells were analyzed. Time is measured in Monte-Carlo (MC) steps. 1 MC step = 10 −4 s

    Techniques Used: Activation Assay, Protein Concentration

    Bcl-2 increases cell-to-cell variability in time-to-MOMP, which contributes significantly to cell-to-cell variability in time-to-death. a The computational model of the intrinsic pathway of apoptosis indicates that Bax2 dimer formation occurs with large cell-to-cell variability, which is modulated by the level of Bcl-2. Note that decrease in Bcl-2 level reduces cell-to-cell variability in time to Bax2 dimer formation. Time is measured in Monte-Carlo (MC) steps. 1 MC step = 10 −4 s. b Mitochondrial depolarization indicates MOMP in SH-SY5Y cells and is displayed as a loss in TMRM fluorescence. Time stamps indicate time after HA14-1 addition. c Non-cumulative frequency distribution for time to initial loss of Δψ m in SH-SY5Y cells ( n ≥ 16 per treatment), based on changes in pixel intensity in the TMRM-sensitive channel. HA14-1-treated HEK293 cells, which are not Bcl-2-dependent, did not respond with the loss of TMRM fluorescence under the same imaging conditions (not shown). d Jurkat T cells were treated with HA14-1 (10 μM) for the time indicated, labeled with FLICA, TMRM and 7-AAD, and analyzed by flow cytometry. Cells with permeabilized plasma membrane (7-AAD positive) were excluded from analysis. Region R2, cells with energized mitochondria and lack of caspase 9 activation; region R3, cells with loss of Δψ m and no or initial caspase 9 activation; region R4, cells with loss of Δψ m and full caspase 9 activation. Note that (i) cell population is losing TMRM fluorescence gradually, rather then in all-or-none manner; (ii) there is a time delay between the initial loss of TMRM fluorescence and caspase 9 activation; (iii) initial caspase 9 activation occurs in cells with minimal TMRM fluorescence. The number of intermediate events between regions R3 and R4 was less then 5%
    Figure Legend Snippet: Bcl-2 increases cell-to-cell variability in time-to-MOMP, which contributes significantly to cell-to-cell variability in time-to-death. a The computational model of the intrinsic pathway of apoptosis indicates that Bax2 dimer formation occurs with large cell-to-cell variability, which is modulated by the level of Bcl-2. Note that decrease in Bcl-2 level reduces cell-to-cell variability in time to Bax2 dimer formation. Time is measured in Monte-Carlo (MC) steps. 1 MC step = 10 −4 s. b Mitochondrial depolarization indicates MOMP in SH-SY5Y cells and is displayed as a loss in TMRM fluorescence. Time stamps indicate time after HA14-1 addition. c Non-cumulative frequency distribution for time to initial loss of Δψ m in SH-SY5Y cells ( n ≥ 16 per treatment), based on changes in pixel intensity in the TMRM-sensitive channel. HA14-1-treated HEK293 cells, which are not Bcl-2-dependent, did not respond with the loss of TMRM fluorescence under the same imaging conditions (not shown). d Jurkat T cells were treated with HA14-1 (10 μM) for the time indicated, labeled with FLICA, TMRM and 7-AAD, and analyzed by flow cytometry. Cells with permeabilized plasma membrane (7-AAD positive) were excluded from analysis. Region R2, cells with energized mitochondria and lack of caspase 9 activation; region R3, cells with loss of Δψ m and no or initial caspase 9 activation; region R4, cells with loss of Δψ m and full caspase 9 activation. Note that (i) cell population is losing TMRM fluorescence gradually, rather then in all-or-none manner; (ii) there is a time delay between the initial loss of TMRM fluorescence and caspase 9 activation; (iii) initial caspase 9 activation occurs in cells with minimal TMRM fluorescence. The number of intermediate events between regions R3 and R4 was less then 5%

    Techniques Used: Fluorescence, Imaging, Labeling, Flow Cytometry, Cytometry, Activation Assay

    Probabilistic computational model of the intrinsic pathway of apoptosis reproduces cell-to-cell variability in time-to-death. a Schematic of the mitochondrial pathway network modeled in this study. MOMP mitochondrial outer membrane permeabilisation, tBid truncated Bid. b Cell-to-cell variability and time to activation of caspase 9 depend on Bcl-2 concentration. Insets indicate the number of Bcl-2 molecules, corresponding to ~0.075 μM (45 molecules), ~0.75 μM (450 molecules), ~3 μM (1800 molecules), and ~7.5 μM (4500 molecules). Time is measured in Monte-Carlo (MC) simulation steps. 1 MC step = 10 −4 s, hence time-scale shown 5 × 10 8 MC steps ~15 h. c Upper panel time-lapse images of SH-SY5Y cells treated with 10 μM HA14-1. Bright field image was taken at time 0. Inset numbers , time (min) after treatment. Dead cells become permeable to propidium iodide (PI). Lower panel the non-cumulative frequency distribution in time-to-death for SH-SY5Y cells treated with HA14-1 at concentrations indicated, as determined by live-cell microscopy ( n > 70 for each condition)
    Figure Legend Snippet: Probabilistic computational model of the intrinsic pathway of apoptosis reproduces cell-to-cell variability in time-to-death. a Schematic of the mitochondrial pathway network modeled in this study. MOMP mitochondrial outer membrane permeabilisation, tBid truncated Bid. b Cell-to-cell variability and time to activation of caspase 9 depend on Bcl-2 concentration. Insets indicate the number of Bcl-2 molecules, corresponding to ~0.075 μM (45 molecules), ~0.75 μM (450 molecules), ~3 μM (1800 molecules), and ~7.5 μM (4500 molecules). Time is measured in Monte-Carlo (MC) simulation steps. 1 MC step = 10 −4 s, hence time-scale shown 5 × 10 8 MC steps ~15 h. c Upper panel time-lapse images of SH-SY5Y cells treated with 10 μM HA14-1. Bright field image was taken at time 0. Inset numbers , time (min) after treatment. Dead cells become permeable to propidium iodide (PI). Lower panel the non-cumulative frequency distribution in time-to-death for SH-SY5Y cells treated with HA14-1 at concentrations indicated, as determined by live-cell microscopy ( n > 70 for each condition)

    Techniques Used: Activation Assay, Concentration Assay, Microscopy

    30) Product Images from "The Neurofilament-Derived Peptide NFL-TBS.40-63 Targets Neural Stem Cells and Affects Their Properties"

    Article Title: The Neurofilament-Derived Peptide NFL-TBS.40-63 Targets Neural Stem Cells and Affects Their Properties

    Journal: Stem Cells Translational Medicine

    doi: 10.5966/sctm.2015-0221

    In vivo localization of the NFL-TBS.40-63 peptide after injection in the right lateral ventricle of adult rats. (A): Confocal microscope localization of the 5-FAM-labeled NFL-TBS.40-63 peptide (green) in the subventricular zone of adult rats 1 and 24 hours after its injection in the right lateral ventricle. Immunofluorescence analysis of vimentin (red; ependymal cells), GFAP (red; subependymal cells), nestin (red; neural stem cells), and DAPI (blue). Scale bars = 10 µm. (B): Confocal microscope localization of the 5-FAM-labeled NFL-TBS.40-63 peptide (green) in different brain areas after injection in the right lateral ventricle. Immunohistochemistry was performed to reveal astrocytes (GFAP; red) and DAPI (nuclei; blue). Scale bars = 50 µm. Abbreviations: 5-FAM, 5-carboxyfluorescein; D, dorsal; DAPI, 4′,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; L, left; LV, lateral ventricle; R, right; SVZ, subventricular zone; V, ventral.
    Figure Legend Snippet: In vivo localization of the NFL-TBS.40-63 peptide after injection in the right lateral ventricle of adult rats. (A): Confocal microscope localization of the 5-FAM-labeled NFL-TBS.40-63 peptide (green) in the subventricular zone of adult rats 1 and 24 hours after its injection in the right lateral ventricle. Immunofluorescence analysis of vimentin (red; ependymal cells), GFAP (red; subependymal cells), nestin (red; neural stem cells), and DAPI (blue). Scale bars = 10 µm. (B): Confocal microscope localization of the 5-FAM-labeled NFL-TBS.40-63 peptide (green) in different brain areas after injection in the right lateral ventricle. Immunohistochemistry was performed to reveal astrocytes (GFAP; red) and DAPI (nuclei; blue). Scale bars = 50 µm. Abbreviations: 5-FAM, 5-carboxyfluorescein; D, dorsal; DAPI, 4′,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; L, left; LV, lateral ventricle; R, right; SVZ, subventricular zone; V, ventral.

    Techniques Used: In Vivo, Injection, Microscopy, Labeling, Immunofluorescence, Immunohistochemistry

    In vitro and in vivo uptake of the NFL-TBS.40-63 peptide in neural stem cells (NSCs) from adult rats. (A): Percentages of adult NSCs that incorporated the 5-FAM-labeled NFL-TBS.40-63 peptide. Data are presented as mean ± SEM. (B): Confocal microscope localization of the 5-FAM-labeled NFL-TBS.40-63 peptide (green) after its injection in the SVZ of adult rats. At 48 hours after peptide injection, immunohistochemistry was performed to reveal neural stem cells (CD133; red), astrocytes (GFAP; red), oligodendrocytes (Olig 2; red), neurons (βIII-tubulin; red), and nuclei (DAPI; blue). White arrows indicate colocalization of the peptide with NSCs. Scale bars = 10 µm. Abbreviations: 5-FAM, 5-carboxyfluorescein; DAPI, 4′,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; LV, lateral ventricle; SVZ, subventricular zone.
    Figure Legend Snippet: In vitro and in vivo uptake of the NFL-TBS.40-63 peptide in neural stem cells (NSCs) from adult rats. (A): Percentages of adult NSCs that incorporated the 5-FAM-labeled NFL-TBS.40-63 peptide. Data are presented as mean ± SEM. (B): Confocal microscope localization of the 5-FAM-labeled NFL-TBS.40-63 peptide (green) after its injection in the SVZ of adult rats. At 48 hours after peptide injection, immunohistochemistry was performed to reveal neural stem cells (CD133; red), astrocytes (GFAP; red), oligodendrocytes (Olig 2; red), neurons (βIII-tubulin; red), and nuclei (DAPI; blue). White arrows indicate colocalization of the peptide with NSCs. Scale bars = 10 µm. Abbreviations: 5-FAM, 5-carboxyfluorescein; DAPI, 4′,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; LV, lateral ventricle; SVZ, subventricular zone.

    Techniques Used: In Vitro, In Vivo, Labeling, Microscopy, Injection, Immunohistochemistry

    The NFL-TBS.40-63 peptide has no detectable effect on the microtubule network and reduces the viability of neural stem cells. (A): The percentages of neural stem cells (NSCs) at G 1 , S, or G 2 /M phases of the cell cycle after treatment with colchicine or increasing concentrations of the NFL-TBS.40-63 peptide. (B): NSCs were incubated with 20 µmol/l 5-FAM-labeled NFL-TBS.40-63 peptide (green) and immunostained to reveal the microtubule network with an anti-α-tubulin (red). The nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). A magnification of a region of interest is included (dotted framed images). Scale bars = 20 µm. (C): Percentages of viable NSCs after treatment with colchicine or increasing concentrations of the NFL-TBS.40-63 peptide. Data are presented as mean ± SEM. ∗, p
    Figure Legend Snippet: The NFL-TBS.40-63 peptide has no detectable effect on the microtubule network and reduces the viability of neural stem cells. (A): The percentages of neural stem cells (NSCs) at G 1 , S, or G 2 /M phases of the cell cycle after treatment with colchicine or increasing concentrations of the NFL-TBS.40-63 peptide. (B): NSCs were incubated with 20 µmol/l 5-FAM-labeled NFL-TBS.40-63 peptide (green) and immunostained to reveal the microtubule network with an anti-α-tubulin (red). The nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). A magnification of a region of interest is included (dotted framed images). Scale bars = 20 µm. (C): Percentages of viable NSCs after treatment with colchicine or increasing concentrations of the NFL-TBS.40-63 peptide. Data are presented as mean ± SEM. ∗, p

    Techniques Used: Incubation, Labeling, Staining

    Uptake of the NFL-TBS.40-63 peptide in neural stem cells from newborn rats. (A): The percentage of neural stem cells (NSCs) incorporating 5-FAM-labeled NFL-TBS.40-63 peptide, with (dark curve) and without (dotted curve) 0.4% trypan blue, was analyzed using the fluorescence-activated cell sorting technique. (B): Confocal microscopy of NSCs incubated with or without 20 µmol/l 5-FAM-labeled NFL-TBS.40-63 peptide (green). Scale bars = 10 µm. (C): Confocal microscopy of neurospheres incubated without (control) or with 20 µmol/l 5-FAM-labeled NFL-TBS.40-63 peptide (green) immunostained with anti-α-tubulin (red) to reveal the microtubule network. The nuclei were stained with DAPI (blue). Scale bars = 20 µm (original magnification ×63) and 5 µm (original magnification ×100). (D): Confocal microscopy of neurospheres incubated with 20 µmol/l 5-FAM-labeled NFL-TBS.40-63 peptide (green) immunostained with an anti-SOX2 antibody (red) to reveal stem cells. The nuclei were stained with DAPI (blue). Scale bars = 50 µm. (E): Percentages of NSCs that incorporate 5-FAM-labeled NFL-TBS.40-63 or scrambled peptides (20 µmol/l). Percentages of NSCs that incorporate 5-FAM-labeled NFL-TBS.40-63 peptide (20 µmol/l), after pretreatment in an ATP-depleted buffer or at 4°C (F) or with different endocytosis and signaling pathway inhibitors (G) . Data are presented as mean ± SEM. ∗∗, p
    Figure Legend Snippet: Uptake of the NFL-TBS.40-63 peptide in neural stem cells from newborn rats. (A): The percentage of neural stem cells (NSCs) incorporating 5-FAM-labeled NFL-TBS.40-63 peptide, with (dark curve) and without (dotted curve) 0.4% trypan blue, was analyzed using the fluorescence-activated cell sorting technique. (B): Confocal microscopy of NSCs incubated with or without 20 µmol/l 5-FAM-labeled NFL-TBS.40-63 peptide (green). Scale bars = 10 µm. (C): Confocal microscopy of neurospheres incubated without (control) or with 20 µmol/l 5-FAM-labeled NFL-TBS.40-63 peptide (green) immunostained with anti-α-tubulin (red) to reveal the microtubule network. The nuclei were stained with DAPI (blue). Scale bars = 20 µm (original magnification ×63) and 5 µm (original magnification ×100). (D): Confocal microscopy of neurospheres incubated with 20 µmol/l 5-FAM-labeled NFL-TBS.40-63 peptide (green) immunostained with an anti-SOX2 antibody (red) to reveal stem cells. The nuclei were stained with DAPI (blue). Scale bars = 50 µm. (E): Percentages of NSCs that incorporate 5-FAM-labeled NFL-TBS.40-63 or scrambled peptides (20 µmol/l). Percentages of NSCs that incorporate 5-FAM-labeled NFL-TBS.40-63 peptide (20 µmol/l), after pretreatment in an ATP-depleted buffer or at 4°C (F) or with different endocytosis and signaling pathway inhibitors (G) . Data are presented as mean ± SEM. ∗∗, p

    Techniques Used: Labeling, Fluorescence, FACS, Confocal Microscopy, Incubation, Staining

    31) Product Images from "Novel monodisperse PEGtide dendrons: design, fabrication and evaluation of mannose receptor-mediated macrophage targeting"

    Article Title: Novel monodisperse PEGtide dendrons: design, fabrication and evaluation of mannose receptor-mediated macrophage targeting

    Journal: Bioconjugate chemistry

    doi: 10.1021/bc400011v

    Synthesis of PEGtide dendrons: (A) G1.0; and (B) G2.0-5.0. The dendrons were synthesized using Fmoc SPPS using following components: Fmoc-Lys(5-FAM)-OH; Fmoc-Lys(Fmoc)-OH; Fmoc-β-Ala-OH; and Fmoc-dPEG 6 -OH.
    Figure Legend Snippet: Synthesis of PEGtide dendrons: (A) G1.0; and (B) G2.0-5.0. The dendrons were synthesized using Fmoc SPPS using following components: Fmoc-Lys(5-FAM)-OH; Fmoc-Lys(Fmoc)-OH; Fmoc-β-Ala-OH; and Fmoc-dPEG 6 -OH.

    Techniques Used: Synthesized

    32) Product Images from "Brain Endothelial Cells Regulate Glucagon-Like Peptide 1 Entry Into the Brain via a Receptor-Mediated Process"

    Article Title: Brain Endothelial Cells Regulate Glucagon-Like Peptide 1 Entry Into the Brain via a Receptor-Mediated Process

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2020.00555

    cAMP agonist increases GLP-1 uptake by RBMECs. (A) RBMECs were incubated with GLP-1-FAM at 10 pM for 3 min with or without 10 μM forskolin. DAPI was used to stain the nucleus. Images was captured using confocal microscope. Fluorescence density was analyzed using ImageJ. (B) quantitative analysis of the images ( n = 6, * p
    Figure Legend Snippet: cAMP agonist increases GLP-1 uptake by RBMECs. (A) RBMECs were incubated with GLP-1-FAM at 10 pM for 3 min with or without 10 μM forskolin. DAPI was used to stain the nucleus. Images was captured using confocal microscope. Fluorescence density was analyzed using ImageJ. (B) quantitative analysis of the images ( n = 6, * p

    Techniques Used: Incubation, Staining, Microscopy, Fluorescence

    33) Product Images from "Functional and structural analyses of N-acylsulfonamide-linked dinucleoside inhibitors of RNase A"

    Article Title: Functional and structural analyses of N-acylsulfonamide-linked dinucleoside inhibitors of RNase A

    Journal: The Febs Journal

    doi: 10.1111/j.1742-4658.2010.07976.x

    (A, B) Schematic and stereo representation of hydrogen bonds in the RNase A complex with N -acylsulfonamide 7 and N -acylsulfonamide 6 , respectively. N -Acylsulfonamide 7 and N -acylsulfonamide 6 , gold; active site residues, pea-green; RNase A, gray. Hydrogen bonds are represented as dashed lines, and water molecules are in cyan. (C, D) Stereo pictures of 2 F o − F c contoured at 1.0 σ for N -acylsulfonamide 7 and N -acylsulfonamide 6 , respectively.
    Figure Legend Snippet: (A, B) Schematic and stereo representation of hydrogen bonds in the RNase A complex with N -acylsulfonamide 7 and N -acylsulfonamide 6 , respectively. N -Acylsulfonamide 7 and N -acylsulfonamide 6 , gold; active site residues, pea-green; RNase A, gray. Hydrogen bonds are represented as dashed lines, and water molecules are in cyan. (C, D) Stereo pictures of 2 F o − F c contoured at 1.0 σ for N -acylsulfonamide 7 and N -acylsulfonamide 6 , respectively.

    Techniques Used:

    Isotherms for the binding of  N -acylsulfonamide-linked dinucleosides to RNase A. Data were fitted to   Eqn (1) . (A)  N -acylsulfonamide  7 ,  K i  = (3.7 ± 0.1) × 10 −4 m . (B)  N -acylsulfonamide  6 ,  K i  = (4.6 ± 0.3) × 10 −4 m .
    Figure Legend Snippet: Isotherms for the binding of N -acylsulfonamide-linked dinucleosides to RNase A. Data were fitted to Eqn (1) . (A) N -acylsulfonamide 7 , K i = (3.7 ± 0.1) × 10 −4 m . (B) N -acylsulfonamide 6 , K i = (4.6 ± 0.3) × 10 −4 m .

    Techniques Used: Binding Assay

    34) Product Images from "Central role for PICALM in amyloid–β blood–brain barrier transcytosis and clearance"

    Article Title: Central role for PICALM in amyloid–β blood–brain barrier transcytosis and clearance

    Journal: Nature neuroscience

    doi: 10.1038/nn.4025

    PICALM/clathrin–dependent endocytosis of Aβ–LRP1 complex by brain endothelial cells a–b , Colocalization of LRP1–Aβ40 complex with PICALM ( a ) and clathrin heavy chain (CHC) ( b ) in human brain endothelial cells (BEC) within 30 s of FAM–Aβ40 (250 nM) treatment. c , Immunostaining for LRP1, PICALM and CHC without Aβ (– Aβ). Dapi, nuclear staining (blue). Insets: higher magnification. Bar=10 µm. d , Quantification of LRP1 puncta colocalized with PICALM in a, c and with CHC in b, c , and FAM–Aβ40 puncta colocalized with LRP1 and PICALM in a, b . Means ± s.d. from 3 primary isolates in triplicate. p
    Figure Legend Snippet: PICALM/clathrin–dependent endocytosis of Aβ–LRP1 complex by brain endothelial cells a–b , Colocalization of LRP1–Aβ40 complex with PICALM ( a ) and clathrin heavy chain (CHC) ( b ) in human brain endothelial cells (BEC) within 30 s of FAM–Aβ40 (250 nM) treatment. c , Immunostaining for LRP1, PICALM and CHC without Aβ (– Aβ). Dapi, nuclear staining (blue). Insets: higher magnification. Bar=10 µm. d , Quantification of LRP1 puncta colocalized with PICALM in a, c and with CHC in b, c , and FAM–Aβ40 puncta colocalized with LRP1 and PICALM in a, b . Means ± s.d. from 3 primary isolates in triplicate. p

    Techniques Used: Immunostaining, Staining

    Diminished Aβ clearance in Picalm +/− mice a , Immunostaining for PICALM (red) and endothelial–specific lectin (blue) in brain microvessels from Picalm +/+ and Picalm +/− mice. b , Relative abundance of PICALM protein compared to β–actin studied by immunoblotting and densitometry analysis in brain microvessels and microvessel–depleted brain homogenates in Picalm +/+ and Picalm +/− mice. P
    Figure Legend Snippet: Diminished Aβ clearance in Picalm +/− mice a , Immunostaining for PICALM (red) and endothelial–specific lectin (blue) in brain microvessels from Picalm +/+ and Picalm +/− mice. b , Relative abundance of PICALM protein compared to β–actin studied by immunoblotting and densitometry analysis in brain microvessels and microvessel–depleted brain homogenates in Picalm +/+ and Picalm +/− mice. P

    Techniques Used: Mouse Assay, Immunostaining

    PICALM reductions in brain capillary endothelium in Alzheimer’s disease a , PICALM and Aβ immunostaining in the prefrontal cortex of an age–matched control (Braak I, left) and AD case (Braak V–VI, right). Bar=20 µm. b , Immunoblotting for PICALM, von Willebrand Factor (vWF), β3–tubulin, glial fibrillar acidic protein (GFAP), and GAPDH (loading control) in isolated microvessels and microvessel–depleted brains from controls (Braak 0–I) and AD cases (Braak V–VI). c , Relative abundance of PICALM in microvessels and microvessel–depleted brains from control and AD cases determined by densitometry analysis relative to GAPDH. Mean ± s.e.m., n=6/group; p
    Figure Legend Snippet: PICALM reductions in brain capillary endothelium in Alzheimer’s disease a , PICALM and Aβ immunostaining in the prefrontal cortex of an age–matched control (Braak I, left) and AD case (Braak V–VI, right). Bar=20 µm. b , Immunoblotting for PICALM, von Willebrand Factor (vWF), β3–tubulin, glial fibrillar acidic protein (GFAP), and GAPDH (loading control) in isolated microvessels and microvessel–depleted brains from controls (Braak 0–I) and AD cases (Braak V–VI). c , Relative abundance of PICALM in microvessels and microvessel–depleted brains from control and AD cases determined by densitometry analysis relative to GAPDH. Mean ± s.e.m., n=6/group; p

    Techniques Used: Immunostaining, Isolation

    35) Product Images from "Brain Endothelial Cells Regulate Glucagon-Like Peptide 1 Entry Into the Brain via a Receptor-Mediated Process"

    Article Title: Brain Endothelial Cells Regulate Glucagon-Like Peptide 1 Entry Into the Brain via a Receptor-Mediated Process

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2020.00555

    GLP-1 and GLP-1 analog exendin-4 enter various brain regions via GLP-1 receptor-mediated process. (A) Protocol. Each rat received saline or exendin-(9-39) (30 nmol/kg/min) for 10 min and then dextran-TRITC (30 pmol/kg/min) + GLP-1-FAM (30 pmol/kg/min) or exendin-4-FAM (30 pmol/kg/min) for 10 min via a carotid artery catheter. Brain tissues were harvested after systemic flush with saline. (B) GLP-1-FAM uptake by the brain ( n = 7, * p
    Figure Legend Snippet: GLP-1 and GLP-1 analog exendin-4 enter various brain regions via GLP-1 receptor-mediated process. (A) Protocol. Each rat received saline or exendin-(9-39) (30 nmol/kg/min) for 10 min and then dextran-TRITC (30 pmol/kg/min) + GLP-1-FAM (30 pmol/kg/min) or exendin-4-FAM (30 pmol/kg/min) for 10 min via a carotid artery catheter. Brain tissues were harvested after systemic flush with saline. (B) GLP-1-FAM uptake by the brain ( n = 7, * p

    Techniques Used:

    36) Product Images from "Two-Component Signal Transduction System CBO0787/CBO0786 Represses Transcription from Botulinum Neurotoxin Promoters in Clostridium botulinum ATCC 3502"

    Article Title: Two-Component Signal Transduction System CBO0787/CBO0786 Represses Transcription from Botulinum Neurotoxin Promoters in Clostridium botulinum ATCC 3502

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003252

    CBO0786 binds to the core promoter −10 region of the ha and ntnh - botA operons. DNase I footprinting analysis of 5′-6-FAM labeled sense strand (A, D) and 5′-HEX labeled antisense strand (B, E) of P ha33 probe (A, B) and P ntnh-botA probe (D, E). Comparison of DNase I digestion in the absence (blue peaks in A and D, green peaks in B and E) or with 10 µM of CBO0786 (red peaks). Protection regions are indicated by square brackets. Protection regions are underlined in sequencing electropherograms of P ha33 probe (C) and P ntnh-botA probe (F) in the sense strand. The consensus −10 regions of the ha and ntnh-botA promoters are indicated.
    Figure Legend Snippet: CBO0786 binds to the core promoter −10 region of the ha and ntnh - botA operons. DNase I footprinting analysis of 5′-6-FAM labeled sense strand (A, D) and 5′-HEX labeled antisense strand (B, E) of P ha33 probe (A, B) and P ntnh-botA probe (D, E). Comparison of DNase I digestion in the absence (blue peaks in A and D, green peaks in B and E) or with 10 µM of CBO0786 (red peaks). Protection regions are indicated by square brackets. Protection regions are underlined in sequencing electropherograms of P ha33 probe (C) and P ntnh-botA probe (F) in the sense strand. The consensus −10 regions of the ha and ntnh-botA promoters are indicated.

    Techniques Used: Footprinting, Labeling, Sequencing

    37) Product Images from "Impact of solid surface hydrophobicity and micrococcal nuclease production on Staphylococcus aureus Newman biofilms"

    Article Title: Impact of solid surface hydrophobicity and micrococcal nuclease production on Staphylococcus aureus Newman biofilms

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-69084-x

    Nuclease activity is reduced during biofilm formation. ( a ) Fluorescence intensity of a FRET-based DNA probe assay in 20 h old planktonic cultures and biofilms of S. aureus Newman WT on glass, silanized glass and Pluronic F-127-coated silanized glass. Dots indicate the mean of 4 different planktonic cultures and 6 biofilms grown with three different bacterial cultures. Lines are least square fits of Eq. ( 1 ) to the measured intensity data. Error bars are eliminated for easy readability. ( b ) Nuclease concentration per CFU was determined in 20 h old planktonic cultures and biofilms of S. aureus Newman WT and S. aureus Newman ∆nuc1 grown on glass, silanized glass and Pluronic F-127-coated silanized glass. Bars indicate the mean of 4 different planktonic cultures and 6 biofilms grown with three different bacterial cultures. Error bars show the standard error of the mean. Statistical significance between WT and mutant strain on the same solid surfaces is indicated with asterisks, ** P ≤ 0.01; *** P ≤ 0.001.
    Figure Legend Snippet: Nuclease activity is reduced during biofilm formation. ( a ) Fluorescence intensity of a FRET-based DNA probe assay in 20 h old planktonic cultures and biofilms of S. aureus Newman WT on glass, silanized glass and Pluronic F-127-coated silanized glass. Dots indicate the mean of 4 different planktonic cultures and 6 biofilms grown with three different bacterial cultures. Lines are least square fits of Eq. ( 1 ) to the measured intensity data. Error bars are eliminated for easy readability. ( b ) Nuclease concentration per CFU was determined in 20 h old planktonic cultures and biofilms of S. aureus Newman WT and S. aureus Newman ∆nuc1 grown on glass, silanized glass and Pluronic F-127-coated silanized glass. Bars indicate the mean of 4 different planktonic cultures and 6 biofilms grown with three different bacterial cultures. Error bars show the standard error of the mean. Statistical significance between WT and mutant strain on the same solid surfaces is indicated with asterisks, ** P ≤ 0.01; *** P ≤ 0.001.

    Techniques Used: Activity Assay, Fluorescence, Concentration Assay, Mutagenesis

    38) Product Images from "Synthetic antimicrobial and LPS-neutralising peptides suppress inflammatory and immune responses in skin cells and promote keratinocyte migration"

    Article Title: Synthetic antimicrobial and LPS-neutralising peptides suppress inflammatory and immune responses in skin cells and promote keratinocyte migration

    Journal: Scientific Reports

    doi: 10.1038/srep31577

    Peptide-induced keratinocyte migration depends on purinergic receptors and metalloproteases. ( A ) HaCaT cells were scratched and stimulated with Pep19-2.5 in the presence or absence of the inhibitors AG1478 (50 nM), marimastat (10 μM) and PPADS (50 μM). TGF-β 1 served as positive control. Images were taken directly after scratching (0 h) and after 20 h and are representative of three independent experiments. ( B ) HaCaT cells were stimulated with Pep19-2.5 and Pep19-4LF in the presence or absence of the inhibitors AG1478 (50 nM), marimastat (10 μM) and PPADS (50 μM). After 24 h EdU incorporation was quantified. Data are normalised to unstimulated cells (assigned as 1.0). Cells incubated with growth medium served as positive control. Data are mean + SD (n = 3).
    Figure Legend Snippet: Peptide-induced keratinocyte migration depends on purinergic receptors and metalloproteases. ( A ) HaCaT cells were scratched and stimulated with Pep19-2.5 in the presence or absence of the inhibitors AG1478 (50 nM), marimastat (10 μM) and PPADS (50 μM). TGF-β 1 served as positive control. Images were taken directly after scratching (0 h) and after 20 h and are representative of three independent experiments. ( B ) HaCaT cells were stimulated with Pep19-2.5 and Pep19-4LF in the presence or absence of the inhibitors AG1478 (50 nM), marimastat (10 μM) and PPADS (50 μM). After 24 h EdU incorporation was quantified. Data are normalised to unstimulated cells (assigned as 1.0). Cells incubated with growth medium served as positive control. Data are mean + SD (n = 3).

    Techniques Used: Migration, Positive Control, Incubation

    39) Product Images from "TNF? Signals via p66Shc to Induce E-Selectin, Promote Leukocyte Transmigration and Enhance Permeability in Human Endothelial Cells"

    Article Title: TNF? Signals via p66Shc to Induce E-Selectin, Promote Leukocyte Transmigration and Enhance Permeability in Human Endothelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0081930

    Role of Ser 36 phosphorylation of p66 Shc in endothelial activation. A . Basal and TNFα-stimulated phosphorylation of p66 Shc on Ser 36 in HUVEC overexpressing the p66 Shc Ala 36 mutant (HUVEC/p66 Shc Ala 36 ). Cells were stimulated with 50 ng/ml TNFα for 0.5 h or left untreated. When indicated, cells were pre-incubated with 30 mM SP600125 or DMSO for 2 h prior to TNFα stimulation. Each pair of representative immunoblots shows p66 Shc phosphorylation on Ser 36 (top left) and Shc protein content (bottom left) in HUVEC/wt, HUVEC/p66 Shc , HUVEC/p66 Shc Ala 36 , and HUVEC/mock, respectively. The ratio of phosphorylated to total p66 Shc protein in the four cell lines is also shown ( right ; HUVEC/wt, filled bars; HUVEC/p66 Shc , open bars; HUVEC/p66 Shc Ala 36 , grey bars; HUVEC/mock, light grey bars). * P
    Figure Legend Snippet: Role of Ser 36 phosphorylation of p66 Shc in endothelial activation. A . Basal and TNFα-stimulated phosphorylation of p66 Shc on Ser 36 in HUVEC overexpressing the p66 Shc Ala 36 mutant (HUVEC/p66 Shc Ala 36 ). Cells were stimulated with 50 ng/ml TNFα for 0.5 h or left untreated. When indicated, cells were pre-incubated with 30 mM SP600125 or DMSO for 2 h prior to TNFα stimulation. Each pair of representative immunoblots shows p66 Shc phosphorylation on Ser 36 (top left) and Shc protein content (bottom left) in HUVEC/wt, HUVEC/p66 Shc , HUVEC/p66 Shc Ala 36 , and HUVEC/mock, respectively. The ratio of phosphorylated to total p66 Shc protein in the four cell lines is also shown ( right ; HUVEC/wt, filled bars; HUVEC/p66 Shc , open bars; HUVEC/p66 Shc Ala 36 , grey bars; HUVEC/mock, light grey bars). * P

    Techniques Used: Activation Assay, Mutagenesis, Incubation, Western Blot

    TNFα-induced phosphorylation of p66 Shc on Ser 36 in HUVEC. A . Dose-response studies. Cells were incubated with TNFα for 0.5 h at the indicated doses or left untreated. Representative immunoblots of p66 Shc phosphorylation on Ser 36 (top left) and Shc protein content (bottom left), and ratio of phosphorylated to total p66 Shc protein (right). Cell lysates were analyzed by immunoblotting with specific antibodies. B . Time-course studies. Cells were incubated with 50 ng/ml TNFα for the indicated times or left untreated. Representative immunoblots of p66 Shc phosphorylation on Ser 36 (top left) and Shc protein content (bottom left), and ratio of phosphorylated to total p66 Shc protein (right). C . Effects of the JNK inhibitor SP600125 on TNFα-induced phosphorylation of p66 Shc on Ser 36 . Cells were pre-treated with 30 mM SP600125 for 2 h and then exposed to 50 ng/ml TNFα for 0.5 h. Representative immunoblots of p66 Shc phosphorylation on Ser 36 (top left) and Shc protein content (middle left), and ratio of phosphorylated to total p66 Shc protein ( right ; untreated cells, black bars; inhibitor-treated cells, grey bars; DMSO-treated cells, white bars). D . Effects of the MEK inhibitor PD98059 on TNFα-induced phosphorylation of p66 Shc on Ser 36 . Cells were pre-treated with 30 mM PD98059 for 2 h and then exposed to 50 ng/ml TNFα for 0.5 h. Representative immunoblots of p66 Shc phosphorylation on Ser 36 (top left) and Shc protein content (middle left), and ratio of phosphorylated to total p66 Shc protein ( right ; untreated cells, black bars; inhibitor-treated cells, grey bars; DMSO-treated cells, white bars). GAPDH protein content was used as loading control. * P
    Figure Legend Snippet: TNFα-induced phosphorylation of p66 Shc on Ser 36 in HUVEC. A . Dose-response studies. Cells were incubated with TNFα for 0.5 h at the indicated doses or left untreated. Representative immunoblots of p66 Shc phosphorylation on Ser 36 (top left) and Shc protein content (bottom left), and ratio of phosphorylated to total p66 Shc protein (right). Cell lysates were analyzed by immunoblotting with specific antibodies. B . Time-course studies. Cells were incubated with 50 ng/ml TNFα for the indicated times or left untreated. Representative immunoblots of p66 Shc phosphorylation on Ser 36 (top left) and Shc protein content (bottom left), and ratio of phosphorylated to total p66 Shc protein (right). C . Effects of the JNK inhibitor SP600125 on TNFα-induced phosphorylation of p66 Shc on Ser 36 . Cells were pre-treated with 30 mM SP600125 for 2 h and then exposed to 50 ng/ml TNFα for 0.5 h. Representative immunoblots of p66 Shc phosphorylation on Ser 36 (top left) and Shc protein content (middle left), and ratio of phosphorylated to total p66 Shc protein ( right ; untreated cells, black bars; inhibitor-treated cells, grey bars; DMSO-treated cells, white bars). D . Effects of the MEK inhibitor PD98059 on TNFα-induced phosphorylation of p66 Shc on Ser 36 . Cells were pre-treated with 30 mM PD98059 for 2 h and then exposed to 50 ng/ml TNFα for 0.5 h. Representative immunoblots of p66 Shc phosphorylation on Ser 36 (top left) and Shc protein content (middle left), and ratio of phosphorylated to total p66 Shc protein ( right ; untreated cells, black bars; inhibitor-treated cells, grey bars; DMSO-treated cells, white bars). GAPDH protein content was used as loading control. * P

    Techniques Used: Incubation, Western Blot

    Role of oxidative stress in p66 Shc signaling. A . Levels of reactive oxygen species (ROS) in HUVEC/p66 Shc and control cells (HUVEC/wt and HUVEC/mock). Cells were pre-incubated with or without 30 mM SP600125 for 2 h and then treated with 50 ng/ml TNFα for 0.5 h or left untreated; ROS levels were evaluated by fluorimetry (HUVEC/wt, filled bars; HUVEC/mock, grey bars; HUVEC/p66 Shc , open bars). * P
    Figure Legend Snippet: Role of oxidative stress in p66 Shc signaling. A . Levels of reactive oxygen species (ROS) in HUVEC/p66 Shc and control cells (HUVEC/wt and HUVEC/mock). Cells were pre-incubated with or without 30 mM SP600125 for 2 h and then treated with 50 ng/ml TNFα for 0.5 h or left untreated; ROS levels were evaluated by fluorimetry (HUVEC/wt, filled bars; HUVEC/mock, grey bars; HUVEC/p66 Shc , open bars). * P

    Techniques Used: Incubation

    40) Product Images from "Type 1 regulatory T cells specific for collagen type II as an efficient cell-based therapy in arthritis"

    Article Title: Type 1 regulatory T cells specific for collagen type II as an efficient cell-based therapy in arthritis

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar4567

    Antigen-specific type 1 regulatory T cells dampen the proliferation of effector T cells. BALB/c mice received injections of carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled, ovalbumin (ova)-specific effector CD4 + cells on the day before subcutaneous immunization with a mixture of ovalbumin and incomplete Freund’s adjuvant. At day 5, either phosphate-buffered saline (PBS) ( n = 7) or 1 × 10 6 ova-specific type 1 regulatory T (ova-Treg) cells ( n = 10) were injected intravenously. The mice received ova injections into their hind paws. Two days afterward, the proliferation of CFSE + KJ1.26 + cells was analyzed by flow cytometry. (A) Representative staining results for KJ1.26 + effector T cells in the draining lymph nodes (DLNs) are graphed. (B) Representative CFSE dilution due to the proliferation of effector KJ1.26 + T cells was analyzed using FlowJo software (TreeStar, Ashland, OR, USA). Graph shows the same numbers of CD4 + KJ1.26 + cells isolated from mice that received injections of saline (gray) or ova-Treg cells (bold). (C) Graphed numbers of KJ1.26 + proliferating cells in the DLNs. (D) CD90.1 congenic BALB/c mice received injections of CFSE-labeled, ova-specific effector CD4 + cells and 1 × 10 7 ova-Treg cells ( n = 8) or PBS ( n = 5). The numbers of ova-Treg cells (CD90.2 + ) in the DLNs are shown. Differences were analyzed by Mann–Whitney U test with 95% confidence intervals.
    Figure Legend Snippet: Antigen-specific type 1 regulatory T cells dampen the proliferation of effector T cells. BALB/c mice received injections of carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled, ovalbumin (ova)-specific effector CD4 + cells on the day before subcutaneous immunization with a mixture of ovalbumin and incomplete Freund’s adjuvant. At day 5, either phosphate-buffered saline (PBS) ( n = 7) or 1 × 10 6 ova-specific type 1 regulatory T (ova-Treg) cells ( n = 10) were injected intravenously. The mice received ova injections into their hind paws. Two days afterward, the proliferation of CFSE + KJ1.26 + cells was analyzed by flow cytometry. (A) Representative staining results for KJ1.26 + effector T cells in the draining lymph nodes (DLNs) are graphed. (B) Representative CFSE dilution due to the proliferation of effector KJ1.26 + T cells was analyzed using FlowJo software (TreeStar, Ashland, OR, USA). Graph shows the same numbers of CD4 + KJ1.26 + cells isolated from mice that received injections of saline (gray) or ova-Treg cells (bold). (C) Graphed numbers of KJ1.26 + proliferating cells in the DLNs. (D) CD90.1 congenic BALB/c mice received injections of CFSE-labeled, ova-specific effector CD4 + cells and 1 × 10 7 ova-Treg cells ( n = 8) or PBS ( n = 5). The numbers of ova-Treg cells (CD90.2 + ) in the DLNs are shown. Differences were analyzed by Mann–Whitney U test with 95% confidence intervals.

    Techniques Used: Mouse Assay, Labeling, Injection, Flow Cytometry, Cytometry, Staining, Software, Isolation, MANN-WHITNEY

    Phenotypic characterization of the collagen type II–specific type 1 regulatory T cell clones. (A) Graphed data of representative fluorescence-activated cell-sorting (FACS) analysis of the selected clones for the expression of T-cell receptor Vβ8 and CD4. (B) Graph illustrating the results of representative FACS analysis of intracellular cytokine staining of collagen type II–specific type 1 regulatory T cell (Col-Treg) clones following 4 hours of polyclonal stimulation. IFN, Interferon; IL, Interleukin. (C) Graph showing the cytokine secretion profile of three representative Col-Treg clones. The cytokine levels were quantified by enzyme-linked immunosorbent assay in the culture supernatants after 48 hours of polyclonal stimulation. The data are expressed as mean ± SEM. (D) Graph describing the immunosuppressive activity of Col-Treg clones, measured in a cell contact–independent assay by carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution, after 3 days of coculture with freshly isolated splenocytes stimulated with anti-CD3 antibody. The data are representative of at least seven clones. Th1, Type 1 T helper cell. (E) Graph of the expression levels of several phenotypic markers following 24 hours of polyclonal stimulation. Values are mean ± SEM of the percentage of positive cells for each marker. The data are representative of three to eighteen clones.
    Figure Legend Snippet: Phenotypic characterization of the collagen type II–specific type 1 regulatory T cell clones. (A) Graphed data of representative fluorescence-activated cell-sorting (FACS) analysis of the selected clones for the expression of T-cell receptor Vβ8 and CD4. (B) Graph illustrating the results of representative FACS analysis of intracellular cytokine staining of collagen type II–specific type 1 regulatory T cell (Col-Treg) clones following 4 hours of polyclonal stimulation. IFN, Interferon; IL, Interleukin. (C) Graph showing the cytokine secretion profile of three representative Col-Treg clones. The cytokine levels were quantified by enzyme-linked immunosorbent assay in the culture supernatants after 48 hours of polyclonal stimulation. The data are expressed as mean ± SEM. (D) Graph describing the immunosuppressive activity of Col-Treg clones, measured in a cell contact–independent assay by carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution, after 3 days of coculture with freshly isolated splenocytes stimulated with anti-CD3 antibody. The data are representative of at least seven clones. Th1, Type 1 T helper cell. (E) Graph of the expression levels of several phenotypic markers following 24 hours of polyclonal stimulation. Values are mean ± SEM of the percentage of positive cells for each marker. The data are representative of three to eighteen clones.

    Techniques Used: Fluorescence, FACS, Clone Assay, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Isolation, Marker

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    Article Snippet: .. PCR amplifications, performed in T1 thermocyclers (Biometra), involved an initial denaturation-step at 95°C for 5 min, followed by 38 cycles of 30 s at 95°C, 40 s at 54.4°C, 60 s at 72°C, followed by a final elongation step of 72°C for 5 min. Amplifications were performed in 25 μl reactions containing 2.5 μl (2 μM each) of the primer set, 2.5 μl dNTPs (2 mM), 3 μl MgCl2 (25 mM), 2.5 μg BSA (Sigma, 10 mg/ml), 1 μl DMSO, ~50 ng DNA, 1 U Firepol Polymerase (Solis Biodyne) and 1 × reaction buffer provided with the enzyme. .. For generating T-RFLP profiles three replicate PCR reactions from each sample were pooled and purified with Invisorb Spin PCRapid Kit (Invitek) before digestion as recommended by .

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    Millipore caspase 3 7
    FG-mediated neurotoxicity is dependent on secreted microglial factors.  (A)  Live cell staining with propidium iodide (PI) of ENGCs for analysis of cellular death after FG treatment for 24–48 h (Scale bar: 15 μm;  Ai ), quantified as a percentage of PI  +  cells in the population ( Aii ).  (B)  Quantification of cell death in ENGCs after treatment with 2.5 mg/ml FG for 24 h + /– hirudin (40 U/ml).  (C)  Analysis of caspase 3/7 activity by FAM-DEVD-FMK live cell staining in ENGCs following exposure to FG (2.5 mg/ml) or staurosporine (STS, 0.5 μM; Scale bar: 20 μm) for 24 h,  Ci . Data were quantified and presented as arbitrary fluorescence units (AFU)/cell/field of view;  Cii ). Confirmation of the presence of microglia in ENGCs with IB 4  cell staining ( Ciii ). All cellular populations were counterstained with Hoechst 33342 (blue).  (D)  Assessment of ENGC death by PI live cell staining after treatment with FG (2.5 mg/ml) or LPS (1 μg/ml) for 24 h before and after microglial ablation ( + LME).  (E)  Analysis of ENGC death by PI live cell staining after exposure to microglial conditioned medium (MGCM) collected from cultures treated for 24 h with FG (0.1–2.5 mg/ml) or untreated (Ctrl). Some MGCM samples from FG treated cultures were boiled to inactivate prior to addition.  (F)  Analysis of ENGC death by PI live cell staining after exposure to MGCM collected from cultures treated for 24 h with FG (2.5 mg/ml) + /– z-VAD-FMK, or untreated (Ctrl). In all graphs, data are the mean ± SEM from at least three independent experiments with internal replicates of at least 3. Significance levels are compared with control condition in each graph unless otherwise indicated,  ∗ p
    Caspase 3 7, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore guava caspase 8 fam caspase 9 sr kit
    Br- LPS induces activation of <t>caspase</t> 8 and 9 in PMNs. (A) Heparinized blood was incubated with 0.3 pmol/mL of Br- LPS or PBS for 30 minutes and stained with anti-active caspase 8 or anti-active caspase 9. PMNs population was analyzed by each caspase marker (B) Heparinized blood samples were treated with Z-VAD-FMK or PBS for 1 hour and then incubated with Br -LPS (1.5 pmol/mL) for 2 hours. PMNs population was analyzed by Annexin V. Geometric means of histograms are displayed as relative units. Experiments were repeated at least three times.
    Guava Caspase 8 Fam Caspase 9 Sr Kit, supplied by Millipore, 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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    92
    Millipore stem loop hairpin rna substrate
    MYC hyperactivation is synthetic lethal with <t>XBP1</t> inhibition. ( A ) Clonogenic growth of MCF10A MYC-ER cells transduced with shRNAs against XBP1 or LacZ and treated with different doses of 4-OHT. Ethanol was used as vehicle for 4-OHT. Changes in colony number were compared with vehicle-treated cells expressing shLacZ . ( B ) Immunoblot of MYC-ER in nuclear extracts of MCF10A MYC-ER cells treated with 4-OHT for 24 hours. ( C ) Chemical structure of 8866. ( D ) XBP1 -splicing assay in 293T cells that were treated with different doses of 8866 in the presence of DMSO or 5 μg/ml TM for 6 hours. ( E ) SUM159 cells were treated with DMSO or 5 μM 8866 in the presence of 5 μg/ml TM for 6 hours. ChIP assays were performed using anti-XBP1s antibody. Data are presented relative to input and shown as mean ± SD of technical triplicates. ( F ) Schematic diagram of fluorescence-based <t>RNA</t> cleavage assay. ( G ) Cytosolic portions of IRE1 protein or RNase A were incubated with hairpin XBP1 RNA substrate in the presence of various doses of 8866. Cleavage reactions were monitored by fluorescence intensity. ( H ) Immunoblot of IRE1 phosphorylation (phos-tag SDS-PAGE), ATF6 cleavage (ATF6p), PERK, and eIF2α phosphorylation in 293T cells treated with different doses of 8866 for 6 hours in the presence of DMSO or 5 μg/ml TM. Images shown are representative of 3 independent experiments. ( I ) Clonogenic growth of MCF10A MYC-ER cells transduced with GFP or XBP1s and treated with DMSO or 5 μM 8866 in the presence of different doses of 4-OHT. Changes in colony number were compared with vehicle-treated (ethanol and DMSO) MCF10A MYC-ER –GFP cells. In A and I , data are presented as mean ± SD of biological triplicates. * P
    Stem Loop Hairpin Rna Substrate, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    FG-mediated neurotoxicity is dependent on secreted microglial factors.  (A)  Live cell staining with propidium iodide (PI) of ENGCs for analysis of cellular death after FG treatment for 24–48 h (Scale bar: 15 μm;  Ai ), quantified as a percentage of PI  +  cells in the population ( Aii ).  (B)  Quantification of cell death in ENGCs after treatment with 2.5 mg/ml FG for 24 h + /– hirudin (40 U/ml).  (C)  Analysis of caspase 3/7 activity by FAM-DEVD-FMK live cell staining in ENGCs following exposure to FG (2.5 mg/ml) or staurosporine (STS, 0.5 μM; Scale bar: 20 μm) for 24 h,  Ci . Data were quantified and presented as arbitrary fluorescence units (AFU)/cell/field of view;  Cii ). Confirmation of the presence of microglia in ENGCs with IB 4  cell staining ( Ciii ). All cellular populations were counterstained with Hoechst 33342 (blue).  (D)  Assessment of ENGC death by PI live cell staining after treatment with FG (2.5 mg/ml) or LPS (1 μg/ml) for 24 h before and after microglial ablation ( + LME).  (E)  Analysis of ENGC death by PI live cell staining after exposure to microglial conditioned medium (MGCM) collected from cultures treated for 24 h with FG (0.1–2.5 mg/ml) or untreated (Ctrl). Some MGCM samples from FG treated cultures were boiled to inactivate prior to addition.  (F)  Analysis of ENGC death by PI live cell staining after exposure to MGCM collected from cultures treated for 24 h with FG (2.5 mg/ml) + /– z-VAD-FMK, or untreated (Ctrl). In all graphs, data are the mean ± SEM from at least three independent experiments with internal replicates of at least 3. Significance levels are compared with control condition in each graph unless otherwise indicated,  ∗ p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Soluble Fibrinogen Triggers Non-cell Autonomous ER Stress-Mediated Microglial-Induced Neurotoxicity

    doi: 10.3389/fncel.2018.00404

    Figure Lengend Snippet: FG-mediated neurotoxicity is dependent on secreted microglial factors. (A) Live cell staining with propidium iodide (PI) of ENGCs for analysis of cellular death after FG treatment for 24–48 h (Scale bar: 15 μm; Ai ), quantified as a percentage of PI + cells in the population ( Aii ). (B) Quantification of cell death in ENGCs after treatment with 2.5 mg/ml FG for 24 h + /– hirudin (40 U/ml). (C) Analysis of caspase 3/7 activity by FAM-DEVD-FMK live cell staining in ENGCs following exposure to FG (2.5 mg/ml) or staurosporine (STS, 0.5 μM; Scale bar: 20 μm) for 24 h, Ci . Data were quantified and presented as arbitrary fluorescence units (AFU)/cell/field of view; Cii ). Confirmation of the presence of microglia in ENGCs with IB 4 cell staining ( Ciii ). All cellular populations were counterstained with Hoechst 33342 (blue). (D) Assessment of ENGC death by PI live cell staining after treatment with FG (2.5 mg/ml) or LPS (1 μg/ml) for 24 h before and after microglial ablation ( + LME). (E) Analysis of ENGC death by PI live cell staining after exposure to microglial conditioned medium (MGCM) collected from cultures treated for 24 h with FG (0.1–2.5 mg/ml) or untreated (Ctrl). Some MGCM samples from FG treated cultures were boiled to inactivate prior to addition. (F) Analysis of ENGC death by PI live cell staining after exposure to MGCM collected from cultures treated for 24 h with FG (2.5 mg/ml) + /– z-VAD-FMK, or untreated (Ctrl). In all graphs, data are the mean ± SEM from at least three independent experiments with internal replicates of at least 3. Significance levels are compared with control condition in each graph unless otherwise indicated, ∗ p

    Article Snippet: Caspase 12 (FITC-ATAD-FMK) activity kits were from Promokine (Heidelberg, Germany) and caspase 3/7 (FAM-DEVD-FMK) activity kits were from Millipore (Watford, United Kingdom).

    Techniques: Staining, Activity Assay, Fluorescence

    Br- LPS induces activation of caspase 8 and 9 in PMNs. (A) Heparinized blood was incubated with 0.3 pmol/mL of Br- LPS or PBS for 30 minutes and stained with anti-active caspase 8 or anti-active caspase 9. PMNs population was analyzed by each caspase marker (B) Heparinized blood samples were treated with Z-VAD-FMK or PBS for 1 hour and then incubated with Br -LPS (1.5 pmol/mL) for 2 hours. PMNs population was analyzed by Annexin V. Geometric means of histograms are displayed as relative units. Experiments were repeated at least three times.

    Journal: PLoS Pathogens

    Article Title: Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide

    doi: 10.1371/journal.ppat.1004853

    Figure Lengend Snippet: Br- LPS induces activation of caspase 8 and 9 in PMNs. (A) Heparinized blood was incubated with 0.3 pmol/mL of Br- LPS or PBS for 30 minutes and stained with anti-active caspase 8 or anti-active caspase 9. PMNs population was analyzed by each caspase marker (B) Heparinized blood samples were treated with Z-VAD-FMK or PBS for 1 hour and then incubated with Br -LPS (1.5 pmol/mL) for 2 hours. PMNs population was analyzed by Annexin V. Geometric means of histograms are displayed as relative units. Experiments were repeated at least three times.

    Article Snippet: Determination of caspase 8 and 9 activation Heparinized human blood (500 μL) was incubated with B . abortus LPS (10 μg/mL) or PBS for 30 minutes under mild agitation and stained directly and incubated with anti-active caspase 8 or anti-active caspase 9 using Guava Caspase 8 FAM & Caspase 9 SR Kit (Millipore) according to manufacturer’s specifications and quantitated by flow cytometry.

    Techniques: Activation Assay, Incubation, Staining, Marker

    MYC hyperactivation is synthetic lethal with XBP1 inhibition. ( A ) Clonogenic growth of MCF10A MYC-ER cells transduced with shRNAs against XBP1 or LacZ and treated with different doses of 4-OHT. Ethanol was used as vehicle for 4-OHT. Changes in colony number were compared with vehicle-treated cells expressing shLacZ . ( B ) Immunoblot of MYC-ER in nuclear extracts of MCF10A MYC-ER cells treated with 4-OHT for 24 hours. ( C ) Chemical structure of 8866. ( D ) XBP1 -splicing assay in 293T cells that were treated with different doses of 8866 in the presence of DMSO or 5 μg/ml TM for 6 hours. ( E ) SUM159 cells were treated with DMSO or 5 μM 8866 in the presence of 5 μg/ml TM for 6 hours. ChIP assays were performed using anti-XBP1s antibody. Data are presented relative to input and shown as mean ± SD of technical triplicates. ( F ) Schematic diagram of fluorescence-based RNA cleavage assay. ( G ) Cytosolic portions of IRE1 protein or RNase A were incubated with hairpin XBP1 RNA substrate in the presence of various doses of 8866. Cleavage reactions were monitored by fluorescence intensity. ( H ) Immunoblot of IRE1 phosphorylation (phos-tag SDS-PAGE), ATF6 cleavage (ATF6p), PERK, and eIF2α phosphorylation in 293T cells treated with different doses of 8866 for 6 hours in the presence of DMSO or 5 μg/ml TM. Images shown are representative of 3 independent experiments. ( I ) Clonogenic growth of MCF10A MYC-ER cells transduced with GFP or XBP1s and treated with DMSO or 5 μM 8866 in the presence of different doses of 4-OHT. Changes in colony number were compared with vehicle-treated (ethanol and DMSO) MCF10A MYC-ER –GFP cells. In A and I , data are presented as mean ± SD of biological triplicates. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Pharmacological targeting of MYC-regulated IRE1/XBP1 pathway suppresses MYC-driven breast cancer

    doi: 10.1172/JCI95873

    Figure Lengend Snippet: MYC hyperactivation is synthetic lethal with XBP1 inhibition. ( A ) Clonogenic growth of MCF10A MYC-ER cells transduced with shRNAs against XBP1 or LacZ and treated with different doses of 4-OHT. Ethanol was used as vehicle for 4-OHT. Changes in colony number were compared with vehicle-treated cells expressing shLacZ . ( B ) Immunoblot of MYC-ER in nuclear extracts of MCF10A MYC-ER cells treated with 4-OHT for 24 hours. ( C ) Chemical structure of 8866. ( D ) XBP1 -splicing assay in 293T cells that were treated with different doses of 8866 in the presence of DMSO or 5 μg/ml TM for 6 hours. ( E ) SUM159 cells were treated with DMSO or 5 μM 8866 in the presence of 5 μg/ml TM for 6 hours. ChIP assays were performed using anti-XBP1s antibody. Data are presented relative to input and shown as mean ± SD of technical triplicates. ( F ) Schematic diagram of fluorescence-based RNA cleavage assay. ( G ) Cytosolic portions of IRE1 protein or RNase A were incubated with hairpin XBP1 RNA substrate in the presence of various doses of 8866. Cleavage reactions were monitored by fluorescence intensity. ( H ) Immunoblot of IRE1 phosphorylation (phos-tag SDS-PAGE), ATF6 cleavage (ATF6p), PERK, and eIF2α phosphorylation in 293T cells treated with different doses of 8866 for 6 hours in the presence of DMSO or 5 μg/ml TM. Images shown are representative of 3 independent experiments. ( I ) Clonogenic growth of MCF10A MYC-ER cells transduced with GFP or XBP1s and treated with DMSO or 5 μM 8866 in the presence of different doses of 4-OHT. Changes in colony number were compared with vehicle-treated (ethanol and DMSO) MCF10A MYC-ER –GFP cells. In A and I , data are presented as mean ± SD of biological triplicates. * P

    Article Snippet: A stem-loop hairpin RNA substrate (5′-6-FAM-CAUGUCCGCAGCGCAUG-BHQ1-3′) mimicking the XBP1 splice site was labeled with 6-FAM and black hole quencher 1 (BHQ1) on its 5′ and 3′ terminus, respectively (MilliporeSigma).

    Techniques: Inhibition, Transduction, Expressing, Splicing Assay, Chromatin Immunoprecipitation, Fluorescence, Cleavage Assay, Incubation, SDS Page