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    Structured Review

    Thermo Fisher rna
    <t>RNA</t> isolation, <t>cDNA</t> synthesis and real-time PCR
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rna - by Bioz Stars, 2022-07
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    Images

    1) Product Images from "Characterization of competence and biofilm development of a Streptocccus sanguinis endocarditis isolate"

    Article Title: Characterization of competence and biofilm development of a Streptocccus sanguinis endocarditis isolate

    Journal: Molecular oral microbiology

    doi: 10.1111/j.2041-1014.2010.00602.x

    RNA isolation, cDNA synthesis and real-time PCR
    Figure Legend Snippet: RNA isolation, cDNA synthesis and real-time PCR

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction

    2) Product Images from "Expression of S100P and Its Novel Binding Partner S100PBPR in Early Pancreatic Cancer"

    Article Title: Expression of S100P and Its Novel Binding Partner S100PBPR in Early Pancreatic Cancer

    Journal:

    doi:

    S100PBPR expression in normal tissues. cDNAs made from RNA samples isolated from normal organs were used as templates for RT-PCR to determine the presence of S100PBPR expression. Samples were run in the following order: lane 1 , universal RNA (positive
    Figure Legend Snippet: S100PBPR expression in normal tissues. cDNAs made from RNA samples isolated from normal organs were used as templates for RT-PCR to determine the presence of S100PBPR expression. Samples were run in the following order: lane 1 , universal RNA (positive

    Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction

    3) Product Images from "Inhibition of HPV-16 E6/E7 immortalization of normal keratinocytes by hairpin ribozymes"

    Article Title: Inhibition of HPV-16 E6/E7 immortalization of normal keratinocytes by hairpin ribozymes

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

    doi:

    Cis-expression of R434 ribozyme inhibits HPV-16E6/E7 in vitro translation. ( A ) Map of HPV-16E6/E7 cis-expression constructs with R434 and R434i ribozymes. PCR-amplified fragments containing the entire HPV-16 E6/E7 genes (nucleotides 97–868) linked to R434 (pCR16E6/E7RZ) or R434i (pCR16E6/E7RZi) ribozymes were cloned in the pCR3.1 vector. The pCR16HH plasmid contains only the HPV-16E6/E7 genes. The relative positions of the Sty I sites used for cloning the ribozymes and the vector poly(A) signal are shown. ( B ) The protein products produced by plasmids pCR16HH, pCR16E6/E7RZ, and pCR16E6/E7RZi were examined by in vitro translation reactions using T7 RNA polymerase and rabbit reticulocyte lysates in the presence of [ 35 S]methionine. ←, the position of E6 and E7 proteins. Luc, luciferase protein reaction control.
    Figure Legend Snippet: Cis-expression of R434 ribozyme inhibits HPV-16E6/E7 in vitro translation. ( A ) Map of HPV-16E6/E7 cis-expression constructs with R434 and R434i ribozymes. PCR-amplified fragments containing the entire HPV-16 E6/E7 genes (nucleotides 97–868) linked to R434 (pCR16E6/E7RZ) or R434i (pCR16E6/E7RZi) ribozymes were cloned in the pCR3.1 vector. The pCR16HH plasmid contains only the HPV-16E6/E7 genes. The relative positions of the Sty I sites used for cloning the ribozymes and the vector poly(A) signal are shown. ( B ) The protein products produced by plasmids pCR16HH, pCR16E6/E7RZ, and pCR16E6/E7RZi were examined by in vitro translation reactions using T7 RNA polymerase and rabbit reticulocyte lysates in the presence of [ 35 S]methionine. ←, the position of E6 and E7 proteins. Luc, luciferase protein reaction control.

    Techniques Used: Expressing, In Vitro, Construct, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Produced, Luciferase

    Short term growth inhibition of HPV-16 E6/E7-transfected HKc by cis-expressed R434. ( A ) HKc were transfected with the pCR16HH, pCR16E6/E7RZ, or pCR16E6/E7RZi constructs and were kept in G418 (200 μg/ml) for 2 wk, and 10 5 cells were seeded for counting in 6-well dishes. The graphics are the mean of three experiments. ( B ) RT-PCR analysis of RNA from HKc transfected with HPV-16 E6/E7 (pCR16HH) and cis-expressed active and inactive ribozymes (pCR16E6/E7RZ and pCR16E6/E7RZi, respectively). Total RNA (1 μg) was subjected to a coupled RT-PCR reaction with primers specific to both sides of the ribozyme cleavage site as described in Material and Methods . A contamination control without reverse transcriptase (control) was included. Separate RT-PCR reactions were performed by using the same RNA sample with primers specific to the human β-actin gene to show RNA integrity. The HPV-16 E6/E7-amplified products (492 and 326 bp) were separated through agarose gel electrophoresis and visualized with ethidium bromide staining. The arrows indicate the position and size of the amplified products.
    Figure Legend Snippet: Short term growth inhibition of HPV-16 E6/E7-transfected HKc by cis-expressed R434. ( A ) HKc were transfected with the pCR16HH, pCR16E6/E7RZ, or pCR16E6/E7RZi constructs and were kept in G418 (200 μg/ml) for 2 wk, and 10 5 cells were seeded for counting in 6-well dishes. The graphics are the mean of three experiments. ( B ) RT-PCR analysis of RNA from HKc transfected with HPV-16 E6/E7 (pCR16HH) and cis-expressed active and inactive ribozymes (pCR16E6/E7RZ and pCR16E6/E7RZi, respectively). Total RNA (1 μg) was subjected to a coupled RT-PCR reaction with primers specific to both sides of the ribozyme cleavage site as described in Material and Methods . A contamination control without reverse transcriptase (control) was included. Separate RT-PCR reactions were performed by using the same RNA sample with primers specific to the human β-actin gene to show RNA integrity. The HPV-16 E6/E7-amplified products (492 and 326 bp) were separated through agarose gel electrophoresis and visualized with ethidium bromide staining. The arrows indicate the position and size of the amplified products.

    Techniques Used: Inhibition, Transfection, Construct, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining

    4) Product Images from "'RNA walk' a novel approach to study RNA-RNA interactions between a small RNA and its target"

    Article Title: 'RNA walk' a novel approach to study RNA-RNA interactions between a small RNA and its target

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp872

    Affinity selection of sRNA-85 in untreated and AMT UV crossed-linked cells. ( A ) Total RNA was prepared from L. collosoma cells (5 × 10 9 ) and subjected to affinity selection using 2′- O -methyl biotinylated oligonucleotide of sRNA-85 as described in ‘Materials and Methods’ section. The affinity-selected product (‘affinity’) and 10% of the total RNA (T) was subjected to primer extension with radiolabeled oligonucleotides complementary to sRNA-85 (lanes 1 and 2) or tRNA Arg (lanes 3 and 4) and separated on a 6% denaturating gel. ( B ) Cells were treated with 0.2 mg ml− 1 AMT, and irradiated at 365 nm with UV light (10 milliWatts cm −2 ) for 60 min. RNA from the same number of irradiated (+; lane 2) or untreated (−; lane 1) cells was subjected to primer extension and separated on a 6% denaturing gel. ( C ) Total RNA from irradiated cells was subjected to either one round (lanes 2 and 5) or two rounds (lanes 3 and 6) of affinity selection and primer extension was preformed with oligonucleotides specific to sRNA-85 and srRNA-2, and compared with 10% of the total RNA used for selection (lanes 1 and 4). ( D ) RNA was prepared from irradiated cells and from control untreated cells as described above. The RNA (from 10 10 cells) was subjected to affinity selection as described in ‘Materials and Methods’ section. The affinity-selected products (lanes 2 and 3) and 2 µg total RNA (lane 1) were subjected to reverse transcription using random primers. The cDNA was amplified by PCR using primers for snoRNA-2. The PCR products were separated on 1.5% agarose gel and detected by ethidium bromide staining.
    Figure Legend Snippet: Affinity selection of sRNA-85 in untreated and AMT UV crossed-linked cells. ( A ) Total RNA was prepared from L. collosoma cells (5 × 10 9 ) and subjected to affinity selection using 2′- O -methyl biotinylated oligonucleotide of sRNA-85 as described in ‘Materials and Methods’ section. The affinity-selected product (‘affinity’) and 10% of the total RNA (T) was subjected to primer extension with radiolabeled oligonucleotides complementary to sRNA-85 (lanes 1 and 2) or tRNA Arg (lanes 3 and 4) and separated on a 6% denaturating gel. ( B ) Cells were treated with 0.2 mg ml− 1 AMT, and irradiated at 365 nm with UV light (10 milliWatts cm −2 ) for 60 min. RNA from the same number of irradiated (+; lane 2) or untreated (−; lane 1) cells was subjected to primer extension and separated on a 6% denaturing gel. ( C ) Total RNA from irradiated cells was subjected to either one round (lanes 2 and 5) or two rounds (lanes 3 and 6) of affinity selection and primer extension was preformed with oligonucleotides specific to sRNA-85 and srRNA-2, and compared with 10% of the total RNA used for selection (lanes 1 and 4). ( D ) RNA was prepared from irradiated cells and from control untreated cells as described above. The RNA (from 10 10 cells) was subjected to affinity selection as described in ‘Materials and Methods’ section. The affinity-selected products (lanes 2 and 3) and 2 µg total RNA (lane 1) were subjected to reverse transcription using random primers. The cDNA was amplified by PCR using primers for snoRNA-2. The PCR products were separated on 1.5% agarose gel and detected by ethidium bromide staining.

    Techniques Used: Selection, Irradiation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    Quantitative real-time PCR analysis of the ‘RNA Walk’ method. Real-time PCR was performed as described in ‘Materials and Methods’ section using cDNA (diluted 1:100 000). Concentration curves were prepared for each domain studied. The curves were used to determine the amount of PCR product amplified from each domain. PCR was performed on DNA prepared from RNA extracted from non-irradiated cells (−UV, gray bars) and from irradiated cells (+UV, black bars). The different rRNA domains examined are indicated and depicted schematically underneath the graph.
    Figure Legend Snippet: Quantitative real-time PCR analysis of the ‘RNA Walk’ method. Real-time PCR was performed as described in ‘Materials and Methods’ section using cDNA (diluted 1:100 000). Concentration curves were prepared for each domain studied. The curves were used to determine the amount of PCR product amplified from each domain. PCR was performed on DNA prepared from RNA extracted from non-irradiated cells (−UV, gray bars) and from irradiated cells (+UV, black bars). The different rRNA domains examined are indicated and depicted schematically underneath the graph.

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

    Identification of the regions where the rRNA interacts with sRNA-85 by the ‘RNA walk’ method. ( A ) Schematic representation of the ‘RNA walk’ approach. Cells were treated with AMT and subjected to UV irradiation; RNA was then prepared by deproteinization and Trizol reagent extraction. RNA was then subjected to affinity selection with anti-sense biotinylated oligonucleotide complementary to sRNA-85. cDNA was prepared from the affinity-selected RNA, and amplified by PCR using primers covering the entire rRNA target. ( B ) PCR analyses of cDNA covering the rRNA. RNA was prepared from irradiated cells and from control untreated cells as described in Figure 1 . The RNA (from 10 10 cells) was subjected to affinity selection as described in ‘Materials and Methods’ section. The affinity-selected product was subjected to reverse transcription using random primers. The cDNA was amplified by PCR using primers dividing the rRNA into 12 ∼500nt domains spanning the entire rRNA. The PCR products were separated on 1.5% agarose gel and detected by ethidium bromide staining. RNA from irradiated and untreated cells is designated by (+) and (−), respectively. The positions of the PCR domains are marked by double-headed arrows on the rRNA subunits (indicated). Stars mark the domains carrying the cross-linked adducts. ( C ) ‘RNA Walk’ using an internal control. ‘RNA walk’ was performed with affinity-selected RNA prepared from irradiated cells (+UV) or non-irradiated cells (−UV). The RNA was spiked with 100 ng of synthetic RNA ( in vitro transcribed luciferase). cDNA was prepared as described in (B) and PCR was performed using primers that amplify the rRNA domain (listed above the lanes) and luciferase mRNA in the same reaction. ( D ) Photoreversal of cross-linking eliminates the block of reverse transcriptase and enables PCR amplification of the cDNA. cDNA was prepared from affinity-selected RNA derived from irradiated cells (lane 2), from untreated cells (lane 1) and from affinity-selected RNA that was photo reversed (lanes 3 and 4). PCR amplification was performed with primers specific to domain 12. ( E ) RNA walk requires both UV and AMT. cDNA was prepared with random primers from affinity-selected RNA derived from untreated cells (lane 1), and cells irradiated in the presence (lane 2) and absence (lane 3) of AMT. PCR amplification was performed with primers specific to domain 12. ( F ) RNA walk stop is confirmed using specific primers. cDNA was prepared from affinity selected (lanes 2 and 4) and total RNA (lanes 1 and 3) from untreated and irradiated cells using a gene specific primer (primer LCLSUβ560AS). PCR amplification was preformed with primers specific to domain 12.
    Figure Legend Snippet: Identification of the regions where the rRNA interacts with sRNA-85 by the ‘RNA walk’ method. ( A ) Schematic representation of the ‘RNA walk’ approach. Cells were treated with AMT and subjected to UV irradiation; RNA was then prepared by deproteinization and Trizol reagent extraction. RNA was then subjected to affinity selection with anti-sense biotinylated oligonucleotide complementary to sRNA-85. cDNA was prepared from the affinity-selected RNA, and amplified by PCR using primers covering the entire rRNA target. ( B ) PCR analyses of cDNA covering the rRNA. RNA was prepared from irradiated cells and from control untreated cells as described in Figure 1 . The RNA (from 10 10 cells) was subjected to affinity selection as described in ‘Materials and Methods’ section. The affinity-selected product was subjected to reverse transcription using random primers. The cDNA was amplified by PCR using primers dividing the rRNA into 12 ∼500nt domains spanning the entire rRNA. The PCR products were separated on 1.5% agarose gel and detected by ethidium bromide staining. RNA from irradiated and untreated cells is designated by (+) and (−), respectively. The positions of the PCR domains are marked by double-headed arrows on the rRNA subunits (indicated). Stars mark the domains carrying the cross-linked adducts. ( C ) ‘RNA Walk’ using an internal control. ‘RNA walk’ was performed with affinity-selected RNA prepared from irradiated cells (+UV) or non-irradiated cells (−UV). The RNA was spiked with 100 ng of synthetic RNA ( in vitro transcribed luciferase). cDNA was prepared as described in (B) and PCR was performed using primers that amplify the rRNA domain (listed above the lanes) and luciferase mRNA in the same reaction. ( D ) Photoreversal of cross-linking eliminates the block of reverse transcriptase and enables PCR amplification of the cDNA. cDNA was prepared from affinity-selected RNA derived from irradiated cells (lane 2), from untreated cells (lane 1) and from affinity-selected RNA that was photo reversed (lanes 3 and 4). PCR amplification was performed with primers specific to domain 12. ( E ) RNA walk requires both UV and AMT. cDNA was prepared with random primers from affinity-selected RNA derived from untreated cells (lane 1), and cells irradiated in the presence (lane 2) and absence (lane 3) of AMT. PCR amplification was performed with primers specific to domain 12. ( F ) RNA walk stop is confirmed using specific primers. cDNA was prepared from affinity selected (lanes 2 and 4) and total RNA (lanes 1 and 3) from untreated and irradiated cells using a gene specific primer (primer LCLSUβ560AS). PCR amplification was preformed with primers specific to domain 12.

    Techniques Used: Irradiation, Selection, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, In Vitro, Luciferase, Blocking Assay, Derivative Assay

    ‘RNA walk’ of mutant sRNA-85 lacking the anti-codon loop. ( A ) Schematic representation of deletion mutation introduced in the sRNA-85. The deletion is indicated by a box. ( B ) Northern analysis of cells carrying the sRNA-85 mutation. The blot was probed with anti-sense oligonucleotide (18288), specific to sRNA-85 that recognizes both wild-type and mutant transcripts. ( C ) Distribution of wild-type and mutant sRNA-85 on RNP particles. Low-salt extracts from cells carrying the mutation in sRNA-85 were layered on continuous 10–30% sucrose gradients containing 100 mM KCl. Gradients were centrifuged for 3 h at 35 000 rpm using SW41 rotor, as described in ‘Materials and Methods’ section. RNA prepared from the sucrose gradient fractions was subjected to northern analysis and the blot was hybridized with the same probe as in (B). S values were determined using standards, i.e. 30, 50 and 70 ribosomes from E. coli and enzyme catalase (10S). The fractions are numbered from top to bottom. The positions of the wild-type and mutated RNA are indicated. Upper panel―long exposure, Lower panel―short exposure. ( D ) Affinity selection of mutant sRNA-85. RNA was prepared from irradiated cells expressing the sRNA-85 mutant as described previously. The RNA (from 10 10 cells) was subjected to affinity selection as described in ‘Materials and Methods’ section. RNA from affinity selected and from total RNA was subjected to primer extension and the products were separated on a 6% polyacrylamide denaturing gel. ( E ) ‘RNA walk’ analysis of sRNA-85 mutant. RNA was prepared from irradiated cells and from control untreated cells expressing the sRNA-85 mutant as described in Figures 1–3. The RNA (from 10 10 cells) was subjected to affinity selection as described in ‘Materials and Methods’ section. The affinity-selected product was subjected to reverse transcription using random primers. The cDNA was amplified by PCR using the primer pairs shown. The PCR products were separated on 1.5% agarose gel and detected by ethidium bromide staining. RNA from irradiated and untreated cells is designated by (+) and (−), respectively. The positions of the PCR domains are marked by double-headed arrows on the rRNA subunits (indicated). Stars mark the domains carrying the cross-linked adducts.
    Figure Legend Snippet: ‘RNA walk’ of mutant sRNA-85 lacking the anti-codon loop. ( A ) Schematic representation of deletion mutation introduced in the sRNA-85. The deletion is indicated by a box. ( B ) Northern analysis of cells carrying the sRNA-85 mutation. The blot was probed with anti-sense oligonucleotide (18288), specific to sRNA-85 that recognizes both wild-type and mutant transcripts. ( C ) Distribution of wild-type and mutant sRNA-85 on RNP particles. Low-salt extracts from cells carrying the mutation in sRNA-85 were layered on continuous 10–30% sucrose gradients containing 100 mM KCl. Gradients were centrifuged for 3 h at 35 000 rpm using SW41 rotor, as described in ‘Materials and Methods’ section. RNA prepared from the sucrose gradient fractions was subjected to northern analysis and the blot was hybridized with the same probe as in (B). S values were determined using standards, i.e. 30, 50 and 70 ribosomes from E. coli and enzyme catalase (10S). The fractions are numbered from top to bottom. The positions of the wild-type and mutated RNA are indicated. Upper panel―long exposure, Lower panel―short exposure. ( D ) Affinity selection of mutant sRNA-85. RNA was prepared from irradiated cells expressing the sRNA-85 mutant as described previously. The RNA (from 10 10 cells) was subjected to affinity selection as described in ‘Materials and Methods’ section. RNA from affinity selected and from total RNA was subjected to primer extension and the products were separated on a 6% polyacrylamide denaturing gel. ( E ) ‘RNA walk’ analysis of sRNA-85 mutant. RNA was prepared from irradiated cells and from control untreated cells expressing the sRNA-85 mutant as described in Figures 1–3. The RNA (from 10 10 cells) was subjected to affinity selection as described in ‘Materials and Methods’ section. The affinity-selected product was subjected to reverse transcription using random primers. The cDNA was amplified by PCR using the primer pairs shown. The PCR products were separated on 1.5% agarose gel and detected by ethidium bromide staining. RNA from irradiated and untreated cells is designated by (+) and (−), respectively. The positions of the PCR domains are marked by double-headed arrows on the rRNA subunits (indicated). Stars mark the domains carrying the cross-linked adducts.

    Techniques Used: Mutagenesis, Northern Blot, Selection, Irradiation, Expressing, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    5) Product Images from "Transcriptome Analysis Reveals Altered Expression of Memory and Neurotransmission Associated Genes in the REM Sleep Deprived Rat Brain"

    Article Title: Transcriptome Analysis Reveals Altered Expression of Memory and Neurotransmission Associated Genes in the REM Sleep Deprived Rat Brain

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00067

    Na–K ATPase activity and expression: (A) Na–K ATPase activity in synaptosomes prepared from rat brains ( n = 4 rats per groups). (B) RNA seq data showing significant up-regulation of ATP1B2 transcripts upon REMSD ( P
    Figure Legend Snippet: Na–K ATPase activity and expression: (A) Na–K ATPase activity in synaptosomes prepared from rat brains ( n = 4 rats per groups). (B) RNA seq data showing significant up-regulation of ATP1B2 transcripts upon REMSD ( P

    Techniques Used: Activity Assay, Expressing, RNA Sequencing Assay

    Validation of rat brain RNA seq data using different controls. Some genes were selected for validation of RNA seq data by qRT-PCR. (A) RNA sequencing results (log 10 FPKM values) showing DEGs involved in sleep and neuronal regulation like OXT, RMRP, LNPEP, AVP, HIST2H4, GRIN2B, PMCH and HCRT were down regulated upon REMSD. Other set of genes up regulated upon REMSD, which includes DBH, NPAS4, EGR1 and SYT2. For validation of these RNA seq results, we have included other additional controls like large platform control (Lrg Plat), recovery control (Rec Ctrl) and Prazosin treatment group (PRZ treated). (B–M) Shows qRT-PCR results for above genes in free moving control (Free Mov), large platform control (Lrg Ctrl), REMSD, recovery control (Rec Ctrl), Prazosin treatment group (PRZ treated) ( n = 5 per group). Statistical analyses were performed using one-way ANOVA followed by Newman–Keuls multiple comparison tests. Data are expressed as the mean ± SEM ( ∗ P
    Figure Legend Snippet: Validation of rat brain RNA seq data using different controls. Some genes were selected for validation of RNA seq data by qRT-PCR. (A) RNA sequencing results (log 10 FPKM values) showing DEGs involved in sleep and neuronal regulation like OXT, RMRP, LNPEP, AVP, HIST2H4, GRIN2B, PMCH and HCRT were down regulated upon REMSD. Other set of genes up regulated upon REMSD, which includes DBH, NPAS4, EGR1 and SYT2. For validation of these RNA seq results, we have included other additional controls like large platform control (Lrg Plat), recovery control (Rec Ctrl) and Prazosin treatment group (PRZ treated). (B–M) Shows qRT-PCR results for above genes in free moving control (Free Mov), large platform control (Lrg Ctrl), REMSD, recovery control (Rec Ctrl), Prazosin treatment group (PRZ treated) ( n = 5 per group). Statistical analyses were performed using one-way ANOVA followed by Newman–Keuls multiple comparison tests. Data are expressed as the mean ± SEM ( ∗ P

    Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR

    6) Product Images from "Oral and Vaginal Epithelial Cell Lines Bind and Transfer Cell-Free Infectious HIV-1 to Permissive Cells but Are Not Productively Infected"

    Article Title: Oral and Vaginal Epithelial Cell Lines Bind and Transfer Cell-Free Infectious HIV-1 to Permissive Cells but Are Not Productively Infected

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0098077

    Different methods used to detect HIV-1 R5 and X4 binding to epithelial cells. (A) Post-lysis detection of p24 gag protein by Western blotting. Primary (gingival) epithelial cells, TR146, FaDu, A431 and TZM-bl cells were incubated overnight (16–24 h) with cell free YU2 (R5) or LAI (X4). After extensive washing to remove unbound virus, normalised total protein lysates were separated by SDS-PAGE and probed for HIV p24 using α-actin as a loading control. (B) Detection of immobilized virus on the cell surface by flow cytometry. Epithelial cells were incubated overnight with cell free virus. Bound virus was detected using a Cy5-labeled anti-human secondary antibody to detect HIV-1 gp120 primary monoclonal on the APC channel. Electronic gates were set around an unlabelled cell control, this area is then set as zero and any cells shifted to the right of the gate are deemed positive. To determine amount of virus bound, virally exposed, labelled cell percentages are subtracted from the uninfected (unexposed) labelled control cell percentages to obtain the % fluorescence values shown. Data are representative of four independent experiments and bars indicate ± standard deviation from the mean. (C) Detection of packaged HIV R5 RNA by amplification of the HIV-1 pol gene using nested PCR. Total RNA was extracted from TR146, FaDu, A431 and TZM-bl cells incubated overnight with cell free YU2 (R5) and used to produce viral cDNA. This was then used as a template in a nested PCR to detect a 2 Kb region of HIV pol. (D) Percentage reduction in detection of immobilized virus on the cell surface by flow cytometry after trypsin treatment. Virally exposed cells are compared with cells labelled with secondary antibody alone. Data set is representative of three independent experiments. * = P
    Figure Legend Snippet: Different methods used to detect HIV-1 R5 and X4 binding to epithelial cells. (A) Post-lysis detection of p24 gag protein by Western blotting. Primary (gingival) epithelial cells, TR146, FaDu, A431 and TZM-bl cells were incubated overnight (16–24 h) with cell free YU2 (R5) or LAI (X4). After extensive washing to remove unbound virus, normalised total protein lysates were separated by SDS-PAGE and probed for HIV p24 using α-actin as a loading control. (B) Detection of immobilized virus on the cell surface by flow cytometry. Epithelial cells were incubated overnight with cell free virus. Bound virus was detected using a Cy5-labeled anti-human secondary antibody to detect HIV-1 gp120 primary monoclonal on the APC channel. Electronic gates were set around an unlabelled cell control, this area is then set as zero and any cells shifted to the right of the gate are deemed positive. To determine amount of virus bound, virally exposed, labelled cell percentages are subtracted from the uninfected (unexposed) labelled control cell percentages to obtain the % fluorescence values shown. Data are representative of four independent experiments and bars indicate ± standard deviation from the mean. (C) Detection of packaged HIV R5 RNA by amplification of the HIV-1 pol gene using nested PCR. Total RNA was extracted from TR146, FaDu, A431 and TZM-bl cells incubated overnight with cell free YU2 (R5) and used to produce viral cDNA. This was then used as a template in a nested PCR to detect a 2 Kb region of HIV pol. (D) Percentage reduction in detection of immobilized virus on the cell surface by flow cytometry after trypsin treatment. Virally exposed cells are compared with cells labelled with secondary antibody alone. Data set is representative of three independent experiments. * = P

    Techniques Used: Binding Assay, Lysis, Western Blot, Incubation, SDS Page, Flow Cytometry, Cytometry, Labeling, Fluorescence, Standard Deviation, Amplification, Nested PCR

    Post-integration HIV-1 mRNA transcription and de novo viral protein production in epithelial cells (MOI: 0.2). (A) Detection of spliced HIV-1 tat mRNA in TR146, FaDu, A431 and TZM-bl control cells by PCR 24 h post-infection with YU2 (R5) or LAI (X4) infectious virus. Equal amounts of total RNA was used to synthesise viral cDNA which was then subjected to PCR using primers designed to span the TAT 1 and 2 exon junctions. (B) p55 gag protein detection in TR146, FaDu, A431 and TZM-bl control cells by Western blot after 24 h infection with R5 (YU2) and LAI (X4) virus. (C) Infection of TR146, FaDu, A431 and NP2-R5/X4 control cells with GFP-linked single-cycle X4, R5 and dual tropic HIV-1 gp160 pseudotyped virus and detection of GFP incorporation into epithelial cell DNA by flow cytometry. Error bars show standard error from the mean. Data are representative of three independent experiments.
    Figure Legend Snippet: Post-integration HIV-1 mRNA transcription and de novo viral protein production in epithelial cells (MOI: 0.2). (A) Detection of spliced HIV-1 tat mRNA in TR146, FaDu, A431 and TZM-bl control cells by PCR 24 h post-infection with YU2 (R5) or LAI (X4) infectious virus. Equal amounts of total RNA was used to synthesise viral cDNA which was then subjected to PCR using primers designed to span the TAT 1 and 2 exon junctions. (B) p55 gag protein detection in TR146, FaDu, A431 and TZM-bl control cells by Western blot after 24 h infection with R5 (YU2) and LAI (X4) virus. (C) Infection of TR146, FaDu, A431 and NP2-R5/X4 control cells with GFP-linked single-cycle X4, R5 and dual tropic HIV-1 gp160 pseudotyped virus and detection of GFP incorporation into epithelial cell DNA by flow cytometry. Error bars show standard error from the mean. Data are representative of three independent experiments.

    Techniques Used: Polymerase Chain Reaction, Infection, Western Blot, Flow Cytometry, Cytometry

    7) Product Images from "Resveratrol modulates the levels of microRNAs targeting genes encoding tumor-suppressors and effectors of TGF? signaling pathway in SW480 cells"

    Article Title: Resveratrol modulates the levels of microRNAs targeting genes encoding tumor-suppressors and effectors of TGF? signaling pathway in SW480 cells

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2010.07.003

    MiR-663 targets TGFβ1 transcripts (A) Schematic representation (not to scale) of the 307 nt-long 3′-untranslated region of TGFβ1 transcripts. The positions of the two sets of overlapping miR-663 target sites (TS1 to TS5) are given in nucleotides. Their sequences (CCCCGCC) are given in black boxes. Nucleotides modified in the two single mutant constructs ( Luc- TGFβ-M663-1 and Luc- TGFβ-M663-2 ) are given below the figure (dashes correspond to conserved nucleotides). Luc-TGFβ-M663-1,2 contains both sets of mutated nucleotides. The first 41 nucleotides of TGFβ1 3′-untranslated region is lacking in Luc- TGFβ-Del-663-1 . (B) Effects of pre-miR-663 on Luc- TGF β constructs. SW480 cells were transfected with wild type Luc- TGF β (WT) constructs both in sense and antisense orientation, with mutated Luc- TGF β constructs, (M663-1, M663-2, and M663-1,2), or with a Luc- TGF β construct bearing a deletion of the first 41 nt of TGFβ1 3′-UTR (Del-663-1), along with either a Control RNA (Pre-miR-Control) or pre-miR-663 . The sequences of the two mutated series of overlapping miR-663 target sites are given in panel A. Values represent the mean ± standard deviation (n = 4). *, Significantly different from Pre-miR-Control, P
    Figure Legend Snippet: MiR-663 targets TGFβ1 transcripts (A) Schematic representation (not to scale) of the 307 nt-long 3′-untranslated region of TGFβ1 transcripts. The positions of the two sets of overlapping miR-663 target sites (TS1 to TS5) are given in nucleotides. Their sequences (CCCCGCC) are given in black boxes. Nucleotides modified in the two single mutant constructs ( Luc- TGFβ-M663-1 and Luc- TGFβ-M663-2 ) are given below the figure (dashes correspond to conserved nucleotides). Luc-TGFβ-M663-1,2 contains both sets of mutated nucleotides. The first 41 nucleotides of TGFβ1 3′-untranslated region is lacking in Luc- TGFβ-Del-663-1 . (B) Effects of pre-miR-663 on Luc- TGF β constructs. SW480 cells were transfected with wild type Luc- TGF β (WT) constructs both in sense and antisense orientation, with mutated Luc- TGF β constructs, (M663-1, M663-2, and M663-1,2), or with a Luc- TGF β construct bearing a deletion of the first 41 nt of TGFβ1 3′-UTR (Del-663-1), along with either a Control RNA (Pre-miR-Control) or pre-miR-663 . The sequences of the two mutated series of overlapping miR-663 target sites are given in panel A. Values represent the mean ± standard deviation (n = 4). *, Significantly different from Pre-miR-Control, P

    Techniques Used: Modification, Mutagenesis, Construct, Transfection, Standard Deviation

    Resveratrol increases the levels of miR-663 as well as those of pri-miR-663 and pre-miR-663 differentially Total RNAs (0.6 μg) extracted from SW480 cells treated for 14 hours with either the vehicle (V) or resveratrol (R) were hybridized with a radiolabeled RNA antisense probe (P1, P2 or U6) in the presence of 4.4 μg of yeast tRNA. The schematic structure of the LO284801 locus, whose transcripts represent miR-663 primary transcripts, is presented on the unscaled top drawing. Open and filled boxes represent miR-663 precursor ( pre-miR-663 ) and mature miR-663 , respectively. P1 protects a 33 nt-long fragment starting 184 nt upstream of pre-miR-663. Pre-miR-663 and miR-663 respectively generate 40 and 22 nt-long protected fragments from P2, which corresponds to one of the miRNA micro-array probes. The relative intensity of the signal following resveratrol treatment is given between parentheses in percent of the vehicle sample, calculated using U6snRNA as an internal control. The efficiency of RNase digestion was assessed in parallel (two left lanes). Samples and controls were from the same gels. C-, no RNase (15 fold dilution); C+, RNase; Nd, non digested; Ns, non specific.
    Figure Legend Snippet: Resveratrol increases the levels of miR-663 as well as those of pri-miR-663 and pre-miR-663 differentially Total RNAs (0.6 μg) extracted from SW480 cells treated for 14 hours with either the vehicle (V) or resveratrol (R) were hybridized with a radiolabeled RNA antisense probe (P1, P2 or U6) in the presence of 4.4 μg of yeast tRNA. The schematic structure of the LO284801 locus, whose transcripts represent miR-663 primary transcripts, is presented on the unscaled top drawing. Open and filled boxes represent miR-663 precursor ( pre-miR-663 ) and mature miR-663 , respectively. P1 protects a 33 nt-long fragment starting 184 nt upstream of pre-miR-663. Pre-miR-663 and miR-663 respectively generate 40 and 22 nt-long protected fragments from P2, which corresponds to one of the miRNA micro-array probes. The relative intensity of the signal following resveratrol treatment is given between parentheses in percent of the vehicle sample, calculated using U6snRNA as an internal control. The efficiency of RNase digestion was assessed in parallel (two left lanes). Samples and controls were from the same gels. C-, no RNase (15 fold dilution); C+, RNase; Nd, non digested; Ns, non specific.

    Techniques Used: Microarray

    Resveratrol decreases the levels of TGFβ1 mainly in a miR-663 -independent manner SW480 cells were transfected with either a control RNA (pre-miR-Control), pre-miR-663 or an antisense miR-663 inhibitory RNA ( 663-I ) before resveratrol treatment. The levels of TGFβ1 and SMAD3 were then assessed on Western blots. The relative levels of TGFβ1and SMAD3 are given in percent of the corresponding Pre-miR-Control Vehicle-treated samples.
    Figure Legend Snippet: Resveratrol decreases the levels of TGFβ1 mainly in a miR-663 -independent manner SW480 cells were transfected with either a control RNA (pre-miR-Control), pre-miR-663 or an antisense miR-663 inhibitory RNA ( 663-I ) before resveratrol treatment. The levels of TGFβ1 and SMAD3 were then assessed on Western blots. The relative levels of TGFβ1and SMAD3 are given in percent of the corresponding Pre-miR-Control Vehicle-treated samples.

    Techniques Used: Transfection, Western Blot

    Resveratrol downregulates TGFβ1 transcripts through miR-663 SW480 cells were transfected with wild type (WT) or mutated (M663-1, M663-2, and M663-1,2) Luc- TGFβ constructs, along with either a Control RNA (Cont.) or a miR-663 antisense inhibitory RNA ( 663-I ) before resveratrol treatment. Bars show the ratios of the relative levels of expression of the different constructs to those of the empty pGL3-Control vector. Values represent the mean ± standard deviation (n = 4). * and **, Significantly different from the corresponding WT / Control, *, P = 0.05, **, P = 0.017 (df = 6).
    Figure Legend Snippet: Resveratrol downregulates TGFβ1 transcripts through miR-663 SW480 cells were transfected with wild type (WT) or mutated (M663-1, M663-2, and M663-1,2) Luc- TGFβ constructs, along with either a Control RNA (Cont.) or a miR-663 antisense inhibitory RNA ( 663-I ) before resveratrol treatment. Bars show the ratios of the relative levels of expression of the different constructs to those of the empty pGL3-Control vector. Values represent the mean ± standard deviation (n = 4). * and **, Significantly different from the corresponding WT / Control, *, P = 0.05, **, P = 0.017 (df = 6).

    Techniques Used: Transfection, Construct, Expressing, Plasmid Preparation, Standard Deviation

    Resveratrol decreases the expression of a SMAD2/SMAD3/SMAD4 luciferase reporter contruct After cotransfection with the SMAD2/SMAD3/SMAD4 luciferase reporter plasmid along with either a Control RNA (pre-miR-Control) or a miR-663 antisense inhibitory RNA ( 663-I ), SW480 cells were mock-treated or treated with TGFβ for 14 hours, and subsequently treated with the vehicle (Veh.) or resveratrol (Res.) for another 14 hours. The Firefly luciferase activity was measured 48 hours after transfection and then normalized to the Renilla luciferase activity. Values represent the mean ± standard deviation (n = 6). * and **, TGFβ1-treated significantly different from the corresponding Mock-treated, *, P
    Figure Legend Snippet: Resveratrol decreases the expression of a SMAD2/SMAD3/SMAD4 luciferase reporter contruct After cotransfection with the SMAD2/SMAD3/SMAD4 luciferase reporter plasmid along with either a Control RNA (pre-miR-Control) or a miR-663 antisense inhibitory RNA ( 663-I ), SW480 cells were mock-treated or treated with TGFβ for 14 hours, and subsequently treated with the vehicle (Veh.) or resveratrol (Res.) for another 14 hours. The Firefly luciferase activity was measured 48 hours after transfection and then normalized to the Renilla luciferase activity. Values represent the mean ± standard deviation (n = 6). * and **, TGFβ1-treated significantly different from the corresponding Mock-treated, *, P

    Techniques Used: Expressing, Luciferase, Cotransfection, Plasmid Preparation, Activity Assay, Transfection, Standard Deviation

    8) Product Images from "Evolutionary Conservation of Primate Lymphocryptovirus MicroRNA Targets"

    Article Title: Evolutionary Conservation of Primate Lymphocryptovirus MicroRNA Targets

    Journal: Journal of Virology

    doi: 10.1128/JVI.02071-13

    Deep sequencing analysis of lymphocryptovirus miRNAs. (A) Distribution of aligned, deep sequencing reads from RISC-immunoprecipitations (RIP-SEQ) or size-selected total RNA (smRNA). Reads from IBL-LCLd3 and Akata-LCLd3 (human LCLs infected with wild-type
    Figure Legend Snippet: Deep sequencing analysis of lymphocryptovirus miRNAs. (A) Distribution of aligned, deep sequencing reads from RISC-immunoprecipitations (RIP-SEQ) or size-selected total RNA (smRNA). Reads from IBL-LCLd3 and Akata-LCLd3 (human LCLs infected with wild-type

    Techniques Used: Sequencing, Infection

    9) Product Images from "Cooperating cancer-gene identification through oncogenic-retrovirus-induced insertional mutagenesis"

    Article Title: Cooperating cancer-gene identification through oncogenic-retrovirus-induced insertional mutagenesis

    Journal: Blood

    doi: 10.1182/blood-2004-12-4840

    Viral integrations decrease Sfpi1 expression and activate Mef2c expression. (A) Real-time RT-PCR analysis of total RNA isolated from spleens of leukemic animals in addition to spleen and bone marrow of normal mice using primers specific for mouse Sfpi1 . Relative gene expression levels were calculated by normalizing to β-actin mRNA levels in the same sample and in normal spleen. (B) Real-time RT-PCR analysis of the same samples using primers specific for mouse Mef2c .
    Figure Legend Snippet: Viral integrations decrease Sfpi1 expression and activate Mef2c expression. (A) Real-time RT-PCR analysis of total RNA isolated from spleens of leukemic animals in addition to spleen and bone marrow of normal mice using primers specific for mouse Sfpi1 . Relative gene expression levels were calculated by normalizing to β-actin mRNA levels in the same sample and in normal spleen. (B) Real-time RT-PCR analysis of the same samples using primers specific for mouse Mef2c .

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

    10) Product Images from ""

    Article Title:

    Journal: Drug Metabolism and Disposition

    doi: 10.1124/dmd.113.051904

    RNA Isolation, cDNA Synthesis, and Real-Time Polymerase Chain Reaction.
    Figure Legend Snippet: RNA Isolation, cDNA Synthesis, and Real-Time Polymerase Chain Reaction.

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction

    11) Product Images from "Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens"

    Article Title: Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0196913

    Exosomal RNA-Seq libraries from archival sera specimens. (A) Bioanalyzer results for 5 independent specimens demonstrate inserted size of exosomal RNAs. (B) Number of mapped reads generated from RNA-Seq libraries for 5 independent specimens with 20 ng exosomal RNAs. RNA-Seq library concentrations were calculated using three different methods: Nanodrop, BioAnalyzer (BioA), and RT-qPCR (qPCR). The Spearman’s rank correlations for Nanodrop versus reads, BioAnalyzer versus reads, and qPCR versus reads are 0.7 ( P = 0.23), 0.6 ( P = 0.35), and 0.7 ( P = 0.23), respectively. (C) Relative abundance of biotypes detected for 5 exosomal RNA-Seq libraries from healthy female subjects. PC = protein coding, PP = processed pseudogene, lincRNA = long intergenic non-coding RNA, UP = unprocessed pseudogene, snRNA = small nuclear RNA, miRNA = microRNA, TEC = to be experimentally confirmed, snoRNA = small nucleolar RNA, TUP = transcribed unprocessed pseudogene.
    Figure Legend Snippet: Exosomal RNA-Seq libraries from archival sera specimens. (A) Bioanalyzer results for 5 independent specimens demonstrate inserted size of exosomal RNAs. (B) Number of mapped reads generated from RNA-Seq libraries for 5 independent specimens with 20 ng exosomal RNAs. RNA-Seq library concentrations were calculated using three different methods: Nanodrop, BioAnalyzer (BioA), and RT-qPCR (qPCR). The Spearman’s rank correlations for Nanodrop versus reads, BioAnalyzer versus reads, and qPCR versus reads are 0.7 ( P = 0.23), 0.6 ( P = 0.35), and 0.7 ( P = 0.23), respectively. (C) Relative abundance of biotypes detected for 5 exosomal RNA-Seq libraries from healthy female subjects. PC = protein coding, PP = processed pseudogene, lincRNA = long intergenic non-coding RNA, UP = unprocessed pseudogene, snRNA = small nuclear RNA, miRNA = microRNA, TEC = to be experimentally confirmed, snoRNA = small nucleolar RNA, TUP = transcribed unprocessed pseudogene.

    Techniques Used: RNA Sequencing Assay, Generated, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    12) Product Images from "The Role of m6A/m-RNA Methylation in Stress Response Regulation"

    Article Title: The Role of m6A/m-RNA Methylation in Stress Response Regulation

    Journal: Neuron

    doi: 10.1016/j.neuron.2018.07.009

    Absolute Regulation of m 6 A/m Methylation Is Site Specific (A) A synthetic RNA oligonucleotide with three internal m 6 A/m sites was used for validation and internal normalization of the m 6 . (B) m 6 A/m-RIP-qPCR detects the methylated spike-in oligonucleotide in a linear fashion without impairing precipitation efficiency for endogenous transcripts in the concentration range used for experiments. Methylated spike-in oligo was added to unfragmented total RNA and precipitated with anti-m 6 A/m antibody (m 6 A/m-RIP) or rabbit IgG (IgG NC). n = 3 technical replicates, normalized expression to 1 fmol input control. Mean ± SEM. (C) m 6 A/m-RIP-qPCR accurately quantifies differential methylation of the spike-in oligo. Spike-in oligo (1 fmol) mixed from fully methylated and fully unmethylated spike-in was added to unfragmented total RNA and precipitated with m 6 A/m-RIP-qPCR. n = 3 technical replicates, normalized to input control. Mean ± SEM. (D) Absolute full-length m 6 . n = 8, mean ± SEM. Significant effects observed in FDR-corrected two-way MANOVA (p
    Figure Legend Snippet: Absolute Regulation of m 6 A/m Methylation Is Site Specific (A) A synthetic RNA oligonucleotide with three internal m 6 A/m sites was used for validation and internal normalization of the m 6 . (B) m 6 A/m-RIP-qPCR detects the methylated spike-in oligonucleotide in a linear fashion without impairing precipitation efficiency for endogenous transcripts in the concentration range used for experiments. Methylated spike-in oligo was added to unfragmented total RNA and precipitated with anti-m 6 A/m antibody (m 6 A/m-RIP) or rabbit IgG (IgG NC). n = 3 technical replicates, normalized expression to 1 fmol input control. Mean ± SEM. (C) m 6 A/m-RIP-qPCR accurately quantifies differential methylation of the spike-in oligo. Spike-in oligo (1 fmol) mixed from fully methylated and fully unmethylated spike-in was added to unfragmented total RNA and precipitated with m 6 A/m-RIP-qPCR. n = 3 technical replicates, normalized to input control. Mean ± SEM. (D) Absolute full-length m 6 . n = 8, mean ± SEM. Significant effects observed in FDR-corrected two-way MANOVA (p

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

    13) Product Images from "miR-122, small RNA annealing and sequence mutations alter the predicted structure of the Hepatitis C virus 5′ UTR RNA to stabilize and promote viral RNA accumulation"

    Article Title: miR-122, small RNA annealing and sequence mutations alter the predicted structure of the Hepatitis C virus 5′ UTR RNA to stabilize and promote viral RNA accumulation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky662

    Wild-type HCV genomic RNA replication is promoted by 5′ UTR targeting siRNAs in Ago2 knockout Huh-7.5 cells. ( A and B ) Show the sequence and western blot data confirming the knockout of the Ago2 gene in two independent cell lines generated using CRISPR/Cas9 technology. Ago2 knockout Huh-7.5 cells line A4 were co-electroporated with wild-type or G33C HCV RNA and the indicated small RNAs and miR-122 antagonist (anti-122) or control antagonist (anti-124). ( C ) Time course transient replication assay of wild-type HCV RNA in Ago2 knockout Huh-7.5 cell lines. Time course transient replication assay of the impact of the siRNA on wild-type HCV RNA in wild-type Huh-7.5 cells ( D ) and Ago2 knockout Huh-7.5 cells ( E ). ( F ) Shows potent siRNA stimulation of transient replication of wild-type HCV RNA in Ago2 knockout Huh-7.5 cells when miR-122 activity is abolished by using a miR-122 antagonist and ( G ) shows potent stimulation of transient replication of G33C by si18-36 G33C in Ago2 knockout cells when miR-122 activity is abolished.
    Figure Legend Snippet: Wild-type HCV genomic RNA replication is promoted by 5′ UTR targeting siRNAs in Ago2 knockout Huh-7.5 cells. ( A and B ) Show the sequence and western blot data confirming the knockout of the Ago2 gene in two independent cell lines generated using CRISPR/Cas9 technology. Ago2 knockout Huh-7.5 cells line A4 were co-electroporated with wild-type or G33C HCV RNA and the indicated small RNAs and miR-122 antagonist (anti-122) or control antagonist (anti-124). ( C ) Time course transient replication assay of wild-type HCV RNA in Ago2 knockout Huh-7.5 cell lines. Time course transient replication assay of the impact of the siRNA on wild-type HCV RNA in wild-type Huh-7.5 cells ( D ) and Ago2 knockout Huh-7.5 cells ( E ). ( F ) Shows potent siRNA stimulation of transient replication of wild-type HCV RNA in Ago2 knockout Huh-7.5 cells when miR-122 activity is abolished by using a miR-122 antagonist and ( G ) shows potent stimulation of transient replication of G33C by si18-36 G33C in Ago2 knockout cells when miR-122 activity is abolished.

    Techniques Used: Knock-Out, Sequencing, Western Blot, Generated, CRISPR, Activity Assay

    Sequence and conservation of the 5′ terminal region of the HCV genome. ( A ) A schematic diagram of the HCV genome highlighting the 5′ terminal sequence, the miR-122 annealing sites and pattern of miR-122 annealing. ( B ) An RNA logo representation of the conservation of this sequence across five virus genotypes.
    Figure Legend Snippet: Sequence and conservation of the 5′ terminal region of the HCV genome. ( A ) A schematic diagram of the HCV genome highlighting the 5′ terminal sequence, the miR-122 annealing sites and pattern of miR-122 annealing. ( B ) An RNA logo representation of the conservation of this sequence across five virus genotypes.

    Techniques Used: Sequencing

    In silico RNA structure analysis of the 5′ UTR region of the HCV genome. RNA Structure prediction algorithms were used to predict the lowest free energy structures formed by the wild-type 5′ UTR sequence in the absence ( A ) or presence ( B ) of miR-122, and the complementary strand at the 3′ terminus ( C ).
    Figure Legend Snippet: In silico RNA structure analysis of the 5′ UTR region of the HCV genome. RNA Structure prediction algorithms were used to predict the lowest free energy structures formed by the wild-type 5′ UTR sequence in the absence ( A ) or presence ( B ) of miR-122, and the complementary strand at the 3′ terminus ( C ).

    Techniques Used: In Silico, RNA Structure Prediction, Sequencing

    Model for the mechanism of miR-122 promotion of HCV replication. ( A ) In the absence of miR-122 annealing the 5′ UTR forms a non-canonical RNA structure that does not support the HCV life cycle. ( B ) Annealing of miR-122 or a perfect match small RNA on or near the miR-122 binding sites, in conjunction with any Ago isoform modifies the 5′ UTR structure to form the canonical structure, protecting it from degradation by host phosphatases and exonucleases, and promoting the virus life cycle. Viral genomes having point mutations that favour the formation of the canonical RNA structure can replicate independently from miR-122 annealing, likely due to dynamic formation of both the canonical and non-canonical structures.
    Figure Legend Snippet: Model for the mechanism of miR-122 promotion of HCV replication. ( A ) In the absence of miR-122 annealing the 5′ UTR forms a non-canonical RNA structure that does not support the HCV life cycle. ( B ) Annealing of miR-122 or a perfect match small RNA on or near the miR-122 binding sites, in conjunction with any Ago isoform modifies the 5′ UTR structure to form the canonical structure, protecting it from degradation by host phosphatases and exonucleases, and promoting the virus life cycle. Viral genomes having point mutations that favour the formation of the canonical RNA structure can replicate independently from miR-122 annealing, likely due to dynamic formation of both the canonical and non-canonical structures.

    Techniques Used: Binding Assay

    Sequences of 5′UTR and transient replication assays of full length viral RNA having mutations to evolutionarily variable sites in siRNA target regions. ( A ) Sequence mutations are shown aligned with the canonical 5′ UTR in the presence of miR-122, and the complementary mutations in the structure of the negative strand 3′ terminus. ( B ) Transient HCV replication assay analysis of the mutant virus RNA in wild-type Huh-7.5 cells. Luciferase expression was measured as a proxy for genome amplification in cells transfected with full length HCV genomic RNA carrying a luciferase reporter gene and the indicated mutation. Data are the average of three independent transfections and error bars represent the standard deviation. The differences in replication fitness of the mutant viruses versus wild-type (miControl) at each time point was analyzed by one-way ANOVA and P -values are indicated as follows * 0.05 to 0.005, ** 0.005 to 0.0001 and ***
    Figure Legend Snippet: Sequences of 5′UTR and transient replication assays of full length viral RNA having mutations to evolutionarily variable sites in siRNA target regions. ( A ) Sequence mutations are shown aligned with the canonical 5′ UTR in the presence of miR-122, and the complementary mutations in the structure of the negative strand 3′ terminus. ( B ) Transient HCV replication assay analysis of the mutant virus RNA in wild-type Huh-7.5 cells. Luciferase expression was measured as a proxy for genome amplification in cells transfected with full length HCV genomic RNA carrying a luciferase reporter gene and the indicated mutation. Data are the average of three independent transfections and error bars represent the standard deviation. The differences in replication fitness of the mutant viruses versus wild-type (miControl) at each time point was analyzed by one-way ANOVA and P -values are indicated as follows * 0.05 to 0.005, ** 0.005 to 0.0001 and ***

    Techniques Used: Sequencing, Mutagenesis, Luciferase, Expressing, Amplification, Transfection, Standard Deviation

    The RNA construct, knockdown efficiency and mutants isolated during the siRNA-directed mutagenesis method. Cells harboring the HCV bicistronic full length replicon RNA shown in ( A ) were repeatedly electroporated with si18-36, si19-37 or si6367, and selected with G418 to pressure the selection of siRNA escape mutants. ( B – D ) Luciferase expression was assessed 3 days after siRNA electroporation for each iteration of the selection process to detect the evolution of knockdown resistance (left panel). Following seven rounds of knockdown and selection, the 5′ UTR sequence was amplified, cloned and sequenced. The sequence and incidence of mutations within the 5′ targeted site is shown for each siRNA used (right panel).
    Figure Legend Snippet: The RNA construct, knockdown efficiency and mutants isolated during the siRNA-directed mutagenesis method. Cells harboring the HCV bicistronic full length replicon RNA shown in ( A ) were repeatedly electroporated with si18-36, si19-37 or si6367, and selected with G418 to pressure the selection of siRNA escape mutants. ( B – D ) Luciferase expression was assessed 3 days after siRNA electroporation for each iteration of the selection process to detect the evolution of knockdown resistance (left panel). Following seven rounds of knockdown and selection, the 5′ UTR sequence was amplified, cloned and sequenced. The sequence and incidence of mutations within the 5′ targeted site is shown for each siRNA used (right panel).

    Techniques Used: Construct, Isolation, Mutagenesis, Selection, Luciferase, Expressing, Electroporation, Sequencing, Amplification, Clone Assay

    Sequence and replication analysis of viruses whose replication was promoted by the siRNA used in the mutation selection method. Viruses selected by si18-36 or si19-37 were tested for promotion by their respective selection siRNA. ( A ) Viruses whose replication was promoted by si18-36 and si19-37 when miR-122 was absent. The indicated viral RNAs were co-transfected with either si18-36, si19-37 or siControl into miR-122-knockout Huh-7.5 cells and replication was assessed based on luciferase expression. ( B ) The 5′ UTR sequences of viruses whose replication was promoted by si18-36 or si19-37, and the specific annealing between si18-36 and C26U are shown. ( C – E ) Time course analysis in wild-type and miR-122-knockout cells of C26U with various small RNAs. C26U RNA was co-electroporated into (C and E) wild-type or (D) miR-122-knockout Huh-7.5 cells with the indicated miRNA, siRNA or miR-122 antagonist (anti122). ( F ) Time course analysis of WT HCV RNA in miR-122-knockout cells with the indicated small RNAs.
    Figure Legend Snippet: Sequence and replication analysis of viruses whose replication was promoted by the siRNA used in the mutation selection method. Viruses selected by si18-36 or si19-37 were tested for promotion by their respective selection siRNA. ( A ) Viruses whose replication was promoted by si18-36 and si19-37 when miR-122 was absent. The indicated viral RNAs were co-transfected with either si18-36, si19-37 or siControl into miR-122-knockout Huh-7.5 cells and replication was assessed based on luciferase expression. ( B ) The 5′ UTR sequences of viruses whose replication was promoted by si18-36 or si19-37, and the specific annealing between si18-36 and C26U are shown. ( C – E ) Time course analysis in wild-type and miR-122-knockout cells of C26U with various small RNAs. C26U RNA was co-electroporated into (C and E) wild-type or (D) miR-122-knockout Huh-7.5 cells with the indicated miRNA, siRNA or miR-122 antagonist (anti122). ( F ) Time course analysis of WT HCV RNA in miR-122-knockout cells with the indicated small RNAs.

    Techniques Used: Sequencing, Mutagenesis, Selection, Transfection, Knock-Out, Luciferase, Expressing

    Sequence and transient replication assays of full length viral RNA having mutations to evolutionarily conserved sites in siRNA target regions. ( A ) Sequence mutations are shown aligned with the canonical 5′ UTR in the presence of miR-122, and the complementary mutations in the structure of the 3′ terminus. ( B ) Transient HCV replication assay analysis of mutant full length viral RNA in wild-type Huh-7.5 cells. Luciferase expression measured 2 days post-transfection was used as a proxy to assess viral genomic RNA amplification in cells transfected with full length HCV genomic RNAs harboring a luciferase reporter gene and the indicated mutation. Data are the average of three independent transfections and error bars represent the standard deviation. The relative replication fitness of the mutant viruses versus wild-type (siControl) was analyzed by one-way ANOVA and P -values are indicated as follows * 0.05 to 0.005, ** 0.005 to 0.0001 and ***
    Figure Legend Snippet: Sequence and transient replication assays of full length viral RNA having mutations to evolutionarily conserved sites in siRNA target regions. ( A ) Sequence mutations are shown aligned with the canonical 5′ UTR in the presence of miR-122, and the complementary mutations in the structure of the 3′ terminus. ( B ) Transient HCV replication assay analysis of mutant full length viral RNA in wild-type Huh-7.5 cells. Luciferase expression measured 2 days post-transfection was used as a proxy to assess viral genomic RNA amplification in cells transfected with full length HCV genomic RNAs harboring a luciferase reporter gene and the indicated mutation. Data are the average of three independent transfections and error bars represent the standard deviation. The relative replication fitness of the mutant viruses versus wild-type (siControl) was analyzed by one-way ANOVA and P -values are indicated as follows * 0.05 to 0.005, ** 0.005 to 0.0001 and ***

    Techniques Used: Sequencing, Mutagenesis, Luciferase, Expressing, Transfection, Amplification, Standard Deviation

    si19-37 annealing stabilizes HCV genomic RNA in Ago2 knockout cells. Ago2 cells were elecroporated with non-replicative HCV RNA and the indicated siRNAs and miRNA antagonists, anti-122 or the control anti-124. RNA was prepared from the cells at the indicated short term time points post-electroporation and probed for HCV and Actin RNA to evaluate HCV RNA stability. A representative northern blot analysis of an HCV RNA stability assay is shown in ( A ) and the quantification of three independent assays is shown in ( B ).
    Figure Legend Snippet: si19-37 annealing stabilizes HCV genomic RNA in Ago2 knockout cells. Ago2 cells were elecroporated with non-replicative HCV RNA and the indicated siRNAs and miRNA antagonists, anti-122 or the control anti-124. RNA was prepared from the cells at the indicated short term time points post-electroporation and probed for HCV and Actin RNA to evaluate HCV RNA stability. A representative northern blot analysis of an HCV RNA stability assay is shown in ( A ) and the quantification of three independent assays is shown in ( B ).

    Techniques Used: Knock-Out, Electroporation, Northern Blot, Stability Assay

    14) Product Images from "Non-homologous recombination between Alu and LINE-1 repeats results in a 91 kb deletion in MERTK causing severe retinitis pigmentosa"

    Article Title: Non-homologous recombination between Alu and LINE-1 repeats results in a 91 kb deletion in MERTK causing severe retinitis pigmentosa

    Journal: Molecular Vision

    doi:

    MERTK c.-8162_1145–1212del, p.? and c.2309A > G, p.Glu770Gly are allelic variants. A : Localization of MERTK -specific primers. B : Reverse transcription PCR (RT–PCR) was performed on cDNA derived from RP116 and control RNA. Using primers 7F and 18R, we could amplify only an allele without a deletion covering exon 7 (1292 bp). PCR with 15F and 18R was used as control (372 bp). Primers sequences and PCR conditions are described in Materials and Methods and Results. C : Sanger sequencing reveals the presence of the heterozygous c.2309A > G mutation in DNA of RP116 (upper panel), the presence of the mutation c.2309G in RNA of RP116 (middle panel), and the presence of a reference A in the control RNA (lower panel).
    Figure Legend Snippet: MERTK c.-8162_1145–1212del, p.? and c.2309A > G, p.Glu770Gly are allelic variants. A : Localization of MERTK -specific primers. B : Reverse transcription PCR (RT–PCR) was performed on cDNA derived from RP116 and control RNA. Using primers 7F and 18R, we could amplify only an allele without a deletion covering exon 7 (1292 bp). PCR with 15F and 18R was used as control (372 bp). Primers sequences and PCR conditions are described in Materials and Methods and Results. C : Sanger sequencing reveals the presence of the heterozygous c.2309A > G mutation in DNA of RP116 (upper panel), the presence of the mutation c.2309G in RNA of RP116 (middle panel), and the presence of a reference A in the control RNA (lower panel).

    Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Sequencing, Mutagenesis

    15) Product Images from "Human papillomavirus E7 induces p63 expression to modulate DNA damage response"

    Article Title: Human papillomavirus E7 induces p63 expression to modulate DNA damage response

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0149-6

    E7 promotes p63 expression at transcriptional level a CaSki cells were transfected with scrambled siRNA or E7-siRNA. Forty-eight hours later, cells were treated with the proteasome inhibitor MG132 (20 μM). The untreated control cells and MG132-treated cells were harvested 4 h later and expression of E7 and p63 were assessed by western blotting. The intensity of the bands was quantified by using the ImageJ software. b Q-PCR was used to quantitate E7 and p63 transcript levels in E7-depleted cells 48 h following transfection. c and d Total RNA from C33A, SiHa, and CaSki cells was extracted and reversed by using random primers. The cDNA was used to analyse the transcript levels of E7 ( c ) and p63 ( d ) by Q-PCR
    Figure Legend Snippet: E7 promotes p63 expression at transcriptional level a CaSki cells were transfected with scrambled siRNA or E7-siRNA. Forty-eight hours later, cells were treated with the proteasome inhibitor MG132 (20 μM). The untreated control cells and MG132-treated cells were harvested 4 h later and expression of E7 and p63 were assessed by western blotting. The intensity of the bands was quantified by using the ImageJ software. b Q-PCR was used to quantitate E7 and p63 transcript levels in E7-depleted cells 48 h following transfection. c and d Total RNA from C33A, SiHa, and CaSki cells was extracted and reversed by using random primers. The cDNA was used to analyse the transcript levels of E7 ( c ) and p63 ( d ) by Q-PCR

    Techniques Used: Expressing, Transfection, Western Blot, Software, Polymerase Chain Reaction

    16) Product Images from "Purification of Highly Active Alphavirus Replication Complexes Demonstrates Altered Fractionation of Multiple Cellular Membranes"

    Article Title: Purification of Highly Active Alphavirus Replication Complexes Demonstrates Altered Fractionation of Multiple Cellular Membranes

    Journal: Journal of Virology

    doi: 10.1128/JVI.01852-17

    Characterization of the in vitro replication activity of the purified RCs. (A) Stability of newly synthesized SFV genomic RNA. After a 60-min replication assay, [ 32 P]CTP incorporation was blocked by the addition of 100 μM 3′-dCTP, and incubation at 30°C was continued for an additional 180 min. The graph on the left represents an assay with 10-fold-diluted S7 and on the right with the purified RCs. At the indicated chase times radioactivity in 42S was quantified (yellow). As a control, a reaction without 3′-dCTP was quantified (blue). In addition, in vitro transcript of Tmed added in the reaction at a 0-min chase was quantified by in-gel hybridization (green). All values are presented as percentages of the values at the 0-min chase. (B) Purified RCs and additional exogenous template. Replication assay reaction mixtures were incubated for 2 h. RCs indicates [ 32 P]CTP incorporation by the purified RCs. RCs + Tmed RNA shows incorporation by the purified RCs after an exogenous Tmed RNA transcript was added. As a control, [ 32 P]CTP incorporation by the P15 membrane fraction from cells transfected with the P1234 polyprotein and Tmed template plasmids is shown, and genomic and subgenomic (SG) Tmed are indicated. The lower panel shows the presence of Tmed, detected by in-gel hybridization. (C) Detergent stability and sensitivity. Purified RCs were treated with 1% Tx-100, followed by an assay to detect replication (upper) and in-gel hybridization to detect the minus-strand template RNA (lower). (D) ssRNA and RF/RI forms of in vitro -synthesized RNAs. After a 2-h replication assay with the purified RCs, RNA was isolated and treated with RNase A/T1 or III under high-salt conditions to specifically digest ssRNA or dsRNA, respectively, and analyzed under denaturing (left) or nondenaturing (right) conditions. (E) Release of newly made RNA. A replication assay was performed with the concentrated purified RCs, and after 60-, 120-, and 180-min replication reactions, aliquots were removed to prepare pellet and supernatant fractions, followed by RNA isolation and analysis in a denaturing agarose gel. The schematic shows how [ 32 P]CTP is incorporated into viral RNA during in vitro RNA synthesis, resulting in dsRNA containing a plus strand synthesized both in cells (indicated by magenta) and in vitro (indicated by orange). After a round of replication and release of the previous plus strand, an RC contains only the in vitro -synthesized plus strand if replication is semiconservative. (F) Increase in the amount of RNA during in vitro replication. After a 4-h replication assay with the purified RCs and unlabeled NTPs, RNA was isolated and genomic RNA was detected by in-gel hybridization. No NTPs indicates a reaction without added NTPs, and ctrl indicates a sample without any incubations before RNA isolation. 42S RNA was quantified, and average percentages from two independent experiments are shown in the table.
    Figure Legend Snippet: Characterization of the in vitro replication activity of the purified RCs. (A) Stability of newly synthesized SFV genomic RNA. After a 60-min replication assay, [ 32 P]CTP incorporation was blocked by the addition of 100 μM 3′-dCTP, and incubation at 30°C was continued for an additional 180 min. The graph on the left represents an assay with 10-fold-diluted S7 and on the right with the purified RCs. At the indicated chase times radioactivity in 42S was quantified (yellow). As a control, a reaction without 3′-dCTP was quantified (blue). In addition, in vitro transcript of Tmed added in the reaction at a 0-min chase was quantified by in-gel hybridization (green). All values are presented as percentages of the values at the 0-min chase. (B) Purified RCs and additional exogenous template. Replication assay reaction mixtures were incubated for 2 h. RCs indicates [ 32 P]CTP incorporation by the purified RCs. RCs + Tmed RNA shows incorporation by the purified RCs after an exogenous Tmed RNA transcript was added. As a control, [ 32 P]CTP incorporation by the P15 membrane fraction from cells transfected with the P1234 polyprotein and Tmed template plasmids is shown, and genomic and subgenomic (SG) Tmed are indicated. The lower panel shows the presence of Tmed, detected by in-gel hybridization. (C) Detergent stability and sensitivity. Purified RCs were treated with 1% Tx-100, followed by an assay to detect replication (upper) and in-gel hybridization to detect the minus-strand template RNA (lower). (D) ssRNA and RF/RI forms of in vitro -synthesized RNAs. After a 2-h replication assay with the purified RCs, RNA was isolated and treated with RNase A/T1 or III under high-salt conditions to specifically digest ssRNA or dsRNA, respectively, and analyzed under denaturing (left) or nondenaturing (right) conditions. (E) Release of newly made RNA. A replication assay was performed with the concentrated purified RCs, and after 60-, 120-, and 180-min replication reactions, aliquots were removed to prepare pellet and supernatant fractions, followed by RNA isolation and analysis in a denaturing agarose gel. The schematic shows how [ 32 P]CTP is incorporated into viral RNA during in vitro RNA synthesis, resulting in dsRNA containing a plus strand synthesized both in cells (indicated by magenta) and in vitro (indicated by orange). After a round of replication and release of the previous plus strand, an RC contains only the in vitro -synthesized plus strand if replication is semiconservative. (F) Increase in the amount of RNA during in vitro replication. After a 4-h replication assay with the purified RCs and unlabeled NTPs, RNA was isolated and genomic RNA was detected by in-gel hybridization. No NTPs indicates a reaction without added NTPs, and ctrl indicates a sample without any incubations before RNA isolation. 42S RNA was quantified, and average percentages from two independent experiments are shown in the table.

    Techniques Used: In Vitro, Activity Assay, Purification, Synthesized, Incubation, Radioactivity, Hybridization, Transfection, Isolation, Agarose Gel Electrophoresis

    17) Product Images from "Type 1 IFN-independent activation of a subset of interferon stimulated genes in West Nile virus Eg101-infected mouse cells"

    Article Title: Type 1 IFN-independent activation of a subset of interferon stimulated genes in West Nile virus Eg101-infected mouse cells

    Journal: Virology

    doi: 10.1016/j.virol.2012.01.006

    IFN beta is produced by WNV Eg101 infected MEFs and induces phosphorylation of STAT1 and STAT2 MEFs were mock-infected or infected with WNV Eg101 at a MOI of 5 for the indicated times or treated with 1000 U/ml of murine IFN beta for 3h. (A) Total RNA from tC3H/He MEFs was extracted and IFN beta mRNA levels were measured by real-time qRT-PCR. (B) Extracellular IFN beta protein levels in tC3H/He MEF culture fluid was measured by ELISA. The values shown are averages from at least two independent experiments performed in triplicate. The error bars represent SDM. (C) tC3H/He cell lysates were analyzed by Western blotting using antibodies specific for the indicated proteins. Actin was used as the loading control. The blots shown are representative of results obtained from three independent experiments. (D) and (E) Comparison of ISG activation by IFN beta in primary and transformed MEFs. Total RNA was extracted from (D) tC3H/He or (E) pC3H/He MEFs infected with WNV Eg101 (MOI 5) for the indicated times, mock-infected or treated with murine IFN beta (1000 U/ml) for 3h. The changes in the levels of Oas1a and Oas1b mRNA were assessed by real-time qRT-PCR. Each experiment was repeated at least two times in triplicate. The mRNA level of each gene was normalized to the level of GAPDH mRNA in that sample and is shown as the fold change over the amount of mRNA in mock samples expressed in relative quantification units (RQU). The error bars represent the calculated SEM (n = 3) and are based on an RQ Min/Max of the 95% confidence level.
    Figure Legend Snippet: IFN beta is produced by WNV Eg101 infected MEFs and induces phosphorylation of STAT1 and STAT2 MEFs were mock-infected or infected with WNV Eg101 at a MOI of 5 for the indicated times or treated with 1000 U/ml of murine IFN beta for 3h. (A) Total RNA from tC3H/He MEFs was extracted and IFN beta mRNA levels were measured by real-time qRT-PCR. (B) Extracellular IFN beta protein levels in tC3H/He MEF culture fluid was measured by ELISA. The values shown are averages from at least two independent experiments performed in triplicate. The error bars represent SDM. (C) tC3H/He cell lysates were analyzed by Western blotting using antibodies specific for the indicated proteins. Actin was used as the loading control. The blots shown are representative of results obtained from three independent experiments. (D) and (E) Comparison of ISG activation by IFN beta in primary and transformed MEFs. Total RNA was extracted from (D) tC3H/He or (E) pC3H/He MEFs infected with WNV Eg101 (MOI 5) for the indicated times, mock-infected or treated with murine IFN beta (1000 U/ml) for 3h. The changes in the levels of Oas1a and Oas1b mRNA were assessed by real-time qRT-PCR. Each experiment was repeated at least two times in triplicate. The mRNA level of each gene was normalized to the level of GAPDH mRNA in that sample and is shown as the fold change over the amount of mRNA in mock samples expressed in relative quantification units (RQU). The error bars represent the calculated SEM (n = 3) and are based on an RQ Min/Max of the 95% confidence level.

    Techniques Used: Produced, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, Transformation Assay

    18) Product Images from "Identification and validation of microRNAs directly regulating the UDP-glucuronosyltransferase 1A subfamily enzymes by a functional genomics approach"

    Article Title: Identification and validation of microRNAs directly regulating the UDP-glucuronosyltransferase 1A subfamily enzymes by a functional genomics approach

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2017.04.017

    Effect of miRNA mimic overexpression on UGT1A1 (A), UGT1A6 (B), and UGT2B7 (C) selective activities and mRNA levels in LS180 human intestinal cells. miRNA mimics or the mimic negative controls were transfected into LS180 cells at 30 nM concentration. 48 hr after transfection, glucuronidation activities (UGT1A1: ezetimibe glucuronidation, Ez-Glu; UGT1A6: 5-hydroxytryptamine glucuronidation, 5HT-Glu; UGT2B7: morphine 3-glucuronidation, M3-Glu) were measured and total RNA isolated for the measurement of expression levels of the respective genes. Glucuronidation activity values and mRNA levels were compared with the average values of cells transfected with the mimic negative controls and non-transfected cells (control). Bars represent mean ± one S.D. from three independent experiments performed in quadruplicate. * p
    Figure Legend Snippet: Effect of miRNA mimic overexpression on UGT1A1 (A), UGT1A6 (B), and UGT2B7 (C) selective activities and mRNA levels in LS180 human intestinal cells. miRNA mimics or the mimic negative controls were transfected into LS180 cells at 30 nM concentration. 48 hr after transfection, glucuronidation activities (UGT1A1: ezetimibe glucuronidation, Ez-Glu; UGT1A6: 5-hydroxytryptamine glucuronidation, 5HT-Glu; UGT2B7: morphine 3-glucuronidation, M3-Glu) were measured and total RNA isolated for the measurement of expression levels of the respective genes. Glucuronidation activity values and mRNA levels were compared with the average values of cells transfected with the mimic negative controls and non-transfected cells (control). Bars represent mean ± one S.D. from three independent experiments performed in quadruplicate. * p

    Techniques Used: Over Expression, Transfection, Concentration Assay, Isolation, Expressing, Activity Assay

    19) Product Images from "Identification of PARP-7 substrates reveals a role for MARylation in microtubule control in ovarian cancer cells"

    Article Title: Identification of PARP-7 substrates reveals a role for MARylation in microtubule control in ovarian cancer cells

    Journal: eLife

    doi: 10.7554/eLife.60481

    RNA-seq analysis of gene expression in ovarian cancer cells following PARP-7 depletion. ( A ) Western blots showing PARP-7 protein levels after siRNA-mediated knockdown (KD) of PARP7 in OVCAR4 cells. Three different siRNAs were used. β-tubulin was used as loading control. ( B ) Heat maps showing the results of RNA-seq assays from OVCAR4 cells upon siRNA-mediated knockdown (KD) of PARP7. Three different siRNAs were used. Results represent fold changes in FPKM values for genes significantly regulated versus the control KD, expressed as log 2 fold change. A fold change ≥1.5 was classified as upregulated, while a fold change ≤−0.5 was classified as downregulated. ( C ) Gene ontology terms enriched for the significantly upregulated genes ( yellow ) and significantly downregulated genes ( blue ) shown in ( B ). The percent of targets from each GO term identified in the analysis is shown, along with the log 10 p-value for the enrichment.
    Figure Legend Snippet: RNA-seq analysis of gene expression in ovarian cancer cells following PARP-7 depletion. ( A ) Western blots showing PARP-7 protein levels after siRNA-mediated knockdown (KD) of PARP7 in OVCAR4 cells. Three different siRNAs were used. β-tubulin was used as loading control. ( B ) Heat maps showing the results of RNA-seq assays from OVCAR4 cells upon siRNA-mediated knockdown (KD) of PARP7. Three different siRNAs were used. Results represent fold changes in FPKM values for genes significantly regulated versus the control KD, expressed as log 2 fold change. A fold change ≥1.5 was classified as upregulated, while a fold change ≤−0.5 was classified as downregulated. ( C ) Gene ontology terms enriched for the significantly upregulated genes ( yellow ) and significantly downregulated genes ( blue ) shown in ( B ). The percent of targets from each GO term identified in the analysis is shown, along with the log 10 p-value for the enrichment.

    Techniques Used: RNA Sequencing Assay, Expressing, Western Blot

    20) Product Images from "SIV replication is directly downregulated by four antiviral miRNAs"

    Article Title: SIV replication is directly downregulated by four antiviral miRNAs

    Journal: Retrovirology

    doi: 10.1186/1742-4690-10-95

    SIV infection increases levels of mature miR- 29a (A), -29b (B), -9 (C) and -146a (D). Macaque macrophages were infected with SIV (MOI 0.05). Cells were harvested at 2, 4, 8, 12, 24 and 48 hours after infection and RNA was isolated. Taqman miRNA RT-qPCR assays were used to measure levels of mature miRNAs. Results were normalized to U6, then to uninfected samples for the individual time points (ΔΔCq method). Values are expressed as fold induction of miRNAs over uninfected controls using the ΔΔCq method and data shown is an average of at least 3 experiments.
    Figure Legend Snippet: SIV infection increases levels of mature miR- 29a (A), -29b (B), -9 (C) and -146a (D). Macaque macrophages were infected with SIV (MOI 0.05). Cells were harvested at 2, 4, 8, 12, 24 and 48 hours after infection and RNA was isolated. Taqman miRNA RT-qPCR assays were used to measure levels of mature miRNAs. Results were normalized to U6, then to uninfected samples for the individual time points (ΔΔCq method). Values are expressed as fold induction of miRNAs over uninfected controls using the ΔΔCq method and data shown is an average of at least 3 experiments.

    Techniques Used: Infection, Isolation, miRNA RT, Real-time Polymerase Chain Reaction

    TNFα increases levels of mature miRNAs and primary miRNA transcripts in human macrophages. Human macrophages were treated with 20 ng/ml TNFα. Cells were harvested at 2, 4, 8, 12 and 24 hours after treatment and RNA isolated. (A-D) Taqman miRNA RT-qPCR assays were used to measure levels of mature miRNAs. (E-H) Taqman pri-miRNA RT-qPCR assays were used to measure levels of primary miRNA transcripts. Results were normalized to U6. Values are expressed as fold induction of miRNAs over uninfected controls using the ΔΔCq method and data shown is an average of at least 3 experiments.
    Figure Legend Snippet: TNFα increases levels of mature miRNAs and primary miRNA transcripts in human macrophages. Human macrophages were treated with 20 ng/ml TNFα. Cells were harvested at 2, 4, 8, 12 and 24 hours after treatment and RNA isolated. (A-D) Taqman miRNA RT-qPCR assays were used to measure levels of mature miRNAs. (E-H) Taqman pri-miRNA RT-qPCR assays were used to measure levels of primary miRNA transcripts. Results were normalized to U6. Values are expressed as fold induction of miRNAs over uninfected controls using the ΔΔCq method and data shown is an average of at least 3 experiments.

    Techniques Used: Isolation, miRNA RT, Real-time Polymerase Chain Reaction

    Four miRNAs reduce levels of full length and multiply spliced viral RNAs. Primary macaque macrophages were transfected with 100 nM of either miRNA mimics or antagonists, then infected with SIV (MOI 0.05) 24 hours after transfection. Cell lysates were collected at 24, 48 and 72 hours post-infection for miRNA experiments and 48 hours for miRNA antagonist experiments. Levels of full length (A, C) and spliced (B) RNA was measured by RT-qPCR. Values are expressed as fold change over SIV-only controls. Results are reported as percent of SIV-only control using the ΔΔCq method.
    Figure Legend Snippet: Four miRNAs reduce levels of full length and multiply spliced viral RNAs. Primary macaque macrophages were transfected with 100 nM of either miRNA mimics or antagonists, then infected with SIV (MOI 0.05) 24 hours after transfection. Cell lysates were collected at 24, 48 and 72 hours post-infection for miRNA experiments and 48 hours for miRNA antagonist experiments. Levels of full length (A, C) and spliced (B) RNA was measured by RT-qPCR. Values are expressed as fold change over SIV-only controls. Results are reported as percent of SIV-only control using the ΔΔCq method.

    Techniques Used: Transfection, Infection, Quantitative RT-PCR

    miRs-29a, -29b, -9 and -146a reduce expression of a luciferase reporter and target predicted binding sites in the 3′ UTR of SIV. (A) SIV nef / U3 - R was cloned into psiCHECK2 plasmid (Promega). 293T cells were co-transfection with 50 nM or 100 nM of each miRNA or scrambled mimic and 100 ng of either psiCHECK+SIV or psiCHECK alone. Control samples were transfected with either psiCHECK or psiCHECK+SIV, and no miRNA mimics. Cells were harvested 24 hours after transfection. Data shown is an average of four experiments and represents miRNA effect on luciferase expression for psiCHECK+SIV over psiCHECK only. (B) WT and mutant biotin-labeled oligos corresponding to SIV RNA sequences containing predicted miRNA binding sites. WT oligo is show on top and mutant on bottom. Boxed bases on WT oligo denote miRNA seed binding site in SIV sequence. Mutant oligo highlighted bases denote the change in seed-binding region. Annotation is based on SIV sequence AY033146. (C) 100 pmol of individual WT or mt oligos were transfected into HeLa cells and lysates collected 24 hours after transfection. Oligos were pulled down using Streptavidin beads and assayed for binding to endogenous cellular miRNAs using Taqman RT-qPCR. Percent input was calculated for each sample (see methods) and data presented as (% input mt )/(% input WT) with % of input of WT set to 100%. In each experiment, the value of the oligo which pulled down the highest percentage of individual endogenous miRNA was set to 100% and binding by other oligos was compared to this value. Data shown is an average of 4 experiments. Statistics represent the comparison of percent of miRNA bound to WT oligo compared to percent of miRNA pulled down by corresponding mt oligo and are reported as a two-tailed t test assuming unequal variance.
    Figure Legend Snippet: miRs-29a, -29b, -9 and -146a reduce expression of a luciferase reporter and target predicted binding sites in the 3′ UTR of SIV. (A) SIV nef / U3 - R was cloned into psiCHECK2 plasmid (Promega). 293T cells were co-transfection with 50 nM or 100 nM of each miRNA or scrambled mimic and 100 ng of either psiCHECK+SIV or psiCHECK alone. Control samples were transfected with either psiCHECK or psiCHECK+SIV, and no miRNA mimics. Cells were harvested 24 hours after transfection. Data shown is an average of four experiments and represents miRNA effect on luciferase expression for psiCHECK+SIV over psiCHECK only. (B) WT and mutant biotin-labeled oligos corresponding to SIV RNA sequences containing predicted miRNA binding sites. WT oligo is show on top and mutant on bottom. Boxed bases on WT oligo denote miRNA seed binding site in SIV sequence. Mutant oligo highlighted bases denote the change in seed-binding region. Annotation is based on SIV sequence AY033146. (C) 100 pmol of individual WT or mt oligos were transfected into HeLa cells and lysates collected 24 hours after transfection. Oligos were pulled down using Streptavidin beads and assayed for binding to endogenous cellular miRNAs using Taqman RT-qPCR. Percent input was calculated for each sample (see methods) and data presented as (% input mt )/(% input WT) with % of input of WT set to 100%. In each experiment, the value of the oligo which pulled down the highest percentage of individual endogenous miRNA was set to 100% and binding by other oligos was compared to this value. Data shown is an average of 4 experiments. Statistics represent the comparison of percent of miRNA bound to WT oligo compared to percent of miRNA pulled down by corresponding mt oligo and are reported as a two-tailed t test assuming unequal variance.

    Techniques Used: Expressing, Luciferase, Binding Assay, Clone Assay, Plasmid Preparation, Cotransfection, Transfection, Mutagenesis, Labeling, Sequencing, Quantitative RT-PCR, Two Tailed Test

    IFNβ increases levels of mature miRNAs and primary miRNA transcripts in human macrophages. Human macrophages were treated with 100 U/ml IFNβ and cells were harvested at 4, 8, 12 and 24 hours after treatment and RNA isolated. (A-D) Taqman miRNA RT-qPCR assays were used to measure levels of mature miRNAs. (E-H) Taqman pri-miRNA RT-qPCR assays were used to measure levels of primary miRNA transcripts. Results were normalized to U6. Values are expressed as fold induction of miRNAs over uninfected controls using the ΔΔCq method and data shown is an average of at least 3 experiments.
    Figure Legend Snippet: IFNβ increases levels of mature miRNAs and primary miRNA transcripts in human macrophages. Human macrophages were treated with 100 U/ml IFNβ and cells were harvested at 4, 8, 12 and 24 hours after treatment and RNA isolated. (A-D) Taqman miRNA RT-qPCR assays were used to measure levels of mature miRNAs. (E-H) Taqman pri-miRNA RT-qPCR assays were used to measure levels of primary miRNA transcripts. Results were normalized to U6. Values are expressed as fold induction of miRNAs over uninfected controls using the ΔΔCq method and data shown is an average of at least 3 experiments.

    Techniques Used: Isolation, miRNA RT, Real-time Polymerase Chain Reaction

    21) Product Images from "Reciprocal regulation of activating and inhibitory Fc? receptors by TLR7/8 activation: Implications for tumor immunotherapy"

    Article Title: Reciprocal regulation of activating and inhibitory Fc? receptors by TLR7/8 activation: Implications for tumor immunotherapy

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-09-2591

    Requirement of secreted factors for R-848-mediated changes in FcγR expression. (A) PBM were pretreated with or without Brefeldin A for 30 minutes and treated for 12 hours with or without 1 µM R848. RNA was extracted and FcγR expression
    Figure Legend Snippet: Requirement of secreted factors for R-848-mediated changes in FcγR expression. (A) PBM were pretreated with or without Brefeldin A for 30 minutes and treated for 12 hours with or without 1 µM R848. RNA was extracted and FcγR expression

    Techniques Used: Expressing

    22) Product Images from "Identification of the E9^E2C cDNA and Functional Characterization of the Gene Product Reveal a New Repressor of Transcription and Replication in Cottontail Rabbit Papillomavirus"

    Article Title: Identification of the E9^E2C cDNA and Functional Characterization of the Gene Product Reveal a New Repressor of Transcription and Replication in Cottontail Rabbit Papillomavirus

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.16.8736-8744.2003

    Detection of CRPV-E9^E2C transcript by reverse transcription-PCR. A total of 500 ng of RNA isolated from papillomas induced by wild-type CRPV-pLAII (WT), CRPV-E9atgmut-pLAII (E9atgmut), or CRPV-E9stop-pLAII (E9stop) was used in a reverse transcription-PCR with primers P1708 (upstream of SD 1751 ) and P3883 (downstream of SA 3714 ) and Ready-To-Go reverse transcription-PCR beads. The correct size of the cDNA fragment derived from the spliced E9^E2C transcript is shown on a 2% agarose gel. The sizes of molecular size markers are given on the right (in base pairs).
    Figure Legend Snippet: Detection of CRPV-E9^E2C transcript by reverse transcription-PCR. A total of 500 ng of RNA isolated from papillomas induced by wild-type CRPV-pLAII (WT), CRPV-E9atgmut-pLAII (E9atgmut), or CRPV-E9stop-pLAII (E9stop) was used in a reverse transcription-PCR with primers P1708 (upstream of SD 1751 ) and P3883 (downstream of SA 3714 ) and Ready-To-Go reverse transcription-PCR beads. The correct size of the cDNA fragment derived from the spliced E9^E2C transcript is shown on a 2% agarose gel. The sizes of molecular size markers are given on the right (in base pairs).

    Techniques Used: Polymerase Chain Reaction, Isolation, Derivative Assay, Agarose Gel Electrophoresis

    23) Product Images from "A Recessive Contiguous Gene Deletion of Chromosome 2p16 Associated with Cystinuria and a Mitochondrial Disease"

    Article Title: A Recessive Contiguous Gene Deletion of Chromosome 2p16 Associated with Cystinuria and a Mitochondrial Disease

    Journal: American Journal of Human Genetics

    doi:

    a, Results of PCR amplifications of the SLC3A1 exons (1–10) and an unrelated gene (N.R.). The patients’ DNA (V10 and V15 in fig. 1 a ) failed to amplify the SLC3A1 exons, whereas good amplification was observed for DNA from a parent (IV1 in fig. 1 a ) and a control. The quality of the patients’ DNA was assessed by its ability to amplify an unrelated gene. A = affected, P = parent, C = unrelated control individual, and M = molecular weight marker X of Amersham. b, Multiplex PCR assay defining the deletion borders. Affected children are indicated by blackened symbols. The PCR reaction included primers a, b, and c, as indicated in fig. 3 a. Primers’ concentrations were 1 mM for primers b and c and 0.5 mM for primer a; annealing temperature was 57°C. The 439-bp product was obtained only if a normal allele was present; the 376-bp product was obtained only if a deletion allele was present. All parents are therefore heterozygotes for the deletion and the wild-type allele. M = the molecular weight marker X of Amersham. c, Results of RT-PCR from lymphoblastoid RNA from an affected individual (A; V10 in fig. 1 a ) compared with control (C) of the SLC3A1, PP2Cβ, and KIAA0436 genes. None of these transcripts are PCR-amplified in the affected individual. The actin gene was included to assess RNA quality. Primers for the PCR were selected on different exons, to avoid amplification from residual DNA in the RNA preparation.
    Figure Legend Snippet: a, Results of PCR amplifications of the SLC3A1 exons (1–10) and an unrelated gene (N.R.). The patients’ DNA (V10 and V15 in fig. 1 a ) failed to amplify the SLC3A1 exons, whereas good amplification was observed for DNA from a parent (IV1 in fig. 1 a ) and a control. The quality of the patients’ DNA was assessed by its ability to amplify an unrelated gene. A = affected, P = parent, C = unrelated control individual, and M = molecular weight marker X of Amersham. b, Multiplex PCR assay defining the deletion borders. Affected children are indicated by blackened symbols. The PCR reaction included primers a, b, and c, as indicated in fig. 3 a. Primers’ concentrations were 1 mM for primers b and c and 0.5 mM for primer a; annealing temperature was 57°C. The 439-bp product was obtained only if a normal allele was present; the 376-bp product was obtained only if a deletion allele was present. All parents are therefore heterozygotes for the deletion and the wild-type allele. M = the molecular weight marker X of Amersham. c, Results of RT-PCR from lymphoblastoid RNA from an affected individual (A; V10 in fig. 1 a ) compared with control (C) of the SLC3A1, PP2Cβ, and KIAA0436 genes. None of these transcripts are PCR-amplified in the affected individual. The actin gene was included to assess RNA quality. Primers for the PCR were selected on different exons, to avoid amplification from residual DNA in the RNA preparation.

    Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight, Marker, Multiplex Assay, Reverse Transcription Polymerase Chain Reaction

    24) Product Images from "MDM4 inhibition: a novel therapeutic strategy to reactivate p53 in hepatoblastoma"

    Article Title: MDM4 inhibition: a novel therapeutic strategy to reactivate p53 in hepatoblastoma

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-82542-4

    Cytotoxic effects of NSC207895 are dependent on MDM4. ( a ) Immunoblotting with HepG2 and HepT1 cells transduced with MDM4 cDNA or vector control (vc) with an antibody recognizing MDM4 (04–1555, Millipore). β-Actin immunoblotting was used as a loading control. Data shown are representative of at least three independent experiments. Full length blots for data shown in a are presented in Supplementary Fig. 11 . ( b ) Bar graphs representing normalized mRNA expression of MDM4 analyzed with qPCR experiments. RNA extracted from HepG2 and HepT1 cells transduced with sh-MDM4-35 or sh-MDM4-37 was compared to that transduced with sh-GFP . ( c ) Cells transduced with MDM4 cDNA or vc were incubated with varying concentrations of NSC207895 for 48 h. MTT assays were performed at 48 h to asses viability. ( d ) Cells transduced with sh-MDM4 or control sh-GFP were grown for 120 h and MTT assays were done at 24 h intervals to assess cell number. Data shown in b, c, and d are representative of at least three independent experiments performed with three replicate wells each time. Error bars represent SD. Student’s t test * P
    Figure Legend Snippet: Cytotoxic effects of NSC207895 are dependent on MDM4. ( a ) Immunoblotting with HepG2 and HepT1 cells transduced with MDM4 cDNA or vector control (vc) with an antibody recognizing MDM4 (04–1555, Millipore). β-Actin immunoblotting was used as a loading control. Data shown are representative of at least three independent experiments. Full length blots for data shown in a are presented in Supplementary Fig. 11 . ( b ) Bar graphs representing normalized mRNA expression of MDM4 analyzed with qPCR experiments. RNA extracted from HepG2 and HepT1 cells transduced with sh-MDM4-35 or sh-MDM4-37 was compared to that transduced with sh-GFP . ( c ) Cells transduced with MDM4 cDNA or vc were incubated with varying concentrations of NSC207895 for 48 h. MTT assays were performed at 48 h to asses viability. ( d ) Cells transduced with sh-MDM4 or control sh-GFP were grown for 120 h and MTT assays were done at 24 h intervals to assess cell number. Data shown in b, c, and d are representative of at least three independent experiments performed with three replicate wells each time. Error bars represent SD. Student’s t test * P

    Techniques Used: Transduction, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Incubation, MTT Assay

    Acute MDM4 inhibition with ATSP-7041 and NSC207895 lead to upregulation of p53 activity and downstream signaling. ( a ) Bar graphs representing normalized mRNA expression of p53 targets Bax , Puma , CDKN1A , and MDM2 analyzed with qPCR experiments. RNA extracted from HepG2 and HepT1 cells treated with 10 μM ATSP-7041 for 24 h was compared to that from untreated cells (0 h). Error bars represent SD. Data shown are representative of at least three independent experiments performed with three replicate wells each time. Student’s t test (two tailed) * P
    Figure Legend Snippet: Acute MDM4 inhibition with ATSP-7041 and NSC207895 lead to upregulation of p53 activity and downstream signaling. ( a ) Bar graphs representing normalized mRNA expression of p53 targets Bax , Puma , CDKN1A , and MDM2 analyzed with qPCR experiments. RNA extracted from HepG2 and HepT1 cells treated with 10 μM ATSP-7041 for 24 h was compared to that from untreated cells (0 h). Error bars represent SD. Data shown are representative of at least three independent experiments performed with three replicate wells each time. Student’s t test (two tailed) * P

    Techniques Used: Inhibition, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

    Prolonged MDM4 inhibition with ATSP-7041 and NSC207895 lead to upregulation of p53 activity and downstream signaling. ( a ) Bar graphs representing normalized mRNA expression of p53 targets Bax , Puma , CDKN1A , and MDM2 analyzed with qPCR experiments. RNA extracted from HepG2 and HepT1 cells treated with the indicated concentrations of ATSP-7041 for 48 h was compared to that from untreated cells (0 h). Error bars represent SD. Data shown are representative of at least three independent experiments performed with three replicate wells each time. Student’s t test (two tailed) * P
    Figure Legend Snippet: Prolonged MDM4 inhibition with ATSP-7041 and NSC207895 lead to upregulation of p53 activity and downstream signaling. ( a ) Bar graphs representing normalized mRNA expression of p53 targets Bax , Puma , CDKN1A , and MDM2 analyzed with qPCR experiments. RNA extracted from HepG2 and HepT1 cells treated with the indicated concentrations of ATSP-7041 for 48 h was compared to that from untreated cells (0 h). Error bars represent SD. Data shown are representative of at least three independent experiments performed with three replicate wells each time. Student’s t test (two tailed) * P

    Techniques Used: Inhibition, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

    Cytotoxic effects of NSC207895 are dependent on p53 signaling. ( a ) Immunoblotting with HepG2 and HepT1 cells transduced with sh-TP53 or control sh-Luc with an antibody recognizing p53. β-Actin immunoblotting was used as a loading control. Data shown are representative of at least three independent experiments. Full length blots for data shown in a are presented in Supplementary Fig. 10 . ( b ) Cells transduced with sh-TP53 or control sh-Luc were incubated with varying indicated concentrations of NSC207895 for 48 h. MTT assays were performed at 48 h to assess viability. ( c ) Bar graphs representing normalized mRNA expression of p53 targets Bax , Puma , CDKN1A , and MDM2 analyzed with qPCR experiments. RNA was extracted from cells transduced with sh-TP53 or control sh-Luc treated with 10 μM NSC207895 for 0 or 4 h. Expression in treated cells (4 h) was compared to that from untreated cells (0 h). Data shown in b and c are representative of at least three independent experiments performed with three replicate wells each time. Error bars represent SD. Student’s t test (two tailed) * P
    Figure Legend Snippet: Cytotoxic effects of NSC207895 are dependent on p53 signaling. ( a ) Immunoblotting with HepG2 and HepT1 cells transduced with sh-TP53 or control sh-Luc with an antibody recognizing p53. β-Actin immunoblotting was used as a loading control. Data shown are representative of at least three independent experiments. Full length blots for data shown in a are presented in Supplementary Fig. 10 . ( b ) Cells transduced with sh-TP53 or control sh-Luc were incubated with varying indicated concentrations of NSC207895 for 48 h. MTT assays were performed at 48 h to assess viability. ( c ) Bar graphs representing normalized mRNA expression of p53 targets Bax , Puma , CDKN1A , and MDM2 analyzed with qPCR experiments. RNA was extracted from cells transduced with sh-TP53 or control sh-Luc treated with 10 μM NSC207895 for 0 or 4 h. Expression in treated cells (4 h) was compared to that from untreated cells (0 h). Data shown in b and c are representative of at least three independent experiments performed with three replicate wells each time. Error bars represent SD. Student’s t test (two tailed) * P

    Techniques Used: Transduction, Incubation, MTT Assay, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

    25) Product Images from "Posttranscriptional Regulation of HIV-1 Gene Expression during Replication and Reactivation from Latency by Nuclear Matrix Protein MATR3"

    Article Title: Posttranscriptional Regulation of HIV-1 Gene Expression during Replication and Reactivation from Latency by Nuclear Matrix Protein MATR3

    Journal: mBio

    doi: 10.1128/mBio.02158-18

    Depletion of MATR3 inhibits HIV-1 Gag expression and nuclear export of unspliced RNA. (A) Optimization of shMATR3 lentivectors. HeLa cells were transduced with lentivectors targeting MATR3 (shMATR3) or with control shRNA (shCTRL). Three days after puromycin selection, MATR3-depleted cells were harvested for immunoblotting. α-Tubulin is the protein loading control. (B) Depletion of MATR3 in Jurkat cells. Lentivectors for MATR3 depletion were used to transduce Jurkat cells, which were processed as indicated above. β-Actin is the loading control. (C) MATR3 knockdown leads to the decrease of HIV-1 Gag expression. Jurkat cells were transduced with shMATR3. Three days after puromycin selection, MATR3-depleted cells were infected with replication-competent HIV-1 NL4.3 virus and harvested 48 h postinfection for immunoblotting. β-Actin is the protein loading control. (D) MATR3 depletion leads to the decrease in the extracellular release of HIV-1 p24. MATR3-depleted Jurkat cells generated as described above were infected with HIV-1 pNL-4.3 virus, and supernatant was harvested 48 h postinfection. Virion production in the supernatant was quantified by HIV-1 p24 ELISA. Average values of three independent experiments are shown, with standard deviations (SD) and P values as described in Materials and Methods. (E) HIV-1 LTR-driven transcription is not affected by MATR3 knockdown. MATR3-depleted Jurkat cells generated as described above were infected with HIV-1 pNL-4.3 luciferase virus (pNL-4.3-R-E-luc) pseudotyped with the VSV-G envelope. Luciferase was measured 48 h postinfection. Relative luciferase expression was normalized to total protein levels as measured by Bradford assay. The results of three independent experiments are shown as mean values ± SD. (F) Nucleocytoplasmic fractionation of Jurkat cells. Following shMATR3 transduction and infection as described above, Jurkat cells were subjected to nucleocytoplasmic fractionation. The cytoplasmic marker protein Hsp90 was detected only in the cytoplasm, while the nuclear marker PARP was detected only in the nuclear fraction. MATR3 was detected only in the nucleus of shCTRL-treated cells and depleted from the nuclear extracts of cells treated with shMATR3_905 and shMATR3_906. The Coomassie blue-stained gel was used as the loading control. (G) HIV-1 unspliced RNA levels are modulated by MATR3. Quantitative analysis of unspliced HIV-1 RNA levels in Jurkat cells treated as above. Unspliced (US) RNA levels were analyzed by quantitative real-time PCR on nuclear (NF) and cytoplasmic (CF) fractions. Data were normalized to GAPDH mRNA expression and presented as fold changes compared to shCTRL. Average values of triplicate independent experiments are shown, with standard deviations (SD) and P values as described in Materials and Methods.
    Figure Legend Snippet: Depletion of MATR3 inhibits HIV-1 Gag expression and nuclear export of unspliced RNA. (A) Optimization of shMATR3 lentivectors. HeLa cells were transduced with lentivectors targeting MATR3 (shMATR3) or with control shRNA (shCTRL). Three days after puromycin selection, MATR3-depleted cells were harvested for immunoblotting. α-Tubulin is the protein loading control. (B) Depletion of MATR3 in Jurkat cells. Lentivectors for MATR3 depletion were used to transduce Jurkat cells, which were processed as indicated above. β-Actin is the loading control. (C) MATR3 knockdown leads to the decrease of HIV-1 Gag expression. Jurkat cells were transduced with shMATR3. Three days after puromycin selection, MATR3-depleted cells were infected with replication-competent HIV-1 NL4.3 virus and harvested 48 h postinfection for immunoblotting. β-Actin is the protein loading control. (D) MATR3 depletion leads to the decrease in the extracellular release of HIV-1 p24. MATR3-depleted Jurkat cells generated as described above were infected with HIV-1 pNL-4.3 virus, and supernatant was harvested 48 h postinfection. Virion production in the supernatant was quantified by HIV-1 p24 ELISA. Average values of three independent experiments are shown, with standard deviations (SD) and P values as described in Materials and Methods. (E) HIV-1 LTR-driven transcription is not affected by MATR3 knockdown. MATR3-depleted Jurkat cells generated as described above were infected with HIV-1 pNL-4.3 luciferase virus (pNL-4.3-R-E-luc) pseudotyped with the VSV-G envelope. Luciferase was measured 48 h postinfection. Relative luciferase expression was normalized to total protein levels as measured by Bradford assay. The results of three independent experiments are shown as mean values ± SD. (F) Nucleocytoplasmic fractionation of Jurkat cells. Following shMATR3 transduction and infection as described above, Jurkat cells were subjected to nucleocytoplasmic fractionation. The cytoplasmic marker protein Hsp90 was detected only in the cytoplasm, while the nuclear marker PARP was detected only in the nuclear fraction. MATR3 was detected only in the nucleus of shCTRL-treated cells and depleted from the nuclear extracts of cells treated with shMATR3_905 and shMATR3_906. The Coomassie blue-stained gel was used as the loading control. (G) HIV-1 unspliced RNA levels are modulated by MATR3. Quantitative analysis of unspliced HIV-1 RNA levels in Jurkat cells treated as above. Unspliced (US) RNA levels were analyzed by quantitative real-time PCR on nuclear (NF) and cytoplasmic (CF) fractions. Data were normalized to GAPDH mRNA expression and presented as fold changes compared to shCTRL. Average values of triplicate independent experiments are shown, with standard deviations (SD) and P values as described in Materials and Methods.

    Techniques Used: Expressing, Transduction, shRNA, Selection, Infection, Generated, Enzyme-linked Immunosorbent Assay, Luciferase, Bradford Assay, Fractionation, Marker, Staining, Real-time Polymerase Chain Reaction

    26) Product Images from "Transcription start site analysis reveals widespread divergent transcription in D. melanogaster and core promoter-encoded enhancer activities"

    Article Title: Transcription start site analysis reveals widespread divergent transcription in D. melanogaster and core promoter-encoded enhancer activities

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky244

    DNA sequence elements impact the stability, directionality and expression strengths of regulatory elements. ( A, B ) Frequencies of RNA processing motifs (A: 5′ splice site, B: polyadenylation site) downstream of CAGE summits broken up by DHS class and strand. Vertical axes show the average number of predicted sites per bp within an increasing window size from the TSS (horizontal axis) in which the motif search was done. 0 indicates the expected hit frequency from random genomic background. ( C ) Histogram of de novo-assembled transcript counts (vertical axis), broken up by number of exons and associated DHS class. ( D ) Fraction of transcribed DHSs (vertical axis) with an identified core promoter element (TATA, Inr, DPE, or MTE) at a given position relative to the major (left panels) and minor (right panels) strand CAGE summits. ( E ) Fraction of transcribed DHSs within each DHS class (vertical axis) associated with at least one out of nine core promoter elements identified on one or both strands. In addition, the fraction of DHSs with no core promoter elements are shown (none). ( F ) Hierarchical Ward agglomerative clustering of motif match scores for the nine considered core promoter elements on major and minor strands of transcribed DHSs. Ten clusters of core promoter element compositions are shown. ( G ) DHS class enrichments, calculated as the fraction of DHSs in each DHS class associated with each core promoter element cluster versus the fraction of total transcribed DHSs, displayed in log 2 for DHS class enrichments for all core promoter clusters.
    Figure Legend Snippet: DNA sequence elements impact the stability, directionality and expression strengths of regulatory elements. ( A, B ) Frequencies of RNA processing motifs (A: 5′ splice site, B: polyadenylation site) downstream of CAGE summits broken up by DHS class and strand. Vertical axes show the average number of predicted sites per bp within an increasing window size from the TSS (horizontal axis) in which the motif search was done. 0 indicates the expected hit frequency from random genomic background. ( C ) Histogram of de novo-assembled transcript counts (vertical axis), broken up by number of exons and associated DHS class. ( D ) Fraction of transcribed DHSs (vertical axis) with an identified core promoter element (TATA, Inr, DPE, or MTE) at a given position relative to the major (left panels) and minor (right panels) strand CAGE summits. ( E ) Fraction of transcribed DHSs within each DHS class (vertical axis) associated with at least one out of nine core promoter elements identified on one or both strands. In addition, the fraction of DHSs with no core promoter elements are shown (none). ( F ) Hierarchical Ward agglomerative clustering of motif match scores for the nine considered core promoter elements on major and minor strands of transcribed DHSs. Ten clusters of core promoter element compositions are shown. ( G ) DHS class enrichments, calculated as the fraction of DHSs in each DHS class associated with each core promoter element cluster versus the fraction of total transcribed DHSs, displayed in log 2 for DHS class enrichments for all core promoter clusters.

    Techniques Used: Sequencing, Expressing

    27) Product Images from "CircAGAP1 promotes tumor progression by sponging miR-15-5p in clear cell renal cell carcinoma"

    Article Title: CircAGAP1 promotes tumor progression by sponging miR-15-5p in clear cell renal cell carcinoma

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-021-01864-3

    Expression and characterization of circAGAP1 in ccRCC. a The expression of circAGAP1 in ccRCC was examined by microarray. b The exonic information of circAGAP1 (circBase ID: hsa_circ_0058792) is demonstrated. Hsa_circ_0058792 is generated by circularization of exons 2–6 of the AGAP1 gene. The back-splicing junction was verified via Sanger sequencing. c Random hexamer or oligo (dT)18 primers were used for the reverse transcription assay. The RNA levels of circAGAP1, linear AGAP1 and GAPDH were detected by RT-qPCR in samples with RNase R (RNase R+) or without RNAse R (RNase R-) treatment. Data are presented as the means ± SEM from three independent experiments. d Expression levels of circAGAP1 in paired ccRCC and adjacent noncancerous tissues ( n = 34). e qRT-PCR analysis of circAGAP1 expression in human ccRCC cell lines (A498, ACHN, CAKI-1 and OS-RC-2) and HK2 cells. f , g qRT-PCR analysis of circAGAP1 expression in human ccRCC cells (A498, ACHN) subjected to either normoxia or hypoxia for 12 h. Data are presented as the means ± SEM from three independent experiments and are expressed as the relative fold-change over normoxic controls. * p
    Figure Legend Snippet: Expression and characterization of circAGAP1 in ccRCC. a The expression of circAGAP1 in ccRCC was examined by microarray. b The exonic information of circAGAP1 (circBase ID: hsa_circ_0058792) is demonstrated. Hsa_circ_0058792 is generated by circularization of exons 2–6 of the AGAP1 gene. The back-splicing junction was verified via Sanger sequencing. c Random hexamer or oligo (dT)18 primers were used for the reverse transcription assay. The RNA levels of circAGAP1, linear AGAP1 and GAPDH were detected by RT-qPCR in samples with RNase R (RNase R+) or without RNAse R (RNase R-) treatment. Data are presented as the means ± SEM from three independent experiments. d Expression levels of circAGAP1 in paired ccRCC and adjacent noncancerous tissues ( n = 34). e qRT-PCR analysis of circAGAP1 expression in human ccRCC cell lines (A498, ACHN, CAKI-1 and OS-RC-2) and HK2 cells. f , g qRT-PCR analysis of circAGAP1 expression in human ccRCC cells (A498, ACHN) subjected to either normoxia or hypoxia for 12 h. Data are presented as the means ± SEM from three independent experiments and are expressed as the relative fold-change over normoxic controls. * p

    Techniques Used: Expressing, Microarray, Generated, Sequencing, Random Hexamer Labeling, Quantitative RT-PCR

    circAGAP1 sponged miR-15a-5p. a The interaction between miR-15a-5p and circAGAP1 was assessed by biotin-coupled probe pull-down assay. Data are presented as the means ± SEM from three independent experiments. b Detection of colocalization of miR-15a-5p and circAGAP1 in A498 and ACHN cells by RNA-FISH assay. Nuclei were stained with DAPI (magnification, × 400). Red, miR-15a-5p; Green, circAGAP1; Blue, DAPI. c Wild-type (WT) and associated mutant (MUT) sequences of the potential miR-15a-5p binding sites in circAGAP1. The effect of the miR-15a-5p mimic on the luciferase activities of WT or MUT circAGAP1 was detected. d Expression of miR-15a-5p in ccRCC and adjacent noncancerous tissues was analyzed ( N = 25, p = 0.0008). The experiments were repeated with at least three biological replicates. ** p
    Figure Legend Snippet: circAGAP1 sponged miR-15a-5p. a The interaction between miR-15a-5p and circAGAP1 was assessed by biotin-coupled probe pull-down assay. Data are presented as the means ± SEM from three independent experiments. b Detection of colocalization of miR-15a-5p and circAGAP1 in A498 and ACHN cells by RNA-FISH assay. Nuclei were stained with DAPI (magnification, × 400). Red, miR-15a-5p; Green, circAGAP1; Blue, DAPI. c Wild-type (WT) and associated mutant (MUT) sequences of the potential miR-15a-5p binding sites in circAGAP1. The effect of the miR-15a-5p mimic on the luciferase activities of WT or MUT circAGAP1 was detected. d Expression of miR-15a-5p in ccRCC and adjacent noncancerous tissues was analyzed ( N = 25, p = 0.0008). The experiments were repeated with at least three biological replicates. ** p

    Techniques Used: Pull Down Assay, Fluorescence In Situ Hybridization, Staining, Mutagenesis, Binding Assay, Luciferase, Expressing

    28) Product Images from "Genome-wide identification and functional analysis of ARF transcription factors in Brassica juncea var. tumida"

    Article Title: Genome-wide identification and functional analysis of ARF transcription factors in Brassica juncea var. tumida

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0232039

    Expression pattern of BjARFs . A. Expression profiles of BjARFs from RNA-seq in different tissues and cultivars. Color represents BjARF expression levels: Log2 (FPKM). The phylogenetic relationship is shown on the left. RNA expression data were selected as follows: leaf (L) of the seedling stage (SS), leaf and stem (S) of the flowing period (FP), leaf and stem of the mature period (MP) from YA1 (yonganxiaoye1), YA2 (yonganxiaoye2) and YA3 (yonganxiaoye3), respectively. B–M. Validation of candidate BjARFs using qRT-PCR, based on altered expression levels. From left to right representing seedling, leaf (VP), tumor stem (VP), tumor stem (FP), flower, and legume. Error bars show the standard error calculated from three biological replicates. Values are presented as the mean ± SEM.
    Figure Legend Snippet: Expression pattern of BjARFs . A. Expression profiles of BjARFs from RNA-seq in different tissues and cultivars. Color represents BjARF expression levels: Log2 (FPKM). The phylogenetic relationship is shown on the left. RNA expression data were selected as follows: leaf (L) of the seedling stage (SS), leaf and stem (S) of the flowing period (FP), leaf and stem of the mature period (MP) from YA1 (yonganxiaoye1), YA2 (yonganxiaoye2) and YA3 (yonganxiaoye3), respectively. B–M. Validation of candidate BjARFs using qRT-PCR, based on altered expression levels. From left to right representing seedling, leaf (VP), tumor stem (VP), tumor stem (FP), flower, and legume. Error bars show the standard error calculated from three biological replicates. Values are presented as the mean ± SEM.

    Techniques Used: Expressing, RNA Sequencing Assay, RNA Expression, Quantitative RT-PCR

    29) Product Images from "The Enterococcus faecium Enterococcal Biofilm Regulator, EbrB, Regulates the esp Operon and Is Implicated in Biofilm Formation and Intestinal Colonization"

    Article Title: The Enterococcus faecium Enterococcal Biofilm Regulator, EbrB, Regulates the esp Operon and Is Implicated in Biofilm Formation and Intestinal Colonization

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065224

    Determination of the esp operon. (A) RT-PCR analysis on synthesized cDNA on RNA obtained from wild-type E1162 using gene specific primers (underlined). Panel 1: intergenic region esp and efmE1162_1544. Panel 2: intergenic region efmE1162_1544 and efmE1162_1543. Panel 3: intergenic region efmE1162_1543 and efmE1162_1542. Panel 4: intergenic region efmE1162_1542 and efmE1162_1541. RT-PCRs were performed on 4 biological replicates represented by lanes 1, 3, 5 and 7. Lanes 2, 4, 6, 8 represent negative controls in which incubation with reverse transcriptase was omitted. Lane 9 is the positive genomic DNA control and lane 10 negative water control. (B) Expression levels of ebrB and the esp operon in ebrB and esp complemented strains. Expression of ebrB, esp, efmE1162_1544, efmE1162_1543, efmE1162_1542 and efmE1162_1541 was analyzed using qRT-PCR on synthesized cDNA from the ebrB complemented strains (E1162▵ ebrB + ebrB and E1162▵ ebrB +pEF25) in blue and esp complemented strains (E1162▵ esp + esp and E1162▵ esp+ pEF25) in red.
    Figure Legend Snippet: Determination of the esp operon. (A) RT-PCR analysis on synthesized cDNA on RNA obtained from wild-type E1162 using gene specific primers (underlined). Panel 1: intergenic region esp and efmE1162_1544. Panel 2: intergenic region efmE1162_1544 and efmE1162_1543. Panel 3: intergenic region efmE1162_1543 and efmE1162_1542. Panel 4: intergenic region efmE1162_1542 and efmE1162_1541. RT-PCRs were performed on 4 biological replicates represented by lanes 1, 3, 5 and 7. Lanes 2, 4, 6, 8 represent negative controls in which incubation with reverse transcriptase was omitted. Lane 9 is the positive genomic DNA control and lane 10 negative water control. (B) Expression levels of ebrB and the esp operon in ebrB and esp complemented strains. Expression of ebrB, esp, efmE1162_1544, efmE1162_1543, efmE1162_1542 and efmE1162_1541 was analyzed using qRT-PCR on synthesized cDNA from the ebrB complemented strains (E1162▵ ebrB + ebrB and E1162▵ ebrB +pEF25) in blue and esp complemented strains (E1162▵ esp + esp and E1162▵ esp+ pEF25) in red.

    Techniques Used: End-sequence Profiling, Reverse Transcription Polymerase Chain Reaction, Synthesized, Incubation, Expressing, Quantitative RT-PCR

    30) Product Images from "Genome-wide characterization of microRNA in foxtail millet (Setaria italica)"

    Article Title: Genome-wide characterization of microRNA in foxtail millet (Setaria italica)

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-13-212

    Verification and characteristics of the novel foxtail millet miRNAs (nov-sit-miRNAs). (A) The nov-sit-miRNAs were validated by stem-loop RT-PCR. The RNA used for stem-loop RT-PCR was isolated from shoots (14-day-old). (B) Nucleotide frequency of novel miRNAs.
    Figure Legend Snippet: Verification and characteristics of the novel foxtail millet miRNAs (nov-sit-miRNAs). (A) The nov-sit-miRNAs were validated by stem-loop RT-PCR. The RNA used for stem-loop RT-PCR was isolated from shoots (14-day-old). (B) Nucleotide frequency of novel miRNAs.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation

    31) Product Images from "MicroRNA-570-3p regulates HuR and cytokine expression in airway epithelial cells"

    Article Title: MicroRNA-570-3p regulates HuR and cytokine expression in airway epithelial cells

    Journal: American Journal of Clinical and Experimental Immunology

    doi:

    RNA isolation, cDNA synthesis, and quantitative real-time PCR
    Figure Legend Snippet: RNA isolation, cDNA synthesis, and quantitative real-time PCR

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction

    Differential effect of miR-570-3p on mRNA stability of selected transcripts. A549 cells were transfected and treated as described previously, followed by addition of Actinomycin D and isolation of RNA at the indicated time points for qPCR analysis of
    Figure Legend Snippet: Differential effect of miR-570-3p on mRNA stability of selected transcripts. A549 cells were transfected and treated as described previously, followed by addition of Actinomycin D and isolation of RNA at the indicated time points for qPCR analysis of

    Techniques Used: Transfection, Isolation, Real-time Polymerase Chain Reaction

    32) Product Images from "TNF-? and CD8+ T Cells Mediate the Beneficial Effects of Nitric Oxide Synthase-2 Deficiency in Pulmonary Paracoccidioidomycosis"

    Article Title: TNF-? and CD8+ T Cells Mediate the Beneficial Effects of Nitric Oxide Synthase-2 Deficiency in Pulmonary Paracoccidioidomycosis

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0002325

    INOS −/− macrophages express high levels of TGF-β arginase1 and TNF-α. Uninfected and P. brasiliensis infected macrophages were used to characterize the expression of iNOS, ARG1, TGF-β, IL-12, TNF-α and IDO mRNA by quantitative Real-Time PCR. Total RNA was extracted using Trizol reagent, reverse transcribed, and cDNA amplified. Real-time PCR was performed using TaqMan universal master mix. Amplified products were normalized to the amount of GAPDH products from macrophages. All analyzes were done with five wells per condition in three independent experiments. The bars depict means ± SEM ** ( P
    Figure Legend Snippet: INOS −/− macrophages express high levels of TGF-β arginase1 and TNF-α. Uninfected and P. brasiliensis infected macrophages were used to characterize the expression of iNOS, ARG1, TGF-β, IL-12, TNF-α and IDO mRNA by quantitative Real-Time PCR. Total RNA was extracted using Trizol reagent, reverse transcribed, and cDNA amplified. Real-time PCR was performed using TaqMan universal master mix. Amplified products were normalized to the amount of GAPDH products from macrophages. All analyzes were done with five wells per condition in three independent experiments. The bars depict means ± SEM ** ( P

    Techniques Used: Infection, Expressing, Real-time Polymerase Chain Reaction, Amplification

    33) Product Images from "RNA-Seq-Based Analysis of Cold Shock Response in Thermoanaerobacter tengcongensis, a Bacterium Harboring a Single Cold Shock Protein Encoding Gene"

    Article Title: RNA-Seq-Based Analysis of Cold Shock Response in Thermoanaerobacter tengcongensis, a Bacterium Harboring a Single Cold Shock Protein Encoding Gene

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093289

    Validation of differentially expressed genes by real-time PCR. Comparison of RNA-seq and real-time PCR measures of changes in expression of eutD (encoding phosphotransacetylase), rpmE (50S ribosomal protein L31), acpP (acyl carrier protein), tte0510 (hypothetical protein), tte2654 (hypothetical protein), tte0106 (hypothetical protein), dnaA (chromosome replication initiator DnaA), tte1002 (hypothetical protein), pspf4 (a transcriptional regulator), and cspC (cold shock protein). All data are shown as means ± SD (n = 3).
    Figure Legend Snippet: Validation of differentially expressed genes by real-time PCR. Comparison of RNA-seq and real-time PCR measures of changes in expression of eutD (encoding phosphotransacetylase), rpmE (50S ribosomal protein L31), acpP (acyl carrier protein), tte0510 (hypothetical protein), tte2654 (hypothetical protein), tte0106 (hypothetical protein), dnaA (chromosome replication initiator DnaA), tte1002 (hypothetical protein), pspf4 (a transcriptional regulator), and cspC (cold shock protein). All data are shown as means ± SD (n = 3).

    Techniques Used: Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Expressing

    34) Product Images from "Unsaturated fatty acids induce mesenchymal stem cells to increase secretion of angiogenic mediators"

    Article Title: Unsaturated fatty acids induce mesenchymal stem cells to increase secretion of angiogenic mediators

    Journal: Journal of Cellular Physiology

    doi: 10.1002/jcp.24013

    Unsaturated fatty acids affect MSC expression of growth factors and cytokines. RNA was extracted from human bone marrow-derived MSC exposed to 20 μM unsaturated fatty acids for 7 days and control MSC cultured without the addition of fatty acids.
    Figure Legend Snippet: Unsaturated fatty acids affect MSC expression of growth factors and cytokines. RNA was extracted from human bone marrow-derived MSC exposed to 20 μM unsaturated fatty acids for 7 days and control MSC cultured without the addition of fatty acids.

    Techniques Used: Expressing, Derivative Assay, Cell Culture

    35) Product Images from "EGF-Activated Grb7 Confers to STAT3-Mediated EPHA4 Gene Expression in Regulating Lung Cancer Progression"

    Article Title: EGF-Activated Grb7 Confers to STAT3-Mediated EPHA4 Gene Expression in Regulating Lung Cancer Progression

    Journal: bioRxiv

    doi: 10.1101/2020.10.24.353268

    EGF-regulated Grb7 phosphorylation in NSCLC aggressiveness. a Cell lysates from serum starved-H460, H1299, A549 human non-small cell lung cancer cell (NSCLC) and HARA human lung squamous carcinoma cell lines with or without EGF (10 ng/ml) treatment were collected and immunoprecipitated with anti-EGFR or anti-Grb7 antibodies followed by Western blot analysis (IB) with an anti-phosphotyrosine antibody to examine the effects of EGF on Grb7 phosphorylation. Cell lysates were also collected and subjected to Western blot with anti-EGFR or anti-Grb7 antibodies. Here, actin was used as a control. A549 cells which were infected with lentiviruses encoding short hairpin Grb7 (shGrb7) were transfected with HA-tagged Grb7 or its truncation mutant, SH2 domain, and subjected to ( b ) cell proliferation assay (for 24 h) by BrdU incorporation and ( c ) cell migration assay (for 8 h) by an modified Boyden chamber assay in the presence of EGF. A549 cells which were infected with lentiviruses encoding short hairpin RNA targeting Grb7 (shGrb7) were subjected to ( d ) anchorage-independent growth assay (for 2 weeks) and ( e ) matrigel invasion assay (for 24 h) in the presence of EGF. HA-overexpressed cells (-) or shLuc-infected (shLuc) cells were used as control cells. All results are shown as the mean ± SEM of at least three independent experiments. Error bars represent ± SEM. * p
    Figure Legend Snippet: EGF-regulated Grb7 phosphorylation in NSCLC aggressiveness. a Cell lysates from serum starved-H460, H1299, A549 human non-small cell lung cancer cell (NSCLC) and HARA human lung squamous carcinoma cell lines with or without EGF (10 ng/ml) treatment were collected and immunoprecipitated with anti-EGFR or anti-Grb7 antibodies followed by Western blot analysis (IB) with an anti-phosphotyrosine antibody to examine the effects of EGF on Grb7 phosphorylation. Cell lysates were also collected and subjected to Western blot with anti-EGFR or anti-Grb7 antibodies. Here, actin was used as a control. A549 cells which were infected with lentiviruses encoding short hairpin Grb7 (shGrb7) were transfected with HA-tagged Grb7 or its truncation mutant, SH2 domain, and subjected to ( b ) cell proliferation assay (for 24 h) by BrdU incorporation and ( c ) cell migration assay (for 8 h) by an modified Boyden chamber assay in the presence of EGF. A549 cells which were infected with lentiviruses encoding short hairpin RNA targeting Grb7 (shGrb7) were subjected to ( d ) anchorage-independent growth assay (for 2 weeks) and ( e ) matrigel invasion assay (for 24 h) in the presence of EGF. HA-overexpressed cells (-) or shLuc-infected (shLuc) cells were used as control cells. All results are shown as the mean ± SEM of at least three independent experiments. Error bars represent ± SEM. * p

    Techniques Used: Immunoprecipitation, Western Blot, Infection, Transfection, Mutagenesis, Proliferation Assay, BrdU Incorporation Assay, Cell Migration Assay, Modification, Boyden Chamber Assay, shRNA, Growth Assay, Invasion Assay

    Grb7 involves in STAT3 phosphorylation in an EGF/EGFR signal-dependent manner. a A549 cells over-expressing HA-tagged Grb7 were serum-starved for 24 h and then stimulated with EGF (10 ng/ml) for 15 min. Cell lysates were collected and subjected to Western blot with anti-STAT3, anti-pTyr705-STAT3, and anti-actin antibodies to examine the effects of Grb7 on EGF-mediated STAT3 phosphorylation. Grb7 expression was visualized by anti-HA antibody. Here, HA-transfected cells were used as control cells. b A549 cells were infected with lentiviruses encoding short hairpin RNA targeting EGFR (shEGFR) or Grb7 (shGrb7) then first serum-starved and stimulated with EGF (10 ng/ml) for 15 min. Cell lysates were collected and immunoprecipitated by an anti-Grb7 antibody followed by Western blotting with an anti-phosphotyrosine antibody. Cell lysates were also collected and subjected to Western blot with anti-STAT3, anti-pTyr705-STAT3 anti-Grb7, and anti-actin antibodies. Here, shLuc-infected A549 cells were used as control cells. WCL, whole cell lysate.
    Figure Legend Snippet: Grb7 involves in STAT3 phosphorylation in an EGF/EGFR signal-dependent manner. a A549 cells over-expressing HA-tagged Grb7 were serum-starved for 24 h and then stimulated with EGF (10 ng/ml) for 15 min. Cell lysates were collected and subjected to Western blot with anti-STAT3, anti-pTyr705-STAT3, and anti-actin antibodies to examine the effects of Grb7 on EGF-mediated STAT3 phosphorylation. Grb7 expression was visualized by anti-HA antibody. Here, HA-transfected cells were used as control cells. b A549 cells were infected with lentiviruses encoding short hairpin RNA targeting EGFR (shEGFR) or Grb7 (shGrb7) then first serum-starved and stimulated with EGF (10 ng/ml) for 15 min. Cell lysates were collected and immunoprecipitated by an anti-Grb7 antibody followed by Western blotting with an anti-phosphotyrosine antibody. Cell lysates were also collected and subjected to Western blot with anti-STAT3, anti-pTyr705-STAT3 anti-Grb7, and anti-actin antibodies. Here, shLuc-infected A549 cells were used as control cells. WCL, whole cell lysate.

    Techniques Used: Expressing, Western Blot, Transfection, Infection, shRNA, Immunoprecipitation

    36) Product Images from "A miR-21 hairpin structure-based gene knockdown vector"

    Article Title: A miR-21 hairpin structure-based gene knockdown vector

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2010.03.047

    Determination of the impact of miR-21 gene flanking sequences on miR-21 expression A. Mouse miR-21 genes with two different flanking sequences (−400 bp to +135 bp and −160 bp to +65bp) were cloned into upstream of the reporter gene EGFP and co-expressed under the control of human UbC promoter in lentiviral vector pCFUW-EGFP, respectively. Meanwhile, the pre-miR-21 with a 160bp upstream and 65bp downstream flanking sequence was cloned into the 3′ UTR of the EGFP reporter gene. B. Mouse pre-miR-21 hairpin structure. C. The relative expressions of pri-miR-21 and mature miR-21 against non-coding small RNA U6 were detected by SYBR-Green real-time PCR in 293FT cells transfected with the control vector pcFUW-EGFP (I), miR-21 expression vector pcFUW-400-miR-21-EGFP (II), pcFUW-160-miR-21-EGFP (III), and pcFUW-EGFP-160-miR-21 (IV). D. EGFP gene expressions were detected using Western blot in control vector pcFUW-EGFP (I) and three different miR-21 lentiviral expression vectors, pcFUW-400-miR-21-EGFP (II), pcFUW-160-miR-21-EGFP (III), and pcFUW-EGFP-160-miR-21 (IV). β-actin was blotted as a loading control.
    Figure Legend Snippet: Determination of the impact of miR-21 gene flanking sequences on miR-21 expression A. Mouse miR-21 genes with two different flanking sequences (−400 bp to +135 bp and −160 bp to +65bp) were cloned into upstream of the reporter gene EGFP and co-expressed under the control of human UbC promoter in lentiviral vector pCFUW-EGFP, respectively. Meanwhile, the pre-miR-21 with a 160bp upstream and 65bp downstream flanking sequence was cloned into the 3′ UTR of the EGFP reporter gene. B. Mouse pre-miR-21 hairpin structure. C. The relative expressions of pri-miR-21 and mature miR-21 against non-coding small RNA U6 were detected by SYBR-Green real-time PCR in 293FT cells transfected with the control vector pcFUW-EGFP (I), miR-21 expression vector pcFUW-400-miR-21-EGFP (II), pcFUW-160-miR-21-EGFP (III), and pcFUW-EGFP-160-miR-21 (IV). D. EGFP gene expressions were detected using Western blot in control vector pcFUW-EGFP (I) and three different miR-21 lentiviral expression vectors, pcFUW-400-miR-21-EGFP (II), pcFUW-160-miR-21-EGFP (III), and pcFUW-EGFP-160-miR-21 (IV). β-actin was blotted as a loading control.

    Techniques Used: Expressing, Clone Assay, Plasmid Preparation, Sequencing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Transfection, Western Blot

    37) Product Images from "Haploinsufficiency for Core Exon Junction Complex Components Disrupts Embryonic Neurogenesis and Causes p53-Mediated Microcephaly"

    Article Title: Haploinsufficiency for Core Exon Junction Complex Components Disrupts Embryonic Neurogenesis and Causes p53-Mediated Microcephaly

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006282

    Transcriptome analyses of E10.5 Magoh , Rbm8a , and Eif4a3 haploinsufficient cortices reveal common downstream pathways. (A) Diagrammatic overview of RNA sequencing analysis of E10.5 neocortices from indicated genotypes. (B) qPCR showing expression of Magoh , Rbm8a , and Eif4a3 in their respective mutant E10.5 cortices. (C) Heatmaps showing z-score transformed normalized expression for all affected transcripts with an FDR corrected p-value, q
    Figure Legend Snippet: Transcriptome analyses of E10.5 Magoh , Rbm8a , and Eif4a3 haploinsufficient cortices reveal common downstream pathways. (A) Diagrammatic overview of RNA sequencing analysis of E10.5 neocortices from indicated genotypes. (B) qPCR showing expression of Magoh , Rbm8a , and Eif4a3 in their respective mutant E10.5 cortices. (C) Heatmaps showing z-score transformed normalized expression for all affected transcripts with an FDR corrected p-value, q

    Techniques Used: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Expressing, Mutagenesis, Transformation Assay

    Transcriptome analyses of E10.5 Magoh germline haploinsufficient brains identifies alterations in ribosome and p53 signaling pathways. (A) Diagrammatic overview of RNA sequencing analysis of E10.5 neocortices (dotted lines) from indicated genotypes. (B) Heatmaps showing z-score transformed normalized expression for control and Magoh Mos2/+ . Genes and samples were clustered using correlation distance with complete linkage. (C) Scatter plot of transcripts significantly upregulated (green dots) and downregulated (red dots) in E10.5 Magoh Mos2/+ cortices ( q
    Figure Legend Snippet: Transcriptome analyses of E10.5 Magoh germline haploinsufficient brains identifies alterations in ribosome and p53 signaling pathways. (A) Diagrammatic overview of RNA sequencing analysis of E10.5 neocortices (dotted lines) from indicated genotypes. (B) Heatmaps showing z-score transformed normalized expression for control and Magoh Mos2/+ . Genes and samples were clustered using correlation distance with complete linkage. (C) Scatter plot of transcripts significantly upregulated (green dots) and downregulated (red dots) in E10.5 Magoh Mos2/+ cortices ( q

    Techniques Used: RNA Sequencing Assay, Transformation Assay, Expressing

    38) Product Images from "Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING"

    Article Title: Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING

    Journal: Nature immunology

    doi: 10.1038/ni.2350

    Fusion induces type I IFN and CXCL10 expression. ( a–c ) VLPs prepared from wt ( a ) or the fusion deficient HSV-1 mutants ΔgH ( b ) and ΔgB( c ) were used to treat PCs (MOI 15) from C57BL6 mice for 4 hours. Expressions of CXCL10, TNF and IFNβ were analyzed by QPCR. Data in a–c represent means and s.e.m. of four independent experiments. ( d ) Wt, ΔgH and ΔgB VLPs were used to treat hMDMs as in panel a and analyzed for CXCL10 expression by QPCR. ( e–f ) HEK293 cells were transfected with expression plasmids for wt HIV Env, F522 Env (FY), or the ΔKS Env. After two days, the HEK293 cells were transferred to cultures with hMDMs. RNA was isolated 8 h later and analyzed for IFNβ and CXCL10 by QPCR. Data in d–f represent means and s.e.m. of data obtained using four independent hMDM donors. ( g–h ) PCs from wt or Myd88 −/− mice ( g ) or an immortalized MyD88 and TRIF double deficient macrophage cell-line ( h ) were treated with cationic liposomes for 4 hours. RNA was isolated and analyzed for CXCL10 and TNF by QPCR. Data in g–h represent means and s.e.m. of five independent experiments. ( i ) BMDCs were treated with PtdCho, DOTAP, or LR-DOPE-DOTAP liposomes for 4 hours. BMDCs were then analyzed for CXCL10 expression by QPCR. Data in i are represent means and s.e.m of two independent experiments.
    Figure Legend Snippet: Fusion induces type I IFN and CXCL10 expression. ( a–c ) VLPs prepared from wt ( a ) or the fusion deficient HSV-1 mutants ΔgH ( b ) and ΔgB( c ) were used to treat PCs (MOI 15) from C57BL6 mice for 4 hours. Expressions of CXCL10, TNF and IFNβ were analyzed by QPCR. Data in a–c represent means and s.e.m. of four independent experiments. ( d ) Wt, ΔgH and ΔgB VLPs were used to treat hMDMs as in panel a and analyzed for CXCL10 expression by QPCR. ( e–f ) HEK293 cells were transfected with expression plasmids for wt HIV Env, F522 Env (FY), or the ΔKS Env. After two days, the HEK293 cells were transferred to cultures with hMDMs. RNA was isolated 8 h later and analyzed for IFNβ and CXCL10 by QPCR. Data in d–f represent means and s.e.m. of data obtained using four independent hMDM donors. ( g–h ) PCs from wt or Myd88 −/− mice ( g ) or an immortalized MyD88 and TRIF double deficient macrophage cell-line ( h ) were treated with cationic liposomes for 4 hours. RNA was isolated and analyzed for CXCL10 and TNF by QPCR. Data in g–h represent means and s.e.m. of five independent experiments. ( i ) BMDCs were treated with PtdCho, DOTAP, or LR-DOPE-DOTAP liposomes for 4 hours. BMDCs were then analyzed for CXCL10 expression by QPCR. Data in i are represent means and s.e.m of two independent experiments.

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Transfection, Isolation

    39) Product Images from "Human Gastric Epithelial Cells Contribute to Gastric Immune Regulation by Providing Retinoic Acid to Dendritic Cells"

    Article Title: Human Gastric Epithelial Cells Contribute to Gastric Immune Regulation by Providing Retinoic Acid to Dendritic Cells

    Journal: Mucosal immunology

    doi: 10.1038/mi.2014.86

    Gastric DCs lack expression of CD103 ( a ) Gating strategy for isolation of gastric and intestinal DCs. ( b ) Phenotype of isolated gastric DCs. Data are representative of n=6–8. ( c ) Intestinal DCs express higher levels of CD103 than gastric DCs. Diamonds: individual samples; bars: mean; P =0.02, Kruskal-Wallis test with Bonferroni correction; n=6–8. ( d, e ) Gastric DCs lack ITGAE ( CD103) gene expression but express similar levels of ZBTB46 . RNA was isolated from FACS-sorted gastric and intestinal dendritic cells (live HLA-DR + /CD45 + /lineage − cells), and expression of ( d ) ITGAE and ( e ) ZBTB46 was analyzed by qRT-PCR. Monocyte-derived DCs (MoDCs; n=1) and elutriated intestinal macrophages (Macs, n=1) were analyzed for comparison. Diamonds: individual samples; bars: mean; lines connect paired samples; n=7. Statistical significance was determined using the Student’s t- test.
    Figure Legend Snippet: Gastric DCs lack expression of CD103 ( a ) Gating strategy for isolation of gastric and intestinal DCs. ( b ) Phenotype of isolated gastric DCs. Data are representative of n=6–8. ( c ) Intestinal DCs express higher levels of CD103 than gastric DCs. Diamonds: individual samples; bars: mean; P =0.02, Kruskal-Wallis test with Bonferroni correction; n=6–8. ( d, e ) Gastric DCs lack ITGAE ( CD103) gene expression but express similar levels of ZBTB46 . RNA was isolated from FACS-sorted gastric and intestinal dendritic cells (live HLA-DR + /CD45 + /lineage − cells), and expression of ( d ) ITGAE and ( e ) ZBTB46 was analyzed by qRT-PCR. Monocyte-derived DCs (MoDCs; n=1) and elutriated intestinal macrophages (Macs, n=1) were analyzed for comparison. Diamonds: individual samples; bars: mean; lines connect paired samples; n=7. Statistical significance was determined using the Student’s t- test.

    Techniques Used: Expressing, Isolation, FACS, Quantitative RT-PCR, Derivative Assay, Magnetic Cell Separation

    Gastric DC synthesize RA as efficiently as intestinal DCs ( a ) ALDH activity of isolated gastric and intestinal DCs and blood monocytes was analyzed with the Aldefluor® assay. Left panels: representative FACS data; grey histograms: DEAB-treated control cells, black outline: untreated samples. Middel panel: Combined data from 5–7 experiments shown as geometric mean fluorescence (normalized to geomean of the DEAB-treated control); Right panel: Combined data from 5–7 experiments shown as % positive cells. ( b, c ) RNA was isolated from FACS-sorted gastric and intestinal dendritic cells (live HLA-DR high /CD45 + /lineage cells), and expression of ( b ) RA biosynthesis genes RDH10 and ALDH1A2, ( c ) RA response genes and TGM2 and CRABP2 and (d) RA receptors RAR-α, RAR-β and RAR-γ was analyzed by qRT-PCR using the random standard curve method. Diamonds: individual samples; bars: mean; lines connect paired samples; n=5–7. Statistical significance was determined using the Student’s t -test.
    Figure Legend Snippet: Gastric DC synthesize RA as efficiently as intestinal DCs ( a ) ALDH activity of isolated gastric and intestinal DCs and blood monocytes was analyzed with the Aldefluor® assay. Left panels: representative FACS data; grey histograms: DEAB-treated control cells, black outline: untreated samples. Middel panel: Combined data from 5–7 experiments shown as geometric mean fluorescence (normalized to geomean of the DEAB-treated control); Right panel: Combined data from 5–7 experiments shown as % positive cells. ( b, c ) RNA was isolated from FACS-sorted gastric and intestinal dendritic cells (live HLA-DR high /CD45 + /lineage cells), and expression of ( b ) RA biosynthesis genes RDH10 and ALDH1A2, ( c ) RA response genes and TGM2 and CRABP2 and (d) RA receptors RAR-α, RAR-β and RAR-γ was analyzed by qRT-PCR using the random standard curve method. Diamonds: individual samples; bars: mean; lines connect paired samples; n=5–7. Statistical significance was determined using the Student’s t -test.

    Techniques Used: Activity Assay, Isolation, FACS, Fluorescence, Expressing, Quantitative RT-PCR

    40) Product Images from "Inactivation of the tumor suppressor PTEN/MMAC1 in advanced human prostate cancer through loss of expression"

    Article Title: Inactivation of the tumor suppressor PTEN/MMAC1 in advanced human prostate cancer through loss of expression

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

    doi:

    Restoration of PTEN/MMAC1 expression by 5-azadeoxycytidine. LuCaP-35 xenograft cells cultured in vitro were treated with 5-azadeoxycytidine at a concentration of 0, 1, or 2 μM, and cells were harvested for RNA after 4 days. Expression of PTEN/MMAC1 was detected by RT-PCR and was visualized by ethidium bromide staining. RT-PCR of PSA is shown as a control for the integrity of RNA in all samples.
    Figure Legend Snippet: Restoration of PTEN/MMAC1 expression by 5-azadeoxycytidine. LuCaP-35 xenograft cells cultured in vitro were treated with 5-azadeoxycytidine at a concentration of 0, 1, or 2 μM, and cells were harvested for RNA after 4 days. Expression of PTEN/MMAC1 was detected by RT-PCR and was visualized by ethidium bromide staining. RT-PCR of PSA is shown as a control for the integrity of RNA in all samples.

    Techniques Used: Expressing, Cell Culture, In Vitro, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Staining

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    Thermo Fisher total rna
    KLF2 regulates endocardial Wnt9b expression and canonical WNT signaling in mesenchymal cells of the developing valve (A) Differential gene expression between E12.5 AV cushions from Klf2 fl/fl mice and Nfatc1 Cre/+ ; Klf2 fl/fl mice was determined using <t>RNA-seq</t> analysis. Heat map displays the top 20 differentially expressed genes with a p-value ≤ 0.05 and an FDR ≤ 0.1. Red stars indicate known Wnt signaling genes. Each replicate consists of 6 AV cushions combined from 6 different litters. (B) Gene ontology analysis of all significant differentially expressed genes between Klf2 fl/fl and Nfatc1 Cre/+ ; Klf2 fl/fl cushions. (C) <t>qPCR</t> measurement of Wnt target gene expression from the indicated E12.5 hearts. Error bars represent ± SEM, * p≤0.05, **p≤0.01, **** p≤0.0001 using an unpaired 2-tailed Student’s t-test (n=3-4 from at least 3 litters). (D) In situ hybridization for Wnt9b (red) in developing control OFT and AV valves between E10.5 and E13.5. DAPI (blue) staining denotes nuclei. Scale bar represents 100μm. (E) In situ hybridization for Wnt9b (red) in developing Nfatc1 Cre/+ ; Klf2 fl/fl and control valves at E13.5. DAPI (blue) staining denotes nuclei. Scale bar represents 100μm. (F) Axin2 CreERT2-tdTomato reporter activity is detected in the indicated developing heart valves using anti-RFP immunostaining (red). Endocardial cells are identified using anti-PECAM staining (green). Scale bars represent 200μm. High magnification images from boxed regions are shown on the right. Scale bars in high magnification images represent 100μm. (G) qPCR of WNT9B and NOS3 .
    Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    KLF2 regulates endocardial Wnt9b expression and canonical WNT signaling in mesenchymal cells of the developing valve (A) Differential gene expression between E12.5 AV cushions from Klf2 fl/fl mice and Nfatc1 Cre/+ ; Klf2 fl/fl mice was determined using RNA-seq analysis. Heat map displays the top 20 differentially expressed genes with a p-value ≤ 0.05 and an FDR ≤ 0.1. Red stars indicate known Wnt signaling genes. Each replicate consists of 6 AV cushions combined from 6 different litters. (B) Gene ontology analysis of all significant differentially expressed genes between Klf2 fl/fl and Nfatc1 Cre/+ ; Klf2 fl/fl cushions. (C) qPCR measurement of Wnt target gene expression from the indicated E12.5 hearts. Error bars represent ± SEM, * p≤0.05, **p≤0.01, **** p≤0.0001 using an unpaired 2-tailed Student’s t-test (n=3-4 from at least 3 litters). (D) In situ hybridization for Wnt9b (red) in developing control OFT and AV valves between E10.5 and E13.5. DAPI (blue) staining denotes nuclei. Scale bar represents 100μm. (E) In situ hybridization for Wnt9b (red) in developing Nfatc1 Cre/+ ; Klf2 fl/fl and control valves at E13.5. DAPI (blue) staining denotes nuclei. Scale bar represents 100μm. (F) Axin2 CreERT2-tdTomato reporter activity is detected in the indicated developing heart valves using anti-RFP immunostaining (red). Endocardial cells are identified using anti-PECAM staining (green). Scale bars represent 200μm. High magnification images from boxed regions are shown on the right. Scale bars in high magnification images represent 100μm. (G) qPCR of WNT9B and NOS3 .

    Journal: Developmental cell

    Article Title: Hemodynamic forces sculpt developing heart valves through a KLF2-WNT9B paracrine signaling axis

    doi: 10.1016/j.devcel.2017.09.023

    Figure Lengend Snippet: KLF2 regulates endocardial Wnt9b expression and canonical WNT signaling in mesenchymal cells of the developing valve (A) Differential gene expression between E12.5 AV cushions from Klf2 fl/fl mice and Nfatc1 Cre/+ ; Klf2 fl/fl mice was determined using RNA-seq analysis. Heat map displays the top 20 differentially expressed genes with a p-value ≤ 0.05 and an FDR ≤ 0.1. Red stars indicate known Wnt signaling genes. Each replicate consists of 6 AV cushions combined from 6 different litters. (B) Gene ontology analysis of all significant differentially expressed genes between Klf2 fl/fl and Nfatc1 Cre/+ ; Klf2 fl/fl cushions. (C) qPCR measurement of Wnt target gene expression from the indicated E12.5 hearts. Error bars represent ± SEM, * p≤0.05, **p≤0.01, **** p≤0.0001 using an unpaired 2-tailed Student’s t-test (n=3-4 from at least 3 litters). (D) In situ hybridization for Wnt9b (red) in developing control OFT and AV valves between E10.5 and E13.5. DAPI (blue) staining denotes nuclei. Scale bar represents 100μm. (E) In situ hybridization for Wnt9b (red) in developing Nfatc1 Cre/+ ; Klf2 fl/fl and control valves at E13.5. DAPI (blue) staining denotes nuclei. Scale bar represents 100μm. (F) Axin2 CreERT2-tdTomato reporter activity is detected in the indicated developing heart valves using anti-RFP immunostaining (red). Endocardial cells are identified using anti-PECAM staining (green). Scale bars represent 200μm. High magnification images from boxed regions are shown on the right. Scale bars in high magnification images represent 100μm. (G) qPCR of WNT9B and NOS3 .

    Article Snippet: Forty-eight hours after infection, total RNA was isolated using TRIzol Reagent (Invitrogen). cDNA was generated from 1 μg total RNA using Superscript III Reverse Transcriptase (Invitrogen). qPCR was performed in Power SYBR Green PCR Master Mix (Applied Biosciences).

    Techniques: Expressing, Mouse Assay, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, In Situ Hybridization, Staining, Activity Assay, Immunostaining

    RNA isolation, cDNA synthesis and real-time PCR

    Journal: Molecular oral microbiology

    Article Title: Characterization of competence and biofilm development of a Streptocccus sanguinis endocarditis isolate

    doi: 10.1111/j.2041-1014.2010.00602.x

    Figure Lengend Snippet: RNA isolation, cDNA synthesis and real-time PCR

    Article Snippet: The concentration and quality of RNA samples were confirmed using NanoDrop spectrophotometer measurements and gel electrophoresis. cDNA was synthesized from 2 μg RNA using the SuperScript II™ Reverse Transcriptase and Random Primers (Invitrogen) according to manufacturer’s instructions.

    Techniques: Isolation, Real-time Polymerase Chain Reaction

    S100PBPR expression in normal tissues. cDNAs made from RNA samples isolated from normal organs were used as templates for RT-PCR to determine the presence of S100PBPR expression. Samples were run in the following order: lane 1 , universal RNA (positive

    Journal:

    Article Title: Expression of S100P and Its Novel Binding Partner S100PBPR in Early Pancreatic Cancer

    doi:

    Figure Lengend Snippet: S100PBPR expression in normal tissues. cDNAs made from RNA samples isolated from normal organs were used as templates for RT-PCR to determine the presence of S100PBPR expression. Samples were run in the following order: lane 1 , universal RNA (positive

    Article Snippet: RNA samples from normal pancreas, brain, breast, spleen, liver, and lung were obtained from Ambion Inc. (Austin, TX). cDNAs were synthesized from 1 μg of total RNA using an oligo dT primer and the Multiscribe reverse transcription kit (Applied Biosystems, Warrington, Cheshire, UK) as instructed by the manufacturer.

    Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction

    Cis-expression of R434 ribozyme inhibits HPV-16E6/E7 in vitro translation. ( A ) Map of HPV-16E6/E7 cis-expression constructs with R434 and R434i ribozymes. PCR-amplified fragments containing the entire HPV-16 E6/E7 genes (nucleotides 97–868) linked to R434 (pCR16E6/E7RZ) or R434i (pCR16E6/E7RZi) ribozymes were cloned in the pCR3.1 vector. The pCR16HH plasmid contains only the HPV-16E6/E7 genes. The relative positions of the Sty I sites used for cloning the ribozymes and the vector poly(A) signal are shown. ( B ) The protein products produced by plasmids pCR16HH, pCR16E6/E7RZ, and pCR16E6/E7RZi were examined by in vitro translation reactions using T7 RNA polymerase and rabbit reticulocyte lysates in the presence of [ 35 S]methionine. ←, the position of E6 and E7 proteins. Luc, luciferase protein reaction control.

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

    Article Title: Inhibition of HPV-16 E6/E7 immortalization of normal keratinocytes by hairpin ribozymes

    doi:

    Figure Lengend Snippet: Cis-expression of R434 ribozyme inhibits HPV-16E6/E7 in vitro translation. ( A ) Map of HPV-16E6/E7 cis-expression constructs with R434 and R434i ribozymes. PCR-amplified fragments containing the entire HPV-16 E6/E7 genes (nucleotides 97–868) linked to R434 (pCR16E6/E7RZ) or R434i (pCR16E6/E7RZi) ribozymes were cloned in the pCR3.1 vector. The pCR16HH plasmid contains only the HPV-16E6/E7 genes. The relative positions of the Sty I sites used for cloning the ribozymes and the vector poly(A) signal are shown. ( B ) The protein products produced by plasmids pCR16HH, pCR16E6/E7RZ, and pCR16E6/E7RZi were examined by in vitro translation reactions using T7 RNA polymerase and rabbit reticulocyte lysates in the presence of [ 35 S]methionine. ←, the position of E6 and E7 proteins. Luc, luciferase protein reaction control.

    Article Snippet: HPV-16 E6/E7 cDNA was produced from 1 μg of total RNA using the Superscript II One Shot kit (Life Technologies).

    Techniques: Expressing, In Vitro, Construct, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Produced, Luciferase

    Short term growth inhibition of HPV-16 E6/E7-transfected HKc by cis-expressed R434. ( A ) HKc were transfected with the pCR16HH, pCR16E6/E7RZ, or pCR16E6/E7RZi constructs and were kept in G418 (200 μg/ml) for 2 wk, and 10 5 cells were seeded for counting in 6-well dishes. The graphics are the mean of three experiments. ( B ) RT-PCR analysis of RNA from HKc transfected with HPV-16 E6/E7 (pCR16HH) and cis-expressed active and inactive ribozymes (pCR16E6/E7RZ and pCR16E6/E7RZi, respectively). Total RNA (1 μg) was subjected to a coupled RT-PCR reaction with primers specific to both sides of the ribozyme cleavage site as described in Material and Methods . A contamination control without reverse transcriptase (control) was included. Separate RT-PCR reactions were performed by using the same RNA sample with primers specific to the human β-actin gene to show RNA integrity. The HPV-16 E6/E7-amplified products (492 and 326 bp) were separated through agarose gel electrophoresis and visualized with ethidium bromide staining. The arrows indicate the position and size of the amplified products.

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

    Article Title: Inhibition of HPV-16 E6/E7 immortalization of normal keratinocytes by hairpin ribozymes

    doi:

    Figure Lengend Snippet: Short term growth inhibition of HPV-16 E6/E7-transfected HKc by cis-expressed R434. ( A ) HKc were transfected with the pCR16HH, pCR16E6/E7RZ, or pCR16E6/E7RZi constructs and were kept in G418 (200 μg/ml) for 2 wk, and 10 5 cells were seeded for counting in 6-well dishes. The graphics are the mean of three experiments. ( B ) RT-PCR analysis of RNA from HKc transfected with HPV-16 E6/E7 (pCR16HH) and cis-expressed active and inactive ribozymes (pCR16E6/E7RZ and pCR16E6/E7RZi, respectively). Total RNA (1 μg) was subjected to a coupled RT-PCR reaction with primers specific to both sides of the ribozyme cleavage site as described in Material and Methods . A contamination control without reverse transcriptase (control) was included. Separate RT-PCR reactions were performed by using the same RNA sample with primers specific to the human β-actin gene to show RNA integrity. The HPV-16 E6/E7-amplified products (492 and 326 bp) were separated through agarose gel electrophoresis and visualized with ethidium bromide staining. The arrows indicate the position and size of the amplified products.

    Article Snippet: HPV-16 E6/E7 cDNA was produced from 1 μg of total RNA using the Superscript II One Shot kit (Life Technologies).

    Techniques: Inhibition, Transfection, Construct, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining