Structured Review

Millipore rna
Log–log scatter plot depicting the normalized average difference (Avg Diff) intensity values for all present called probe sets used to monitor expression in E.coli when grown to mid log phase in rich LB medium. Diagonal lines in the graph represent 2-, 3-, 5- and 10-old variation between the compared expression experiments. ( A ) Scatter plot comparing the levels of expression in duplicate experiments using the <t>cDNA</t> sample preparation method and ( B ) scatter plot comparing the levels of expression in duplicate experiments using the direct labeling of enriched <t>RNA</t> method. ( C ) Scatter plot comparing the expression levels from the direct labeled enriched RNA method with the cDNA sample.
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Images

1) Product Images from "Prokaryotic RNA preparation methods useful for high density array analysis: comparison of two approaches"

Article Title: Prokaryotic RNA preparation methods useful for high density array analysis: comparison of two approaches

Journal: Nucleic Acids Research

doi:

Log–log scatter plot depicting the normalized average difference (Avg Diff) intensity values for all present called probe sets used to monitor expression in E.coli when grown to mid log phase in rich LB medium. Diagonal lines in the graph represent 2-, 3-, 5- and 10-old variation between the compared expression experiments. ( A ) Scatter plot comparing the levels of expression in duplicate experiments using the cDNA sample preparation method and ( B ) scatter plot comparing the levels of expression in duplicate experiments using the direct labeling of enriched RNA method. ( C ) Scatter plot comparing the expression levels from the direct labeled enriched RNA method with the cDNA sample.
Figure Legend Snippet: Log–log scatter plot depicting the normalized average difference (Avg Diff) intensity values for all present called probe sets used to monitor expression in E.coli when grown to mid log phase in rich LB medium. Diagonal lines in the graph represent 2-, 3-, 5- and 10-old variation between the compared expression experiments. ( A ) Scatter plot comparing the levels of expression in duplicate experiments using the cDNA sample preparation method and ( B ) scatter plot comparing the levels of expression in duplicate experiments using the direct labeling of enriched RNA method. ( C ) Scatter plot comparing the expression levels from the direct labeled enriched RNA method with the cDNA sample.

Techniques Used: Expressing, Sample Prep, Labeling

Slot blot analysis of selected genes. Total RNA and synthesized cDNA was spotted in equivalent amounts (1 µg). After hybridization with a labeled PCR fragment and staining, the intensities were measured. The relative change in intensities is shown as the fold change.
Figure Legend Snippet: Slot blot analysis of selected genes. Total RNA and synthesized cDNA was spotted in equivalent amounts (1 µg). After hybridization with a labeled PCR fragment and staining, the intensities were measured. The relative change in intensities is shown as the fold change.

Techniques Used: Dot Blot, Synthesized, Hybridization, Labeling, Polymerase Chain Reaction, Staining

2) Product Images from "Refined RIP-seq protocol for epitranscriptome analysis with low input materials"

Article Title: Refined RIP-seq protocol for epitranscriptome analysis with low input materials

Journal: PLoS Biology

doi: 10.1371/journal.pbio.2006092

Low/high salt-washing method outperforms competitive elution method. (A) Schematic diagram of 3 strategies of m 6 A MeRIP. (B) S/N ratio of GLuc/CLuc was highest in Method II using low/high salt washing. An RNA mixture containing equal amounts of the m 6 A modified control RNA GLuc, the unmodified control RNA CLuc, and NEB antibody were used for m6A MeRIP. (C) S/N ratio of GLuc/CLuc was further increased in a second round of IP using Method II. S/N ratio of GLuc/CLuc (panel D) and SETD7 / GAPDH . CLuc, unmodified control RNA; GLuc, m 6 A-modified control RNA; IP, immunoprecipitation; m 6 A, N6-Methyladenosine; m 6 A MeRIP, m 6 A RNA immunoprecipitation followed by high-throughput sequencing; NEB, New England Biolabs; S/N, signal-to-noise.
Figure Legend Snippet: Low/high salt-washing method outperforms competitive elution method. (A) Schematic diagram of 3 strategies of m 6 A MeRIP. (B) S/N ratio of GLuc/CLuc was highest in Method II using low/high salt washing. An RNA mixture containing equal amounts of the m 6 A modified control RNA GLuc, the unmodified control RNA CLuc, and NEB antibody were used for m6A MeRIP. (C) S/N ratio of GLuc/CLuc was further increased in a second round of IP using Method II. S/N ratio of GLuc/CLuc (panel D) and SETD7 / GAPDH . CLuc, unmodified control RNA; GLuc, m 6 A-modified control RNA; IP, immunoprecipitation; m 6 A, N6-Methyladenosine; m 6 A MeRIP, m 6 A RNA immunoprecipitation followed by high-throughput sequencing; NEB, New England Biolabs; S/N, signal-to-noise.

Techniques Used: Modification, Immunoprecipitation, Next-Generation Sequencing

Comparison of 3 different m 6 A antibodies for MeRIP. (A) S/N ratio of GLuc/CLuc and SETD7 / GAPDH with different antibodies. The amount of 32 μg total RNA from human lung cancer cell line A549 with spiked-in control RNA GLuc and CLuc was used for m 6 A MeRIP using Method II. (B) m 6 A peak signals of SETD7 transcripts in 3 MeRIP-seq libraries. (C) Overlap of m 6 A peaks from the SySy, NEB, and Millipore libraries. (D) Number of m 6 A peaks called by subsampling to different read depths with different antibodies. (E) The percentages of m 6 A peaks in 5 nonoverlapping transcript segments: TSS; 5’UTR; CDS; stop codon; and 3’UTR. (F) Metagene profiles depicting sequence coverage in windows surrounding the TSS and stop codon demonstrated that m 6 . CDS, coding sequence; CLuc, unmodified control RNA; GLuc, m 6 A-modified control RNA; m 6 A, N6-Methyladenosine; m 6 A MeRIP, m 6 A RNA immunoprecipitation followed by high-throughput sequencing; NEB, New England Biolabs; S/N, signal-to-noise; SySy, Synaptic Systems; TSS, transcription start site; UTR, untranslated region.
Figure Legend Snippet: Comparison of 3 different m 6 A antibodies for MeRIP. (A) S/N ratio of GLuc/CLuc and SETD7 / GAPDH with different antibodies. The amount of 32 μg total RNA from human lung cancer cell line A549 with spiked-in control RNA GLuc and CLuc was used for m 6 A MeRIP using Method II. (B) m 6 A peak signals of SETD7 transcripts in 3 MeRIP-seq libraries. (C) Overlap of m 6 A peaks from the SySy, NEB, and Millipore libraries. (D) Number of m 6 A peaks called by subsampling to different read depths with different antibodies. (E) The percentages of m 6 A peaks in 5 nonoverlapping transcript segments: TSS; 5’UTR; CDS; stop codon; and 3’UTR. (F) Metagene profiles depicting sequence coverage in windows surrounding the TSS and stop codon demonstrated that m 6 . CDS, coding sequence; CLuc, unmodified control RNA; GLuc, m 6 A-modified control RNA; m 6 A, N6-Methyladenosine; m 6 A MeRIP, m 6 A RNA immunoprecipitation followed by high-throughput sequencing; NEB, New England Biolabs; S/N, signal-to-noise; SySy, Synaptic Systems; TSS, transcription start site; UTR, untranslated region.

Techniques Used: Sequencing, Modification, Immunoprecipitation, Next-Generation Sequencing

Optimized m 6 A MeRIP-seq protocol worked well starting with 2 μg total RNA. (A) MeRIP efficiency decreases with the reduction of starting RNA amount. (B) At the same sequencing depth, the total number of m 6 A peaks identified increased with the increase of starting RNA amount. The “unique” and “overlapped with 2 μg” peaks indicate the peak number compared to 2 μg. (C) The average RNA expression level of the transcripts with unique m 6 A peaks identified in the 32-μg library was significantly lower than that of overlapping m 6 A peaks; *** p
Figure Legend Snippet: Optimized m 6 A MeRIP-seq protocol worked well starting with 2 μg total RNA. (A) MeRIP efficiency decreases with the reduction of starting RNA amount. (B) At the same sequencing depth, the total number of m 6 A peaks identified increased with the increase of starting RNA amount. The “unique” and “overlapped with 2 μg” peaks indicate the peak number compared to 2 μg. (C) The average RNA expression level of the transcripts with unique m 6 A peaks identified in the 32-μg library was significantly lower than that of overlapping m 6 A peaks; *** p

Techniques Used: Sequencing, RNA Expression

3) Product Images from "Nucleic Acid Content in Crustacean Zooplankton: Bridging Metabolic and Stoichiometric Predictions"

Article Title: Nucleic Acid Content in Crustacean Zooplankton: Bridging Metabolic and Stoichiometric Predictions

Journal: PLoS ONE

doi: 10.1371/journal.pone.0086493

Body size, nucleic acid content, RNA:DNA ratio and total phosphorus content for investigated zooplankton species. (A) Body size, (B) nucleic acid (NA) content (% of dry weight, %NA), (C) RNA:DNA ratio, (D) total phosphorus (P) content (% of dry weight, %P) for each crustacean species of copepods [ Cyclops (C.) abyssorum , Diaptomus (D.) cyaneus , Eudiaptomus (E.) vulgaris , Mixodiaptomus (M.) laciniatus ] and cladocerans [ Alona (A.) affinis, Daphnia (D.) longispina , Daphnia (D.) pulicaria ]. Insets represent (A) body size, (B) %NA, (C) RNA:DNA ratio, and (D) %P for species grouped into copepods (copepoda) and cladocerans (cladocera). Columns are mean values, and error bars are standard deviations. Note that lack of %P data for Eudiaptomus vulgaris was due to the inability to collect a sufficient number of individuals for reliable estimations.
Figure Legend Snippet: Body size, nucleic acid content, RNA:DNA ratio and total phosphorus content for investigated zooplankton species. (A) Body size, (B) nucleic acid (NA) content (% of dry weight, %NA), (C) RNA:DNA ratio, (D) total phosphorus (P) content (% of dry weight, %P) for each crustacean species of copepods [ Cyclops (C.) abyssorum , Diaptomus (D.) cyaneus , Eudiaptomus (E.) vulgaris , Mixodiaptomus (M.) laciniatus ] and cladocerans [ Alona (A.) affinis, Daphnia (D.) longispina , Daphnia (D.) pulicaria ]. Insets represent (A) body size, (B) %NA, (C) RNA:DNA ratio, and (D) %P for species grouped into copepods (copepoda) and cladocerans (cladocera). Columns are mean values, and error bars are standard deviations. Note that lack of %P data for Eudiaptomus vulgaris was due to the inability to collect a sufficient number of individuals for reliable estimations.

Techniques Used:

Inter- and intraspecific variabilities in nucleic acid content, RNA:DNA ratio, and total phosphorus content. The diagram illustrates the variability (error bars) in (A) nucleic acid (NA) content (% of dry weight, %NA), (B) RNA:DNA ratio, and (C) total phosphorus (P) content (% of dry weight, %P). Interspecific and interstage variabilities were obtained by calculating the standard deviation of means for all species and stages of the copepod Mixodiaptomus laciniatus , respectively. Intrastage variability for Mixodiaptomus laciniatus was obtained by calculating mean stage-specific standard deviations. Circles are mean values.
Figure Legend Snippet: Inter- and intraspecific variabilities in nucleic acid content, RNA:DNA ratio, and total phosphorus content. The diagram illustrates the variability (error bars) in (A) nucleic acid (NA) content (% of dry weight, %NA), (B) RNA:DNA ratio, and (C) total phosphorus (P) content (% of dry weight, %P). Interspecific and interstage variabilities were obtained by calculating the standard deviation of means for all species and stages of the copepod Mixodiaptomus laciniatus , respectively. Intrastage variability for Mixodiaptomus laciniatus was obtained by calculating mean stage-specific standard deviations. Circles are mean values.

Techniques Used: Standard Deviation

Nucleic acid content, RNA:DNA ratio, total phosphorus content, and relative phosphorus investment indices for the copepod Mixodiaptomus laciniatus . (A) Nucleic acid (NA) content (% of dry weight, %NA), (B) RNA:DNA ratio, (C) total phosphorus (P) content (% of dry weight, %P), and (D) relative P investment index (RPII) for RNA (RPII RNA ), DNA (RPII DNA ), and total NAs (RPII TNAs ) of Mixodiaptomus laciniatus stages. Insets represent these variables as a function of gender in adulthood and reproductive status in adult females. Columns in A-C and circles in D are mean values. Error bars represent standard deviations for nauplius (NI-NVI), copepodite (CI-CV) and adult (ADUm, adult male; ADUf, adult female; Non-oviger. ADUf, non-ovigerous adult female; Oviger. ADUf, ovigerous adult female) stages.
Figure Legend Snippet: Nucleic acid content, RNA:DNA ratio, total phosphorus content, and relative phosphorus investment indices for the copepod Mixodiaptomus laciniatus . (A) Nucleic acid (NA) content (% of dry weight, %NA), (B) RNA:DNA ratio, (C) total phosphorus (P) content (% of dry weight, %P), and (D) relative P investment index (RPII) for RNA (RPII RNA ), DNA (RPII DNA ), and total NAs (RPII TNAs ) of Mixodiaptomus laciniatus stages. Insets represent these variables as a function of gender in adulthood and reproductive status in adult females. Columns in A-C and circles in D are mean values. Error bars represent standard deviations for nauplius (NI-NVI), copepodite (CI-CV) and adult (ADUm, adult male; ADUf, adult female; Non-oviger. ADUf, non-ovigerous adult female; Oviger. ADUf, ovigerous adult female) stages.

Techniques Used:

4) Product Images from "m1A and m1G Potently Disrupt A-RNA Structure Due to the Intrinsic Instability of Hoogsteen Base Pairs"

Article Title: m1A and m1G Potently Disrupt A-RNA Structure Due to the Intrinsic Instability of Hoogsteen Base Pairs

Journal: Nature structural & molecular biology

doi: 10.1038/nsmb.3270

Different propensities to form HG bps in B-DNA and A-RNA enable contrasting roles at the genome and transcriptome level. ( a ) In DNA, m 1 dA or m 1 dG damage is absorbed as HG bps that can be recognized by repair enzymes (in red). Had B-DNA lacked the ability to form HG bps, damage could result in duplex melting and genomic instability. In RNA, post-transcriptional modifications resulting in m 1 rA and m 1 rG block both WC and HG pairing, melting or modulating RNA secondary structure to favor functional states or effect epigenetic regulation. Had A-RNA had the ability to form HG, the m 1 rA and m 1 rG would form HG bps and potentially fail to more significantly alter RNA structure and function. ( b ) Highly conserved m 1 rA9 in human mitochondrial tRNA Lys blocks rA–rU WC base pairing and stabilizes native tRNA structure in which m 1 rA9 is in a single strand 58 . The m 1 rA9 modification would not stabilize native tRNA structure if it were simply absorbed as a HG bp. ( c ) Highly conserved m 1 rG37 next to the anti-codon loop 37 blocks base pairing between m 1 rG37 and the first rC in the codon and prevents +1 frameshifting in tRNA Pro , which could occur if m 1 rG37 formed stable HG bp with rC. ( d ) Proposed mechanism for m 1 rA enhanced translation through destabilization of secondary structure in the 5′ UTR of mRNA.
Figure Legend Snippet: Different propensities to form HG bps in B-DNA and A-RNA enable contrasting roles at the genome and transcriptome level. ( a ) In DNA, m 1 dA or m 1 dG damage is absorbed as HG bps that can be recognized by repair enzymes (in red). Had B-DNA lacked the ability to form HG bps, damage could result in duplex melting and genomic instability. In RNA, post-transcriptional modifications resulting in m 1 rA and m 1 rG block both WC and HG pairing, melting or modulating RNA secondary structure to favor functional states or effect epigenetic regulation. Had A-RNA had the ability to form HG, the m 1 rA and m 1 rG would form HG bps and potentially fail to more significantly alter RNA structure and function. ( b ) Highly conserved m 1 rA9 in human mitochondrial tRNA Lys blocks rA–rU WC base pairing and stabilizes native tRNA structure in which m 1 rA9 is in a single strand 58 . The m 1 rA9 modification would not stabilize native tRNA structure if it were simply absorbed as a HG bp. ( c ) Highly conserved m 1 rG37 next to the anti-codon loop 37 blocks base pairing between m 1 rG37 and the first rC in the codon and prevents +1 frameshifting in tRNA Pro , which could occur if m 1 rG37 formed stable HG bp with rC. ( d ) Proposed mechanism for m 1 rA enhanced translation through destabilization of secondary structure in the 5′ UTR of mRNA.

Techniques Used: Blocking Assay, Functional Assay, Modification

Absence of detectable WC⇄HG exchange in A-RNA by NMR relaxation dispersion. ( a ) Comparison of A-form RNA (violet) and B-form (blue) DNA double helices. ( b ) WC and HG bps in dynamic equilibrium in B-DNA. Sites used for RD measurements are highlighted in orange. ( c ) A 6 -DNA and hp-A 6 -RNA duplexes with bps targeted in RD measurements highlighted. ( d ) Off-resonance RD profiles showing R 2 + R ex as a function of spin lock offset (Ω 2π −1 Hz, where Ω = Ω obs – ω RF ) and power (ω SL 2π −1 Hz, in insets). Error bars represent experimental uncertainty (one s.d.) estimated from mono-exponential fitting using a Monte-Carlo based method (Methods). Solid line represents a fit to two-state exchange 6 .
Figure Legend Snippet: Absence of detectable WC⇄HG exchange in A-RNA by NMR relaxation dispersion. ( a ) Comparison of A-form RNA (violet) and B-form (blue) DNA double helices. ( b ) WC and HG bps in dynamic equilibrium in B-DNA. Sites used for RD measurements are highlighted in orange. ( c ) A 6 -DNA and hp-A 6 -RNA duplexes with bps targeted in RD measurements highlighted. ( d ) Off-resonance RD profiles showing R 2 + R ex as a function of spin lock offset (Ω 2π −1 Hz, where Ω = Ω obs – ω RF ) and power (ω SL 2π −1 Hz, in insets). Error bars represent experimental uncertainty (one s.d.) estimated from mono-exponential fitting using a Monte-Carlo based method (Methods). Solid line represents a fit to two-state exchange 6 .

Techniques Used: Nuclear Magnetic Resonance

m 1 A and m 1 G do not form HG bps and disrupt A-RNA structure. ( a ) N 1 -methylated purines trap HG bps in B-DNA. NMR chemical shift probes of HG bps are in orange and of purine methylation state in cyan. Arrows indicate characteristic HG NOE cross-peaks. ( b ) Duplexes containing m 1 A or m 1 G (turquoise circles). syn or anti purines deduced by NMR are shown as open and filled letters, respectively. HG and partially melted bps as deduced by NMR are indicated using open and dashed lines, respectively. Residues showing significant chemical shift perturbations or line-broadening due to m 1 A or m 1 G are colored orange and grey, respectively. ( c ) m 1 A or m 1 G induced purine-C1′ chemical shift perturbations (Δω = ω modified – ω unmodified ) in A-RNA (violet) and B-DNA (blue). Shown for comparison are Δω = ω HG – ω WC measured for transient dA–dT HG bps by RD (“RD”) in unmodified DNA duplexes (error bars showing one s.d.) and computed for adenine residues using DFT (Methods). ( d ) NOESY H1′–H8 cross-peaks showing syn purine bases in B-DNA but not A-RNA. Shown for reference is the cytosine base H5–H6 NOE with inter-atomic distance ≈2.5 Å. ( e ) 1D 1 H spectra showing the imino/amino resonances expected for HG type H-bonds in A 6 -DNA m1A and A 6 -DNA m1G but not in methylated RNA at 5°C and 15°C, respectively. ( f ) Example showing m 1 G induced loss of a WC imino resonance (highlighted in a circle) in A 6 -RNA but not A 6 -DNA in 2D NMR spectra. ( g ) Example downfield shifted carbon chemical shifts induced by m 1 A. ( h ) Free energy (ΔΔG) and enthalpy (ΔΔH) destabilization due to m 1 A and m 1 G in DNA (blue) and RNA (violet) duplexes measured by UV melting experiments with error bars, one s.d. (n = 3 independent measurements) (Methods and Supplementary Table 2 ).
Figure Legend Snippet: m 1 A and m 1 G do not form HG bps and disrupt A-RNA structure. ( a ) N 1 -methylated purines trap HG bps in B-DNA. NMR chemical shift probes of HG bps are in orange and of purine methylation state in cyan. Arrows indicate characteristic HG NOE cross-peaks. ( b ) Duplexes containing m 1 A or m 1 G (turquoise circles). syn or anti purines deduced by NMR are shown as open and filled letters, respectively. HG and partially melted bps as deduced by NMR are indicated using open and dashed lines, respectively. Residues showing significant chemical shift perturbations or line-broadening due to m 1 A or m 1 G are colored orange and grey, respectively. ( c ) m 1 A or m 1 G induced purine-C1′ chemical shift perturbations (Δω = ω modified – ω unmodified ) in A-RNA (violet) and B-DNA (blue). Shown for comparison are Δω = ω HG – ω WC measured for transient dA–dT HG bps by RD (“RD”) in unmodified DNA duplexes (error bars showing one s.d.) and computed for adenine residues using DFT (Methods). ( d ) NOESY H1′–H8 cross-peaks showing syn purine bases in B-DNA but not A-RNA. Shown for reference is the cytosine base H5–H6 NOE with inter-atomic distance ≈2.5 Å. ( e ) 1D 1 H spectra showing the imino/amino resonances expected for HG type H-bonds in A 6 -DNA m1A and A 6 -DNA m1G but not in methylated RNA at 5°C and 15°C, respectively. ( f ) Example showing m 1 G induced loss of a WC imino resonance (highlighted in a circle) in A 6 -RNA but not A 6 -DNA in 2D NMR spectra. ( g ) Example downfield shifted carbon chemical shifts induced by m 1 A. ( h ) Free energy (ΔΔG) and enthalpy (ΔΔH) destabilization due to m 1 A and m 1 G in DNA (blue) and RNA (violet) duplexes measured by UV melting experiments with error bars, one s.d. (n = 3 independent measurements) (Methods and Supplementary Table 2 ).

Techniques Used: Methylation, Nuclear Magnetic Resonance, Modification

Source of HG instability in A-RNA. ( a ) Comparison of RD profiles measured in A 6 -DNA rA , A 6 -DNA rG , and A 6 -DNA. Error bars correspond to one s.d. estimated from mono-exponential fitting using a Monte-Carlo based method (Methods). Note that the larger R 2 value in A 6 -DNA rA A16-C8 as compared to A 6 -DNA likely reflects decreased flexibility in rA16. Exchange parameters are shown in Supplementary Fig. 4b . ( b ) Inter-atomic distances (in Å) with unfavorable steric contacts in pink when rotating the purine base 180° around the glycoside bond in WC bps derived from idealized B-DNA and A-RNA duplexes (Methods) to adopt a syn conformation. Shown below are corresponding distance distributions in WC bps derived from X-ray structures of A-RNA (total 146) and B-DNA (total 159) duplexes before (solid line) and following (dashed line) 180° rotation of the purine base. The inter-atomic cut-off distance (grey line) was defined based on the van der Waals radii. ( c ) Relative interaction energy versus χ-angle from biased MD trajectories of A 6 -DNA (blue) and hp-A 6 -RNA (violet). ( d ) Simulation time (ns) versus the global RMSD (Methods) for single A 6 -DNA m1A and hp-A 6 -RNA m1A trajectories depicting the destabilization of the RNA strand within the time of the simulation.
Figure Legend Snippet: Source of HG instability in A-RNA. ( a ) Comparison of RD profiles measured in A 6 -DNA rA , A 6 -DNA rG , and A 6 -DNA. Error bars correspond to one s.d. estimated from mono-exponential fitting using a Monte-Carlo based method (Methods). Note that the larger R 2 value in A 6 -DNA rA A16-C8 as compared to A 6 -DNA likely reflects decreased flexibility in rA16. Exchange parameters are shown in Supplementary Fig. 4b . ( b ) Inter-atomic distances (in Å) with unfavorable steric contacts in pink when rotating the purine base 180° around the glycoside bond in WC bps derived from idealized B-DNA and A-RNA duplexes (Methods) to adopt a syn conformation. Shown below are corresponding distance distributions in WC bps derived from X-ray structures of A-RNA (total 146) and B-DNA (total 159) duplexes before (solid line) and following (dashed line) 180° rotation of the purine base. The inter-atomic cut-off distance (grey line) was defined based on the van der Waals radii. ( c ) Relative interaction energy versus χ-angle from biased MD trajectories of A 6 -DNA (blue) and hp-A 6 -RNA (violet). ( d ) Simulation time (ns) versus the global RMSD (Methods) for single A 6 -DNA m1A and hp-A 6 -RNA m1A trajectories depicting the destabilization of the RNA strand within the time of the simulation.

Techniques Used: Derivative Assay

5) Product Images from "RNA Folding Affects the Recruitment of SR Proteins by Mouse and Human Polypurinic Enhancer Elements in the Fibronectin EDA Exon"

Article Title: RNA Folding Affects the Recruitment of SR Proteins by Mouse and Human Polypurinic Enhancer Elements in the Fibronectin EDA Exon

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.3.1387-1400.2004

UV cross-linking analysis of the wild-type human and mouse sequences (hTot and mTot) and ESE deletion-carrying mutants (hΔ2e and mΔA) with HeLa nuclear extract. Each RNA was labeled with [α- 32 P]UTP and then incubated with approximately 150 μg of HeLa nuclear extract before being subjected to UV cross-linking and digestion with RNase. Samples were then run on an SDS-11% PAGE gel and exposed to BioMax autoradiographic film. The electrophoretic mobility of prestained markers (Broad Range; New England Biolabs) is shown on the left (A). IP was then performed with equal amounts of each UV cross-linked sample shown in panel A with specific MAbs against different SR proteins: SF2/ASF (B, MAb 96), the phosphorylated RS domain (C, MAb 1H4), and SC35 (D, anti-SC35). The mobility of the SR proteins is indicated on the left. The SF2/ASF antibody immunoprecipitates more than one protein band owing to the presence of differently phosphorylated forms, as specified by the manufacturer.
Figure Legend Snippet: UV cross-linking analysis of the wild-type human and mouse sequences (hTot and mTot) and ESE deletion-carrying mutants (hΔ2e and mΔA) with HeLa nuclear extract. Each RNA was labeled with [α- 32 P]UTP and then incubated with approximately 150 μg of HeLa nuclear extract before being subjected to UV cross-linking and digestion with RNase. Samples were then run on an SDS-11% PAGE gel and exposed to BioMax autoradiographic film. The electrophoretic mobility of prestained markers (Broad Range; New England Biolabs) is shown on the left (A). IP was then performed with equal amounts of each UV cross-linked sample shown in panel A with specific MAbs against different SR proteins: SF2/ASF (B, MAb 96), the phosphorylated RS domain (C, MAb 1H4), and SC35 (D, anti-SC35). The mobility of the SR proteins is indicated on the left. The SF2/ASF antibody immunoprecipitates more than one protein band owing to the presence of differently phosphorylated forms, as specified by the manufacturer.

Techniques Used: Labeling, Incubation, Polyacrylamide Gel Electrophoresis

Secondary-structure analysis of the ESE region in the mTot (A), mΔB5 (B), and mΔB6 (C) constructs. The in vitro-transcribed RNAs were enzymatically digested with S1 nuclease and RNases T1 and V1 and reverse transcribed, and the RT products were separated on a sequencing polyacrylamide gel. No enzyme was added to the reaction mixture in lane N. The regions containing the ESE and ESS elements are shown by a vertical line. The upper part of each panel shows the enzymatic analysis of RNA templates of mTot, mΔB5, and mΔB6 constructs. The bottom part reports the cleavages on the optimized secondary-structure predictions. Squares, circles, and triangles indicate S1 nuclease and RNase T1 and V1 cleavage sites, respectively. Black, shaded, and white symbols indicate high, medium, and low cleavage intensities, respectively. The arrow indicates the position of G150, which is present in a stem position in the mTot RNA but shifts to a loop configuration in the mΔB6 mutant.
Figure Legend Snippet: Secondary-structure analysis of the ESE region in the mTot (A), mΔB5 (B), and mΔB6 (C) constructs. The in vitro-transcribed RNAs were enzymatically digested with S1 nuclease and RNases T1 and V1 and reverse transcribed, and the RT products were separated on a sequencing polyacrylamide gel. No enzyme was added to the reaction mixture in lane N. The regions containing the ESE and ESS elements are shown by a vertical line. The upper part of each panel shows the enzymatic analysis of RNA templates of mTot, mΔB5, and mΔB6 constructs. The bottom part reports the cleavages on the optimized secondary-structure predictions. Squares, circles, and triangles indicate S1 nuclease and RNase T1 and V1 cleavage sites, respectively. Black, shaded, and white symbols indicate high, medium, and low cleavage intensities, respectively. The arrow indicates the position of G150, which is present in a stem position in the mTot RNA but shifts to a loop configuration in the mΔB6 mutant.

Techniques Used: Construct, In Vitro, Sequencing, Mutagenesis

Enzymatic determination of the RNA secondary structure of the mouse EDA exon. In vitro-transcribed mTot RNA was enzymatically digested with S1 nuclease and RNases T1 and V1 and reverse transcribed with an antisense primer. The RT products were separated on a sequencing polyacrylamide gel. A sequencing reaction (numbered according to the exon length) performed with the same primer was run in parallel with the cleavages in order to precisely determine the cleavage sites. In order to cover the whole exon length, short (A) and long (B) runs were performed. Squares, circles, and triangles indicate S1 nuclease and RNase T1 and V1 cleavage sites, respectively. Black, shaded, and white symbols indicate high, medium, and low cleavage intensities, respectively. No enzyme was added to the reaction mixture in lane N. The mESS and mESE positions are marked on the right of each sequencing reaction.
Figure Legend Snippet: Enzymatic determination of the RNA secondary structure of the mouse EDA exon. In vitro-transcribed mTot RNA was enzymatically digested with S1 nuclease and RNases T1 and V1 and reverse transcribed with an antisense primer. The RT products were separated on a sequencing polyacrylamide gel. A sequencing reaction (numbered according to the exon length) performed with the same primer was run in parallel with the cleavages in order to precisely determine the cleavage sites. In order to cover the whole exon length, short (A) and long (B) runs were performed. Squares, circles, and triangles indicate S1 nuclease and RNase T1 and V1 cleavage sites, respectively. Black, shaded, and white symbols indicate high, medium, and low cleavage intensities, respectively. No enzyme was added to the reaction mixture in lane N. The mESS and mESE positions are marked on the right of each sequencing reaction.

Techniques Used: In Vitro, Sequencing

Comparison of the RNA secondary structure of the human (A) and mouse (B) wild-type EDA sequences. The two structures were optimized by computer-assisted RNA modeling, and the respective ESE and ESS elements are circled to facilitate their localization. Squares, circles, and triangles indicate S1 nuclease and RNase T1 and V1 cleavage sites, respectively. Black, shaded, and white symbols indicate high, medium, and low cleavage intensities, respectively. The asterisks in the mouse secondary-structure model mark the nucleotide differences between the mouse and human nucleotide sequences.
Figure Legend Snippet: Comparison of the RNA secondary structure of the human (A) and mouse (B) wild-type EDA sequences. The two structures were optimized by computer-assisted RNA modeling, and the respective ESE and ESS elements are circled to facilitate their localization. Squares, circles, and triangles indicate S1 nuclease and RNase T1 and V1 cleavage sites, respectively. Black, shaded, and white symbols indicate high, medium, and low cleavage intensities, respectively. The asterisks in the mouse secondary-structure model mark the nucleotide differences between the mouse and human nucleotide sequences.

Techniques Used:

UV cross-linking analysis of human and mouse mutants with HeLa nuclear extract. Each RNA was labeled with [α- 32 P]UTP and then incubated with HeLa nuclear extract before being subjected to UV cross-linking and digestion with RNase. Samples were then run on an SDS-11% PAGE gel and exposed to BioMax autoradiographic film. The electrophoretic mobility of prestained molecular size markers (Broad Range; New England Biolabs) is shown on the left (A). IP was then performed with the same amount of each UV cross-linked sample shown in panel A with specific MAbs against different SR proteins: SF2/ASF (B, MAb 96), the phosphorylated RS domain (C, MAb 1H4), and SC35 (D, anti-SC35). The mobility of the SR proteins is indicated on the left. The SF2/ASF antibody immunoprecipitates more than one protein band owing to the presence of differently phosphorylated forms, as specified by the manufacturer.
Figure Legend Snippet: UV cross-linking analysis of human and mouse mutants with HeLa nuclear extract. Each RNA was labeled with [α- 32 P]UTP and then incubated with HeLa nuclear extract before being subjected to UV cross-linking and digestion with RNase. Samples were then run on an SDS-11% PAGE gel and exposed to BioMax autoradiographic film. The electrophoretic mobility of prestained molecular size markers (Broad Range; New England Biolabs) is shown on the left (A). IP was then performed with the same amount of each UV cross-linked sample shown in panel A with specific MAbs against different SR proteins: SF2/ASF (B, MAb 96), the phosphorylated RS domain (C, MAb 1H4), and SC35 (D, anti-SC35). The mobility of the SR proteins is indicated on the left. The SF2/ASF antibody immunoprecipitates more than one protein band owing to the presence of differently phosphorylated forms, as specified by the manufacturer.

Techniques Used: Labeling, Incubation, Polyacrylamide Gel Electrophoresis

6) Product Images from "RNA Folding Affects the Recruitment of SR Proteins by Mouse and Human Polypurinic Enhancer Elements in the Fibronectin EDA Exon"

Article Title: RNA Folding Affects the Recruitment of SR Proteins by Mouse and Human Polypurinic Enhancer Elements in the Fibronectin EDA Exon

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.3.1387-1400.2004

UV cross-linking analysis of the wild-type human and mouse sequences (hTot and mTot) and ESE deletion-carrying mutants (hΔ2e and mΔA) with HeLa nuclear extract. Each RNA was labeled with [α- 32 P]UTP and then incubated with approximately 150 μg of HeLa nuclear extract before being subjected to UV cross-linking and digestion with RNase. Samples were then run on an SDS-11% PAGE gel and exposed to BioMax autoradiographic film. The electrophoretic mobility of prestained markers (Broad Range; New England Biolabs) is shown on the left (A). IP was then performed with equal amounts of each UV cross-linked sample shown in panel A with specific MAbs against different SR proteins: SF2/ASF (B, MAb 96), the phosphorylated RS domain (C, MAb 1H4), and SC35 (D, anti-SC35). The mobility of the SR proteins is indicated on the left. The SF2/ASF antibody immunoprecipitates more than one protein band owing to the presence of differently phosphorylated forms, as specified by the manufacturer.
Figure Legend Snippet: UV cross-linking analysis of the wild-type human and mouse sequences (hTot and mTot) and ESE deletion-carrying mutants (hΔ2e and mΔA) with HeLa nuclear extract. Each RNA was labeled with [α- 32 P]UTP and then incubated with approximately 150 μg of HeLa nuclear extract before being subjected to UV cross-linking and digestion with RNase. Samples were then run on an SDS-11% PAGE gel and exposed to BioMax autoradiographic film. The electrophoretic mobility of prestained markers (Broad Range; New England Biolabs) is shown on the left (A). IP was then performed with equal amounts of each UV cross-linked sample shown in panel A with specific MAbs against different SR proteins: SF2/ASF (B, MAb 96), the phosphorylated RS domain (C, MAb 1H4), and SC35 (D, anti-SC35). The mobility of the SR proteins is indicated on the left. The SF2/ASF antibody immunoprecipitates more than one protein band owing to the presence of differently phosphorylated forms, as specified by the manufacturer.

Techniques Used: Labeling, Incubation, Polyacrylamide Gel Electrophoresis

Secondary-structure analysis of the ESE region in the mTot (A), mΔB5 (B), and mΔB6 (C) constructs. The in vitro-transcribed RNAs were enzymatically digested with S1 nuclease and RNases T1 and V1 and reverse transcribed, and the RT products were separated on a sequencing polyacrylamide gel. No enzyme was added to the reaction mixture in lane N. The regions containing the ESE and ESS elements are shown by a vertical line. The upper part of each panel shows the enzymatic analysis of RNA templates of mTot, mΔB5, and mΔB6 constructs. The bottom part reports the cleavages on the optimized secondary-structure predictions. Squares, circles, and triangles indicate S1 nuclease and RNase T1 and V1 cleavage sites, respectively. Black, shaded, and white symbols indicate high, medium, and low cleavage intensities, respectively. The arrow indicates the position of G150, which is present in a stem position in the mTot RNA but shifts to a loop configuration in the mΔB6 mutant.
Figure Legend Snippet: Secondary-structure analysis of the ESE region in the mTot (A), mΔB5 (B), and mΔB6 (C) constructs. The in vitro-transcribed RNAs were enzymatically digested with S1 nuclease and RNases T1 and V1 and reverse transcribed, and the RT products were separated on a sequencing polyacrylamide gel. No enzyme was added to the reaction mixture in lane N. The regions containing the ESE and ESS elements are shown by a vertical line. The upper part of each panel shows the enzymatic analysis of RNA templates of mTot, mΔB5, and mΔB6 constructs. The bottom part reports the cleavages on the optimized secondary-structure predictions. Squares, circles, and triangles indicate S1 nuclease and RNase T1 and V1 cleavage sites, respectively. Black, shaded, and white symbols indicate high, medium, and low cleavage intensities, respectively. The arrow indicates the position of G150, which is present in a stem position in the mTot RNA but shifts to a loop configuration in the mΔB6 mutant.

Techniques Used: Construct, In Vitro, Sequencing, Mutagenesis

Enzymatic determination of the RNA secondary structure of the mouse EDA exon. In vitro-transcribed mTot RNA was enzymatically digested with S1 nuclease and RNases T1 and V1 and reverse transcribed with an antisense primer. The RT products were separated on a sequencing polyacrylamide gel. A sequencing reaction (numbered according to the exon length) performed with the same primer was run in parallel with the cleavages in order to precisely determine the cleavage sites. In order to cover the whole exon length, short (A) and long (B) runs were performed. Squares, circles, and triangles indicate S1 nuclease and RNase T1 and V1 cleavage sites, respectively. Black, shaded, and white symbols indicate high, medium, and low cleavage intensities, respectively. No enzyme was added to the reaction mixture in lane N. The mESS and mESE positions are marked on the right of each sequencing reaction.
Figure Legend Snippet: Enzymatic determination of the RNA secondary structure of the mouse EDA exon. In vitro-transcribed mTot RNA was enzymatically digested with S1 nuclease and RNases T1 and V1 and reverse transcribed with an antisense primer. The RT products were separated on a sequencing polyacrylamide gel. A sequencing reaction (numbered according to the exon length) performed with the same primer was run in parallel with the cleavages in order to precisely determine the cleavage sites. In order to cover the whole exon length, short (A) and long (B) runs were performed. Squares, circles, and triangles indicate S1 nuclease and RNase T1 and V1 cleavage sites, respectively. Black, shaded, and white symbols indicate high, medium, and low cleavage intensities, respectively. No enzyme was added to the reaction mixture in lane N. The mESS and mESE positions are marked on the right of each sequencing reaction.

Techniques Used: In Vitro, Sequencing

Comparison of the RNA secondary structure of the human (A) and mouse (B) wild-type EDA sequences. The two structures were optimized by computer-assisted RNA modeling, and the respective ESE and ESS elements are circled to facilitate their localization. Squares, circles, and triangles indicate S1 nuclease and RNase T1 and V1 cleavage sites, respectively. Black, shaded, and white symbols indicate high, medium, and low cleavage intensities, respectively. The asterisks in the mouse secondary-structure model mark the nucleotide differences between the mouse and human nucleotide sequences.
Figure Legend Snippet: Comparison of the RNA secondary structure of the human (A) and mouse (B) wild-type EDA sequences. The two structures were optimized by computer-assisted RNA modeling, and the respective ESE and ESS elements are circled to facilitate their localization. Squares, circles, and triangles indicate S1 nuclease and RNase T1 and V1 cleavage sites, respectively. Black, shaded, and white symbols indicate high, medium, and low cleavage intensities, respectively. The asterisks in the mouse secondary-structure model mark the nucleotide differences between the mouse and human nucleotide sequences.

Techniques Used:

UV cross-linking analysis of human and mouse mutants with HeLa nuclear extract. Each RNA was labeled with [α- 32 P]UTP and then incubated with HeLa nuclear extract before being subjected to UV cross-linking and digestion with RNase. Samples were then run on an SDS-11% PAGE gel and exposed to BioMax autoradiographic film. The electrophoretic mobility of prestained molecular size markers (Broad Range; New England Biolabs) is shown on the left (A). IP was then performed with the same amount of each UV cross-linked sample shown in panel A with specific MAbs against different SR proteins: SF2/ASF (B, MAb 96), the phosphorylated RS domain (C, MAb 1H4), and SC35 (D, anti-SC35). The mobility of the SR proteins is indicated on the left. The SF2/ASF antibody immunoprecipitates more than one protein band owing to the presence of differently phosphorylated forms, as specified by the manufacturer.
Figure Legend Snippet: UV cross-linking analysis of human and mouse mutants with HeLa nuclear extract. Each RNA was labeled with [α- 32 P]UTP and then incubated with HeLa nuclear extract before being subjected to UV cross-linking and digestion with RNase. Samples were then run on an SDS-11% PAGE gel and exposed to BioMax autoradiographic film. The electrophoretic mobility of prestained molecular size markers (Broad Range; New England Biolabs) is shown on the left (A). IP was then performed with the same amount of each UV cross-linked sample shown in panel A with specific MAbs against different SR proteins: SF2/ASF (B, MAb 96), the phosphorylated RS domain (C, MAb 1H4), and SC35 (D, anti-SC35). The mobility of the SR proteins is indicated on the left. The SF2/ASF antibody immunoprecipitates more than one protein band owing to the presence of differently phosphorylated forms, as specified by the manufacturer.

Techniques Used: Labeling, Incubation, Polyacrylamide Gel Electrophoresis

7) Product Images from "Prolonged Induction of miR-212/132 and REST Expression in Rat Striatum Following Cocaine Self-Administration"

Article Title: Prolonged Induction of miR-212/132 and REST Expression in Rat Striatum Following Cocaine Self-Administration

Journal: Molecular Neurobiology

doi: 10.1007/s12035-016-9817-2

A putative mechanism for the interaction between miRNA and the Ago2, Pum2, and REST proteins. Transcription : (i) REST interacts with mSin3A and CoREST, which, in turn, recruits the HDAC complex (HDAC1 and HDAC2). Additionally, CoREST recruits LSD1. The HDACs deacetylate and LSD1 demethylates the lysine residues of the nucleosomal histone core; these events condense the chromatin and limit the accessibility to the DNA, resulting in the inhibition of translation (indicated schematically by an X) [ 54 ]. (ii) REST recruits TET3 and stimulates its hydroxylase activity to form 5hmC. Subsequently, TET3 interacts with histone writers (e.g., NSD3, NSD2, and SETD2) and mediates histone H3 methylation [ 55 ]. This mechanism may be involved in the expression of either miR-212/132 cluster or miR-132 alone . miRNA biogenesis and function : (i) Ago2 interacts with Dicer and TRBP to efficiently catalyze pre-miRNA cleavage; (ii) Pum2 mediates the activity of the RISC complex in neurons [ 56 ], and, together with RISC and miRNAs, Ago2 participates in gene silencing. Neuronal activity : (i) Under low-activity conditions, Ago2 and its associated RNA-binding proteins (FMR1 and Pum2) suppress protein production by blocking cap-dependent translation. (ii) Following synaptic stimulation, posttranslational modifications of Ago2, FRM1, and Pum2 (?) are involved in the remodeling of the miRISC complex, leading to efficient mRNA translation [ 56 ]. Protein relocalization : (i) P-bodies may regulate local transcription in the synapses by managing miRNA-mediated regulation of mRNA transcription. In the P-bodies, the mRNAs are targeted for storage and/or degradation. Upon synapse activation, a portion of the mRNAs stored in the dendritic P-bodies may reenter the translational pathway [ 57 ]. Phosphorylation of the Ago2 protein (at Ser-387) facilitates its localization to the P-body [ 58 ]. (ii) Stress granules (SGs) are generated in response to stressful environmental conditions and are another reservoir of mRNAs. SGs contain mRNAs, translation initiation factors (e.g., eIF3, eIF4A, and eIF4G), small ribosomal subunits and poly(A) binding protein (PABP) proteins as well as the mRNA decay machinery. OS represses the translation of important neuronal genes and influences Ago2 and Pum2 accumulation in the SGs
Figure Legend Snippet: A putative mechanism for the interaction between miRNA and the Ago2, Pum2, and REST proteins. Transcription : (i) REST interacts with mSin3A and CoREST, which, in turn, recruits the HDAC complex (HDAC1 and HDAC2). Additionally, CoREST recruits LSD1. The HDACs deacetylate and LSD1 demethylates the lysine residues of the nucleosomal histone core; these events condense the chromatin and limit the accessibility to the DNA, resulting in the inhibition of translation (indicated schematically by an X) [ 54 ]. (ii) REST recruits TET3 and stimulates its hydroxylase activity to form 5hmC. Subsequently, TET3 interacts with histone writers (e.g., NSD3, NSD2, and SETD2) and mediates histone H3 methylation [ 55 ]. This mechanism may be involved in the expression of either miR-212/132 cluster or miR-132 alone . miRNA biogenesis and function : (i) Ago2 interacts with Dicer and TRBP to efficiently catalyze pre-miRNA cleavage; (ii) Pum2 mediates the activity of the RISC complex in neurons [ 56 ], and, together with RISC and miRNAs, Ago2 participates in gene silencing. Neuronal activity : (i) Under low-activity conditions, Ago2 and its associated RNA-binding proteins (FMR1 and Pum2) suppress protein production by blocking cap-dependent translation. (ii) Following synaptic stimulation, posttranslational modifications of Ago2, FRM1, and Pum2 (?) are involved in the remodeling of the miRISC complex, leading to efficient mRNA translation [ 56 ]. Protein relocalization : (i) P-bodies may regulate local transcription in the synapses by managing miRNA-mediated regulation of mRNA transcription. In the P-bodies, the mRNAs are targeted for storage and/or degradation. Upon synapse activation, a portion of the mRNAs stored in the dendritic P-bodies may reenter the translational pathway [ 57 ]. Phosphorylation of the Ago2 protein (at Ser-387) facilitates its localization to the P-body [ 58 ]. (ii) Stress granules (SGs) are generated in response to stressful environmental conditions and are another reservoir of mRNAs. SGs contain mRNAs, translation initiation factors (e.g., eIF3, eIF4A, and eIF4G), small ribosomal subunits and poly(A) binding protein (PABP) proteins as well as the mRNA decay machinery. OS represses the translation of important neuronal genes and influences Ago2 and Pum2 accumulation in the SGs

Techniques Used: Inhibition, Activity Assay, Methylation, Expressing, RNA Binding Assay, Blocking Assay, Activation Assay, Generated, Binding Assay

8) Product Images from "P-TEFb Activation by RBM7 Shapes a Pro-survival Transcriptional Response to Genotoxic Stress"

Article Title: P-TEFb Activation by RBM7 Shapes a Pro-survival Transcriptional Response to Genotoxic Stress

Journal: Molecular Cell

doi: 10.1016/j.molcel.2019.01.033

Active P-TEFb Is Vital for the Pol II Transcriptional Response to Genotoxic Stress (A) (Top) Schematic depicting major steps in the generation of 4sU-labeled transcripts (4sU RNA) for 4sU-seq. (Bottom) Pie charts showing the fractions of DE protein-coding genes (mRNA) in 4-NQO-treated HeLa cells as assessed by 4sU-seq (n = 2). (B) Bar charts showing the number of DE classes of transcripts in HeLa cells as assessed by 4sU-seq (n = 2). The degrees of differential expression are presented according to the legend. Conditions with (in hours) and without (−) 4-NQO or FP are shown. (C) Boxplots indicating the distribution of gene lengths for upregulated and downregulated protein-coding genes. Median gene length for each group is shown. (D) Top Molecular and Cellular Functions categories of the 4FP gene set as identified by IPA. The number of affected genes per category is shown on the right. (E) RT-qPCR of the indicated DNA damage-induced unspliced (pre-mRNA), uaRNA, and eRNA transcripts. HeLa cells were treated as indicated by the legend. Results were normalized to the DMSO control and are presented as the mean ± SEM (n = 3). ∗ p
Figure Legend Snippet: Active P-TEFb Is Vital for the Pol II Transcriptional Response to Genotoxic Stress (A) (Top) Schematic depicting major steps in the generation of 4sU-labeled transcripts (4sU RNA) for 4sU-seq. (Bottom) Pie charts showing the fractions of DE protein-coding genes (mRNA) in 4-NQO-treated HeLa cells as assessed by 4sU-seq (n = 2). (B) Bar charts showing the number of DE classes of transcripts in HeLa cells as assessed by 4sU-seq (n = 2). The degrees of differential expression are presented according to the legend. Conditions with (in hours) and without (−) 4-NQO or FP are shown. (C) Boxplots indicating the distribution of gene lengths for upregulated and downregulated protein-coding genes. Median gene length for each group is shown. (D) Top Molecular and Cellular Functions categories of the 4FP gene set as identified by IPA. The number of affected genes per category is shown on the right. (E) RT-qPCR of the indicated DNA damage-induced unspliced (pre-mRNA), uaRNA, and eRNA transcripts. HeLa cells were treated as indicated by the legend. Results were normalized to the DMSO control and are presented as the mean ± SEM (n = 3). ∗ p

Techniques Used: Labeling, Expressing, Indirect Immunoperoxidase Assay, Quantitative RT-PCR

Genotoxic Stress Induces the Interaction of RBM7 with 7SK (A) Distribution charts of unique tags derived from the F-RBM7 libraries based on percentages of the total iCLIP reads and mapped to the indicated RNA classes. Charts on the right show distribution of the indicated types of ncRNA. (B) F-RBM7 iCLIP reads mapped to 7SK. Positions of the four stem-loops (SL1–4) are shown below the iCLIP reads and on a 7SK secondary structure model. (C) RIP-qPCR of 7SK in wild-type and mRNP1 F-RBM7 IP from whole-cell extracts (WCEs) of HEK293 cells. RBM7 with RRM (in pink) and the position of RNP1 (white stripe) is shown on top. (D) RIP-qPCR of 7SK in F-RBM7 IP from WCE of HEK293 cells. Conditions with (red bars; in hours) and without (blue bars) 4-NQO are shown. Results in (C) and (D) are presented as the mean ± SEM (n = 3). ∗∗ A–S1C.
Figure Legend Snippet: Genotoxic Stress Induces the Interaction of RBM7 with 7SK (A) Distribution charts of unique tags derived from the F-RBM7 libraries based on percentages of the total iCLIP reads and mapped to the indicated RNA classes. Charts on the right show distribution of the indicated types of ncRNA. (B) F-RBM7 iCLIP reads mapped to 7SK. Positions of the four stem-loops (SL1–4) are shown below the iCLIP reads and on a 7SK secondary structure model. (C) RIP-qPCR of 7SK in wild-type and mRNP1 F-RBM7 IP from whole-cell extracts (WCEs) of HEK293 cells. RBM7 with RRM (in pink) and the position of RNP1 (white stripe) is shown on top. (D) RIP-qPCR of 7SK in F-RBM7 IP from WCE of HEK293 cells. Conditions with (red bars; in hours) and without (blue bars) 4-NQO are shown. Results in (C) and (D) are presented as the mean ± SEM (n = 3). ∗∗ A–S1C.

Techniques Used: Derivative Assay, Real-time Polymerase Chain Reaction

9) Product Images from "Screening for Small-Molecule Modulators of Long Noncoding RNA-Protein Interactions Using AlphaScreen"

Article Title: Screening for Small-Molecule Modulators of Long Noncoding RNA-Protein Interactions Using AlphaScreen

Journal: Journal of Biomolecular Screening

doi: 10.1177/1087057115594187

Determining negative and positive RNA controls for EZH2 interactions. EZH2 reportedly binds many RNAs without well-defined protein-binding motifs. As a negative control, a nonrelated Renilla luciferase transcript was tested that did show some affinity for the EZH2 protein in the ( A ) Alphascreen assay ( n = 3) as well as in the ( B ) RNA EMSA assay. This finding is consistent with reports of EZH2 promiscuity, including the ability of this enzyme to bind nonmammalian transcripts with low affinity. 16 ( C ) The ability of biotinylated lncRNAs BDNF -AS and HOTAIR to interact with EZH2 is measured in the AlphaScreen assay. Both lncRNAs are titrated in EZH2 (4 nM). EZH2 interacts with both lncRNAs in a concentration-dependent manner ( n = 3). HOTAIR serves as a positive RNA control and biologically important screen.
Figure Legend Snippet: Determining negative and positive RNA controls for EZH2 interactions. EZH2 reportedly binds many RNAs without well-defined protein-binding motifs. As a negative control, a nonrelated Renilla luciferase transcript was tested that did show some affinity for the EZH2 protein in the ( A ) Alphascreen assay ( n = 3) as well as in the ( B ) RNA EMSA assay. This finding is consistent with reports of EZH2 promiscuity, including the ability of this enzyme to bind nonmammalian transcripts with low affinity. 16 ( C ) The ability of biotinylated lncRNAs BDNF -AS and HOTAIR to interact with EZH2 is measured in the AlphaScreen assay. Both lncRNAs are titrated in EZH2 (4 nM). EZH2 interacts with both lncRNAs in a concentration-dependent manner ( n = 3). HOTAIR serves as a positive RNA control and biologically important screen.

Techniques Used: Protein Binding, Negative Control, Luciferase, Amplified Luminescent Proximity Homogenous Assay, Concentration Assay

( A ) Mechanism of the BDNF -AS–EZH2 interaction. The BDNF -AS transcript interacts with EZH2 (RNA-protein interaction), guiding this ubiquitously expressed epigenetic enzyme to the BDNF locus (RNA-chromatin interaction) where EZH2 is able to epigenetically silence BDNF gene expression. Inhibition of the BDNF -AS–EZH2 interaction can prevent EZH2 recruitment to the BDNF promoter and results in up-regulation of the BDNF gene. ( B ) Schematic of AlphaScreen adapted to quantify lncRNA-protein interactions. Following the incubation of biotinylated long noncoding RNA BDNF -AS with Flag-tagged EZH2 protein, anti-flag tagged acceptor beads and streptavidin-coated donor beads are added to each well. Upon excitation of the donor beads at 680 nm, ambient oxygen is elevated to an excited state and excites nearby acceptor beads, resulting in a measurable emission at 570 nm that is used to quantify the assay.
Figure Legend Snippet: ( A ) Mechanism of the BDNF -AS–EZH2 interaction. The BDNF -AS transcript interacts with EZH2 (RNA-protein interaction), guiding this ubiquitously expressed epigenetic enzyme to the BDNF locus (RNA-chromatin interaction) where EZH2 is able to epigenetically silence BDNF gene expression. Inhibition of the BDNF -AS–EZH2 interaction can prevent EZH2 recruitment to the BDNF promoter and results in up-regulation of the BDNF gene. ( B ) Schematic of AlphaScreen adapted to quantify lncRNA-protein interactions. Following the incubation of biotinylated long noncoding RNA BDNF -AS with Flag-tagged EZH2 protein, anti-flag tagged acceptor beads and streptavidin-coated donor beads are added to each well. Upon excitation of the donor beads at 680 nm, ambient oxygen is elevated to an excited state and excites nearby acceptor beads, resulting in a measurable emission at 570 nm that is used to quantify the assay.

Techniques Used: Expressing, Inhibition, Amplified Luminescent Proximity Homogenous Assay, Incubation

Optimization of AlphaScreen assay conditions. ( A ) Long noncoding RNA and protein concentrations were optimized by titrating BDNF -AS in fixed concentrations of EZH2 protein. The hook effect was observed at all concentrations of protein, and therefore, the maximal signal or the hook point is graphed. Data represent triplicates of a single experiment that was repeated three independent times. ( B ) AlphaScreen acceptor and donor beads were cross-titrated to determine optimal bead concentrations to produce maximal assay signal ( n = 3).
Figure Legend Snippet: Optimization of AlphaScreen assay conditions. ( A ) Long noncoding RNA and protein concentrations were optimized by titrating BDNF -AS in fixed concentrations of EZH2 protein. The hook effect was observed at all concentrations of protein, and therefore, the maximal signal or the hook point is graphed. Data represent triplicates of a single experiment that was repeated three independent times. ( B ) AlphaScreen acceptor and donor beads were cross-titrated to determine optimal bead concentrations to produce maximal assay signal ( n = 3).

Techniques Used: Amplified Luminescent Proximity Homogenous Assay

Secondary assays to test the effect of ellipticine on the target of BDNF -AS–EZH2, BDNF, in vitro. ( A ) HEK293 cells were treated for 48 h with ellipticine (1 µM) before RNA was extracted to measure changes in BDNF gene expression normalized to GAPDH (~threefold up-regulation in BDNF mRNA, p
Figure Legend Snippet: Secondary assays to test the effect of ellipticine on the target of BDNF -AS–EZH2, BDNF, in vitro. ( A ) HEK293 cells were treated for 48 h with ellipticine (1 µM) before RNA was extracted to measure changes in BDNF gene expression normalized to GAPDH (~threefold up-regulation in BDNF mRNA, p

Techniques Used: In Vitro, Expressing

10) Product Images from "Activation of the protein kinase PKR by short double-stranded RNAs with single-stranded tails"

Article Title: Activation of the protein kinase PKR by short double-stranded RNAs with single-stranded tails

Journal: RNA

doi: 10.1261/rna.7150804

Selection experiment using a partially structured library. ( A ). ( B ) Randomized library constructed to search for RNAs capable of binding to PKR. Eighteen nucleotides were randomized, with nine positions opposite the other nine (bold font). “RT” denotes the binding site for the reverse transcription primer. The five 3 ′ -most nucleotides were only present in the run-off transcripts from the final clones (see Materials and Methods). ( C ). C59(9,15) and C83(9,15) are representative clones after 11 rounds of selection with p20; both have 5 ′ - and 3 ′ -single-stranded tails of 9 and 15 nt, respectively. Concentrations of p20 were 0, 0.01, 0.05, 0.1, 0.2, 0.25, 0.5, 1.0, 2.0, and 5.0 μM. Positions of unbound p*RNA and bound complexes are noted. Binding of RNA to the dsRBD increases with the number of selection rounds, with affinities similar to that of TAR RNA. Similar results were found for in vitro selections for binding to the K296R mutant of full-length PKR (not shown).
Figure Legend Snippet: Selection experiment using a partially structured library. ( A ). ( B ) Randomized library constructed to search for RNAs capable of binding to PKR. Eighteen nucleotides were randomized, with nine positions opposite the other nine (bold font). “RT” denotes the binding site for the reverse transcription primer. The five 3 ′ -most nucleotides were only present in the run-off transcripts from the final clones (see Materials and Methods). ( C ). C59(9,15) and C83(9,15) are representative clones after 11 rounds of selection with p20; both have 5 ′ - and 3 ′ -single-stranded tails of 9 and 15 nt, respectively. Concentrations of p20 were 0, 0.01, 0.05, 0.1, 0.2, 0.25, 0.5, 1.0, 2.0, and 5.0 μM. Positions of unbound p*RNA and bound complexes are noted. Binding of RNA to the dsRBD increases with the number of selection rounds, with affinities similar to that of TAR RNA. Similar results were found for in vitro selections for binding to the K296R mutant of full-length PKR (not shown).

Techniques Used: Selection, Construct, Binding Assay, Clone Assay, In Vitro, Mutagenesis

Effects of varying the length of the 5 ′ - and 3 ′ -tails on PKR phosphorylation activity. The lengths of the 5 ′ - and 3 ′ -tails flanking the stem–loop of C26 are provided in the figure. ( A ) Effects of tail length on PKR activity. In vitro activation assays of PKR on denaturing 10% SDS-polyacrylamide gels are shown. Concentrations of RNAs for the first five samples were 0, 0.1, 0.3, 1.0, and 3.0 μM; in a few instances, the 3.0-μM point was replaced with 5 μM RNA, which does not affect the conclusions. Concentrations of the sixth sample, C26(9,10), were 0.1, 0.3, 1.0, and 2.0 μM; the 0-μM point was run on another portion of this gel (not shown) and had 0% activity. The position of phosphorylated PKR is noted. Normalized phosphorylation activities are presented under the gels. The figure is a composite of five different gels. For each gel, a separate activation assay was performed using C26(9,11) (only one of which is shown) and used to normalize the activity of the sample on that particular gel. Flanking sequence is seen to have a marked effect on PKR activity. ( B ) Effects of tail length on binding to p20. Native 15% polyacrylamide gel run at 13°C is shown. Concentrations of p20 were 0, 0.03, 0.1, 0.3, 1.0, 2.0, and 3.0 μM. Positions of unbound p*RNA and bound complexes are noted. ( C ) Representative plots of fraction RNA bound versus p20 concentration for data in panel B . Symbols are as follows: C26(6,6) (•), C26(9,9) (○), C26(9,10) (▪), and C26(9,11) (□). Values of K d and f max were determined by nonlinear least squares fitting (Kaleidagraph, Synergy Software) to a simple hyperbolic equation (see Materials and Methods), except in the case of C26(6,6) where the value of f max ), although its nature is not entirely clear. The microshift above the main unbound p*RNA was scored as free RNA, which biases the K d toward higher values. Because the nature of the microshift is unclear, the following K d values must be treated as semiquantitative. Values of f max and K d from the fits were as follows: C26(6,6) 0.2 (fixed in fit) and 2 μM; C26(9,9) 0.25 and 0.3 μM; C26(9,10) 0.3 and 0.3 μM; C26(9,11) 0.5 and 0.2 μM. Binding is only weakly dependent on the length of flanking sequence. ( D ) Inhibition of PKR activity by C26(9,9). In vitro activation assays of PKR on a denaturing 10% SDS-polyacrylamide gel are shown. C26(9,11) at 0.3 μM was challenged with increasing concentrations (0.3, 1.0, and 2.0 μM) of C26(9,9) RNA. Normalized phosphorylation activities are presented under the gels. A weak activator of PKR can serve as an inhibitor. ( E ) Effects of having only one tail on PKR activity. In vitro activation assays of PKR on a denaturing 10% SDS-polyacrylamide gel are shown. C26(0,15) has a 15-nt 3 ′ -tail but no 5 ′ -tail, while C26(15,0) has a 15-nt 5 ′ -tail but no 3 ′ -tail; see Materials and Methods for exact sequences. Concentrations of tail-deleted RNAs were 0.1, 0.3, 1.0, and 2.0 μM, and the concentration of C26(9,11) was 0.3 μM. Normalized phosphorylation activities are presented under the gel. Flanking sequence on either the 5 ′ - or 3 ′ -end is sufficient to activate PKR.
Figure Legend Snippet: Effects of varying the length of the 5 ′ - and 3 ′ -tails on PKR phosphorylation activity. The lengths of the 5 ′ - and 3 ′ -tails flanking the stem–loop of C26 are provided in the figure. ( A ) Effects of tail length on PKR activity. In vitro activation assays of PKR on denaturing 10% SDS-polyacrylamide gels are shown. Concentrations of RNAs for the first five samples were 0, 0.1, 0.3, 1.0, and 3.0 μM; in a few instances, the 3.0-μM point was replaced with 5 μM RNA, which does not affect the conclusions. Concentrations of the sixth sample, C26(9,10), were 0.1, 0.3, 1.0, and 2.0 μM; the 0-μM point was run on another portion of this gel (not shown) and had 0% activity. The position of phosphorylated PKR is noted. Normalized phosphorylation activities are presented under the gels. The figure is a composite of five different gels. For each gel, a separate activation assay was performed using C26(9,11) (only one of which is shown) and used to normalize the activity of the sample on that particular gel. Flanking sequence is seen to have a marked effect on PKR activity. ( B ) Effects of tail length on binding to p20. Native 15% polyacrylamide gel run at 13°C is shown. Concentrations of p20 were 0, 0.03, 0.1, 0.3, 1.0, 2.0, and 3.0 μM. Positions of unbound p*RNA and bound complexes are noted. ( C ) Representative plots of fraction RNA bound versus p20 concentration for data in panel B . Symbols are as follows: C26(6,6) (•), C26(9,9) (○), C26(9,10) (▪), and C26(9,11) (□). Values of K d and f max were determined by nonlinear least squares fitting (Kaleidagraph, Synergy Software) to a simple hyperbolic equation (see Materials and Methods), except in the case of C26(6,6) where the value of f max ), although its nature is not entirely clear. The microshift above the main unbound p*RNA was scored as free RNA, which biases the K d toward higher values. Because the nature of the microshift is unclear, the following K d values must be treated as semiquantitative. Values of f max and K d from the fits were as follows: C26(6,6) 0.2 (fixed in fit) and 2 μM; C26(9,9) 0.25 and 0.3 μM; C26(9,10) 0.3 and 0.3 μM; C26(9,11) 0.5 and 0.2 μM. Binding is only weakly dependent on the length of flanking sequence. ( D ) Inhibition of PKR activity by C26(9,9). In vitro activation assays of PKR on a denaturing 10% SDS-polyacrylamide gel are shown. C26(9,11) at 0.3 μM was challenged with increasing concentrations (0.3, 1.0, and 2.0 μM) of C26(9,9) RNA. Normalized phosphorylation activities are presented under the gels. A weak activator of PKR can serve as an inhibitor. ( E ) Effects of having only one tail on PKR activity. In vitro activation assays of PKR on a denaturing 10% SDS-polyacrylamide gel are shown. C26(0,15) has a 15-nt 3 ′ -tail but no 5 ′ -tail, while C26(15,0) has a 15-nt 5 ′ -tail but no 3 ′ -tail; see Materials and Methods for exact sequences. Concentrations of tail-deleted RNAs were 0.1, 0.3, 1.0, and 2.0 μM, and the concentration of C26(9,11) was 0.3 μM. Normalized phosphorylation activities are presented under the gel. Flanking sequence on either the 5 ′ - or 3 ′ -end is sufficient to activate PKR.

Techniques Used: Activity Assay, In Vitro, Activation Assay, Sequencing, Binding Assay, Concentration Assay, Software, Inhibition

11) Product Images from "Structure of a TrmA–RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m5U methyltransferases"

Article Title: Structure of a TrmA–RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m5U methyltransferases

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

doi: 10.1073/pnas.0802247105

RNA–protein interactions. ( A ) Diagram of the secondary structure and interactions of the bound RNA. ( B ) Stereoview of a stick representation of T-loop bases U54, U55, C56, and G57 bound to the TrmA active site. Putative hydrogen bonds between the target U and the protein are shown with red dotted lines, and hydrogen bonds between the other bases and the protein are shown with green dotted lines. ( C ) Close-up of the interactions between the target uridine and the amino acid side chains overlaid with F o − F c map (4.5 σ) calculated with U54 and surrounding side chains omitted.
Figure Legend Snippet: RNA–protein interactions. ( A ) Diagram of the secondary structure and interactions of the bound RNA. ( B ) Stereoview of a stick representation of T-loop bases U54, U55, C56, and G57 bound to the TrmA active site. Putative hydrogen bonds between the target U and the protein are shown with red dotted lines, and hydrogen bonds between the other bases and the protein are shown with green dotted lines. ( C ) Close-up of the interactions between the target uridine and the amino acid side chains overlaid with F o − F c map (4.5 σ) calculated with U54 and surrounding side chains omitted.

Techniques Used:

Sequence alignment of TrmA and RumA. Conserved residues are white on a red background, and conservative substitutions are red on a white background. The secondary structures of TrmA and RumA are shown above and below their sequences, respectively (coil = helix, arrow = β-strand, T = turn) and are colored purple, yellow, and blue for the cross-over segment, RNA binding, and catalytic domains, respectively. Conserved motifs are marked with blue dashes under the sequence alignment.
Figure Legend Snippet: Sequence alignment of TrmA and RumA. Conserved residues are white on a red background, and conservative substitutions are red on a white background. The secondary structures of TrmA and RumA are shown above and below their sequences, respectively (coil = helix, arrow = β-strand, T = turn) and are colored purple, yellow, and blue for the cross-over segment, RNA binding, and catalytic domains, respectively. Conserved motifs are marked with blue dashes under the sequence alignment.

Techniques Used: Sequencing, RNA Binding Assay

Mechanism and substrates of TrmA. ( A ) The proposed catalytic mechanism of RNA m 5 ). ( B ) Consensus sequence of the T arm of tRNA derived from the sequences of E. coli ). Blue indicates conserved and yellow indicates nonconserved bases in the T arm. The target uridine is red. The sequence of the T arm used in this study is indicated next to the base numbering. Circles indicate the additional base pair added to the original T loop sequence. 2′-Se-Me-U is indicated as U se . ( C ) Side view of TrmA–T arm structure.
Figure Legend Snippet: Mechanism and substrates of TrmA. ( A ) The proposed catalytic mechanism of RNA m 5 ). ( B ) Consensus sequence of the T arm of tRNA derived from the sequences of E. coli ). Blue indicates conserved and yellow indicates nonconserved bases in the T arm. The target uridine is red. The sequence of the T arm used in this study is indicated next to the base numbering. Circles indicate the additional base pair added to the original T loop sequence. 2′-Se-Me-U is indicated as U se . ( C ) Side view of TrmA–T arm structure.

Techniques Used: Sequencing, Derivative Assay

Substrate binding in TrmA and RumA. ( A ) Comparison of loop conformations of the T-arm analog from the TrmA–RNA structure (pink and red), with the T loop of unbound tRNA Phe ). U54 is shown in stick representation. ( B ). U1939 is shown as sticks. ( C ) Overlap of the RNA loops bound to the active sites of TrmA (pink, bases 53–58) and RumA (blue, bases 1938–1942). ( D ) ( Left ) Surface representation of TrmA bound to its 19-mer substrate (magenta), with the RumA 37-mer substrate superposed in the yellow cartoon. ( Right ) Surface representation of RumA bound to its substrate (yellow), with the TrmA substrate superposed (magenta). RNA binding, catalytic, and OB-fold domains are colored blue, gray, and green, respectively.
Figure Legend Snippet: Substrate binding in TrmA and RumA. ( A ) Comparison of loop conformations of the T-arm analog from the TrmA–RNA structure (pink and red), with the T loop of unbound tRNA Phe ). U54 is shown in stick representation. ( B ). U1939 is shown as sticks. ( C ) Overlap of the RNA loops bound to the active sites of TrmA (pink, bases 53–58) and RumA (blue, bases 1938–1942). ( D ) ( Left ) Surface representation of TrmA bound to its 19-mer substrate (magenta), with the RumA 37-mer substrate superposed in the yellow cartoon. ( Right ) Surface representation of RumA bound to its substrate (yellow), with the TrmA substrate superposed (magenta). RNA binding, catalytic, and OB-fold domains are colored blue, gray, and green, respectively.

Techniques Used: Binding Assay, RNA Binding Assay

12) Product Images from "Toll-like receptor 9 suppresses lupus disease in Fas-sufficient MRL Mice"

Article Title: Toll-like receptor 9 suppresses lupus disease in Fas-sufficient MRL Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0173471

Autoantibody production in Tlr9 -/- MRL/+ mice. (A) Serum anti-nucleosome IgG autoantibodies were measured by ELISA and are expressed relative to a PL2-3 standard. (B) Serum anti-Sm IgG autoantibodies were measured by ELISA and are expressed relative to a Y2 standard. (C) Serum anti-RNA IgG autoantibodies were measured by ELISA and are expressed relative to a BWR4 standard. (D) Serum kappa anti-IgG2a rheumatoid factor autoantibodies were measured by ELISA and are expressed relative to a 400tμ23 standard. * p
Figure Legend Snippet: Autoantibody production in Tlr9 -/- MRL/+ mice. (A) Serum anti-nucleosome IgG autoantibodies were measured by ELISA and are expressed relative to a PL2-3 standard. (B) Serum anti-Sm IgG autoantibodies were measured by ELISA and are expressed relative to a Y2 standard. (C) Serum anti-RNA IgG autoantibodies were measured by ELISA and are expressed relative to a BWR4 standard. (D) Serum kappa anti-IgG2a rheumatoid factor autoantibodies were measured by ELISA and are expressed relative to a 400tμ23 standard. * p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

13) Product Images from "Nuclear S100A7 Is Associated with Poor Prognosis in Head and Neck Cancer"

Article Title: Nuclear S100A7 Is Associated with Poor Prognosis in Head and Neck Cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0011939

Verification of S100A7 expression in tissues. (a) RT-PCR analysis of S100A7 in oral normal mucosa, squamous cell hyperplasia, dysplasia and HNSCC tissues. For RT-PCR analysis and Western blot analysis, we used normal (n = 5), hyperplasia (n = 5), dysplasia (n = 5) and HNSCC (n = 5) tissues. Panel shows increased levels of S100A7 transcripts in oral lesions -squamous cell hyperplasia (H), dysplasia (D) and HNSCC (T) compared with the oral normal mucosa (N) that showed basal levels of S100A7 transcripts. β-actin used as a control to normalize the quantity of RNA used for each RT-PCR reaction is shown in the lower panel. (b) Western blot analysis of S100A7 in oral normal mucosa (N), squamous cell hyperplasia (H), dysplasia (D) and HNSCC tissues. Equal amount of protein lysates from these tissues were electrophoresed on 12% SDS-PAGE and transferred to PVDF membrane. The membrane was incubated with respective primary and secondary antibodies as described in the Methods section and the signal detected by enhanced chemiluminescence method. Panel shows increased expression of S100A7 protein in oral lesions - squamous cell hyperplasia (H), dysplasia (D) and HNSCC (T) compared with oral normal mucosa (N). GAPDH was used as loading control.
Figure Legend Snippet: Verification of S100A7 expression in tissues. (a) RT-PCR analysis of S100A7 in oral normal mucosa, squamous cell hyperplasia, dysplasia and HNSCC tissues. For RT-PCR analysis and Western blot analysis, we used normal (n = 5), hyperplasia (n = 5), dysplasia (n = 5) and HNSCC (n = 5) tissues. Panel shows increased levels of S100A7 transcripts in oral lesions -squamous cell hyperplasia (H), dysplasia (D) and HNSCC (T) compared with the oral normal mucosa (N) that showed basal levels of S100A7 transcripts. β-actin used as a control to normalize the quantity of RNA used for each RT-PCR reaction is shown in the lower panel. (b) Western blot analysis of S100A7 in oral normal mucosa (N), squamous cell hyperplasia (H), dysplasia (D) and HNSCC tissues. Equal amount of protein lysates from these tissues were electrophoresed on 12% SDS-PAGE and transferred to PVDF membrane. The membrane was incubated with respective primary and secondary antibodies as described in the Methods section and the signal detected by enhanced chemiluminescence method. Panel shows increased expression of S100A7 protein in oral lesions - squamous cell hyperplasia (H), dysplasia (D) and HNSCC (T) compared with oral normal mucosa (N). GAPDH was used as loading control.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, SDS Page, Incubation

14) Product Images from "A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation"

Article Title: A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation

Journal: Molecular Neurodegeneration

doi: 10.1186/1750-1326-3-4

Cellular levels of RNF182 modulate the rates of cell death . N2a cells were transiently transfected with RNF182/pcDNA3.1/myc-his plasmid or mouse RNF182 on-target plus smart pool siRNAs. Cells were collected for total RNA extractions 24–48 h after transfections. Trypan Blue exclusion assay was performed 24 h after transfection or 16 h after a 7 h OGD treatment of the siRNA transfected samples. A . Over expression of RNF182 mRNA was assessed by RT-PCR. In: lane 1 – negative PCR control, lane 2 – mock transfection, lane 3-transfection with RNF182/pcDNA3.1/myc-his plasmid. B . Percentage of cell death before and after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ
Figure Legend Snippet: Cellular levels of RNF182 modulate the rates of cell death . N2a cells were transiently transfected with RNF182/pcDNA3.1/myc-his plasmid or mouse RNF182 on-target plus smart pool siRNAs. Cells were collected for total RNA extractions 24–48 h after transfections. Trypan Blue exclusion assay was performed 24 h after transfection or 16 h after a 7 h OGD treatment of the siRNA transfected samples. A . Over expression of RNF182 mRNA was assessed by RT-PCR. In: lane 1 – negative PCR control, lane 2 – mock transfection, lane 3-transfection with RNF182/pcDNA3.1/myc-his plasmid. B . Percentage of cell death before and after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ

Techniques Used: Transfection, Plasmid Preparation, Trypan Blue Exclusion Assay, Over Expression, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

RNF182 targets ATP6V0C for proteosome degradation . N2a cells were transiently transfected with empty pEGFP-N1 or pCMV-Tag1 vector alone, pRNF182*EGFP or pCMV-Tag1-ATP6V0C alone, or pRNF182*EGFP and pCMV-Tag1-ATP6V0C simultaneously. Cells were collected for Trypan Blue exclusion assay as well as total RNA and protein extractions 24 h after transfection or treated with 30 μM MG132 for 8 h prior to total RNA and protein extractions. A . Over-expression of ATP6V0C in RNF182 transfected cells did not change the percentage of cell death caused by RNF182 over-expression. This figure shows the percentage of cell death before and after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisks indicate a significant difference (ρ
Figure Legend Snippet: RNF182 targets ATP6V0C for proteosome degradation . N2a cells were transiently transfected with empty pEGFP-N1 or pCMV-Tag1 vector alone, pRNF182*EGFP or pCMV-Tag1-ATP6V0C alone, or pRNF182*EGFP and pCMV-Tag1-ATP6V0C simultaneously. Cells were collected for Trypan Blue exclusion assay as well as total RNA and protein extractions 24 h after transfection or treated with 30 μM MG132 for 8 h prior to total RNA and protein extractions. A . Over-expression of ATP6V0C in RNF182 transfected cells did not change the percentage of cell death caused by RNF182 over-expression. This figure shows the percentage of cell death before and after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisks indicate a significant difference (ρ

Techniques Used: Transfection, Plasmid Preparation, Trypan Blue Exclusion Assay, Over Expression

15) Product Images from "Antagonistic actions of two human Pan3 isoforms on global mRNA turnover"

Article Title: Antagonistic actions of two human Pan3 isoforms on global mRNA turnover

Journal: RNA

doi: 10.1261/rna.061556.117

Effects of Pan3 isoform knockdowns on individual mRNA decay pathways in vivo. ( A – C ) Northern blots showing the decay of the β-globin mRNA (BBB; A ), β-globin mRNA carrying the c- fos ARE (BBB+ARE; B ), or β-globin mRNA carrying three consecutive let-7 binding sites (BBB+3xlet7; C ) in human BEAS-2B cells transfected with control siRNA, Pan3L-specific siRNA, Pan3S-specific siRNA, or both Pan3 siRNAs (Pan3S+L). The tTA-expressing BEAS-2B-19 cells were first transfected with the indicated siRNAs and then cotransfected with plasmids encoding the indicated reporter mRNA and a constitutively expressed α-globin/GAPDH hybrid mRNA that served as a control (ctrl) mRNA. Transcription of the reporter mRNA was transiently induced for 2 h by tetracycline removal. RNA samples were prepared at the indicated time points after tetracycline was replenished. Poly(A) − RNA samples were prepared in vitro by treating RNA samples from early time points with oligo(dT) and RNase H. RT-PCR and agarose gel electrophoresis were performed to assess the Pan3 knockdown efficiency; GAPDH served as the loading control. Semi-log plots illustrate decay kinetics of BBB+ARE ( B ) and BBB+3xlet7 ( C ) mRNAs after knocking down the Pan3 isoforms. Decay curves and RNA half-lives ( t 1/2 ) were obtained by least squares regression of the fraction of mRNA remaining as a function of time, using data from two separate time-course experiments with reproducible results. ( D ) Dual luciferase assay showing the effects of Pan3 isoform knockdowns on miRNA-mediated gene silencing. ( Upper left ) Schematic diagram of renilla luciferase (RL) mRNAs carrying three copies of the WT or mutant let-7 binding sites in the 3′UTR (RL+3xlet7 or RL+3xlet7mut). ( Lower left ) RT-PCR and agarose gel electrophoresis were performed to assess the Pan3 knockdown efficiency; GAPDH served as the loading control. ( Right ) Results of dual luciferase assays. Firefly luciferase (FL) activity, derived from the same plasmid carrying the RL reporter gene, was used for normalization. The relative fold changes were measured by comparing the RL/FL activity detected under different knockdown (KD) conditions as indicated with that detected in cells expressing the corresponding reporter mRNA under control KD (set as 1). All data represent the average ±SD ( n = 3).
Figure Legend Snippet: Effects of Pan3 isoform knockdowns on individual mRNA decay pathways in vivo. ( A – C ) Northern blots showing the decay of the β-globin mRNA (BBB; A ), β-globin mRNA carrying the c- fos ARE (BBB+ARE; B ), or β-globin mRNA carrying three consecutive let-7 binding sites (BBB+3xlet7; C ) in human BEAS-2B cells transfected with control siRNA, Pan3L-specific siRNA, Pan3S-specific siRNA, or both Pan3 siRNAs (Pan3S+L). The tTA-expressing BEAS-2B-19 cells were first transfected with the indicated siRNAs and then cotransfected with plasmids encoding the indicated reporter mRNA and a constitutively expressed α-globin/GAPDH hybrid mRNA that served as a control (ctrl) mRNA. Transcription of the reporter mRNA was transiently induced for 2 h by tetracycline removal. RNA samples were prepared at the indicated time points after tetracycline was replenished. Poly(A) − RNA samples were prepared in vitro by treating RNA samples from early time points with oligo(dT) and RNase H. RT-PCR and agarose gel electrophoresis were performed to assess the Pan3 knockdown efficiency; GAPDH served as the loading control. Semi-log plots illustrate decay kinetics of BBB+ARE ( B ) and BBB+3xlet7 ( C ) mRNAs after knocking down the Pan3 isoforms. Decay curves and RNA half-lives ( t 1/2 ) were obtained by least squares regression of the fraction of mRNA remaining as a function of time, using data from two separate time-course experiments with reproducible results. ( D ) Dual luciferase assay showing the effects of Pan3 isoform knockdowns on miRNA-mediated gene silencing. ( Upper left ) Schematic diagram of renilla luciferase (RL) mRNAs carrying three copies of the WT or mutant let-7 binding sites in the 3′UTR (RL+3xlet7 or RL+3xlet7mut). ( Lower left ) RT-PCR and agarose gel electrophoresis were performed to assess the Pan3 knockdown efficiency; GAPDH served as the loading control. ( Right ) Results of dual luciferase assays. Firefly luciferase (FL) activity, derived from the same plasmid carrying the RL reporter gene, was used for normalization. The relative fold changes were measured by comparing the RL/FL activity detected under different knockdown (KD) conditions as indicated with that detected in cells expressing the corresponding reporter mRNA under control KD (set as 1). All data represent the average ±SD ( n = 3).

Techniques Used: In Vivo, Northern Blot, Binding Assay, Transfection, Expressing, In Vitro, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Luciferase, Mutagenesis, Activity Assay, Derivative Assay, Plasmid Preparation

Effects of Pan3 isoforms and their domain truncations on Pan2 deadenylase activity in vivo. NIH3T3 cells were transfected with a plasmid coding for HA-Pan2 alone or together with a plasmid coding for one of the HA-tagged WT or truncated Pan3 isoforms. β-Globin mRNA constitutively expressed ( A ) or transiently induced ( B – D ) in the same cells served as a reporter transcript. Total cytoplasmic RNA was extracted for Northern blot analysis to check poly(A) size distribution of the β-globin reporter mRNA ( upper panels of A , B ; upper left panels of C , D ). Poly(A) – RNA samples were prepared with oligo(dT) and RNase H treatment. Levels of the indicated HA-tagged proteins were visualized by Western blot analysis ( lower panels of A , B ; lower left panels of C , D ). The poly(A) size distribution profiles ( middle panels of A , B ; right panels of C , D ) were obtained by scanning each lane of the Northern blots using ImageJ software. Red arrows ( A , B ) highlight changes of the poly(A) size distribution profile. Red and blue dashed lines ( C , D ) serve as a reference to compare the effects caused by different Pan3 domain truncations on the poly(A) size distribution.
Figure Legend Snippet: Effects of Pan3 isoforms and their domain truncations on Pan2 deadenylase activity in vivo. NIH3T3 cells were transfected with a plasmid coding for HA-Pan2 alone or together with a plasmid coding for one of the HA-tagged WT or truncated Pan3 isoforms. β-Globin mRNA constitutively expressed ( A ) or transiently induced ( B – D ) in the same cells served as a reporter transcript. Total cytoplasmic RNA was extracted for Northern blot analysis to check poly(A) size distribution of the β-globin reporter mRNA ( upper panels of A , B ; upper left panels of C , D ). Poly(A) – RNA samples were prepared with oligo(dT) and RNase H treatment. Levels of the indicated HA-tagged proteins were visualized by Western blot analysis ( lower panels of A , B ; lower left panels of C , D ). The poly(A) size distribution profiles ( middle panels of A , B ; right panels of C , D ) were obtained by scanning each lane of the Northern blots using ImageJ software. Red arrows ( A , B ) highlight changes of the poly(A) size distribution profile. Red and blue dashed lines ( C , D ) serve as a reference to compare the effects caused by different Pan3 domain truncations on the poly(A) size distribution.

Techniques Used: Activity Assay, In Vivo, Transfection, Plasmid Preparation, Northern Blot, Western Blot, Software

16) Product Images from "Temperature Regulation of the Hemin Storage (Hms+) Phenotype of Yersinia pestis Is Posttranscriptional"

Article Title: Temperature Regulation of the Hemin Storage (Hms+) Phenotype of Yersinia pestis Is Posttranscriptional

Journal: Journal of Bacteriology

doi: 10.1128/JB.186.6.1638-1647.2004

RNA dot blot. Total RNA from cells of KIM6+ (Hms + ), KIM6 (Δ pgm ), and KIM6-2051+ ( hmsT2051 ::mini- kan ) cultured at 26 or 37°C were transferred to nylon membranes and hybridized against probes for hmsH (A), hmsS (B), or hmsT (C). RNase indicates hybridization against 1 μg of RNA treated with RNase A.
Figure Legend Snippet: RNA dot blot. Total RNA from cells of KIM6+ (Hms + ), KIM6 (Δ pgm ), and KIM6-2051+ ( hmsT2051 ::mini- kan ) cultured at 26 or 37°C were transferred to nylon membranes and hybridized against probes for hmsH (A), hmsS (B), or hmsT (C). RNase indicates hybridization against 1 μg of RNA treated with RNase A.

Techniques Used: Dot Blot, Cell Culture, Hybridization

17) Product Images from "Reticuloendotheliosis Virus Strain T Induces miR-155, Which Targets JARID2 and Promotes Cell Survival ▿"

Article Title: Reticuloendotheliosis Virus Strain T Induces miR-155, Which Targets JARID2 and Promotes Cell Survival ▿

Journal:

doi: 10.1128/JVI.01182-09

miR-155 is upregulated in v-rel- and c-myc -induced B-cell lymphomas and by REV-T. (A) Total RNA from cells and tissues was harvested, and miR-155 levels were assayed by RNase protection. KBMC and CM758 are v-rel -derived tumor cell lines. CEFs were infected
Figure Legend Snippet: miR-155 is upregulated in v-rel- and c-myc -induced B-cell lymphomas and by REV-T. (A) Total RNA from cells and tissues was harvested, and miR-155 levels were assayed by RNase protection. KBMC and CM758 are v-rel -derived tumor cell lines. CEFs were infected

Techniques Used: Derivative Assay, Infection

18) Product Images from "Viral unmasking of cellular 5S rRNA pseudogene transcripts induces RIG-I mediated immunity"

Article Title: Viral unmasking of cellular 5S rRNA pseudogene transcripts induces RIG-I mediated immunity

Journal: Nature immunology

doi: 10.1038/s41590-017-0005-y

RNA5SP141 activates RIG-I. ( a ) qRT-PCR analysis of the indicated cytokine and ISG transcripts (vertical axes) in HEK 293T cells transfected with 200 fmol or 1 pmol in vitro -transcribed RNA5SP141 , or in vitro –transcribed RNA corresponding to the rabies virus leader sequence (RABV Le ; positive control) or an internal rabies virus sequence (RABV INT ; negative control). ( b ) Native PAGE and SDS-PAGE of lysates from HEK 293T cells transfected with 125 pmol of the indicated in vitro transcripts or infected with 50 HAU/ml SeV for 16 h, or left untreated (Mock). Endogenous IRF3 and actin were detected by immunoblot (IB) with anti-IRF3 and anti-actin, respectively. ( c ) Left: ISRE-luciferase reporter activity in HEK 293T cells transfected for 30 h with non-targeting control siRNA (si.Ctrl) or siRNAs targeting RIG-I or MDA5 (si.RIG-I or si.MDA5), and subsequently mock-transfected or transfected with 500 ng of a DNA construct encoding U6 promoter-expressed RNA5SP141 or RABV INT (U6-5SP141 or U6-RABV INT ) for 18 h. Treatment with 1 µg/ml HMW-poly(I:C) served as a control. Middle, right: Knockdown efficiency of endogenous RIG-I ( DDX58 ) and MDA5 ( IFIH1 ) was confirmed by qRT-PCR. ( d ) Left: qRT-PCR analysis of IFNB1 mRNA in NHLF cells that were transfected with the indicated siRNAs and U6 promoter-expressed DNA constructs as in (c). Treatment with 0.05 µg/mL HMW-poly(I:C) served as a control. Middle, right: Knockdown efficiency of endogenous RIG-I ( DDX58 ) and MDA5 ( IFIH1 ) was confirmed by qRT-PCR. ( e ) ISRE-luciferase reporter activity in HEK 293T cells transfected for 18 h with 500 fmol in vitro –transcribed RNA5SP141 or RABV Le , which had been pre-treated with calf alkaline phosphate (CIP), or left untreated. ( f ) Quantification of hydrolyzed [γ- 32 P]ATP by RIG-I incubated for the indicated times with 250 nM of in vitro -transcribed RNA5SP141 . Incubation of RIG-I with in vitro –transcribed RABV Le , or no RNA, served as positive and negative controls, respectively. Free phosphate was separated from unhydrolyzed ATP by thin layer chromatography, and the percentage of hydrolyzed ATP in each sample was calculated. Data are representative of two ( b–f ) or three ( a ) independent experiments (mean and s.d. of n = 2 biological replicates in a , n = 3 biological replicates in b–e , n = 3 technical replicates in f ).
Figure Legend Snippet: RNA5SP141 activates RIG-I. ( a ) qRT-PCR analysis of the indicated cytokine and ISG transcripts (vertical axes) in HEK 293T cells transfected with 200 fmol or 1 pmol in vitro -transcribed RNA5SP141 , or in vitro –transcribed RNA corresponding to the rabies virus leader sequence (RABV Le ; positive control) or an internal rabies virus sequence (RABV INT ; negative control). ( b ) Native PAGE and SDS-PAGE of lysates from HEK 293T cells transfected with 125 pmol of the indicated in vitro transcripts or infected with 50 HAU/ml SeV for 16 h, or left untreated (Mock). Endogenous IRF3 and actin were detected by immunoblot (IB) with anti-IRF3 and anti-actin, respectively. ( c ) Left: ISRE-luciferase reporter activity in HEK 293T cells transfected for 30 h with non-targeting control siRNA (si.Ctrl) or siRNAs targeting RIG-I or MDA5 (si.RIG-I or si.MDA5), and subsequently mock-transfected or transfected with 500 ng of a DNA construct encoding U6 promoter-expressed RNA5SP141 or RABV INT (U6-5SP141 or U6-RABV INT ) for 18 h. Treatment with 1 µg/ml HMW-poly(I:C) served as a control. Middle, right: Knockdown efficiency of endogenous RIG-I ( DDX58 ) and MDA5 ( IFIH1 ) was confirmed by qRT-PCR. ( d ) Left: qRT-PCR analysis of IFNB1 mRNA in NHLF cells that were transfected with the indicated siRNAs and U6 promoter-expressed DNA constructs as in (c). Treatment with 0.05 µg/mL HMW-poly(I:C) served as a control. Middle, right: Knockdown efficiency of endogenous RIG-I ( DDX58 ) and MDA5 ( IFIH1 ) was confirmed by qRT-PCR. ( e ) ISRE-luciferase reporter activity in HEK 293T cells transfected for 18 h with 500 fmol in vitro –transcribed RNA5SP141 or RABV Le , which had been pre-treated with calf alkaline phosphate (CIP), or left untreated. ( f ) Quantification of hydrolyzed [γ- 32 P]ATP by RIG-I incubated for the indicated times with 250 nM of in vitro -transcribed RNA5SP141 . Incubation of RIG-I with in vitro –transcribed RABV Le , or no RNA, served as positive and negative controls, respectively. Free phosphate was separated from unhydrolyzed ATP by thin layer chromatography, and the percentage of hydrolyzed ATP in each sample was calculated. Data are representative of two ( b–f ) or three ( a ) independent experiments (mean and s.d. of n = 2 biological replicates in a , n = 3 biological replicates in b–e , n = 3 technical replicates in f ).

Techniques Used: Quantitative RT-PCR, Transfection, In Vitro, Sequencing, Positive Control, Negative Control, Clear Native PAGE, SDS Page, Infection, Luciferase, Activity Assay, Construct, Incubation, Thin Layer Chromatography

RNA5SP141 is relocalized from the nucleus to the cytoplasm during HSV-1 infection. ( a ) Left: Relative abundance of RNA5SP141 transcripts in the cytoplasmic and nuclear fractions of HEK 293T cells that were infected with HSV-1 WT (MOI 1) for 16 h, or left uninfected (Mock), determined by cytoplasmic-nuclear fractionation assay and qRT-PCR. Analysis of the relative abundance of RNU2-1, MALAT1 , and NEAT1 RNA served as controls. Right: IB analysis of Lamin A/C and β-Tubulin confirmed the purity of the nuclear and cytoplasmic fraction, respectively. IB analysis of whole cell lysates (WCL) with anti-HSV-1 infected cell protein 8 (ICP8) and anti-actin served as infection and loading controls, respectively. Left margin, size in kilodaltons (kDa). ( b ) Left: Relative abundance of RNA5SP141 transcripts in the cytoplasmic and nuclear fractions of HEK 293T cells that were infected with SeV (50 HAU/ml) for 16 h or left uninfected (Mock), determined by fractionation assay and qRT-PCR analysis as described in (a). Right: IB analysis of cytoplasmic and nuclear fractions as in (a). IB analysis of WCL with anti-SeV confirmed efficient infection. Data are representative of two independent experiments.
Figure Legend Snippet: RNA5SP141 is relocalized from the nucleus to the cytoplasm during HSV-1 infection. ( a ) Left: Relative abundance of RNA5SP141 transcripts in the cytoplasmic and nuclear fractions of HEK 293T cells that were infected with HSV-1 WT (MOI 1) for 16 h, or left uninfected (Mock), determined by cytoplasmic-nuclear fractionation assay and qRT-PCR. Analysis of the relative abundance of RNU2-1, MALAT1 , and NEAT1 RNA served as controls. Right: IB analysis of Lamin A/C and β-Tubulin confirmed the purity of the nuclear and cytoplasmic fraction, respectively. IB analysis of whole cell lysates (WCL) with anti-HSV-1 infected cell protein 8 (ICP8) and anti-actin served as infection and loading controls, respectively. Left margin, size in kilodaltons (kDa). ( b ) Left: Relative abundance of RNA5SP141 transcripts in the cytoplasmic and nuclear fractions of HEK 293T cells that were infected with SeV (50 HAU/ml) for 16 h or left uninfected (Mock), determined by fractionation assay and qRT-PCR analysis as described in (a). Right: IB analysis of cytoplasmic and nuclear fractions as in (a). IB analysis of WCL with anti-SeV confirmed efficient infection. Data are representative of two independent experiments.

Techniques Used: Infection, Fractionation, Quantitative RT-PCR

Downregulation of TST and MRPL18 by HSV-1 allows RIG-I activation. ( a ) Binding of biotinylated in vitro -transcribed RNA5SP141 to FLAG-tagged RPL5, MRPL18, and TST in transiently transfected HEK 293T cells, assessed by streptavidin pulldown (Strep-PD) and IB with anti-FLAG. WCLs were probed by IB with anti-FLAG and anti-actin. Biotinylated 5S rRNA and a scrambled random RNA (Scrambled) served as positive and negative controls, respectively. ( b ) Relative mRNA expression of RPL5, MRPL18 , and TST in HEK 293T cells infected with HSV-1 (MOI 1) for 16 h as compared to uninfected cells, determined by RNAseq. Red boundaries represent ±2-fold change in gene expression. ( c ) qRT-PCR analysis of RPL5, MRPL18 , and TST mRNA in HEK 293T cells infected with HSV-1 (MOI 1) for the indicated times. ( d ) IB analysis of endogenous RPL5, MRPL18, and TST proteins in the WCLs of HEK 293T cells infected with HSV-1 WT (revertant) or HSV-1 Δvhs (both MOI 10) for the indicated times. IB analysis of HSV-1 ICP8 and cellular Actin served as infection and loading controls, respectively. ( e ) HEK 293T cells were transfected with FLAG-RIG-I or FLAG-GFP. 24 h later, cells were infected with HSV-1 WT (revertant) or HSV-1 Δvhs (both MOI 10) for 16 h, or left uninfected (Mock). RNA bound to FLAG-RIG-I or FLAG-GFP was precipitated from cell lysates using anti-FLAG PD as described in Figure 1a , followed by qRT-PCR analysis to assess bound RNA5SP141 transcripts. ( f ) qRT-PCR analysis of IFNB1 mRNA in NHLF cells infected with HSV-1 WT (revertant) or HSV-1 Δvhs (both MOI 1) for 16 h and 24 h, or left uninfected (Mock). ( g ) qRT-PCR analysis of IFNB1 transcripts in HEK 293T cells transfected with the indicated siRNAs for 30 h and then transfected with either no RNA (Mock) or 1 pmol of in vitro –transcribed RNA5SP141 for 16 h. ( h ) IFN-β luciferase reporter activity in HEK 293T cells transfected for 30 h with the indicated amounts of plasmids expressing FLAG-tagged RPL5, MRPL18, or TST and subsequently transfected with 1 pmol of RNA5SP141 for 16 h to stimulate RIG-I signaling. Expression of FLAG-tagged proteins was confirmed in the WCL by IB with anti-FLAG. Data are representative of two ( a, c-f ), one ( b ) or three ( g, h ) independent experiments (mean and s.d. of n = 3 technical replicates in e , n = 2 biological replicates in c, f , and h , or n = 3 biological replicates in g ). * P
Figure Legend Snippet: Downregulation of TST and MRPL18 by HSV-1 allows RIG-I activation. ( a ) Binding of biotinylated in vitro -transcribed RNA5SP141 to FLAG-tagged RPL5, MRPL18, and TST in transiently transfected HEK 293T cells, assessed by streptavidin pulldown (Strep-PD) and IB with anti-FLAG. WCLs were probed by IB with anti-FLAG and anti-actin. Biotinylated 5S rRNA and a scrambled random RNA (Scrambled) served as positive and negative controls, respectively. ( b ) Relative mRNA expression of RPL5, MRPL18 , and TST in HEK 293T cells infected with HSV-1 (MOI 1) for 16 h as compared to uninfected cells, determined by RNAseq. Red boundaries represent ±2-fold change in gene expression. ( c ) qRT-PCR analysis of RPL5, MRPL18 , and TST mRNA in HEK 293T cells infected with HSV-1 (MOI 1) for the indicated times. ( d ) IB analysis of endogenous RPL5, MRPL18, and TST proteins in the WCLs of HEK 293T cells infected with HSV-1 WT (revertant) or HSV-1 Δvhs (both MOI 10) for the indicated times. IB analysis of HSV-1 ICP8 and cellular Actin served as infection and loading controls, respectively. ( e ) HEK 293T cells were transfected with FLAG-RIG-I or FLAG-GFP. 24 h later, cells were infected with HSV-1 WT (revertant) or HSV-1 Δvhs (both MOI 10) for 16 h, or left uninfected (Mock). RNA bound to FLAG-RIG-I or FLAG-GFP was precipitated from cell lysates using anti-FLAG PD as described in Figure 1a , followed by qRT-PCR analysis to assess bound RNA5SP141 transcripts. ( f ) qRT-PCR analysis of IFNB1 mRNA in NHLF cells infected with HSV-1 WT (revertant) or HSV-1 Δvhs (both MOI 1) for 16 h and 24 h, or left uninfected (Mock). ( g ) qRT-PCR analysis of IFNB1 transcripts in HEK 293T cells transfected with the indicated siRNAs for 30 h and then transfected with either no RNA (Mock) or 1 pmol of in vitro –transcribed RNA5SP141 for 16 h. ( h ) IFN-β luciferase reporter activity in HEK 293T cells transfected for 30 h with the indicated amounts of plasmids expressing FLAG-tagged RPL5, MRPL18, or TST and subsequently transfected with 1 pmol of RNA5SP141 for 16 h to stimulate RIG-I signaling. Expression of FLAG-tagged proteins was confirmed in the WCL by IB with anti-FLAG. Data are representative of two ( a, c-f ), one ( b ) or three ( g, h ) independent experiments (mean and s.d. of n = 3 technical replicates in e , n = 2 biological replicates in c, f , and h , or n = 3 biological replicates in g ). * P

Techniques Used: Activation Assay, Binding Assay, In Vitro, Transfection, Expressing, Infection, Quantitative RT-PCR, Luciferase, Activity Assay

Endogenous non-coding RNAs co-immunoprecipitate with RIG-I during HSV-1 infection. ( a ) Schematic representation of the experimental setup for isolation and identification of RNAs from FLAG-RIG-I- or FLAG-GFP-precipitates. RNAseq, next-generation RNA sequencing. ( b ) ISRE-luciferase reporter activity in HEK 293T cells transfected for 18 h with 7 μl RNA from FLAG-RIG-I- or FLAG-GFP-precipitates from uninfected (Mock) or HSV-1 mut -infected cells, isolated as described in (a). RNA from FLAG-RIG-I- or FLAG-GFP-precipitates from HEK 293T cells infected with SeV (50 HAU/ml) served as control. Luciferase activity is presented as fold induction relative to the values for FLAG-GFP-precipitates from uninfected cells, set to 1. ( c ) Relative enrichment of HSV-1–derived or human-derived transcripts in FLAG-RIG-I precipitates from HSV-1 mut -infected cells, determined by RNAseq. Relative enrichment (log 2 ) of transcripts was calculated by comparing the abundance of transcripts in FLAG-RIG-I precipitates to FLAG-GFP precipitates. Data are from two independent experiments. ( d ) Relative enrichment (log 10 ) of human transcripts in FLAG-RIG-I precipitates from HSV-1 mut -infected cells. The 9 most highly-enriched non-coding transcripts are shown. rRNA pseudogene (red); long non-coding RNA (lncRNA, green); small nucleolar RNA (snoRNA, yellow); small Cajal body-specific RNAs (scaRNA, purple). ( e ) Relative enrichment of the 10 most highly-enriched human transcripts (both non-coding and coding) in FLAG-RIG-I precipitates from HSV-1 mut -infected cells, as compared to uninfected cells. ( f ) Quantitative RT-PCR (qRT-PCR) analysis of RNA5SP141 transcripts from RNA isolated as described in (a) from HEK 293T cells transfected with FLAG-RIG-I or FLAG-GFP and infected with HSV-1 WT or HSV-1 mut (both MOI 1), or left uninfected (Mock). Data are representative of two independent experiments ( b,f ; mean and s.d. of n = 3 technical replicates) or are from two independent experiments ( c-e ). ** P
Figure Legend Snippet: Endogenous non-coding RNAs co-immunoprecipitate with RIG-I during HSV-1 infection. ( a ) Schematic representation of the experimental setup for isolation and identification of RNAs from FLAG-RIG-I- or FLAG-GFP-precipitates. RNAseq, next-generation RNA sequencing. ( b ) ISRE-luciferase reporter activity in HEK 293T cells transfected for 18 h with 7 μl RNA from FLAG-RIG-I- or FLAG-GFP-precipitates from uninfected (Mock) or HSV-1 mut -infected cells, isolated as described in (a). RNA from FLAG-RIG-I- or FLAG-GFP-precipitates from HEK 293T cells infected with SeV (50 HAU/ml) served as control. Luciferase activity is presented as fold induction relative to the values for FLAG-GFP-precipitates from uninfected cells, set to 1. ( c ) Relative enrichment of HSV-1–derived or human-derived transcripts in FLAG-RIG-I precipitates from HSV-1 mut -infected cells, determined by RNAseq. Relative enrichment (log 2 ) of transcripts was calculated by comparing the abundance of transcripts in FLAG-RIG-I precipitates to FLAG-GFP precipitates. Data are from two independent experiments. ( d ) Relative enrichment (log 10 ) of human transcripts in FLAG-RIG-I precipitates from HSV-1 mut -infected cells. The 9 most highly-enriched non-coding transcripts are shown. rRNA pseudogene (red); long non-coding RNA (lncRNA, green); small nucleolar RNA (snoRNA, yellow); small Cajal body-specific RNAs (scaRNA, purple). ( e ) Relative enrichment of the 10 most highly-enriched human transcripts (both non-coding and coding) in FLAG-RIG-I precipitates from HSV-1 mut -infected cells, as compared to uninfected cells. ( f ) Quantitative RT-PCR (qRT-PCR) analysis of RNA5SP141 transcripts from RNA isolated as described in (a) from HEK 293T cells transfected with FLAG-RIG-I or FLAG-GFP and infected with HSV-1 WT or HSV-1 mut (both MOI 1), or left uninfected (Mock). Data are representative of two independent experiments ( b,f ; mean and s.d. of n = 3 technical replicates) or are from two independent experiments ( c-e ). ** P

Techniques Used: Infection, Isolation, RNA Sequencing Assay, Luciferase, Activity Assay, Transfection, Derivative Assay, Quantitative RT-PCR

19) 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

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

20) 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

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

21) Product Images from "An Essential Function for the ATR-Activation-Domain (AAD) of TopBP1 in Mouse Development and Cellular Senescence"

Article Title: An Essential Function for the ATR-Activation-Domain (AAD) of TopBP1 in Mouse Development and Cellular Senescence

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003702

AAD mutation induces premature cellular senescence. ( A ) GFP+ TopBP1 ki/+ cells were sorted 24 hr after transfection and cultured. Images show the cell density and morphology at D1 and D5. Enlargement shows a representative area of TopBP1 ki/− cells from D5. ( B ) SA- β - galactosidase staining of cells 6 days after shRNA transfection shown in blue. ( C ) Quantification of SA-β- galactosidase positive cells from B. The data represent the mean ± SD of at least 500 cells from 2 independent experiments. P value: Student's t -test. ( D ) Semi-quantitative RT-PCR analysis of RNA isolated from D5 cultures from A. The expression level (indicated on top of each sample) was estimated by quantification normalized to the level of GAPDH and then correlated with GFP-shLuc transfected cells. Two independent experiments were performed which showed equivalent results.
Figure Legend Snippet: AAD mutation induces premature cellular senescence. ( A ) GFP+ TopBP1 ki/+ cells were sorted 24 hr after transfection and cultured. Images show the cell density and morphology at D1 and D5. Enlargement shows a representative area of TopBP1 ki/− cells from D5. ( B ) SA- β - galactosidase staining of cells 6 days after shRNA transfection shown in blue. ( C ) Quantification of SA-β- galactosidase positive cells from B. The data represent the mean ± SD of at least 500 cells from 2 independent experiments. P value: Student's t -test. ( D ) Semi-quantitative RT-PCR analysis of RNA isolated from D5 cultures from A. The expression level (indicated on top of each sample) was estimated by quantification normalized to the level of GAPDH and then correlated with GFP-shLuc transfected cells. Two independent experiments were performed which showed equivalent results.

Techniques Used: Mutagenesis, Transfection, Cell Culture, Staining, shRNA, Quantitative RT-PCR, Isolation, Expressing

22) Product Images from "Brassinosteroids modulate ABA-induced stomatal closure in Arabidopsis"

Article Title: Brassinosteroids modulate ABA-induced stomatal closure in Arabidopsis

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/erw385

High concentrations of BL inhibited ABA-induced NO production. (A) Relative expression of NIA1 and NIA2 in response to ABA and BL alone, and in combination. qRT-PCR analyses were performed in triplicate using RNA isolated from 10-d-old wild type Col-0 seedlings. Data were normalized to the expression of ubiquitin . Experiments were independently repeated twice. Error bars indicate standard error. Values labeled with different letters (roman and italic, respectively) are statistically different analysed by one-way ANOVA ( P
Figure Legend Snippet: High concentrations of BL inhibited ABA-induced NO production. (A) Relative expression of NIA1 and NIA2 in response to ABA and BL alone, and in combination. qRT-PCR analyses were performed in triplicate using RNA isolated from 10-d-old wild type Col-0 seedlings. Data were normalized to the expression of ubiquitin . Experiments were independently repeated twice. Error bars indicate standard error. Values labeled with different letters (roman and italic, respectively) are statistically different analysed by one-way ANOVA ( P

Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Labeling

High concentrations of BL inhibited ABA-induced ROS production. (A) Relative expression of AtrbohD and AtrbohF in response to ABA and BL alone, and as a co-treatment. qRT-PCR analyses were performed in triplicate using RNA isolated from 10-d-old wild type Col-0 seedlings. Data were normalized to the expression of ubiquitin . Experiments were independently repeated twice. Error bars indicate standard error. Values labeled with different letters (roman and italic, respectively) are statistically different analysed by one-way ANOVA ( P
Figure Legend Snippet: High concentrations of BL inhibited ABA-induced ROS production. (A) Relative expression of AtrbohD and AtrbohF in response to ABA and BL alone, and as a co-treatment. qRT-PCR analyses were performed in triplicate using RNA isolated from 10-d-old wild type Col-0 seedlings. Data were normalized to the expression of ubiquitin . Experiments were independently repeated twice. Error bars indicate standard error. Values labeled with different letters (roman and italic, respectively) are statistically different analysed by one-way ANOVA ( P

Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Labeling

23) Product Images from "The terminal enzymes of cholesterol synthesis, DHCR24 and DHCR7, interact physically and functionally [S]"

Article Title: The terminal enzymes of cholesterol synthesis, DHCR24 and DHCR7, interact physically and functionally [S]

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M056986

DHCR24 knockdown decreases DHCR7 activity. CHO-7 cells were transfected with control, hamster-specific DHCR24 , or DHCR7 siRNA (25 nM) for 24 h. A: The cells were washed with PBS and refed fresh media overnight. Total RNA was harvested and reverse transcribed
Figure Legend Snippet: DHCR24 knockdown decreases DHCR7 activity. CHO-7 cells were transfected with control, hamster-specific DHCR24 , or DHCR7 siRNA (25 nM) for 24 h. A: The cells were washed with PBS and refed fresh media overnight. Total RNA was harvested and reverse transcribed

Techniques Used: Activity Assay, Transfection

24) Product Images from "The Negative Effects of KPN00353 on Glycerol Kinase and Microaerobic 1,3-Propanediol Production in Klebsiella pneumoniae"

Article Title: The Negative Effects of KPN00353 on Glycerol Kinase and Microaerobic 1,3-Propanediol Production in Klebsiella pneumoniae

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.02441

Schematic diagram of the genetic organization of KPN00353 (A), KPN00352 (B), KPN00351 (C), KPN00350 (D), KPN00349 (E), KPN00348 (F) and KPN00354 (G) in K. pneumoniae MGH 78578. Arrows denote the direction of transcription. Each line indicates a junction (i, ii, iii, iv, and v) between the ORFs. The RNA extracted from wild-type K. pneumoniae was analyzed by RT-PCR (a) or PCR (b). The genomic DNA of the wild-type strain was analyzed by PCR as a positive control (c).
Figure Legend Snippet: Schematic diagram of the genetic organization of KPN00353 (A), KPN00352 (B), KPN00351 (C), KPN00350 (D), KPN00349 (E), KPN00348 (F) and KPN00354 (G) in K. pneumoniae MGH 78578. Arrows denote the direction of transcription. Each line indicates a junction (i, ii, iii, iv, and v) between the ORFs. The RNA extracted from wild-type K. pneumoniae was analyzed by RT-PCR (a) or PCR (b). The genomic DNA of the wild-type strain was analyzed by PCR as a positive control (c).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Positive Control

25) Product Images from "Transcriptional Organization and Dynamic Expression of the hbpCAD Genes, Which Encode the First Three Enzymes for 2-Hydroxybiphenyl Degradation in Pseudomonas azelaica HBP1"

Article Title: Transcriptional Organization and Dynamic Expression of the hbpCAD Genes, Which Encode the First Three Enzymes for 2-Hydroxybiphenyl Degradation in Pseudomonas azelaica HBP1

Journal: Journal of Bacteriology

doi: 10.1128/JB.183-1.270-279.2001

Northern analysis of the different hbp transcripts. RNA was isolated from a carbon-limited glucose-grown chemostat culture of P. azelaica HBP1, 1 min before and 3, 7, 13, 23, and 30 min after a shift to an immediate pulse of 0.5 mM 2-HBP and subsequent medium change to glucose plus 0.5 mM 2-HBP. From each sample, approximately 1 (A to C) or 8 μg (D) of RNA was blotted in each lane and hybridized with radioactively labeled probes against hbpC (A), hbpA (B), hbpD (C), or hbpR (D). Black bars, locations and sizes of the gene-specific probes. Transcript sizes are marked on the right. The presence of two bands in panel D is an artifact caused by the abundance of 16S rRNA migrating slightly below the hbpR transcript.
Figure Legend Snippet: Northern analysis of the different hbp transcripts. RNA was isolated from a carbon-limited glucose-grown chemostat culture of P. azelaica HBP1, 1 min before and 3, 7, 13, 23, and 30 min after a shift to an immediate pulse of 0.5 mM 2-HBP and subsequent medium change to glucose plus 0.5 mM 2-HBP. From each sample, approximately 1 (A to C) or 8 μg (D) of RNA was blotted in each lane and hybridized with radioactively labeled probes against hbpC (A), hbpA (B), hbpD (C), or hbpR (D). Black bars, locations and sizes of the gene-specific probes. Transcript sizes are marked on the right. The presence of two bands in panel D is an artifact caused by the abundance of 16S rRNA migrating slightly below the hbpR transcript.

Techniques Used: Northern Blot, Isolation, Labeling

Mapping of the in vivo transcriptional start sites of the hbpC (A) and the hbpD (B) genes by primer extension analysis of RNA isolated from a glucose-grown chemostat culture of P. azelaica HBP1 1 min before and 7 min after induction with 0.5 mM 2-HBP. The primer extension products were run next to products of sequence reactions performed with the same primer. +1, transcriptional start site. An expanded view of the (complementary) nucleotide sequence (5′-to-3′ direction) surrounding the ς 54 ).
Figure Legend Snippet: Mapping of the in vivo transcriptional start sites of the hbpC (A) and the hbpD (B) genes by primer extension analysis of RNA isolated from a glucose-grown chemostat culture of P. azelaica HBP1 1 min before and 7 min after induction with 0.5 mM 2-HBP. The primer extension products were run next to products of sequence reactions performed with the same primer. +1, transcriptional start site. An expanded view of the (complementary) nucleotide sequence (5′-to-3′ direction) surrounding the ς 54 ).

Techniques Used: In Vivo, Isolation, Sequencing

26) Product Images from "In Situ Dimerization of Multiple Wild Type and Mutant Zinc Transporters in Live Cells Using Bimolecular Fluorescence Complementation"

Article Title: In Situ Dimerization of Multiple Wild Type and Mutant Zinc Transporters in Live Cells Using Bimolecular Fluorescence Complementation

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.533786

Endogenous expression of ZnT1–7 in MCF-7 cells. RNA was purified from MCF-7 cells and reverse transcribed into cDNA. RT-PCR was performed using primers targeted to the ORFs of ZnT1–7. PCR products were resolved on 2% agarose gel, and a
Figure Legend Snippet: Endogenous expression of ZnT1–7 in MCF-7 cells. RNA was purified from MCF-7 cells and reverse transcribed into cDNA. RT-PCR was performed using primers targeted to the ORFs of ZnT1–7. PCR products were resolved on 2% agarose gel, and a

Techniques Used: Expressing, Purification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis

27) Product Images from "Chromatin Landscape Distinguishes the Genomic Loci of Hundreds of Androgen-Receptor-Associated LincRNAs From the Loci of Non-associated LincRNAs"

Article Title: Chromatin Landscape Distinguishes the Genomic Loci of Hundreds of Androgen-Receptor-Associated LincRNAs From the Loci of Non-associated LincRNAs

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2018.00132

Validation by RIP-qPCR of a set of ARA-lincRNAs, which were detected in the RIP-seq assay as associated to AR. LNCaP cells treated with 0.1 nM androgen (red lines) or with vehicle control (blue lines) were assayed as indicated on the x -axis. The amount of the indicated lincRNA that was co-immunoprecipitated with antiAR antibody or with IgG from non-immunized rabbit (negative control) was measured by RT-qPCR in three different biological replicates (each represented with a different symbol), and the corresponding points for antiAR and IgG for each replicate are connected with a straight line. The results are shown as % input RNA (mean ± SEM) of three technical replicates for each individual biological replicate. For the four lincRNA genes in the experiment with androgen whose experimental points are connected with red dotted lines, the amount of RIP material was only enough for two technical replicates each, and the enrichment t -test was not applied. Red and blue solid lines = significant difference between antiAR and IgG ( p
Figure Legend Snippet: Validation by RIP-qPCR of a set of ARA-lincRNAs, which were detected in the RIP-seq assay as associated to AR. LNCaP cells treated with 0.1 nM androgen (red lines) or with vehicle control (blue lines) were assayed as indicated on the x -axis. The amount of the indicated lincRNA that was co-immunoprecipitated with antiAR antibody or with IgG from non-immunized rabbit (negative control) was measured by RT-qPCR in three different biological replicates (each represented with a different symbol), and the corresponding points for antiAR and IgG for each replicate are connected with a straight line. The results are shown as % input RNA (mean ± SEM) of three technical replicates for each individual biological replicate. For the four lincRNA genes in the experiment with androgen whose experimental points are connected with red dotted lines, the amount of RIP material was only enough for two technical replicates each, and the enrichment t -test was not applied. Red and blue solid lines = significant difference between antiAR and IgG ( p

Techniques Used: Real-time Polymerase Chain Reaction, Acetylene Reduction Assay, Immunoprecipitation, Negative Control, Quantitative RT-PCR

28) Product Images from "LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation"

Article Title: LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0175849

Immunofluorescence and LC-MS-MS experiments measure the demethylation activity of FTO in vivo. (a) Immunofluorescence analysis of 5hmdC level generated from 5mdC in FTO or TET2 overexpressed Hela cells. Cells were stained with anti-5hmdC antibody (green), showed that 5hmdC signal is obvious in the TET2 overpexpressed cells, instead of TET2 mutant or FTO gene transfected cells. Nuclei are stained by DAPI. Scale bar: 0–50 μm. (b) LC-MS-MS quantification analysis showed percentage of m6A/A in mRNA and total RNA isolated from control and FTO overexpressed cells. (c) LC-MS-MS quantification analysis showed percentage of 5hmdC/dC, 5hmdC/5mdC in DNA isolated from control, FTO and TET2 overexpressed cells. *p
Figure Legend Snippet: Immunofluorescence and LC-MS-MS experiments measure the demethylation activity of FTO in vivo. (a) Immunofluorescence analysis of 5hmdC level generated from 5mdC in FTO or TET2 overexpressed Hela cells. Cells were stained with anti-5hmdC antibody (green), showed that 5hmdC signal is obvious in the TET2 overpexpressed cells, instead of TET2 mutant or FTO gene transfected cells. Nuclei are stained by DAPI. Scale bar: 0–50 μm. (b) LC-MS-MS quantification analysis showed percentage of m6A/A in mRNA and total RNA isolated from control and FTO overexpressed cells. (c) LC-MS-MS quantification analysis showed percentage of 5hmdC/dC, 5hmdC/5mdC in DNA isolated from control, FTO and TET2 overexpressed cells. *p

Techniques Used: Immunofluorescence, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Activity Assay, In Vivo, Generated, Staining, Mutagenesis, Transfection, Isolation

29) Product Images from "Top3? is an RNA topoisomerase that works with Fragile X syndrome protein to promote synapse formation"

Article Title: Top3? is an RNA topoisomerase that works with Fragile X syndrome protein to promote synapse formation

Journal: Nature neuroscience

doi: 10.1038/nn.3479

Top3β binds coding regions of mRNAs, and its bound mRNAs are enriched with match FMRP targets (a) Autoradiograph of a SDS-PAGE gel from the HITS-CLIP assay shows that a significant amount of 32 P-labeled RNA was crosslinked to HF-Top3β. A mock immunoprecipitation was performed using HeLa cells that do not express HF-Top3β. The RNA in gel slices marked H (high) and L (low) were extracted, reverse-transcribed, and sequenced; but only data from H are presented. (b) Histogram illustrates that Top3β binding sites, represented by sequence tags identified by HITS-CLIP, preferentially map to exons, but not introns. The number of sequence tags was counted at specific distances from exon start (left graph) and exon end (right graph) and then converted to tag density per 1 Kb. Data for longer exons are included in Supplementary Fig. 5 . (c) A graph showing that Top3β binding sites on mRNAs are enriched in open-reading frames (ORFs), but not in 5′ or 3′ untranslated regions (UTRs). Peaks are locations with > 15 tags. (d) A ranked plot shows that genes containing higher frequency of Top3β tags are enriched with FMRP targets (red line) than those with lower frequency of Top3β tags. The small inlet graph shows that the ratio between the number of FMRP targets in the top 1000 mRNAs with highest Top3β tag frequency vs. that in the bottom 1000 mRNAs with lowest Top3β tag frequency. The enrichment for HuR was included for comparison. The statistical analyses used Chi-square test. (e) A Venn diagram shows the number of top Top3β targets that overlap those of FMRP 13 . These common targets are listed in Table S2 . (f) A graph shows the top gene ontology (GO) terms that are enriched in the common targets of Top3β and FMRP. The GO terms are ranked by –log ( P values). The boxes highlight the categories that may be relevant to functions of Top3β and FMRP in neurons. The genes of each category are listed in Supplementary Table 4 .
Figure Legend Snippet: Top3β binds coding regions of mRNAs, and its bound mRNAs are enriched with match FMRP targets (a) Autoradiograph of a SDS-PAGE gel from the HITS-CLIP assay shows that a significant amount of 32 P-labeled RNA was crosslinked to HF-Top3β. A mock immunoprecipitation was performed using HeLa cells that do not express HF-Top3β. The RNA in gel slices marked H (high) and L (low) were extracted, reverse-transcribed, and sequenced; but only data from H are presented. (b) Histogram illustrates that Top3β binding sites, represented by sequence tags identified by HITS-CLIP, preferentially map to exons, but not introns. The number of sequence tags was counted at specific distances from exon start (left graph) and exon end (right graph) and then converted to tag density per 1 Kb. Data for longer exons are included in Supplementary Fig. 5 . (c) A graph showing that Top3β binding sites on mRNAs are enriched in open-reading frames (ORFs), but not in 5′ or 3′ untranslated regions (UTRs). Peaks are locations with > 15 tags. (d) A ranked plot shows that genes containing higher frequency of Top3β tags are enriched with FMRP targets (red line) than those with lower frequency of Top3β tags. The small inlet graph shows that the ratio between the number of FMRP targets in the top 1000 mRNAs with highest Top3β tag frequency vs. that in the bottom 1000 mRNAs with lowest Top3β tag frequency. The enrichment for HuR was included for comparison. The statistical analyses used Chi-square test. (e) A Venn diagram shows the number of top Top3β targets that overlap those of FMRP 13 . These common targets are listed in Table S2 . (f) A graph shows the top gene ontology (GO) terms that are enriched in the common targets of Top3β and FMRP. The GO terms are ranked by –log ( P values). The boxes highlight the categories that may be relevant to functions of Top3β and FMRP in neurons. The genes of each category are listed in Supplementary Table 4 .

Techniques Used: Autoradiography, SDS Page, Cross-linking Immunoprecipitation, Labeling, Immunoprecipitation, Binding Assay, Sequencing

Drosophila Top3β and dFmr1 work in the same pathway to promote expression of ptk2/FAK (a) Representative immunofluorescence images, and (b) their quantification, show that the level of ptk2/FAK protein (green) is reduced in NMJs of the 3 rd instar larvae of Top3β and dFmr1 single and double mutant flies. Presynapses of NMJs and axons were labeled with anti-HRP antibody (Red), whereas postsynapses were labeled with anti-DLG antibody (Blue). The FAK staining of the entire NMJ area, which was marked by DLG staining [arrows in (a)], was quantified by using Imaris imaging software and shown in (b). Eighteen sets of the NMJs from wild-type or top3β mutants were stained and quantified. The p -values in the graph were calculated using Student' t-test. Error bars represent standard errors of means (s.e.m.). (c and d) Quantification of immunofluorescence signals of HRP and DLG to serve as comparisons. The representative images shown have been repeated at least twice, and the results are reproducible. (e) A model to illustrate how Top3β -TDRD3 complex may work antagonistically with FMRP to regulate translation of an mRNA. An mRNA may become topologically constrained by circularization through protein-mediated interactions between its 5′-UTR and 3′-poly A tail. The mRNA may contain local duplex regions or hairpin structures. When ribosomes or RNA helicases unwind such structures, it may create topological stress that enhances ribosomal stalling which is facilitated by FMRP binding. Top3β may reduce the topological stress and thus antagonize FMRP-mediated ribosomal stalling. Our biochemistry experiments showed that a large fraction of FMRP in cells does not associate with Top3β-TDRD3. We hypothesize that this fraction of FMRP antagonizes Top3β action. (f) A model that illustrates how Top3β -TDRD3 may cooperate with FMRP to enhance mRNA translation. Our biochemistry experiments show that a fraction of Top3β-TDRD3 complex associates with FMRP and vise versa. We hypothesize that this fraction of FMRP may enhance Top3β-TDRD3 to bind its target mRNA, reduce topological stress, and stimulate mRNA translation.
Figure Legend Snippet: Drosophila Top3β and dFmr1 work in the same pathway to promote expression of ptk2/FAK (a) Representative immunofluorescence images, and (b) their quantification, show that the level of ptk2/FAK protein (green) is reduced in NMJs of the 3 rd instar larvae of Top3β and dFmr1 single and double mutant flies. Presynapses of NMJs and axons were labeled with anti-HRP antibody (Red), whereas postsynapses were labeled with anti-DLG antibody (Blue). The FAK staining of the entire NMJ area, which was marked by DLG staining [arrows in (a)], was quantified by using Imaris imaging software and shown in (b). Eighteen sets of the NMJs from wild-type or top3β mutants were stained and quantified. The p -values in the graph were calculated using Student' t-test. Error bars represent standard errors of means (s.e.m.). (c and d) Quantification of immunofluorescence signals of HRP and DLG to serve as comparisons. The representative images shown have been repeated at least twice, and the results are reproducible. (e) A model to illustrate how Top3β -TDRD3 complex may work antagonistically with FMRP to regulate translation of an mRNA. An mRNA may become topologically constrained by circularization through protein-mediated interactions between its 5′-UTR and 3′-poly A tail. The mRNA may contain local duplex regions or hairpin structures. When ribosomes or RNA helicases unwind such structures, it may create topological stress that enhances ribosomal stalling which is facilitated by FMRP binding. Top3β may reduce the topological stress and thus antagonize FMRP-mediated ribosomal stalling. Our biochemistry experiments showed that a large fraction of FMRP in cells does not associate with Top3β-TDRD3. We hypothesize that this fraction of FMRP antagonizes Top3β action. (f) A model that illustrates how Top3β -TDRD3 may cooperate with FMRP to enhance mRNA translation. Our biochemistry experiments show that a fraction of Top3β-TDRD3 complex associates with FMRP and vise versa. We hypothesize that this fraction of FMRP may enhance Top3β-TDRD3 to bind its target mRNA, reduce topological stress, and stimulate mRNA translation.

Techniques Used: Expressing, Immunofluorescence, Mutagenesis, Labeling, Staining, Imaging, Software, Binding Assay

Top3β has RNA topoisomerase activity that depends on a conserved RGG RNA-binding motif (a) Schematic representation of an RNA topoisomerase assay modified from a previous publication 2 . A synthetic circular RNA substrate contains two pairs of complementary regions (red and green) separated by single-stranded spacers (black). Through strand passage reactions, this substrate is converted to a knot in which the two pairs of complementary regions can form normal double helices. (b) Silver-stained SDS gel showing purified recombinant wildtype or Y336F mutant HF-Top3β proteins. This mutation is known to inactivate topoisomerase activity on DNA. (c) Autoradiographs from the RNA topoisomerase assay show that Top3β, but not its catalytic mutant or Top3α, has RNA topoisomerase activity. The reaction contains 1 nM 32 P-labeled circular RNA substrate and increasing concentrations of wildtype or Y336F Top3β mutant (0.05 nM, 0.1 nM, 0.2 nM, 0.4 nM, 0.8 nM or 1.6 nM), or Top3α (0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM or 30 nM). A ladder of RNA markers and the purified RNA knot were loaded on the left of every panel. The linear breakdown products of cyclic RNA exist in all reactions. A darker exposure of the autoradiographs shows that a small amount of catenane was also generated in the reaction ( Supplementary Fig. 4a ). (d) Top panel: schematic representations showing that Top3β, but not Top3α, contains a RGG motif. Bottom panel: the alignment of RGG motifs of Top3β from several higher eukaryotes. The RGG boxes are indicated by underline. Arginine and Glycine are indicated by red and green letters, respectively. Conserved regions are highlighted in yellow. (e,f) Gel-shift assay (e) and its quantification (f) show that a fusion protein containing MBP and the RGG motif of Top3β (MBP-RGG) preferentially binds RNA compared to DNA. Reaction contains 1 nM single-stranded (ss) or double-stranded (ds) RNA or DNA. MBP-TDRD3 1-187 was included as a negative control. (g,h) RNA topoisomerase assay (g, right panel) and its quantification (h) show that the RGG motif of Top3β is important for its RNA topoisomerase activity; (g, left panel), silver-stained SDS gel of the purified wild-type or RGG motif-deleted Top3β. The representative images shown have been repeated at least twice, and the results are reproducible.
Figure Legend Snippet: Top3β has RNA topoisomerase activity that depends on a conserved RGG RNA-binding motif (a) Schematic representation of an RNA topoisomerase assay modified from a previous publication 2 . A synthetic circular RNA substrate contains two pairs of complementary regions (red and green) separated by single-stranded spacers (black). Through strand passage reactions, this substrate is converted to a knot in which the two pairs of complementary regions can form normal double helices. (b) Silver-stained SDS gel showing purified recombinant wildtype or Y336F mutant HF-Top3β proteins. This mutation is known to inactivate topoisomerase activity on DNA. (c) Autoradiographs from the RNA topoisomerase assay show that Top3β, but not its catalytic mutant or Top3α, has RNA topoisomerase activity. The reaction contains 1 nM 32 P-labeled circular RNA substrate and increasing concentrations of wildtype or Y336F Top3β mutant (0.05 nM, 0.1 nM, 0.2 nM, 0.4 nM, 0.8 nM or 1.6 nM), or Top3α (0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM or 30 nM). A ladder of RNA markers and the purified RNA knot were loaded on the left of every panel. The linear breakdown products of cyclic RNA exist in all reactions. A darker exposure of the autoradiographs shows that a small amount of catenane was also generated in the reaction ( Supplementary Fig. 4a ). (d) Top panel: schematic representations showing that Top3β, but not Top3α, contains a RGG motif. Bottom panel: the alignment of RGG motifs of Top3β from several higher eukaryotes. The RGG boxes are indicated by underline. Arginine and Glycine are indicated by red and green letters, respectively. Conserved regions are highlighted in yellow. (e,f) Gel-shift assay (e) and its quantification (f) show that a fusion protein containing MBP and the RGG motif of Top3β (MBP-RGG) preferentially binds RNA compared to DNA. Reaction contains 1 nM single-stranded (ss) or double-stranded (ds) RNA or DNA. MBP-TDRD3 1-187 was included as a negative control. (g,h) RNA topoisomerase assay (g, right panel) and its quantification (h) show that the RGG motif of Top3β is important for its RNA topoisomerase activity; (g, left panel), silver-stained SDS gel of the purified wild-type or RGG motif-deleted Top3β. The representative images shown have been repeated at least twice, and the results are reproducible.

Techniques Used: Activity Assay, RNA Binding Assay, Modification, Staining, SDS-Gel, Purification, Recombinant, Mutagenesis, Labeling, Generated, Electrophoretic Mobility Shift Assay, Negative Control

30) Product Images from "Formation of 53BP1 foci and ATM activation under oxidative stress is facilitated by RNA:DNA hybrids and loss of ATM-53BP1 expression promotes photoreceptor cell survival in mice"

Article Title: Formation of 53BP1 foci and ATM activation under oxidative stress is facilitated by RNA:DNA hybrids and loss of ATM-53BP1 expression promotes photoreceptor cell survival in mice

Journal: F1000Research

doi: 10.12688/f1000research.15579.1

RNA:DNA-hybrids accumulate in photoreceptor nuclei. ( a ) Labelled diagram shows stratified organization of retinal layers and cell types. ( b ) Immunofluorescence with DNA:RNA hybrid specific S9.6 antibody of mice retinal cells. Tissue was proteolysed and disintegrated for staining (see Methods). Photoreceptors (outer nuclear layer cells) can be identified by typical inverted chromatin, as seen by DAPI stain.
Figure Legend Snippet: RNA:DNA-hybrids accumulate in photoreceptor nuclei. ( a ) Labelled diagram shows stratified organization of retinal layers and cell types. ( b ) Immunofluorescence with DNA:RNA hybrid specific S9.6 antibody of mice retinal cells. Tissue was proteolysed and disintegrated for staining (see Methods). Photoreceptors (outer nuclear layer cells) can be identified by typical inverted chromatin, as seen by DAPI stain.

Techniques Used: Immunofluorescence, Mouse Assay, Staining

RNA:DNA hybrids promote 53BP1 foci formation. ( a ) Immunofluorescence using the anti-flag and anti-53BP1 antibody in cells expressing Flag-tagged hybrid-binding (HB) domain or flag-tagged active RNaseH1. 53BP1 foci were quantified in cells expressing HB-domain or RNaseH1. Column bars represent the mean of n number of cells (described on each column), from three independent experiments. Error bars represent SEM. *P ≤ 0.05; **P
Figure Legend Snippet: RNA:DNA hybrids promote 53BP1 foci formation. ( a ) Immunofluorescence using the anti-flag and anti-53BP1 antibody in cells expressing Flag-tagged hybrid-binding (HB) domain or flag-tagged active RNaseH1. 53BP1 foci were quantified in cells expressing HB-domain or RNaseH1. Column bars represent the mean of n number of cells (described on each column), from three independent experiments. Error bars represent SEM. *P ≤ 0.05; **P

Techniques Used: Immunofluorescence, Expressing, Binding Assay

RNA:DNA hybrids promote ATM activation. ( a ) Western blot of cell extracts treated with H 2 O 2 in presence of ATM inhibitor or ectopic RNaseH1 expression. Loading control is Coomassie-equivalent staining of gels, before transfer (detailed in the Methods). ( b ) Immunofluorescence of cells with antibody against ATM phosphorylated on Ser1981. Images are representative of n=4 (for a), and n=3 (for b) independent experiments.
Figure Legend Snippet: RNA:DNA hybrids promote ATM activation. ( a ) Western blot of cell extracts treated with H 2 O 2 in presence of ATM inhibitor or ectopic RNaseH1 expression. Loading control is Coomassie-equivalent staining of gels, before transfer (detailed in the Methods). ( b ) Immunofluorescence of cells with antibody against ATM phosphorylated on Ser1981. Images are representative of n=4 (for a), and n=3 (for b) independent experiments.

Techniques Used: Activation Assay, Western Blot, Expressing, Staining, Immunofluorescence

RNA:DNA hybrid formation is central to ATM-53BP1 repair pathway. ( a ) Immunofluorescence using the anti-flag to quantify nuclear hybrid-binding (HB) domain foci signal in pre-permeabilized RPE cells and quantification of HB domain signal in cells treated with siATM, ( b ) Western blot showing ATM depletion. ( c ) Immunofluorescence and HB-domain foci quantification in cells treated with ATM inhibitor. ( d ) Quantitation of γH2AX foci in cells expressing HB domain or active RNAseH1, in presence or absence of H 2 O 2 dependent oxidative stress. siC, non-targeted control siLuciferease RNA. Column bars represent the mean of n number of cells (described on each column) from three independent experiments. Error bars represent SEM. *P ≤ 0.05; **P
Figure Legend Snippet: RNA:DNA hybrid formation is central to ATM-53BP1 repair pathway. ( a ) Immunofluorescence using the anti-flag to quantify nuclear hybrid-binding (HB) domain foci signal in pre-permeabilized RPE cells and quantification of HB domain signal in cells treated with siATM, ( b ) Western blot showing ATM depletion. ( c ) Immunofluorescence and HB-domain foci quantification in cells treated with ATM inhibitor. ( d ) Quantitation of γH2AX foci in cells expressing HB domain or active RNAseH1, in presence or absence of H 2 O 2 dependent oxidative stress. siC, non-targeted control siLuciferease RNA. Column bars represent the mean of n number of cells (described on each column) from three independent experiments. Error bars represent SEM. *P ≤ 0.05; **P

Techniques Used: Immunofluorescence, Binding Assay, Western Blot, Quantitation Assay, Expressing

Loss of functional PRPF31 induce RNA:DNA hybrid dependent genomic instability but not in mice retinal neurons. ( a and b ) γH2AX and 53BP1 foci analysis in PRPF31 siRNA-transfected RPE-1 cells. (c and d) γH2AX and 53BP1 foci analysis in vasculo-stromal fraction derived primary cells from Prpf31 +/A216P mice ( Prpf31-ki) . ( e ) γH2AX and 53BP1 foci analysis in retina from Prpf31 +/A216P mice on postnatal day 20. All column bars represent the mean. For ( a - d ) “n”, mentioned on respective column, signify number of cells analyzed from two independent experiments. For ( e ) n=16 for each column and signify number of retinal sections analyzed; acquired from n=4 eyes. Error bars represent Standard error of Mean (SEM). *P≤0.05; **P
Figure Legend Snippet: Loss of functional PRPF31 induce RNA:DNA hybrid dependent genomic instability but not in mice retinal neurons. ( a and b ) γH2AX and 53BP1 foci analysis in PRPF31 siRNA-transfected RPE-1 cells. (c and d) γH2AX and 53BP1 foci analysis in vasculo-stromal fraction derived primary cells from Prpf31 +/A216P mice ( Prpf31-ki) . ( e ) γH2AX and 53BP1 foci analysis in retina from Prpf31 +/A216P mice on postnatal day 20. All column bars represent the mean. For ( a - d ) “n”, mentioned on respective column, signify number of cells analyzed from two independent experiments. For ( e ) n=16 for each column and signify number of retinal sections analyzed; acquired from n=4 eyes. Error bars represent Standard error of Mean (SEM). *P≤0.05; **P

Techniques Used: Functional Assay, Mouse Assay, Transfection, Derivative Assay

31) Product Images from "Long Noncoding RNA UCA1 Regulates PRL-3 Expression by Sponging MicroRNA-495 to Promote the Progression of Gastric Cancer"

Article Title: Long Noncoding RNA UCA1 Regulates PRL-3 Expression by Sponging MicroRNA-495 to Promote the Progression of Gastric Cancer

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2019.10.020

UCA1 Competitively Binds to miR-495 to Upregulate PRL-3 (A) The targeting relationship between UCA1 and miR-495 predicted by the bioinformatics website. (B) The targeting relationship between UCA1 and miR-495 verified by dual-Luc reporter gene assay. (C) The targeting relationship between PRL-3 and miR-495 predicted by the bioinformatics website. (D) The targeting relationship between PRL-3 and miR-495 verified by dual-Luc reporter gene assay. (E) miR-495 binds to UCA1 by RNA pull-down assessment. (F) miR-495 binds to UCA1 by RIP assay. (G) miR-495 was negatively correlated with UCA1. (H) PRL-3 was negatively correlated with miR-495. (I) Expressions of UCA1, miR-495, and PRL-3 detected by qRT-PCR in each group. *p
Figure Legend Snippet: UCA1 Competitively Binds to miR-495 to Upregulate PRL-3 (A) The targeting relationship between UCA1 and miR-495 predicted by the bioinformatics website. (B) The targeting relationship between UCA1 and miR-495 verified by dual-Luc reporter gene assay. (C) The targeting relationship between PRL-3 and miR-495 predicted by the bioinformatics website. (D) The targeting relationship between PRL-3 and miR-495 verified by dual-Luc reporter gene assay. (E) miR-495 binds to UCA1 by RNA pull-down assessment. (F) miR-495 binds to UCA1 by RIP assay. (G) miR-495 was negatively correlated with UCA1. (H) PRL-3 was negatively correlated with miR-495. (I) Expressions of UCA1, miR-495, and PRL-3 detected by qRT-PCR in each group. *p

Techniques Used: Reporter Gene Assay, Quantitative RT-PCR

32) Product Images from "Genomic and transcriptomic alterations in Leishmania donovani lines experimentally resistant to antileishmanial drugs"

Article Title: Genomic and transcriptomic alterations in Leishmania donovani lines experimentally resistant to antileishmanial drugs

Journal: International Journal for Parasitology: Drugs and Drug Resistance

doi: 10.1016/j.ijpddr.2018.04.002

Gene expression analysis of PAD, SMT, D-LDH, and BCAT genes in different L. donovani lines. Total RNA of L. donovani WT line, S, A and P resistant lines, and WT + Lexsy, A + Lexsy, WT + PAD, A + SMT, WT + D-LDH, WT + BCAT, WT + D-LDH + BCAT and WT + BCAT + D-LDH transfected lines, was extracted from promastigotes grown at log-phase as described in Materials and Methods. RT-qPCR expression values of the genes in each line were normalized with the expression of GAPDH. The relative expression of each gene was calculated as the fold-change between the resistant lines and the WT line (which was set to 1.0) (A) or transfected lines with PAD, SMT or D-LDH, BCAT, and WT + Lexsy, WT + Lexsy + pIR1SAT or A + Lexsy (set to 1.0) (B), see Material and Methods for further details. Results shown are the means ± SD from two independent experiments. In all cases, significant differences versus the controls were determined using Student's t -test ( p
Figure Legend Snippet: Gene expression analysis of PAD, SMT, D-LDH, and BCAT genes in different L. donovani lines. Total RNA of L. donovani WT line, S, A and P resistant lines, and WT + Lexsy, A + Lexsy, WT + PAD, A + SMT, WT + D-LDH, WT + BCAT, WT + D-LDH + BCAT and WT + BCAT + D-LDH transfected lines, was extracted from promastigotes grown at log-phase as described in Materials and Methods. RT-qPCR expression values of the genes in each line were normalized with the expression of GAPDH. The relative expression of each gene was calculated as the fold-change between the resistant lines and the WT line (which was set to 1.0) (A) or transfected lines with PAD, SMT or D-LDH, BCAT, and WT + Lexsy, WT + Lexsy + pIR1SAT or A + Lexsy (set to 1.0) (B), see Material and Methods for further details. Results shown are the means ± SD from two independent experiments. In all cases, significant differences versus the controls were determined using Student's t -test ( p

Techniques Used: Expressing, Transfection, Quantitative RT-PCR

33) Product Images from "Identification and characterization of two forms of mouse MUTYH proteins encoded by alternatively spliced transcripts"

Article Title: Identification and characterization of two forms of mouse MUTYH proteins encoded by alternatively spliced transcripts

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkh214

The expression of Mutyh mRNA in various mouse tissues. cDNAs were prepared from total RNA extracted from various mouse tissues, and the common 3′-region for the three types of Mutyh mRNA including exons 13–17 were amplified with a set of primers of mY5A2 + mY3TGA (top panel). The relative amount of total Mutyh mRNA in each tissue to that in thymus, which was normalized by the level of Gapdh mRNA (middle panel), is shown under each lane. Data from amplification for 32 cycles are shown, which achieved a linear range of amplification. The 5′-parts of three types of Mutyh cDNA (type a, b, c) were competitively amplified with a primer set of mY5B1 + mY3A1 for each tissue, and data from amplification for 40 cycles are shown (bottom panel).
Figure Legend Snippet: The expression of Mutyh mRNA in various mouse tissues. cDNAs were prepared from total RNA extracted from various mouse tissues, and the common 3′-region for the three types of Mutyh mRNA including exons 13–17 were amplified with a set of primers of mY5A2 + mY3TGA (top panel). The relative amount of total Mutyh mRNA in each tissue to that in thymus, which was normalized by the level of Gapdh mRNA (middle panel), is shown under each lane. Data from amplification for 32 cycles are shown, which achieved a linear range of amplification. The 5′-parts of three types of Mutyh cDNA (type a, b, c) were competitively amplified with a primer set of mY5B1 + mY3A1 for each tissue, and data from amplification for 40 cycles are shown (bottom panel).

Techniques Used: Expressing, Amplification

Two forms of mMUTYH protein encoded by the alternatively spliced transcripts. ( A ) In vitro translation of type b and type c Mutyh mRNAs. RNAs synthesized from pT7Blue plasmids carrying type b and type c Mutyh cDNA or human MUTYH ) by T7 RNA polymerase were translated using rabbit reticulocyte lysate, and translation products were subjected to a western blot analysis with anti-hMUTYH antibody. No template, during in vitro translation, template RNA was omitted. An arrow indicates 50 kDa mMUTYHα encoded by type b mRNA, and an arrowhead indicates the 47 kDa mMUTYHβ encoded by type c mRNA, respectively. ( B ) The detection of two forms of MUTYH in mouse ES cells. Whole cell extracts prepared from wild-type ES cell line, CCE28 cells, MUTYH-null YDK15 cells, and YDKα or YDKβ cells to which an expression construct for type b or type c cDNA was stably introduced, respectively, were subjected to western blot analysis with anti-hMUTYH antibody. An arrow indicates mMUTYHα and an arrowhead indicates mMUTYHβ, respectively. ( C ) The detection of two forms of mMUTYH protein in mouse thymocytes. Thymocyte extracts prepared from two independent wild-type (lanes 1, 2) and MUTYH-null mice ( Mutyh –/– ; lanes 3, 4) were subjected to western blot analysis with anti-hMUTYH antibody (top panel), or with anti-mMUTYHβN (middle panel). An arrow indicates mMUTYHα and an arrowhead indicates mMUTYHβ, respectively. Stained filter with Coomassie Brilliant Blue is shown (bottom) for loading control.
Figure Legend Snippet: Two forms of mMUTYH protein encoded by the alternatively spliced transcripts. ( A ) In vitro translation of type b and type c Mutyh mRNAs. RNAs synthesized from pT7Blue plasmids carrying type b and type c Mutyh cDNA or human MUTYH ) by T7 RNA polymerase were translated using rabbit reticulocyte lysate, and translation products were subjected to a western blot analysis with anti-hMUTYH antibody. No template, during in vitro translation, template RNA was omitted. An arrow indicates 50 kDa mMUTYHα encoded by type b mRNA, and an arrowhead indicates the 47 kDa mMUTYHβ encoded by type c mRNA, respectively. ( B ) The detection of two forms of MUTYH in mouse ES cells. Whole cell extracts prepared from wild-type ES cell line, CCE28 cells, MUTYH-null YDK15 cells, and YDKα or YDKβ cells to which an expression construct for type b or type c cDNA was stably introduced, respectively, were subjected to western blot analysis with anti-hMUTYH antibody. An arrow indicates mMUTYHα and an arrowhead indicates mMUTYHβ, respectively. ( C ) The detection of two forms of mMUTYH protein in mouse thymocytes. Thymocyte extracts prepared from two independent wild-type (lanes 1, 2) and MUTYH-null mice ( Mutyh –/– ; lanes 3, 4) were subjected to western blot analysis with anti-hMUTYH antibody (top panel), or with anti-mMUTYHβN (middle panel). An arrow indicates mMUTYHα and an arrowhead indicates mMUTYHβ, respectively. Stained filter with Coomassie Brilliant Blue is shown (bottom) for loading control.

Techniques Used: In Vitro, Synthesized, Western Blot, Expressing, Construct, Stable Transfection, Mouse Assay, Staining

34) Product Images from "Regulation of Fibronectin EDA Exon Alternative Splicing: Possible Role of RNA Secondary Structure for Enhancer Display"

Article Title: Regulation of Fibronectin EDA Exon Alternative Splicing: Possible Role of RNA Secondary Structure for Enhancer Display

Journal: Molecular and Cellular Biology

doi:

Enzymatic determination of the RNA secondary structure of the EDA exon. (A) Enzymatic analysis of RNA templates of the Δ4, Tot, and Δ2e constructs. In vitro-transcribed RNAs were enzymatically digested with S1 nuclease and T 1 and V 1 RNases and reverse transcribed, and the RT products were separated on a polyacrylamide sequencing gel as described in Materials and Methods. A sequencing reaction with the same primer was run in parallel to precisely determine the cleavage sites. Squares, circles, and triangles indicate S1 nuclease and RNase T 1 and V 1 ). The ESE and ESS elements are circled.
Figure Legend Snippet: Enzymatic determination of the RNA secondary structure of the EDA exon. (A) Enzymatic analysis of RNA templates of the Δ4, Tot, and Δ2e constructs. In vitro-transcribed RNAs were enzymatically digested with S1 nuclease and T 1 and V 1 RNases and reverse transcribed, and the RT products were separated on a polyacrylamide sequencing gel as described in Materials and Methods. A sequencing reaction with the same primer was run in parallel to precisely determine the cleavage sites. Squares, circles, and triangles indicate S1 nuclease and RNase T 1 and V 1 ). The ESE and ESS elements are circled.

Techniques Used: Construct, In Vitro, Sequencing

35) Product Images from "Phosphorylation of RIG-I by Casein Kinase II Inhibits Its Antiviral Response ▿"

Article Title: Phosphorylation of RIG-I by Casein Kinase II Inhibits Its Antiviral Response ▿

Journal: Journal of Virology

doi: 10.1128/JVI.01734-10

HCV infection results in the dephosphorylation of RIG-I. (A) Specificity of the anti-phospho-antibody. Bacterially expressed RIG-I was incubated with (lane 1) or without (lane 2) CK2α in a buffer containing 100 μM ATP. The mixture was subjected to SDS-PAGE after 1 h of incubation at 37°C, followed by immunoblotting with antibodies against phosphorylated S854 to 855 and T770 peptides of RIG-I, in the presence of either related phosphorylated peptide or nonphosphorylated peptide. (Bottom) The anti-phospho-antibody was generated by the synthetic peptides. (B) 293T cells (top) and HUVEC (bottom) were infected with SeV for the indicated periods of time. Total protein was extracted and subjected to SDS-PAGE, followed by immunoblotting with antibodies against phosphorylated S854 to 855 and T770 peptides of RIG-I, RIG-I, and actin. (C) Huh7 cells were infected with HCV for the indicated periods of time. Total protein was extracted and subjected to SDS-PAGE, followed by immunoblotting with antibodies against phosphorylated S854 to 855 and T770 peptides of RIG-I, RIG-I, and actin. (D, top) Huh7 cells (1 × 10 5 ) were transfected with CK2α-RNAi double-stranded oligonucleotides (2 μg/ml). A total of 36 h after transfection, cells were infected with HCV. Total RNA was extracted at 24 h postinfection and analyzed by real-time PCR for the expression of HCV NS5A and CK2α. (Bottom) Result from Western blotting with specific anti-CK2α and anti-actin antibodies for normalization.
Figure Legend Snippet: HCV infection results in the dephosphorylation of RIG-I. (A) Specificity of the anti-phospho-antibody. Bacterially expressed RIG-I was incubated with (lane 1) or without (lane 2) CK2α in a buffer containing 100 μM ATP. The mixture was subjected to SDS-PAGE after 1 h of incubation at 37°C, followed by immunoblotting with antibodies against phosphorylated S854 to 855 and T770 peptides of RIG-I, in the presence of either related phosphorylated peptide or nonphosphorylated peptide. (Bottom) The anti-phospho-antibody was generated by the synthetic peptides. (B) 293T cells (top) and HUVEC (bottom) were infected with SeV for the indicated periods of time. Total protein was extracted and subjected to SDS-PAGE, followed by immunoblotting with antibodies against phosphorylated S854 to 855 and T770 peptides of RIG-I, RIG-I, and actin. (C) Huh7 cells were infected with HCV for the indicated periods of time. Total protein was extracted and subjected to SDS-PAGE, followed by immunoblotting with antibodies against phosphorylated S854 to 855 and T770 peptides of RIG-I, RIG-I, and actin. (D, top) Huh7 cells (1 × 10 5 ) were transfected with CK2α-RNAi double-stranded oligonucleotides (2 μg/ml). A total of 36 h after transfection, cells were infected with HCV. Total RNA was extracted at 24 h postinfection and analyzed by real-time PCR for the expression of HCV NS5A and CK2α. (Bottom) Result from Western blotting with specific anti-CK2α and anti-actin antibodies for normalization.

Techniques Used: Infection, De-Phosphorylation Assay, Incubation, SDS Page, Generated, Transfection, Real-time Polymerase Chain Reaction, Expressing, Western Blot

CK2α inhibits RIG-I signaling. (A) 293T cells were transfected with different luciferase constructs or expression plasmids containing the genes indicated. Luciferase assays were performed 18 h after transfection. Values are shown as the means ± SD from one representative experiment. The results were obtained from three independent experiments. Rel. Luc. Acti., relative luciferase activity. (B) The indicated amounts of CK2α/β or the CK2α K68M/β mutant were transfected into 293T cells and HUVEC for 16 h, respectively. Cells were treated with SeV or AdV for 8 h. Total RNA was extracted and analyzed by real-time PCR for the expression of IFN-β. (C) 293T cells (top) and HUVEC (bottom) were transfected with CK2α K68M/β or empty plasmids and then pretreated with increasing concentrations of CK2 inhibitor DMAT for 2 h, followed by SeV or AdV infection for 8 h. Total RNA was extracted and analyzed by real-time PCR for the expression of IFN-β. (D) 293T cells (1 × 10 5 ) were transfected with CK2α (2 μg) and CK2β (2 μg) expression plasmids together with an empty control plasmid (2 μg) or with CK2α (2 μg) and CK2β (2 μg) expression plasmids with a RIG-I (2 μg) expression plasmid. At 24 h after transfection, cells were infected with VSV (MOI of 10), and supernatants were harvested at 24 h postinfection. Supernatants were analyzed for VSV production using standard plaque assays. Plaques were counted, and titers were calculated as the number of PFU/ml. (E) 293T cells were pretreated with the indicated concentrations of CK2 inhibitor DMAT for 2 h, followed by the infection of VSV (MOI of 10) for 24 h. Supernatants were analyzed for VSV production using standard plaque assays. (F, left) 293T cells (1 × 10 5 ) were transfected with CK2α-RNAi (2 μg/ml) double-strand oligonucleotides and infected with VSV at 36 h after transfection. Supernatants were analyzed for VSV production using standard plaque assays. (Right) Result from Western blotting with specific anti-CK2α and antiactin antibodies for normalization. All statistic data are represented as the means ± SD. The results were obtained from three independent experiments.
Figure Legend Snippet: CK2α inhibits RIG-I signaling. (A) 293T cells were transfected with different luciferase constructs or expression plasmids containing the genes indicated. Luciferase assays were performed 18 h after transfection. Values are shown as the means ± SD from one representative experiment. The results were obtained from three independent experiments. Rel. Luc. Acti., relative luciferase activity. (B) The indicated amounts of CK2α/β or the CK2α K68M/β mutant were transfected into 293T cells and HUVEC for 16 h, respectively. Cells were treated with SeV or AdV for 8 h. Total RNA was extracted and analyzed by real-time PCR for the expression of IFN-β. (C) 293T cells (top) and HUVEC (bottom) were transfected with CK2α K68M/β or empty plasmids and then pretreated with increasing concentrations of CK2 inhibitor DMAT for 2 h, followed by SeV or AdV infection for 8 h. Total RNA was extracted and analyzed by real-time PCR for the expression of IFN-β. (D) 293T cells (1 × 10 5 ) were transfected with CK2α (2 μg) and CK2β (2 μg) expression plasmids together with an empty control plasmid (2 μg) or with CK2α (2 μg) and CK2β (2 μg) expression plasmids with a RIG-I (2 μg) expression plasmid. At 24 h after transfection, cells were infected with VSV (MOI of 10), and supernatants were harvested at 24 h postinfection. Supernatants were analyzed for VSV production using standard plaque assays. Plaques were counted, and titers were calculated as the number of PFU/ml. (E) 293T cells were pretreated with the indicated concentrations of CK2 inhibitor DMAT for 2 h, followed by the infection of VSV (MOI of 10) for 24 h. Supernatants were analyzed for VSV production using standard plaque assays. (F, left) 293T cells (1 × 10 5 ) were transfected with CK2α-RNAi (2 μg/ml) double-strand oligonucleotides and infected with VSV at 36 h after transfection. Supernatants were analyzed for VSV production using standard plaque assays. (Right) Result from Western blotting with specific anti-CK2α and antiactin antibodies for normalization. All statistic data are represented as the means ± SD. The results were obtained from three independent experiments.

Techniques Used: Transfection, Luciferase, Construct, Expressing, Activity Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Infection, Plasmid Preparation, Western Blot

Dephosphorylation of threonine 770 and serine 854 and 855 results in the activation of RIG-I. (A) 293T cells were transfected with the indicated luciferase construct and plasmids containing RIG-I or its variants. The luciferase assay was performed 18 h after transfection. Values are shown as the means ± SD from one representative experiment. The results were obtained from three independent experiments. (B) 293T cells were transfected with the plasmids containing RIG-I or its variants for 24 h. Total RNA was extracted and analyzed by real-time PCR for the expression of IFN-β and actin. (C) Bacterially expressed His-tagged RIG-I fragments were incubated with GST-CK2 in buffer containing 100 μM unlabeled ATP and 5 μCi of [γ- 32 P]ATP. SDS-PAGE was performed with the above-described mixture after the reaction. (Top) Phosphorylation was analyzed by autoradiography. (Bottom) The loading protein was stained with Coomassie blue. (D) The bacterially expressed His-tagged RIG-I RD and its mutants were incubated with GST-CK2 in buffer containing 100 μM unlabeled ATP and 5 μCi of [γ- 32 P]ATP. SDS-PAGE was performed with the above-described mixture after the reaction. (Top) Phosphorylation was analyzed by autoradiography. (Bottom) The loading protein was stained with Coomassie blue. (E) 293T cells were transfected with the plasmids containing the Flag-tagged RIG-I RD and its mutants for 24 h. The ectopic protein was immunoprecipitated by anti-Flag antibody and immunoblotted with antiphosphoserine or antiphosphothreonine and anti-Flag antibody. (F, top) 293T cells were pretreated with increasing concentrations of the serine/threonine-specific phosphatase inhibitor OA for 2 h, followed by infection with SeV for 8 h. Total RNA was extracted and analyzed by real-time PCR for the expression of IFN-β. (Bottom) The same experiment was then performed with HUVEC. (G) Phosphopeptide sequences identified by mass spectrometry. *, phosphorylation sites.
Figure Legend Snippet: Dephosphorylation of threonine 770 and serine 854 and 855 results in the activation of RIG-I. (A) 293T cells were transfected with the indicated luciferase construct and plasmids containing RIG-I or its variants. The luciferase assay was performed 18 h after transfection. Values are shown as the means ± SD from one representative experiment. The results were obtained from three independent experiments. (B) 293T cells were transfected with the plasmids containing RIG-I or its variants for 24 h. Total RNA was extracted and analyzed by real-time PCR for the expression of IFN-β and actin. (C) Bacterially expressed His-tagged RIG-I fragments were incubated with GST-CK2 in buffer containing 100 μM unlabeled ATP and 5 μCi of [γ- 32 P]ATP. SDS-PAGE was performed with the above-described mixture after the reaction. (Top) Phosphorylation was analyzed by autoradiography. (Bottom) The loading protein was stained with Coomassie blue. (D) The bacterially expressed His-tagged RIG-I RD and its mutants were incubated with GST-CK2 in buffer containing 100 μM unlabeled ATP and 5 μCi of [γ- 32 P]ATP. SDS-PAGE was performed with the above-described mixture after the reaction. (Top) Phosphorylation was analyzed by autoradiography. (Bottom) The loading protein was stained with Coomassie blue. (E) 293T cells were transfected with the plasmids containing the Flag-tagged RIG-I RD and its mutants for 24 h. The ectopic protein was immunoprecipitated by anti-Flag antibody and immunoblotted with antiphosphoserine or antiphosphothreonine and anti-Flag antibody. (F, top) 293T cells were pretreated with increasing concentrations of the serine/threonine-specific phosphatase inhibitor OA for 2 h, followed by infection with SeV for 8 h. Total RNA was extracted and analyzed by real-time PCR for the expression of IFN-β. (Bottom) The same experiment was then performed with HUVEC. (G) Phosphopeptide sequences identified by mass spectrometry. *, phosphorylation sites.

Techniques Used: De-Phosphorylation Assay, Activation Assay, Transfection, Luciferase, Construct, Real-time Polymerase Chain Reaction, Expressing, Incubation, SDS Page, Autoradiography, Staining, Immunoprecipitation, Infection, Mass Spectrometry

36) Product Images from "The RNA targetome of Staphylococcus aureus non-coding RNA RsaA: impact on cell surface properties and defense mechanisms"

Article Title: The RNA targetome of Staphylococcus aureus non-coding RNA RsaA: impact on cell surface properties and defense mechanisms

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx219

The MS2-RsaA variant is normally expressed, correctly retained by affinity chromatography and functional. ( A ) Northern blots showing the expression of RsaA and the MS2-RsaA variant in HG001 WT and HG001-Δ rsaA strains. Total RNAs were prepared after 2, 4 and 6 h of culture in BHI medium at 37°C. Hybridization against 5S RNA was used as loading control. ( B ) Northern blot targeting RsaA, MS2-RsaA, mgr A or hu performed on RNAs purified after MS2 chromatography affinity; 1 μg of total RNA was loaded on a 2% agarose gel. CE: crude extract; FT : Flow-through ; W : Washing ; EL : Elution. mgr A* denotes a short fragment of the mgr A mRNA (below 274 nts) that was specifically detected by the mgr A probe in the elution fraction. This RNA fragment most likely represented a degradation product of mgr A mRNA containing the sequences interacting with RsaA. ( C ) In vitro translation assay performed with PURESYSTEM. The reactions were performed with 10 pmol of wild-type (WT) mgr A mRNA and in the presence of increasing quantities of WT RsaA or MS2-RsaA. The proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (10%) and were revealed using a FLAG-specific antibody. *unspecific protein revealed with the FLAG-antibody, this protein was used as an internal loading control.
Figure Legend Snippet: The MS2-RsaA variant is normally expressed, correctly retained by affinity chromatography and functional. ( A ) Northern blots showing the expression of RsaA and the MS2-RsaA variant in HG001 WT and HG001-Δ rsaA strains. Total RNAs were prepared after 2, 4 and 6 h of culture in BHI medium at 37°C. Hybridization against 5S RNA was used as loading control. ( B ) Northern blot targeting RsaA, MS2-RsaA, mgr A or hu performed on RNAs purified after MS2 chromatography affinity; 1 μg of total RNA was loaded on a 2% agarose gel. CE: crude extract; FT : Flow-through ; W : Washing ; EL : Elution. mgr A* denotes a short fragment of the mgr A mRNA (below 274 nts) that was specifically detected by the mgr A probe in the elution fraction. This RNA fragment most likely represented a degradation product of mgr A mRNA containing the sequences interacting with RsaA. ( C ) In vitro translation assay performed with PURESYSTEM. The reactions were performed with 10 pmol of wild-type (WT) mgr A mRNA and in the presence of increasing quantities of WT RsaA or MS2-RsaA. The proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (10%) and were revealed using a FLAG-specific antibody. *unspecific protein revealed with the FLAG-antibody, this protein was used as an internal loading control.

Techniques Used: Variant Assay, Affinity Chromatography, Functional Assay, Northern Blot, Expressing, Hybridization, Purification, Chromatography, Agarose Gel Electrophoresis, Flow Cytometry, In Vitro, Polyacrylamide Gel Electrophoresis

The expression of target mRNAs was quantified by RT-qPCR. The data were normalized to the level of gyr B mRNA expression from total RNA extracts prepared from in vitro culture to the late-exponential phase (6 h) of HG001 (WT), the Δ rsa A mutant strain and the complemented strain with a plasmid expressing RsaA under its own promoter.
Figure Legend Snippet: The expression of target mRNAs was quantified by RT-qPCR. The data were normalized to the level of gyr B mRNA expression from total RNA extracts prepared from in vitro culture to the late-exponential phase (6 h) of HG001 (WT), the Δ rsa A mutant strain and the complemented strain with a plasmid expressing RsaA under its own promoter.

Techniques Used: Expressing, Quantitative RT-PCR, In Vitro, Mutagenesis, Plasmid Preparation

Schematic drawing summarizing the regulatory networks involving RsaA and its mRNA targets in Staphylococcus aureus . RsaA activated by σ B binds to mgr A, flr and ssa A mRNAs to inhibit their translation. The role of RsaA interaction with HG001_01977 is still unclear. Arrows are for activation, bars for repression. In blue are the transcriptional protein regulators, in red the regulatory RNA and in grey the virulence factors. Red lines corresponded to post-transcriptional regulation and black lines to transcriptional regulation. Dotted line represented the regulatory events for which direct regulation is not yet demonstrated.
Figure Legend Snippet: Schematic drawing summarizing the regulatory networks involving RsaA and its mRNA targets in Staphylococcus aureus . RsaA activated by σ B binds to mgr A, flr and ssa A mRNAs to inhibit their translation. The role of RsaA interaction with HG001_01977 is still unclear. Arrows are for activation, bars for repression. In blue are the transcriptional protein regulators, in red the regulatory RNA and in grey the virulence factors. Red lines corresponded to post-transcriptional regulation and black lines to transcriptional regulation. Dotted line represented the regulatory events for which direct regulation is not yet demonstrated.

Techniques Used: Activation Assay

37) Product Images from "Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism"

Article Title: Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism

Journal: RNA

doi: 10.1261/rna.1230209

Cis -splicing of poly A polymerase ( PAP ) and ATP-dependent DEAD/H RNA helicases ( ADRH ) is regulated by PTB1 but not by PTB2. Levels of PAP and ADRH precursor and mature mRNA detected by RT-PCR. cDNA was prepared as described in Materials and Methods.
Figure Legend Snippet: Cis -splicing of poly A polymerase ( PAP ) and ATP-dependent DEAD/H RNA helicases ( ADRH ) is regulated by PTB1 but not by PTB2. Levels of PAP and ADRH precursor and mature mRNA detected by RT-PCR. cDNA was prepared as described in Materials and Methods.

Techniques Used: Reverse Transcription Polymerase Chain Reaction

Validation of the microarray data by RT-PCR. cDNA was prepared from total RNA (1 μg) derived from uninduced cells (−Tet) or cells after 3 d of silencing (+Tet), as described in Materials and Methods. An aliquot of the cDNA (1/50 to 1/100)
Figure Legend Snippet: Validation of the microarray data by RT-PCR. cDNA was prepared from total RNA (1 μg) derived from uninduced cells (−Tet) or cells after 3 d of silencing (+Tet), as described in Materials and Methods. An aliquot of the cDNA (1/50 to 1/100)

Techniques Used: Microarray, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

38) Product Images from "Perilipin 1 Mediates Lipid Metabolism Homeostasis and Inhibits Inflammatory Cytokine Synthesis in Bovine Adipocytes"

Article Title: Perilipin 1 Mediates Lipid Metabolism Homeostasis and Inhibits Inflammatory Cytokine Synthesis in Bovine Adipocytes

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00467

Perilipin-1 (PLIN1) silencing increased the expression and synthesis of inflammatory cytokines. Adipocytes were transfected with siPLIN1 and NC with or without LPS. Nine replicate samples were used for each condition ( N = 9). (A–C) The mRNA expression levels of IL-1β, IL-6, and TNF-α. (D–F) Supernatant concentrations of IL-1β, IL-6, and TNF-α. (C) Control group. Abbreviations: NC, negative control siRNA; siPLIN1, small interfering RNA of PLIN1; LPS, lipopolysaccharide; LPS + siPLIN1, adipocytes transfected with siPLIN1 and treated with LPS. The data presented are the mean ± SEM. (A–D) The same letter indicates no significant difference ( p > 0.05), whereas different letters indicate a significant difference ( p
Figure Legend Snippet: Perilipin-1 (PLIN1) silencing increased the expression and synthesis of inflammatory cytokines. Adipocytes were transfected with siPLIN1 and NC with or without LPS. Nine replicate samples were used for each condition ( N = 9). (A–C) The mRNA expression levels of IL-1β, IL-6, and TNF-α. (D–F) Supernatant concentrations of IL-1β, IL-6, and TNF-α. (C) Control group. Abbreviations: NC, negative control siRNA; siPLIN1, small interfering RNA of PLIN1; LPS, lipopolysaccharide; LPS + siPLIN1, adipocytes transfected with siPLIN1 and treated with LPS. The data presented are the mean ± SEM. (A–D) The same letter indicates no significant difference ( p > 0.05), whereas different letters indicate a significant difference ( p

Techniques Used: Expressing, Transfection, Negative Control, Small Interfering RNA

Perilipin 1 (PLIN1) influenced the TAG content in cow adipocytes. Adipocytes were transfected with PLIN1 overexpression adenovirus (Ad-PLIN1), GFP adenovirus (Ad-GFP, a negative control group compared with Ad-PLIN1), small interfering RNA of PLIN1 (siPLIN1), and negative control siRNA. Nine replicate samples were used for each condition ( N = 9). (A) Intracellular TG content in overexpression treatment. (B) Intracellular TG content in silencing treatment. (C) The Oil red O staining value. (D) Oil red O staining results (20×). Abbreviations: C, control group; Ad-GFP, GFP adenovirus; Ad-PLIN1, PLIN1 overexpression adenovirus; NC, negative control of siPLIN1; siPLIN1, small interfering RNA of PLIN1. The data presented are the mean ± SEM. (A–C) The same letter indicates no significant difference ( p > 0.05), whereas different letters indicate a significant difference ( p
Figure Legend Snippet: Perilipin 1 (PLIN1) influenced the TAG content in cow adipocytes. Adipocytes were transfected with PLIN1 overexpression adenovirus (Ad-PLIN1), GFP adenovirus (Ad-GFP, a negative control group compared with Ad-PLIN1), small interfering RNA of PLIN1 (siPLIN1), and negative control siRNA. Nine replicate samples were used for each condition ( N = 9). (A) Intracellular TG content in overexpression treatment. (B) Intracellular TG content in silencing treatment. (C) The Oil red O staining value. (D) Oil red O staining results (20×). Abbreviations: C, control group; Ad-GFP, GFP adenovirus; Ad-PLIN1, PLIN1 overexpression adenovirus; NC, negative control of siPLIN1; siPLIN1, small interfering RNA of PLIN1. The data presented are the mean ± SEM. (A–C) The same letter indicates no significant difference ( p > 0.05), whereas different letters indicate a significant difference ( p

Techniques Used: Transfection, Over Expression, Negative Control, Small Interfering RNA, Staining

Perilipin-1 (PLIN1) silencing inhibited fatty acids and TG synthesis and increased the TG lipolysis in cow adipocytes. Adipocytes were transfected with siPLIN1 and NC. Nine replicate samples were used for each condition ( N = 9). (A–G) The mRNA expression levels of PLIN1 (A) , SREBP-1c (B) , ACC1 (C) , SCD1 (D) , FAS (E) , DGAT1 (F) , and DGAT2 (G) in adipocytes. (H) Western blot analysis of PLIN1, SREBP-1c, ACC1, SCD1, and FAS. (I–H) : the mRNA expression levels of hormone-sensitive lipase (I) , CGI-58 for adipose triglyceride lipase (ATGL) (J) , and MGLL (K) in adipocytes. (L) The protein expression levels of PLIN1, SREBP-1c, ACC1, FAS, and ATGL. (C) Control group. NC, negative control siRNA; siPLIN1, small interfering RNA of PLIN1. The data presented are the mean ± SEM. (A,B) The same letter indicates no significant difference ( p > 0.05), whereas different letters indicate a significant difference ( p
Figure Legend Snippet: Perilipin-1 (PLIN1) silencing inhibited fatty acids and TG synthesis and increased the TG lipolysis in cow adipocytes. Adipocytes were transfected with siPLIN1 and NC. Nine replicate samples were used for each condition ( N = 9). (A–G) The mRNA expression levels of PLIN1 (A) , SREBP-1c (B) , ACC1 (C) , SCD1 (D) , FAS (E) , DGAT1 (F) , and DGAT2 (G) in adipocytes. (H) Western blot analysis of PLIN1, SREBP-1c, ACC1, SCD1, and FAS. (I–H) : the mRNA expression levels of hormone-sensitive lipase (I) , CGI-58 for adipose triglyceride lipase (ATGL) (J) , and MGLL (K) in adipocytes. (L) The protein expression levels of PLIN1, SREBP-1c, ACC1, FAS, and ATGL. (C) Control group. NC, negative control siRNA; siPLIN1, small interfering RNA of PLIN1. The data presented are the mean ± SEM. (A,B) The same letter indicates no significant difference ( p > 0.05), whereas different letters indicate a significant difference ( p

Techniques Used: Transfection, Expressing, Western Blot, Negative Control, Small Interfering RNA

The effect of perilipin-1 (PLIN1) on NF-κB signaling pathway in dairy cow adipocytes. Adipocytes were transfected with Ad-PLIN1, Ad-GFP, siPLIN1, and NC with or without LPS. Nine replicate samples were used for each condition ( N = 9). (A,B) The mRNA expression levels of NF-κB p65. (C,D) Western blot analysis of key molecules of the NF-κB signaling pathway in dairy cow adipocytes. (E,F) The phosphorylation levels of IκB (p-IκB/IκB) and NF-κB p65 (p-p65/p65). (C) Control group. Abbreviations: Ad-GFP, GFP adenovirus; Ad-PLIN1, PLIN1 overexpression adenovirus; LPS, lipopolysaccharide; NC, negative control siRNA; siPLIN1, small interfering RNA of PLIN1. The data presented are the mean ± SEM. (A–D) The same letter indicates no significant difference ( p > 0.05), whereas different letters indicate a significant difference ( p
Figure Legend Snippet: The effect of perilipin-1 (PLIN1) on NF-κB signaling pathway in dairy cow adipocytes. Adipocytes were transfected with Ad-PLIN1, Ad-GFP, siPLIN1, and NC with or without LPS. Nine replicate samples were used for each condition ( N = 9). (A,B) The mRNA expression levels of NF-κB p65. (C,D) Western blot analysis of key molecules of the NF-κB signaling pathway in dairy cow adipocytes. (E,F) The phosphorylation levels of IκB (p-IκB/IκB) and NF-κB p65 (p-p65/p65). (C) Control group. Abbreviations: Ad-GFP, GFP adenovirus; Ad-PLIN1, PLIN1 overexpression adenovirus; LPS, lipopolysaccharide; NC, negative control siRNA; siPLIN1, small interfering RNA of PLIN1. The data presented are the mean ± SEM. (A–D) The same letter indicates no significant difference ( p > 0.05), whereas different letters indicate a significant difference ( p

Techniques Used: Transfection, Expressing, Western Blot, Over Expression, Negative Control, Small Interfering RNA

39) Product Images from "Inclusion bodies of human parainfluenza virus type 3 inhibit antiviral stress granule formation by shielding viral RNAs"

Article Title: Inclusion bodies of human parainfluenza virus type 3 inhibit antiviral stress granule formation by shielding viral RNAs

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006948

HPIV3 viral RNA triggers SG formation. (A and B) HeLa cells were transfected with the indicated RNA samples from mock-infected or HPIV3-infected MK2 cells, treated with pIC for 12 h or AS for 1 h. (A) Cells were immunostained for TIA-1 (green) and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (B) Cell lysates were analyzed via western blot using anti-phosphorylated PKR, anti-PKR, anti-phosphorylated eIF2α, anti-eIF2α, and anti-GAPDH antibodies. (C and D) HeLa cells were transfected with the RNA from HPIV3-infected MK2 cells, the respective PolyA + RNA fraction, or the respective PolyA - RNA fraction for 12 h. (C) Cells were immunostained for TIA-1 (green) and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (D) The percentage of cells containing SGs was quantified in three independent experiments. (E and F) HeLa cells were transfected with the respective PolyA + RNA fraction from HPIV3-infected MK2 cells for 12 h and subsequently mock-treated or treated with CHX for another 1 h, 2 h, or 3 h. (E) Cells were immunostained for TIA-1 (green) and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (F) The percentage of cells containing SGs was quantified in three independent experiments. Data are represented as means ±SD. Student’s t test: * P
Figure Legend Snippet: HPIV3 viral RNA triggers SG formation. (A and B) HeLa cells were transfected with the indicated RNA samples from mock-infected or HPIV3-infected MK2 cells, treated with pIC for 12 h or AS for 1 h. (A) Cells were immunostained for TIA-1 (green) and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (B) Cell lysates were analyzed via western blot using anti-phosphorylated PKR, anti-PKR, anti-phosphorylated eIF2α, anti-eIF2α, and anti-GAPDH antibodies. (C and D) HeLa cells were transfected with the RNA from HPIV3-infected MK2 cells, the respective PolyA + RNA fraction, or the respective PolyA - RNA fraction for 12 h. (C) Cells were immunostained for TIA-1 (green) and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (D) The percentage of cells containing SGs was quantified in three independent experiments. (E and F) HeLa cells were transfected with the respective PolyA + RNA fraction from HPIV3-infected MK2 cells for 12 h and subsequently mock-treated or treated with CHX for another 1 h, 2 h, or 3 h. (E) Cells were immunostained for TIA-1 (green) and G3BP (red). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. (F) The percentage of cells containing SGs was quantified in three independent experiments. Data are represented as means ±SD. Student’s t test: * P

Techniques Used: Transfection, Infection, Staining, Western Blot

40) Product Images from "Recognition of Lipoproteins by Toll-like Receptor 2 and DNA by the AIM2 Inflammasome Is Responsible for Production of Interleukin-1β by Virulent Suilysin-Negative Streptococcus suis Serotype 2"

Article Title: Recognition of Lipoproteins by Toll-like Receptor 2 and DNA by the AIM2 Inflammasome Is Responsible for Production of Interleukin-1β by Virulent Suilysin-Negative Streptococcus suis Serotype 2

Journal: Pathogens

doi: 10.3390/pathogens9020147

Role of SLY-negative S. suis strain 89-1591 components in bmDC-produced IL-1β. ( A ) IL-1β production by bmDCs following 16 h of infection with 1 × 10 6 CFU of strain 89-1591 or its capsular polysaccharide-deficient mutant (89-1591∆ cpsF ). ( B ) IL-1β production 24 h following activation of wild-type (WT) or TLR2 -/- bmDCs with 30 μg/mL of lipoteichoic acid (LTA) extracts from strain 89-1591 or its lgt -deficient mutant (89-1591Δ lgt ). Non-stimulated cells served as negative control (C-). ( C ) IL-1β production by bmDCs following phagosomal delivery of 1 μg of S. suis RNA or DNA. Cells stimulated with elution buffer served as negative control (C-). Data are expressed as mean ± SEM ( n = 3). * ( p
Figure Legend Snippet: Role of SLY-negative S. suis strain 89-1591 components in bmDC-produced IL-1β. ( A ) IL-1β production by bmDCs following 16 h of infection with 1 × 10 6 CFU of strain 89-1591 or its capsular polysaccharide-deficient mutant (89-1591∆ cpsF ). ( B ) IL-1β production 24 h following activation of wild-type (WT) or TLR2 -/- bmDCs with 30 μg/mL of lipoteichoic acid (LTA) extracts from strain 89-1591 or its lgt -deficient mutant (89-1591Δ lgt ). Non-stimulated cells served as negative control (C-). ( C ) IL-1β production by bmDCs following phagosomal delivery of 1 μg of S. suis RNA or DNA. Cells stimulated with elution buffer served as negative control (C-). Data are expressed as mean ± SEM ( n = 3). * ( p

Techniques Used: Produced, Infection, Mutagenesis, Activation Assay, Negative Control

Proposed model of the mechanisms involved in SLY-negative virulent S. suis strain 89-1591-induced IL-1β production by bmDCs. Bacterial recognition by bmDCs requires MyD88-dependent signaling and partial involvement of TLR2 via recognition of lipoproteins (LPs). Following internalization, bacterial DNA and RNA can also induce IL-1β production. S. suis recognition then leads to activation of the NF-κB, MEK and JNK pathways. Finally, activation of the NLRP3 and AIM2 inflammasomes, the latter by bacterial DNA following elevated internalization, is involved in caspase-1-dependent processing of IL-1β.
Figure Legend Snippet: Proposed model of the mechanisms involved in SLY-negative virulent S. suis strain 89-1591-induced IL-1β production by bmDCs. Bacterial recognition by bmDCs requires MyD88-dependent signaling and partial involvement of TLR2 via recognition of lipoproteins (LPs). Following internalization, bacterial DNA and RNA can also induce IL-1β production. S. suis recognition then leads to activation of the NF-κB, MEK and JNK pathways. Finally, activation of the NLRP3 and AIM2 inflammasomes, the latter by bacterial DNA following elevated internalization, is involved in caspase-1-dependent processing of IL-1β.

Techniques Used: Activation Assay

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Fluorescence:

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Isolation:

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Article Snippet: .. Total RNA was isolated from MCF7 cells grown in E2-free medium using the GenElute mammalian total RNA miniprep kit (Sigma). .. RNA was reverse transcribed into first-strand cDNA using MMLV reverse transcriptase (Promega).

Immunoprecipitation:

Article Title: A novel miR-371a-5p-mediated pathway, leading to BAG3 upregulation in cardiomyocytes in response to epinephrine, is lost in Takotsubo cardiomyopathy
Article Snippet: .. HEK293 cells were lysed and the RNA-associated proteins were immunoprecipitated (IP) with anti-AGO2 Antibody (Millipore, 03-110). .. The precipitated RNA was retro-transcribed and measured by quantitative real-time PCR (Roche, Universal Probe Library System) by normalization to the non-IP control (Input).

Article Title: ICF-specific DNMT3B dysfunction interferes with intragenic regulation of mRNA transcription and alternative splicing
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Article Title: ILF3 is a substrate of SPOP for regulating serine biosynthesis in colorectal cancer
Article Snippet: .. RNA immunoprecipitation RNA immunoprecipitation was performed using Magna RIP RNA-Binding Protein Immunoprecipitation Kit (17-701, Millipore) according to manufacturer’s instructions. .. mRNA stability analysis DLD1 cells were treated with scramble or ILF3 shRNA followed by treatment of Actinomycin D (10 mg/mL) for 0, 3, 6, 9, or 12 h followed by Trizol RNA extraction.

other:

Article Title: Refined RIP-seq protocol for epitranscriptome analysis with low input materials
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Sequencing:

Article Title: ICF-specific DNMT3B dysfunction interferes with intragenic regulation of mRNA transcription and alternative splicing
Article Snippet: .. RNA immunoprecipitation (RIP) sequencing and data processing Native and cross-linked RNA immunoprecipitation experiments were performed using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore) and the Magna Nuclear RIP (Cross-Linked) RNA-Binding Protein Immunoprecipitation Kit (Millipore), respectively, according to the manufacturer's instructions. .. Antibodies for RIP assays were anti-DNMT3B (Diagenode), anti-DNMT3A (Abcam) and anti-hnRNP-LL (Aviva).

RNA Binding Assay:

Article Title: ICF-specific DNMT3B dysfunction interferes with intragenic regulation of mRNA transcription and alternative splicing
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Article Title: ILF3 is a substrate of SPOP for regulating serine biosynthesis in colorectal cancer
Article Snippet: .. RNA immunoprecipitation RNA immunoprecipitation was performed using Magna RIP RNA-Binding Protein Immunoprecipitation Kit (17-701, Millipore) according to manufacturer’s instructions. .. mRNA stability analysis DLD1 cells were treated with scramble or ILF3 shRNA followed by treatment of Actinomycin D (10 mg/mL) for 0, 3, 6, 9, or 12 h followed by Trizol RNA extraction.

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    Millipore rna binding protein immunoprecipitation rip
    ZEB1-AS1 downregulates miR-200b , upregulates fascin-1 ( FSCN1 ), and promotes cell migration and invasion. a Luciferase assays were performed in T24, RT4, and 293 T cells co-transfected for 24 h with miR-negative control (NC) or miR-200b and a plasmid containing wild-type or mutant-type ZEB1-AS1 3′untranslated region (UTR) upstream the luciferase gene. Firefly luciferase activity of each sample was normalized by Renilla luciferase activity. Data were analyzed by T-test. b <t>RNA-binding</t> protein <t>immunoprecipitation</t> <t>(RIP)</t> assays with anti-AGO2 antibodies were performed in T24 and RT4 cells transiently transfected with miR-200b ; ZEB1-AS1 levels were detected by quantitative PCR (qPCR); 10% input was used as positive control and RIP with anti-IgG antibodies served as negative control. Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) was used as the internal control. Data were analyzed by T-test. c Transfection efficiency of ZEB1-AS1 knockdown (si1, si2) and overexpression (OE) in T24 and RT4 cells was detected by qPCR. Data were analyzed by T-test. d and e The levels of miR-200b ( d ) and FSCN1 ( e ) were measured by qPCR after ZEB1-AS1 knocked down or overexpressed in T24 and RT4 cells. Data were analyzed by T-test. f E-cadherin, N-cadherin, vimentin, and FSCN1 protein expression in T24 and RT4 cells in which ZEB1-AS1 had been knocked down or overexpressed. Data were analyzed by T-test. ( g ) FSCN1 levels in T24 cells co-transfected with miR-NC or miR-200b and with an empty vector (EV) or a plasmid overexpressing ZEB1-AS1 . Data were analyzed by T-test. h and i Transwell assays (without or with Matrigel) to detect cell migration ( h ) and invasion ( i ) of T24 and RT4 after ZEB1-AS1 silencing or overexpression. j and k Transwell assays (without or with Matrigel) to detect cell migration ( j ) and invasion ( k ) of T24 and RT4 co-transfected with miR-NC or miR-200b and with an EV or a plasmid overexpressing ZEB1-AS1 . Data were analyzed by T-test. Data are presented as the mean ± standard deviation (SD). * P
    Rna Binding Protein Immunoprecipitation Rip, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore rna immunoprecipitation assay siha cells
    LncRNA LINCRNA is located in nucleus of <t>SiHa</t> cells. A, Prediction of LINC01305 subcellular localization in different cells through lncatlas.crg analysis. B, Verification of LINC01305 subcellular localization in SiHa cells (×400) whereby the nucleus was represented by blue, green represented the fluorescence localization of LINC01305, and the ruler = 25 μm. lncRNA LINC01305, long non‐coding <t>RNA</t> LINC01305; DAPI, Diamidino‐phenylindole
    Rna Immunoprecipitation Assay Siha Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rna immunoprecipitation rip
    XIAP-AS1 enhances XIAP transcription. (A) XIAP-AS1 and XIAP expression was detected with qRT-PCR and western blot, respectively, in the BGC823 shRNA- XIAP-AS1 or shScramble stable cell lines. (B) The XIAP-AS1 expression vector and the control vector were transfected into MNK28 cells, and XIAP-AS1 and XIAP expression was detected after 48 h. The data are presented as the median ± standard error (SE). * P ≤0.05, ** P ≤0.01. (C) siRNA against XIAP and scrambled siRNA were transfected into BGC823 cells, and XIAP-AS1 expression was detected by qRT-PCR after 48 h. Data are presented as the median ± standard error (SE). * P ≤0.05, ** P ≤0.01. (D) BGC823 or SGC7901 cells were collected and 10% of extracts was used as input and the rest in triplicate was used in <t>RNA</t> <t>immunoprecipitation</t> for Sp1, AP-1 and rabbit normal IgG. The co-immunoprecipitated RNA and input RNA was extracted and the fold enrichment of XIAP-AS1 was determined with qRT-PCR. AP-1 and rabbit normal IgG used as control. (E) Extracts derived from the BGC823 shRNA- XIAP-AS1 and shScramble stable cell lines were incubated with rabbit anti-Sp1, rabbit anti-AP-1 or rabbit normal IgG antibodies, and the immune complexes were precipitated with magnetic beads conjugated to Protein G. A total of 10% of the whole cell extract was used as the internal control. DNA was extracted from the immunoprecipitated complexes, and expression of the XIAP promoter was determined by real-time PCR. Data are presented as the median ± standard error (SE).
    Rna Immunoprecipitation Rip, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ZEB1-AS1 downregulates miR-200b , upregulates fascin-1 ( FSCN1 ), and promotes cell migration and invasion. a Luciferase assays were performed in T24, RT4, and 293 T cells co-transfected for 24 h with miR-negative control (NC) or miR-200b and a plasmid containing wild-type or mutant-type ZEB1-AS1 3′untranslated region (UTR) upstream the luciferase gene. Firefly luciferase activity of each sample was normalized by Renilla luciferase activity. Data were analyzed by T-test. b RNA-binding protein immunoprecipitation (RIP) assays with anti-AGO2 antibodies were performed in T24 and RT4 cells transiently transfected with miR-200b ; ZEB1-AS1 levels were detected by quantitative PCR (qPCR); 10% input was used as positive control and RIP with anti-IgG antibodies served as negative control. Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) was used as the internal control. Data were analyzed by T-test. c Transfection efficiency of ZEB1-AS1 knockdown (si1, si2) and overexpression (OE) in T24 and RT4 cells was detected by qPCR. Data were analyzed by T-test. d and e The levels of miR-200b ( d ) and FSCN1 ( e ) were measured by qPCR after ZEB1-AS1 knocked down or overexpressed in T24 and RT4 cells. Data were analyzed by T-test. f E-cadherin, N-cadherin, vimentin, and FSCN1 protein expression in T24 and RT4 cells in which ZEB1-AS1 had been knocked down or overexpressed. Data were analyzed by T-test. ( g ) FSCN1 levels in T24 cells co-transfected with miR-NC or miR-200b and with an empty vector (EV) or a plasmid overexpressing ZEB1-AS1 . Data were analyzed by T-test. h and i Transwell assays (without or with Matrigel) to detect cell migration ( h ) and invasion ( i ) of T24 and RT4 after ZEB1-AS1 silencing or overexpression. j and k Transwell assays (without or with Matrigel) to detect cell migration ( j ) and invasion ( k ) of T24 and RT4 co-transfected with miR-NC or miR-200b and with an EV or a plasmid overexpressing ZEB1-AS1 . Data were analyzed by T-test. Data are presented as the mean ± standard deviation (SD). * P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Long non-coding RNA ZEB1-AS1 regulates miR-200b/FSCN1 signaling and enhances migration and invasion induced by TGF-β1 in bladder cancer cells

    doi: 10.1186/s13046-019-1102-6

    Figure Lengend Snippet: ZEB1-AS1 downregulates miR-200b , upregulates fascin-1 ( FSCN1 ), and promotes cell migration and invasion. a Luciferase assays were performed in T24, RT4, and 293 T cells co-transfected for 24 h with miR-negative control (NC) or miR-200b and a plasmid containing wild-type or mutant-type ZEB1-AS1 3′untranslated region (UTR) upstream the luciferase gene. Firefly luciferase activity of each sample was normalized by Renilla luciferase activity. Data were analyzed by T-test. b RNA-binding protein immunoprecipitation (RIP) assays with anti-AGO2 antibodies were performed in T24 and RT4 cells transiently transfected with miR-200b ; ZEB1-AS1 levels were detected by quantitative PCR (qPCR); 10% input was used as positive control and RIP with anti-IgG antibodies served as negative control. Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) was used as the internal control. Data were analyzed by T-test. c Transfection efficiency of ZEB1-AS1 knockdown (si1, si2) and overexpression (OE) in T24 and RT4 cells was detected by qPCR. Data were analyzed by T-test. d and e The levels of miR-200b ( d ) and FSCN1 ( e ) were measured by qPCR after ZEB1-AS1 knocked down or overexpressed in T24 and RT4 cells. Data were analyzed by T-test. f E-cadherin, N-cadherin, vimentin, and FSCN1 protein expression in T24 and RT4 cells in which ZEB1-AS1 had been knocked down or overexpressed. Data were analyzed by T-test. ( g ) FSCN1 levels in T24 cells co-transfected with miR-NC or miR-200b and with an empty vector (EV) or a plasmid overexpressing ZEB1-AS1 . Data were analyzed by T-test. h and i Transwell assays (without or with Matrigel) to detect cell migration ( h ) and invasion ( i ) of T24 and RT4 after ZEB1-AS1 silencing or overexpression. j and k Transwell assays (without or with Matrigel) to detect cell migration ( j ) and invasion ( k ) of T24 and RT4 co-transfected with miR-NC or miR-200b and with an EV or a plasmid overexpressing ZEB1-AS1 . Data were analyzed by T-test. Data are presented as the mean ± standard deviation (SD). * P

    Article Snippet: RNA-binding protein immunoprecipitation (RIP) The Magna RIP kit (Millipore) was used for RIP assays with the anti-Ago2 antibody (Abcam), according to the manufacturer’s recommendations.

    Techniques: Migration, Luciferase, Transfection, Negative Control, Plasmid Preparation, Mutagenesis, Activity Assay, RNA Binding Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Over Expression, Expressing, Standard Deviation

    LncRNA LINCRNA is located in nucleus of SiHa cells. A, Prediction of LINC01305 subcellular localization in different cells through lncatlas.crg analysis. B, Verification of LINC01305 subcellular localization in SiHa cells (×400) whereby the nucleus was represented by blue, green represented the fluorescence localization of LINC01305, and the ruler = 25 μm. lncRNA LINC01305, long non‐coding RNA LINC01305; DAPI, Diamidino‐phenylindole

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: LncRNA LINC01305 silencing inhibits cell epithelial‐mesenchymal transition in cervical cancer by inhibiting TNXB‐mediated PI3K/Akt signalling pathway, et al. LncRNA LINC01305 silencing inhibits cell epithelial‐mesenchymal transition in cervical cancer by inhibiting TNXB‐mediated PI3K/Akt signalling pathway

    doi: 10.1111/jcmm.14161

    Figure Lengend Snippet: LncRNA LINCRNA is located in nucleus of SiHa cells. A, Prediction of LINC01305 subcellular localization in different cells through lncatlas.crg analysis. B, Verification of LINC01305 subcellular localization in SiHa cells (×400) whereby the nucleus was represented by blue, green represented the fluorescence localization of LINC01305, and the ruler = 25 μm. lncRNA LINC01305, long non‐coding RNA LINC01305; DAPI, Diamidino‐phenylindole

    Article Snippet: 2.3 RNA immunoprecipitation assay SiHa cells (2 × 107 ) were selected and treated according to Magna RNA immunoprecipitation (RIP) TM RNA‐Binding Protein Immunoprecipitation Kit (Millipore Corp., Billerica, MA, USA).

    Techniques: Fluorescence

    XIAP-AS1 enhances XIAP transcription. (A) XIAP-AS1 and XIAP expression was detected with qRT-PCR and western blot, respectively, in the BGC823 shRNA- XIAP-AS1 or shScramble stable cell lines. (B) The XIAP-AS1 expression vector and the control vector were transfected into MNK28 cells, and XIAP-AS1 and XIAP expression was detected after 48 h. The data are presented as the median ± standard error (SE). * P ≤0.05, ** P ≤0.01. (C) siRNA against XIAP and scrambled siRNA were transfected into BGC823 cells, and XIAP-AS1 expression was detected by qRT-PCR after 48 h. Data are presented as the median ± standard error (SE). * P ≤0.05, ** P ≤0.01. (D) BGC823 or SGC7901 cells were collected and 10% of extracts was used as input and the rest in triplicate was used in RNA immunoprecipitation for Sp1, AP-1 and rabbit normal IgG. The co-immunoprecipitated RNA and input RNA was extracted and the fold enrichment of XIAP-AS1 was determined with qRT-PCR. AP-1 and rabbit normal IgG used as control. (E) Extracts derived from the BGC823 shRNA- XIAP-AS1 and shScramble stable cell lines were incubated with rabbit anti-Sp1, rabbit anti-AP-1 or rabbit normal IgG antibodies, and the immune complexes were precipitated with magnetic beads conjugated to Protein G. A total of 10% of the whole cell extract was used as the internal control. DNA was extracted from the immunoprecipitated complexes, and expression of the XIAP promoter was determined by real-time PCR. Data are presented as the median ± standard error (SE).

    Journal: PLoS ONE

    Article Title: The long noncoding RNA XIAP-AS1 promotes XIAP transcription by XIAP-AS1 interacting with Sp1 in gastric cancer cells

    doi: 10.1371/journal.pone.0182433

    Figure Lengend Snippet: XIAP-AS1 enhances XIAP transcription. (A) XIAP-AS1 and XIAP expression was detected with qRT-PCR and western blot, respectively, in the BGC823 shRNA- XIAP-AS1 or shScramble stable cell lines. (B) The XIAP-AS1 expression vector and the control vector were transfected into MNK28 cells, and XIAP-AS1 and XIAP expression was detected after 48 h. The data are presented as the median ± standard error (SE). * P ≤0.05, ** P ≤0.01. (C) siRNA against XIAP and scrambled siRNA were transfected into BGC823 cells, and XIAP-AS1 expression was detected by qRT-PCR after 48 h. Data are presented as the median ± standard error (SE). * P ≤0.05, ** P ≤0.01. (D) BGC823 or SGC7901 cells were collected and 10% of extracts was used as input and the rest in triplicate was used in RNA immunoprecipitation for Sp1, AP-1 and rabbit normal IgG. The co-immunoprecipitated RNA and input RNA was extracted and the fold enrichment of XIAP-AS1 was determined with qRT-PCR. AP-1 and rabbit normal IgG used as control. (E) Extracts derived from the BGC823 shRNA- XIAP-AS1 and shScramble stable cell lines were incubated with rabbit anti-Sp1, rabbit anti-AP-1 or rabbit normal IgG antibodies, and the immune complexes were precipitated with magnetic beads conjugated to Protein G. A total of 10% of the whole cell extract was used as the internal control. DNA was extracted from the immunoprecipitated complexes, and expression of the XIAP promoter was determined by real-time PCR. Data are presented as the median ± standard error (SE).

    Article Snippet: RNA immunoprecipitation (RIP) The RIP experiments were conducted using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA, USA) according to the manufacturer's instructions.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, shRNA, Stable Transfection, Plasmid Preparation, Transfection, Immunoprecipitation, Derivative Assay, Incubation, Magnetic Beads, Real-time Polymerase Chain Reaction

    SNHG12 acts as a miR-181a sponge (A) The binding site of miR-181a within the SNHG12. (B) The levels of nuclear control transcript (MALAT1), cytoplasmic control transcript (Tubulin), and SNHG12 were determined by qRT-PCR in nuclear and cytoplasmic fractions and normalized to levels of external RNA. (C and D) Luciferase activity in A549/DDP cells co-transfected with the wipe-type or mutant SNHG12 reporters (SNHG12-Wt or SNHG12-Mut) and (miR-181a mimics or miR-Con) or (anti-miR-181a or anti-miR-Con). (E) Cellular lysates from A549/DDP cells were used for RNA immunoprecipitation (RIP) with Ago2 antibody. Detection of SNHG12 and miR-181a using qRT-PCR. (F) The levels of SNHG12 were detected in A549/DDP cells after pcDNA-SNHG12 or pcDNA-SNHG12-Mut or SNHG12 siRNAs transfection. (G) qRT-PCR analysis was performed to detect the expression of miR-181a in A549/DDP cells after pcDNA-SNHG12 or pcDNA-SNHG12-Mut or SNHG12 siRNAs transfection. (H) Negative correlation between SNHG12 and miR-181a expressions in NSCLC tumor tissues. * P

    Journal: Oncotarget

    Article Title: LncRNA SNHG12 contributes to multidrug resistance through activating the MAPK/Slug pathway by sponging miR-181a in non-small cell lung cancer

    doi: 10.18632/oncotarget.20475

    Figure Lengend Snippet: SNHG12 acts as a miR-181a sponge (A) The binding site of miR-181a within the SNHG12. (B) The levels of nuclear control transcript (MALAT1), cytoplasmic control transcript (Tubulin), and SNHG12 were determined by qRT-PCR in nuclear and cytoplasmic fractions and normalized to levels of external RNA. (C and D) Luciferase activity in A549/DDP cells co-transfected with the wipe-type or mutant SNHG12 reporters (SNHG12-Wt or SNHG12-Mut) and (miR-181a mimics or miR-Con) or (anti-miR-181a or anti-miR-Con). (E) Cellular lysates from A549/DDP cells were used for RNA immunoprecipitation (RIP) with Ago2 antibody. Detection of SNHG12 and miR-181a using qRT-PCR. (F) The levels of SNHG12 were detected in A549/DDP cells after pcDNA-SNHG12 or pcDNA-SNHG12-Mut or SNHG12 siRNAs transfection. (G) qRT-PCR analysis was performed to detect the expression of miR-181a in A549/DDP cells after pcDNA-SNHG12 or pcDNA-SNHG12-Mut or SNHG12 siRNAs transfection. (H) Negative correlation between SNHG12 and miR-181a expressions in NSCLC tumor tissues. * P

    Article Snippet: RNA immunoprecipitation (RIP) assay RNA immunoprecipitation was performed using the Magna RIP Kit (Millipore, Billerica, MA, USA).

    Techniques: Binding Assay, Quantitative RT-PCR, Luciferase, Activity Assay, Transfection, Mutagenesis, Immunoprecipitation, Expressing