in vitro rna transcription  (Thermo Fisher)


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    Structured Review

    Thermo Fisher in vitro rna transcription
    The number of clinical specimens that were positive for SARS-CoV-2 <t>RNA</t> by the <t>COVID-19-RdRp/Hel</t> assay or RdRp-P2 assay on different days after symptom onset from nasopharyngeal aspirates/swabs and/or throat swabs (A), saliva specimens (B), sputum specimens (C), plasma specimens (D), and feces or rectal swabs (E).
    In Vitro Rna Transcription, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Improved Molecular Diagnosis of COVID-19 by the Novel, Highly Sensitive and Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-PCR Assay Validated In Vitro and with Clinical Specimens"

    Article Title: Improved Molecular Diagnosis of COVID-19 by the Novel, Highly Sensitive and Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-PCR Assay Validated In Vitro and with Clinical Specimens

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.00310-20

    The number of clinical specimens that were positive for SARS-CoV-2 RNA by the COVID-19-RdRp/Hel assay or RdRp-P2 assay on different days after symptom onset from nasopharyngeal aspirates/swabs and/or throat swabs (A), saliva specimens (B), sputum specimens (C), plasma specimens (D), and feces or rectal swabs (E).
    Figure Legend Snippet: The number of clinical specimens that were positive for SARS-CoV-2 RNA by the COVID-19-RdRp/Hel assay or RdRp-P2 assay on different days after symptom onset from nasopharyngeal aspirates/swabs and/or throat swabs (A), saliva specimens (B), sputum specimens (C), plasma specimens (D), and feces or rectal swabs (E).

    Techniques Used:

    2) Product Images from "Arterivirus Nsp1 Modulates the Accumulation of Minus-Strand Templates to Control the Relative Abundance of Viral mRNAs"

    Article Title: Arterivirus Nsp1 Modulates the Accumulation of Minus-Strand Templates to Control the Relative Abundance of Viral mRNAs

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000772

    Minus-strand RNA accumulation is also modulated by mutations in nsp1. (A–D) Analysis and quantification of EAV minus-strand accumulation by a two-cycle RNase protection assay. (A) Schematic representation of the nested set of viral minus-strand RNA [(−)RNA] species produced in EAV-infected cells. The anti-leader sequence is depicted in light green. The in vitro-transcribed plus-strand probes used for detection of (−)RNA1 (pRNA1), (−)RNA6 (pRNA6) and (−) RNA7 (pRNA7) are shown. pRNA6 and pRNA7 target the leader-body junction sequences of (−)RNA6 and (−)RNA7, respectively. Note that hybridization with pRNA1 results in the protection of a single fragment, while the probes for (−)RNAs 6 and 7 each protect three fragments – one derived from the full-length sg minus strand, and two fragments derived in part from partial hybridization of these probes to larger viral (−)RNAs in which the target sequences are noncontiguous (exemplified for pRNA6). For simplicity, non-EAV sequences present near the termini of the three probes were omitted from the scheme. (B) Viral (−)RNA accumulation was analyzed at 11 h post-transfection for the ZCH, A1 and A4 mutants, and a wt control. Protected fragments were resolved on denaturing 5% polyacrylamide/8M urea gels and visualized by phosphorimaging. The constructs analyzed are labeled above the lanes (M, mock-transfected cells; (−), no-RNase control that shows a band corresponding to 0.2 fmol of the full-length probe). Sizes (nt) of RNA markers have been indicated on the left. The single 327-nt protected fragment resulting from hybridization with the positive-sense probe for RNA1(−) is indicated. The probes for subgenome-length minus strands protected fragments derived from the full-length (−)RNA6 and (−)RNA7 (327 nt and 319 nt, respectively; denoted with LB), as well as from the (−)RNA6 and (−)RNA7 body sequences (188 nt and 180 nt, respectively; denoted with B) and the anti-leader sequence (139 nt; denoted with L). The presence of two bands in the size range of the anti-leader fragment has been described previously [55] . (C) The relative levels of minus-strand accumulation were quantified by phosphorimaging. For (−)RNAs 6 and 7, only the bands resulting from protection of full-length sg minus strands (denoted with LB in panel [B]) were quantified. The values correspond to the means from three independent transfections that were normalized to the level of accumulation of each minus-strand RNA in the wt control, which was set at 1. Intracellular RNA from the same transfection samples for which plus-strand accumulation was quantified ( Fig. 4B ) was used. Genomic minus-strand RNA levels are represented as dark blue bars. Error bars denote standard deviation. (D) The ratio of plus-strand to minus-strand accumulation for RNAs 1, 6 and 7 was calculated using the mean relative values obtained in Fig. 4B and Fig. 5C .
    Figure Legend Snippet: Minus-strand RNA accumulation is also modulated by mutations in nsp1. (A–D) Analysis and quantification of EAV minus-strand accumulation by a two-cycle RNase protection assay. (A) Schematic representation of the nested set of viral minus-strand RNA [(−)RNA] species produced in EAV-infected cells. The anti-leader sequence is depicted in light green. The in vitro-transcribed plus-strand probes used for detection of (−)RNA1 (pRNA1), (−)RNA6 (pRNA6) and (−) RNA7 (pRNA7) are shown. pRNA6 and pRNA7 target the leader-body junction sequences of (−)RNA6 and (−)RNA7, respectively. Note that hybridization with pRNA1 results in the protection of a single fragment, while the probes for (−)RNAs 6 and 7 each protect three fragments – one derived from the full-length sg minus strand, and two fragments derived in part from partial hybridization of these probes to larger viral (−)RNAs in which the target sequences are noncontiguous (exemplified for pRNA6). For simplicity, non-EAV sequences present near the termini of the three probes were omitted from the scheme. (B) Viral (−)RNA accumulation was analyzed at 11 h post-transfection for the ZCH, A1 and A4 mutants, and a wt control. Protected fragments were resolved on denaturing 5% polyacrylamide/8M urea gels and visualized by phosphorimaging. The constructs analyzed are labeled above the lanes (M, mock-transfected cells; (−), no-RNase control that shows a band corresponding to 0.2 fmol of the full-length probe). Sizes (nt) of RNA markers have been indicated on the left. The single 327-nt protected fragment resulting from hybridization with the positive-sense probe for RNA1(−) is indicated. The probes for subgenome-length minus strands protected fragments derived from the full-length (−)RNA6 and (−)RNA7 (327 nt and 319 nt, respectively; denoted with LB), as well as from the (−)RNA6 and (−)RNA7 body sequences (188 nt and 180 nt, respectively; denoted with B) and the anti-leader sequence (139 nt; denoted with L). The presence of two bands in the size range of the anti-leader fragment has been described previously [55] . (C) The relative levels of minus-strand accumulation were quantified by phosphorimaging. For (−)RNAs 6 and 7, only the bands resulting from protection of full-length sg minus strands (denoted with LB in panel [B]) were quantified. The values correspond to the means from three independent transfections that were normalized to the level of accumulation of each minus-strand RNA in the wt control, which was set at 1. Intracellular RNA from the same transfection samples for which plus-strand accumulation was quantified ( Fig. 4B ) was used. Genomic minus-strand RNA levels are represented as dark blue bars. Error bars denote standard deviation. (D) The ratio of plus-strand to minus-strand accumulation for RNAs 1, 6 and 7 was calculated using the mean relative values obtained in Fig. 4B and Fig. 5C .

    Techniques Used: Rnase Protection Assay, Produced, Infection, Sequencing, In Vitro, Hybridization, Derivative Assay, Transfection, Construct, Labeling, Standard Deviation

    Organization and expression of the polycistronic EAV +RNA genome. (A) Top: EAV genome organization, showing the 5′-proximal replicase open reading frames (ORFs), as well as the downstream ORFs encoding the viral structural proteins envelope (E), membrane (M), nucleocapsid (N), and glycoproteins (GP) 2–5 and the 3′ poly(A) tail (A n ). Bottom: overview of the pp1a and pp1ab replicase polyproteins that result from genome translation, which requires an ORF1a/1b ribosomal frameshift (RFS) to produce pp1ab. Arrowheads represent sites cleaved by the three virus-encoded proteases (open for autoproteolytically processed ones, closed for sites processed by the main proteinase in nsp4). The resulting nonstructural proteins (nsp) are numbered. The key viral enzymatic domains such as the nsp1 papain-like cysteine proteinase β (PCP), nsp2 cysteine proteinase (CP), nsp4 serine proteinase (SP), nsp9 viral RNA-dependent RNA polymerase (RdRp), nsp10 helicase (Hel), and nsp11 endoribonuclease (Ne) are indicated. (B) Overview of viral mRNA species produced in EAV-infected cells. The ORFs expressed from the respective mRNAs are shown in gray, and the 5′ leader sequence is depicted in dark red. The orange boxes indicate the positions of transcription-regulating sequences (TRS). The gel hybridization image on the right is representative of the wild-type accumulation levels of the seven EAV mRNAs at the time point used for analysis in the study (see text for details). The amount of each mRNA, determined by quantitative phosphorimager analysis, is indicated as percentage of the total amount of viral mRNA. (C) Model for EAV replication and transcription. Continuous minus-strand RNA synthesis yields a genome-length minus strand template for genome replication, a process for which nsp1 is dispensable. Discontinuous minus-strand RNA synthesis results in a nested set of subgenome-length minus strands that serve as templates for sg mRNA synthesis (see text for details). Nsp1 is crucial for this process, which is also guided by a base pairing interaction between the TRS complement [(−)TRS] at the 3′ end of the nascent minus-strand and the genomic leader TRS, present in a RNA hairpin structure (LTH).
    Figure Legend Snippet: Organization and expression of the polycistronic EAV +RNA genome. (A) Top: EAV genome organization, showing the 5′-proximal replicase open reading frames (ORFs), as well as the downstream ORFs encoding the viral structural proteins envelope (E), membrane (M), nucleocapsid (N), and glycoproteins (GP) 2–5 and the 3′ poly(A) tail (A n ). Bottom: overview of the pp1a and pp1ab replicase polyproteins that result from genome translation, which requires an ORF1a/1b ribosomal frameshift (RFS) to produce pp1ab. Arrowheads represent sites cleaved by the three virus-encoded proteases (open for autoproteolytically processed ones, closed for sites processed by the main proteinase in nsp4). The resulting nonstructural proteins (nsp) are numbered. The key viral enzymatic domains such as the nsp1 papain-like cysteine proteinase β (PCP), nsp2 cysteine proteinase (CP), nsp4 serine proteinase (SP), nsp9 viral RNA-dependent RNA polymerase (RdRp), nsp10 helicase (Hel), and nsp11 endoribonuclease (Ne) are indicated. (B) Overview of viral mRNA species produced in EAV-infected cells. The ORFs expressed from the respective mRNAs are shown in gray, and the 5′ leader sequence is depicted in dark red. The orange boxes indicate the positions of transcription-regulating sequences (TRS). The gel hybridization image on the right is representative of the wild-type accumulation levels of the seven EAV mRNAs at the time point used for analysis in the study (see text for details). The amount of each mRNA, determined by quantitative phosphorimager analysis, is indicated as percentage of the total amount of viral mRNA. (C) Model for EAV replication and transcription. Continuous minus-strand RNA synthesis yields a genome-length minus strand template for genome replication, a process for which nsp1 is dispensable. Discontinuous minus-strand RNA synthesis results in a nested set of subgenome-length minus strands that serve as templates for sg mRNA synthesis (see text for details). Nsp1 is crucial for this process, which is also guided by a base pairing interaction between the TRS complement [(−)TRS] at the 3′ end of the nascent minus-strand and the genomic leader TRS, present in a RNA hairpin structure (LTH).

    Techniques Used: Expressing, Produced, Infection, Sequencing, Hybridization

    Second-site mutations in nsp1 moderate species-specific defects in mRNA accumulation. (A–D) Gel hybridization analysis and quantification of EAV-specific mRNA accumulation. (A) BHK-21 cells were transfected with ZCH and A1 mutants, reconstructed pseudorevertants, and wt controls. The positions of the originally mutated amino acid clusters are indicated with arrows; the open circle denotes the position of second-site mutations. Viral mRNAs were analyzed 11 h post-transfection by gel hybridization as described above. (B) For ZCH+A29D and A1+A29K, the accumulation levels of each viral mRNA were quantified by phosphorimaging and the values were normalized to the wt level of accumulation of each corresponding viral mRNA from the same experiment, set at 1. Genomic RNA levels are shown as blue bars. The relative values correspond to the means from three independent transfections and error bars denote the standard deviation. The relative accumulation levels of viral mRNAs at 11 h post-transfection for the ZCH and A1 mutants are derived from Fig. 4B and are represented here to facilitate comparison between mutant and pseudorevertant phenotypes. (C) BHK-21 cells were transfected with the A4 mutant, reconstructed pseudorevertants and a wt control. The positions of the originally mutated amino acid cluster and the second-site mutations are indicated as in (A). Viral mRNAs were analyzed 11 h post-transfection. (D) For A4+G47A, A4+E112K, and A4+T196K, quantification of relative viral mRNA accumulation levels was performed as described in (B). Similarly, the relative accumulation levels of viral mRNAs at 11 h post-transfection for the A4 mutant derived from Fig. 4B are represented to facilitate comparison.
    Figure Legend Snippet: Second-site mutations in nsp1 moderate species-specific defects in mRNA accumulation. (A–D) Gel hybridization analysis and quantification of EAV-specific mRNA accumulation. (A) BHK-21 cells were transfected with ZCH and A1 mutants, reconstructed pseudorevertants, and wt controls. The positions of the originally mutated amino acid clusters are indicated with arrows; the open circle denotes the position of second-site mutations. Viral mRNAs were analyzed 11 h post-transfection by gel hybridization as described above. (B) For ZCH+A29D and A1+A29K, the accumulation levels of each viral mRNA were quantified by phosphorimaging and the values were normalized to the wt level of accumulation of each corresponding viral mRNA from the same experiment, set at 1. Genomic RNA levels are shown as blue bars. The relative values correspond to the means from three independent transfections and error bars denote the standard deviation. The relative accumulation levels of viral mRNAs at 11 h post-transfection for the ZCH and A1 mutants are derived from Fig. 4B and are represented here to facilitate comparison between mutant and pseudorevertant phenotypes. (C) BHK-21 cells were transfected with the A4 mutant, reconstructed pseudorevertants and a wt control. The positions of the originally mutated amino acid cluster and the second-site mutations are indicated as in (A). Viral mRNAs were analyzed 11 h post-transfection. (D) For A4+G47A, A4+E112K, and A4+T196K, quantification of relative viral mRNA accumulation levels was performed as described in (B). Similarly, the relative accumulation levels of viral mRNAs at 11 h post-transfection for the A4 mutant derived from Fig. 4B are represented to facilitate comparison.

    Techniques Used: Hybridization, Transfection, Standard Deviation, Derivative Assay, Mutagenesis

    Importance of nsp1 subdomains for transcription and virus production. (A, B). Analysis of EAV-specific mRNA accumulation by gel hybridization. The domain organization of nsp1 is depicted as in Fig. 2 and the positions of the clusters of amino acid mutations analyzed are indicated with arrows. BHK-21 cells were transfected with RNA transcribed from wt or selected mutant EAV full-length cDNA clones. Total intracellular RNA was isolated at 11 h post-transfection and resolved by denaturing formaldehyde electrophoresis. Equal loading of samples was confirmed by ethidium bromide staining of ribosomal RNA (data not shown). EAV-specific mRNAs were detected by hybridization of the gel with a 32 P-labelled probe complementary to the 3′-end of the viral genome and subsequent phosphorimaging. The positions of the EAV genome (RNA1) and the six sg mRNAs (RNA2 to RNA7) are indicated. (C) Plaque phenotype and virus titers of the Z2 and A3 mutants. Plaque assays were performed on BHK-21 using cell culture supernatants harvested 24 h after transfection. Cells were incubated under a semi-solid overlay at 39.5°C for 72 h, fixed and stained with crystal violet. Virus titers represent an average of three independent experiments. Pfu, plaque-forming units.
    Figure Legend Snippet: Importance of nsp1 subdomains for transcription and virus production. (A, B). Analysis of EAV-specific mRNA accumulation by gel hybridization. The domain organization of nsp1 is depicted as in Fig. 2 and the positions of the clusters of amino acid mutations analyzed are indicated with arrows. BHK-21 cells were transfected with RNA transcribed from wt or selected mutant EAV full-length cDNA clones. Total intracellular RNA was isolated at 11 h post-transfection and resolved by denaturing formaldehyde electrophoresis. Equal loading of samples was confirmed by ethidium bromide staining of ribosomal RNA (data not shown). EAV-specific mRNAs were detected by hybridization of the gel with a 32 P-labelled probe complementary to the 3′-end of the viral genome and subsequent phosphorimaging. The positions of the EAV genome (RNA1) and the six sg mRNAs (RNA2 to RNA7) are indicated. (C) Plaque phenotype and virus titers of the Z2 and A3 mutants. Plaque assays were performed on BHK-21 using cell culture supernatants harvested 24 h after transfection. Cells were incubated under a semi-solid overlay at 39.5°C for 72 h, fixed and stained with crystal violet. Virus titers represent an average of three independent experiments. Pfu, plaque-forming units.

    Techniques Used: Hybridization, Transfection, Mutagenesis, Clone Assay, Isolation, Electrophoresis, Staining, Cell Culture, Incubation

    Multiple mutations in nsp1 exert species-specific effects on viral mRNA accumulation. (A, B) Gel hybridization analysis and quantification of EAV-specific mRNA accumulation in cells transfected with the ZCH, A1, A4 mutant or a wt control. (A) Viral mRNA accumulation was analyzed at 11 h post-transfection by gel hybridization as described in the legend to Fig. 3 . (B) The accumulation levels of each viral mRNA in the nsp1 mutants were quantified by phosphorimaging in the linear range of exposure and normalized to the level of accumulation of each corresponding viral mRNA in the wt control, which was set at 1. Genomic RNA levels are represented as blue bars. The relative values correspond to the means from three independent transfections and error bars denote standard deviation.
    Figure Legend Snippet: Multiple mutations in nsp1 exert species-specific effects on viral mRNA accumulation. (A, B) Gel hybridization analysis and quantification of EAV-specific mRNA accumulation in cells transfected with the ZCH, A1, A4 mutant or a wt control. (A) Viral mRNA accumulation was analyzed at 11 h post-transfection by gel hybridization as described in the legend to Fig. 3 . (B) The accumulation levels of each viral mRNA in the nsp1 mutants were quantified by phosphorimaging in the linear range of exposure and normalized to the level of accumulation of each corresponding viral mRNA in the wt control, which was set at 1. Genomic RNA levels are represented as blue bars. The relative values correspond to the means from three independent transfections and error bars denote standard deviation.

    Techniques Used: Hybridization, Transfection, Mutagenesis, Standard Deviation

    3) Product Images from "Cis-acting structural element in 5′ UTR is essential for infectivity of porcine reproductive and respiratory syndrome virus"

    Article Title: Cis-acting structural element in 5′ UTR is essential for infectivity of porcine reproductive and respiratory syndrome virus

    Journal: Virus Research

    doi: 10.1016/j.virusres.2011.08.018

    Schematic representation of serial deletions of the PRRSV 5′ UTR. The upper panel showed the PRRSV genomic organization, which had 9 encoding ORFs denoted by gray box, flanked by 5′, 3′ UTR and poly (A) tail. Group I represented pAPRRSD1 and SD3, which were used as T7 promoter-driven RNA-launched mutant analysis. Group II represented the CMV promoter-driven DNA-launched serial deletion mutants designated as pAPRRSM x , x denoted the number of nucleotides (also in hyphen) deleted, using pAPRRSM19 and SM190 as representative. Group III represented pAPRRSMzg3 and Mzg6. White boxes indicated the authentic PRRSV sequence of 5′ UTR. Hyphens showed deleted nucleotides at the very beginning of 5′ UTR. Sph I was the restriction endonuclease recognition site introduced between the CMV and T7 promoter, indicated by arrowhead. Letters of lowercase type were modification in T7 promoter.
    Figure Legend Snippet: Schematic representation of serial deletions of the PRRSV 5′ UTR. The upper panel showed the PRRSV genomic organization, which had 9 encoding ORFs denoted by gray box, flanked by 5′, 3′ UTR and poly (A) tail. Group I represented pAPRRSD1 and SD3, which were used as T7 promoter-driven RNA-launched mutant analysis. Group II represented the CMV promoter-driven DNA-launched serial deletion mutants designated as pAPRRSM x , x denoted the number of nucleotides (also in hyphen) deleted, using pAPRRSM19 and SM190 as representative. Group III represented pAPRRSMzg3 and Mzg6. White boxes indicated the authentic PRRSV sequence of 5′ UTR. Hyphens showed deleted nucleotides at the very beginning of 5′ UTR. Sph I was the restriction endonuclease recognition site introduced between the CMV and T7 promoter, indicated by arrowhead. Letters of lowercase type were modification in T7 promoter.

    Techniques Used: Mutagenesis, Sequencing, Modification

    The cDNA sequence alignment for genetically stable, exogenous AU-rich sequences located in the 5′ proximal regions of mutant viruses. (A) Schematic representation of the filling-in cDNA sequences at the utmost 5′ end of the mutant viral genome. “cactatagg” for in vitro synthetic RNA APRRSM10, M12, 14,16 was denoted in boldface and lowercase type, while the sequence of APRRSMzg3 and Mzg6 was “gcgtccc”. T7 promoter sequence “taatacgactcactatagg” was also shown. The different kinds of exogenous AU-rich sequences obtained by 5′ RACE were denoted follows. The tag “S1”, “S2” and “S3” indicated restorations using nonviral foreign sequences. Hyphens indicated the deleted nucleotide sequences. The numbers of clones for nucleotide sequencing were indicated in right lane. (B) The sequence stability was detected via 5′ RACE, using vAPRRM12S2, vAPRRSM14S1 and vAPRRSMzg3S2 as representatives. The 5′ terminal sequences of mutant virus for every passage (P1–P5) were indicated in boldface and lowercase type and were stable among in vitro cell culture passage.
    Figure Legend Snippet: The cDNA sequence alignment for genetically stable, exogenous AU-rich sequences located in the 5′ proximal regions of mutant viruses. (A) Schematic representation of the filling-in cDNA sequences at the utmost 5′ end of the mutant viral genome. “cactatagg” for in vitro synthetic RNA APRRSM10, M12, 14,16 was denoted in boldface and lowercase type, while the sequence of APRRSMzg3 and Mzg6 was “gcgtccc”. T7 promoter sequence “taatacgactcactatagg” was also shown. The different kinds of exogenous AU-rich sequences obtained by 5′ RACE were denoted follows. The tag “S1”, “S2” and “S3” indicated restorations using nonviral foreign sequences. Hyphens indicated the deleted nucleotide sequences. The numbers of clones for nucleotide sequencing were indicated in right lane. (B) The sequence stability was detected via 5′ RACE, using vAPRRM12S2, vAPRRSM14S1 and vAPRRSMzg3S2 as representatives. The 5′ terminal sequences of mutant virus for every passage (P1–P5) were indicated in boldface and lowercase type and were stable among in vitro cell culture passage.

    Techniques Used: Sequencing, Mutagenesis, In Vitro, Clone Assay, Cell Culture

    4) Product Images from "A twist in the tail: SHAPE mapping of long-range interactions and structural rearrangements of RNA elements involved in HCV replication"

    Article Title: A twist in the tail: SHAPE mapping of long-range interactions and structural rearrangements of RNA elements involved in HCV replication

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks370

    SHAPE mapping RNA structures in the HCV genome. ( A ) Schematic diagram of the genome of HCV J6/JFH-1 (top) and Con1b-luc-rep (below) indicating the location of relevant stem–loop structures (SL) using both standardized positional references ( 19 ) and naming schemes from previous publications ( 11 , 18 , 22 ). The dotted lines above each genome diagram indicate the in vitro transcripts used as templates for SHAPE mapping. ( B ) Representative SHAPE gels generated from full-length (top) and truncated (bottom) Con1b–luc–rep templates. In each case, a conventional Sanger sequencing reaction is adjacent and below primer extension reactions of templates treated (+) or untreated (−) with NMIA. Open block arrows indicate the bioinformatically predicted duplexed regions with filled rectangles (terminal loop = black, sub-terminal bulge loop = grey) representing unpaired regions. Note that when interpreting SHAPE autoradiographs the primer extension product terminates at the base before the uncopyable, chemically acylated and exposed nucleotide. ( C ) Schematic representation of the structure and context of Con1b–luc–rep SL9266. Numbering relates to reference H77 sequence ( 19 ). Bases in black are the area shown in the autoradiographs above.
    Figure Legend Snippet: SHAPE mapping RNA structures in the HCV genome. ( A ) Schematic diagram of the genome of HCV J6/JFH-1 (top) and Con1b-luc-rep (below) indicating the location of relevant stem–loop structures (SL) using both standardized positional references ( 19 ) and naming schemes from previous publications ( 11 , 18 , 22 ). The dotted lines above each genome diagram indicate the in vitro transcripts used as templates for SHAPE mapping. ( B ) Representative SHAPE gels generated from full-length (top) and truncated (bottom) Con1b–luc–rep templates. In each case, a conventional Sanger sequencing reaction is adjacent and below primer extension reactions of templates treated (+) or untreated (−) with NMIA. Open block arrows indicate the bioinformatically predicted duplexed regions with filled rectangles (terminal loop = black, sub-terminal bulge loop = grey) representing unpaired regions. Note that when interpreting SHAPE autoradiographs the primer extension product terminates at the base before the uncopyable, chemically acylated and exposed nucleotide. ( C ) Schematic representation of the structure and context of Con1b–luc–rep SL9266. Numbering relates to reference H77 sequence ( 19 ). Bases in black are the area shown in the autoradiographs above.

    Techniques Used: In Vitro, Generated, Sequencing, Blocking Assay

    Comparative SHAPE analysis of native RNA structures. ( A ) SHAPE mapping of J6/JFH-1 and Con1b–luc–rep SL9266. The bars represent the relative exposure of the numbered nucleotides across SL9266 with the standard error of four independent gels (two short templates, two long) indicated. Duplexed and loop regions of SL9266 are indicated using the schematic representation used in Figure 1 B. Long-range interacting regions are indicated with grey (upstream) and black (‘kissing loop’) bars. ( B ) RNA structure representation of the data presented in Figure 2 A, colour coded to highlight relative exposure of the terminal loop of Con1b–luc–rep. ( C ) Comparative analysis of the 9110 region, SL9266 and SL9571 (left to right, respectively) of native unmodified templates of Con1b–luc–rep (blue) and J6/JFH-1 (red). Duplexed and loop regions of RNA structures indicated as described in Figure 2 A.
    Figure Legend Snippet: Comparative SHAPE analysis of native RNA structures. ( A ) SHAPE mapping of J6/JFH-1 and Con1b–luc–rep SL9266. The bars represent the relative exposure of the numbered nucleotides across SL9266 with the standard error of four independent gels (two short templates, two long) indicated. Duplexed and loop regions of SL9266 are indicated using the schematic representation used in Figure 1 B. Long-range interacting regions are indicated with grey (upstream) and black (‘kissing loop’) bars. ( B ) RNA structure representation of the data presented in Figure 2 A, colour coded to highlight relative exposure of the terminal loop of Con1b–luc–rep. ( C ) Comparative analysis of the 9110 region, SL9266 and SL9571 (left to right, respectively) of native unmodified templates of Con1b–luc–rep (blue) and J6/JFH-1 (red). Duplexed and loop regions of RNA structures indicated as described in Figure 2 A.

    Techniques Used:

    Recovery and infectivity in Huh 7.5 cells of J6/JFH-1 bearing mutations in SL9266 and interacting regions. RNA generated in vitro was electroporated into Huh 7.5 cells. HCV-infected cells were quantified following immunofluorescence staining for NS5A 72 h post-transfection (shaded bars) or 72 h after passage of transfected cell supernatant to a fresh cell monolayer of Huh 7.5 cells (open bars). Results are expressed as focus forming units per millilitre (ffu/ml) and represent the average of three independent assays (error bars indicate the standard error from the mean). Mutations present in the genome are indicated on the X axis. WT = an unmodified J6/JFH-1 template, GND = a template bearing a GDD to GND mutation within the active site of the NS5B RNA-dependent RNA polymerase.
    Figure Legend Snippet: Recovery and infectivity in Huh 7.5 cells of J6/JFH-1 bearing mutations in SL9266 and interacting regions. RNA generated in vitro was electroporated into Huh 7.5 cells. HCV-infected cells were quantified following immunofluorescence staining for NS5A 72 h post-transfection (shaded bars) or 72 h after passage of transfected cell supernatant to a fresh cell monolayer of Huh 7.5 cells (open bars). Results are expressed as focus forming units per millilitre (ffu/ml) and represent the average of three independent assays (error bars indicate the standard error from the mean). Mutations present in the genome are indicated on the X axis. WT = an unmodified J6/JFH-1 template, GND = a template bearing a GDD to GND mutation within the active site of the NS5B RNA-dependent RNA polymerase.

    Techniques Used: Infection, Generated, In Vitro, Immunofluorescence, Staining, Transfection, Mutagenesis

    5) Product Images from "Both Sphingomyelin and Cholesterol in the Host Cell Membrane Are Essential for Rubella Virus Entry"

    Article Title: Both Sphingomyelin and Cholesterol in the Host Cell Membrane Are Essential for Rubella Virus Entry

    Journal: Journal of Virology

    doi: 10.1128/JVI.01130-17

    Effects of SMase, myriocin, HPA-12, and IFN on RuV replicon systems. (A) Structure of the subgenomic replicon RuV-Luc genome (HS-Rep-C-P2R). P2R, a reporter fusion protein composed of puromycin N -acetyl-transferase (Puro), the foot-and-mouth disease virus 2A self-cleavage domain, and Renilla luciferase (RLuc); IRES, internal ribosome entry site sequence of encephalomyocarditis virus; P150 and P90, RuV nonstructural proteins; C, RuV capsid protein; AG1, monomeric Aami-Green1. (B) The replicon RuV-Luc genome was synthesized in vitro , and Vero cells were transfected with the in vitro -synthesized replicon RuV-Luc genome. At 4 h posttransfection, the cells were left untreated or treated with SMase (150 mU/ml) and incubated for 72 h at 35°C. The luciferase activity in the cells was then measured. (C) RuV-RNA replicon cells (Vero-HS-Rep-C-P2R cells) were left untreated or treated with SMase (150 mU/ml), myriocin (100 nM), HPA-12 (5 μM), or IFN (100 units/ml) and incubated for 2 days at 35°C. The Renilla luciferase activity in the cells was then measured. For panels B and C, the average luciferase activity in untreated cells (−) was set to 100%. The asterisk indicates a significant difference based on a t test ( P
    Figure Legend Snippet: Effects of SMase, myriocin, HPA-12, and IFN on RuV replicon systems. (A) Structure of the subgenomic replicon RuV-Luc genome (HS-Rep-C-P2R). P2R, a reporter fusion protein composed of puromycin N -acetyl-transferase (Puro), the foot-and-mouth disease virus 2A self-cleavage domain, and Renilla luciferase (RLuc); IRES, internal ribosome entry site sequence of encephalomyocarditis virus; P150 and P90, RuV nonstructural proteins; C, RuV capsid protein; AG1, monomeric Aami-Green1. (B) The replicon RuV-Luc genome was synthesized in vitro , and Vero cells were transfected with the in vitro -synthesized replicon RuV-Luc genome. At 4 h posttransfection, the cells were left untreated or treated with SMase (150 mU/ml) and incubated for 72 h at 35°C. The luciferase activity in the cells was then measured. (C) RuV-RNA replicon cells (Vero-HS-Rep-C-P2R cells) were left untreated or treated with SMase (150 mU/ml), myriocin (100 nM), HPA-12 (5 μM), or IFN (100 units/ml) and incubated for 2 days at 35°C. The Renilla luciferase activity in the cells was then measured. For panels B and C, the average luciferase activity in untreated cells (−) was set to 100%. The asterisk indicates a significant difference based on a t test ( P

    Techniques Used: Luciferase, Sequencing, Synthesized, In Vitro, Transfection, Incubation, Activity Assay

    6) Product Images from "SmD3 Regulates Intronic Noncoding RNA Biogenesis"

    Article Title: SmD3 Regulates Intronic Noncoding RNA Biogenesis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00022-12

    snoRNA-containing intron lariats are decreased in 6H2 cells. (A) WT and 6H2 CHO cells were untreated or treated with palm for 9 h. Nuclear RNA was isolated and reverse transcribed using random hexamers. rpL13a intron lariat abundance was determined by
    Figure Legend Snippet: snoRNA-containing intron lariats are decreased in 6H2 cells. (A) WT and 6H2 CHO cells were untreated or treated with palm for 9 h. Nuclear RNA was isolated and reverse transcribed using random hexamers. rpL13a intron lariat abundance was determined by

    Techniques Used: Isolation

    7) Product Images from "Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells"

    Article Title: Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells

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

    doi: 10.1073/pnas.1218464110

    EAV lacking PLP2 DUB activity elicits an enhanced innate immune response. ELFs were infected with EAV encoding WT or mutant PLP2 at m.o.i. 0.25 and at 20 and 24 hpi RNA was isolated for real-time qRT-PCR measurement of the levels of ( A ) IFN-β
    Figure Legend Snippet: EAV lacking PLP2 DUB activity elicits an enhanced innate immune response. ELFs were infected with EAV encoding WT or mutant PLP2 at m.o.i. 0.25 and at 20 and 24 hpi RNA was isolated for real-time qRT-PCR measurement of the levels of ( A ) IFN-β

    Techniques Used: Activity Assay, Infection, Mutagenesis, Isolation, Quantitative RT-PCR

    EAV PLP2 mutants display similar replication kinetics as WT virus. Equine lung fibroblasts were infected with WT or mutant EAV at m.o.i. 0.5 ( A ) or 5 ( B–D ). ( A and B ) At the indicated time points, total RNA was isolated for qRT-PCR measurement
    Figure Legend Snippet: EAV PLP2 mutants display similar replication kinetics as WT virus. Equine lung fibroblasts were infected with WT or mutant EAV at m.o.i. 0.5 ( A ) or 5 ( B–D ). ( A and B ) At the indicated time points, total RNA was isolated for qRT-PCR measurement

    Techniques Used: Infection, Mutagenesis, Isolation, Quantitative RT-PCR

    8) Product Images from "NF-κB activation triggers NK-cell stimulation by monocyte-derived dendritic cells"

    Article Title: NF-κB activation triggers NK-cell stimulation by monocyte-derived dendritic cells

    Journal: Therapeutic Advances in Medical Oncology

    doi: 10.1177/1758835919891622

    caIKKβ-DCs induce NK cells to secrete pro-inflammatory cytokines. Cytokine-matured dendritic cells (DCs) were either electroporated with RNA encoding caIKKβ or as a control were mock electroporated. (a) Transfected DCs were co-cultured 2–4 h after electroporation with fresh autologous peripheral blood mononuclear cells (PBMCs) at a ratio of 1:2 (final concentrations: 1 × 10 6 DCs/ml and 2 × 10 6 PBMCs/ml) or 1:10 (final concentrations: 2 × 10 5 DCs/ml and 2 × 10 6 PBMCs/ml). As controls, PBMCs and DCs were cultured alone. Secretion of IL-12p70, TNFα, and IFNγ was measured in the supernatant by Cytometric Bead Array after 24 h and 48 h of co-incubation. Average cytokine concentrations with SEM are shown from 7 (24 h) or 4 (48 h) different donors; for original data see Supplemental Table S3 . (b) Transfected DCs were co-cultured with fresh autologous NK cells at a ratio of 5:1 (final concentrations: 1 × 10 6 DCs/ml and 2 × 10 5 NK cells/ml) or 1:1 (final concentrations: 1 × 10 6 DCs/ml and 1 × 10 6 NK cells/ml). As controls, NK cells and DCs were cultured alone. Cytokine secretion was measured as described in (a). Average cytokine concentrations are shown from 5 (24 h) or 4 (48 h) different donors; for original data see Supplemental Table S4 . p values were calculated to the respective mock condition with the paired Student’s t test, **** p ⩽ 0.0001, *** p ⩽ 0.001, ** p ⩽ 0.01, * p ⩽ 0.05, numbers indicate p values of 0.05 ⩽ p ⩽ 0.1.
    Figure Legend Snippet: caIKKβ-DCs induce NK cells to secrete pro-inflammatory cytokines. Cytokine-matured dendritic cells (DCs) were either electroporated with RNA encoding caIKKβ or as a control were mock electroporated. (a) Transfected DCs were co-cultured 2–4 h after electroporation with fresh autologous peripheral blood mononuclear cells (PBMCs) at a ratio of 1:2 (final concentrations: 1 × 10 6 DCs/ml and 2 × 10 6 PBMCs/ml) or 1:10 (final concentrations: 2 × 10 5 DCs/ml and 2 × 10 6 PBMCs/ml). As controls, PBMCs and DCs were cultured alone. Secretion of IL-12p70, TNFα, and IFNγ was measured in the supernatant by Cytometric Bead Array after 24 h and 48 h of co-incubation. Average cytokine concentrations with SEM are shown from 7 (24 h) or 4 (48 h) different donors; for original data see Supplemental Table S3 . (b) Transfected DCs were co-cultured with fresh autologous NK cells at a ratio of 5:1 (final concentrations: 1 × 10 6 DCs/ml and 2 × 10 5 NK cells/ml) or 1:1 (final concentrations: 1 × 10 6 DCs/ml and 1 × 10 6 NK cells/ml). As controls, NK cells and DCs were cultured alone. Cytokine secretion was measured as described in (a). Average cytokine concentrations are shown from 5 (24 h) or 4 (48 h) different donors; for original data see Supplemental Table S4 . p values were calculated to the respective mock condition with the paired Student’s t test, **** p ⩽ 0.0001, *** p ⩽ 0.001, ** p ⩽ 0.01, * p ⩽ 0.05, numbers indicate p values of 0.05 ⩽ p ⩽ 0.1.

    Techniques Used: Transfection, Cell Culture, Electroporation, Incubation

    Stimulation of peripheral blood mononuclear cells (PBMCs) with caIKKβ-DCs leads to activation of both NK cells and CD8 + T cells. Cytokine-matured dendritic cells (DCs) were electroporated either with caIKKβ-RNA or, as a control, were mock electroporated. Transfected DCs were then either loaded with a CD8 + T-cell epitope from the melanoma antigen MelanA (MelA pept) or were left untreated (no pept). These DCs were co-cultured with fresh autologous PMBCs at a ratio of 1:10 (final concentrations: 2 × 10 5 DCs/ml and 2 × 10 6 PBMC/ml) and incubated for 1 week. (a) MelanA-specific CD8 + T cells were measured by peptide-HLA-tetramer staining. To identify CD8 + T cells the gating strategy shown in Supplemental Figure S8A – E was used. The percentage of MelanA-specific CD8 + T cells out of all CD8 + T cells was calculated. Dot plots from a representative donor out of four individual donors is shown; for all original data, see Supplemental Table S7 . (b) The expression of CD69 on NK cells (using the gating strategy shown in Supplemental Figure S8A – D to identify NK cells) was determined for each condition via flow cytometry. The average MFI of four different donors with the SEM is shown; for original data, see Supplemental Table S8 . p values were calculated to the respective mock condition with paired Student’s t test. ** p ⩽ 0.01, * p ⩽ 0.05.
    Figure Legend Snippet: Stimulation of peripheral blood mononuclear cells (PBMCs) with caIKKβ-DCs leads to activation of both NK cells and CD8 + T cells. Cytokine-matured dendritic cells (DCs) were electroporated either with caIKKβ-RNA or, as a control, were mock electroporated. Transfected DCs were then either loaded with a CD8 + T-cell epitope from the melanoma antigen MelanA (MelA pept) or were left untreated (no pept). These DCs were co-cultured with fresh autologous PMBCs at a ratio of 1:10 (final concentrations: 2 × 10 5 DCs/ml and 2 × 10 6 PBMC/ml) and incubated for 1 week. (a) MelanA-specific CD8 + T cells were measured by peptide-HLA-tetramer staining. To identify CD8 + T cells the gating strategy shown in Supplemental Figure S8A – E was used. The percentage of MelanA-specific CD8 + T cells out of all CD8 + T cells was calculated. Dot plots from a representative donor out of four individual donors is shown; for all original data, see Supplemental Table S7 . (b) The expression of CD69 on NK cells (using the gating strategy shown in Supplemental Figure S8A – D to identify NK cells) was determined for each condition via flow cytometry. The average MFI of four different donors with the SEM is shown; for original data, see Supplemental Table S8 . p values were calculated to the respective mock condition with paired Student’s t test. ** p ⩽ 0.01, * p ⩽ 0.05.

    Techniques Used: Activation Assay, Transfection, Cell Culture, Incubation, Staining, Expressing, Flow Cytometry, Cytometry

    Cell–cell interaction is necessary for best NK-cell activation by caIKKβ-DCs. (a) Cytokine-matured dendritic cells (DCs) were electroporated with caIKKβ-RNA or, as a negative control, were mock electroporated. A transwell assay was carried out, to analyze whether cell–cell interaction between DCs and NK cells was required, using a membrane allowing transfer of soluble factors while separating cell populations, 2–4 h after electroporation. DCs and fresh autologous peripheral blood mononuclear cells (PBMCs) were either completely separated through a 0.4 µm pore sized membrane (I = mock DCs, II = caIKKβ-DCs) or were co-cultured in the lower compartment (III.2) and separated from further PBMCs in the upper (III.1). Each condition was incubated for 48 h. (b) Surface marker expressions (CD54, CD69, and CD25) on NK cells (using the gating strategy shown in Supplemental Figure S2 ) were determined for each condition as described in (a) by flow cytometry. Average values of 4 (I) or 5 (II and III) different donors with SEM are shown; for original data, see Supplemental Table S5 . (c) The concentrations of IL-12p70, TNFα, and IFNγ in the supernatants from each condition were measured by Cytometric Bead Array. Average values of 4 (I) or 5 (II and III) different donors with SEM are shown; for original data, see Supplemental Table S6 . All donors were analyzed in independent experiments. p values were evaluated using paired Student’s t test, * p ⩽ 0.05, numbers indicate p values of 0.05 ⩽ p ⩽ 0.1 in (b) and (c).
    Figure Legend Snippet: Cell–cell interaction is necessary for best NK-cell activation by caIKKβ-DCs. (a) Cytokine-matured dendritic cells (DCs) were electroporated with caIKKβ-RNA or, as a negative control, were mock electroporated. A transwell assay was carried out, to analyze whether cell–cell interaction between DCs and NK cells was required, using a membrane allowing transfer of soluble factors while separating cell populations, 2–4 h after electroporation. DCs and fresh autologous peripheral blood mononuclear cells (PBMCs) were either completely separated through a 0.4 µm pore sized membrane (I = mock DCs, II = caIKKβ-DCs) or were co-cultured in the lower compartment (III.2) and separated from further PBMCs in the upper (III.1). Each condition was incubated for 48 h. (b) Surface marker expressions (CD54, CD69, and CD25) on NK cells (using the gating strategy shown in Supplemental Figure S2 ) were determined for each condition as described in (a) by flow cytometry. Average values of 4 (I) or 5 (II and III) different donors with SEM are shown; for original data, see Supplemental Table S5 . (c) The concentrations of IL-12p70, TNFα, and IFNγ in the supernatants from each condition were measured by Cytometric Bead Array. Average values of 4 (I) or 5 (II and III) different donors with SEM are shown; for original data, see Supplemental Table S6 . All donors were analyzed in independent experiments. p values were evaluated using paired Student’s t test, * p ⩽ 0.05, numbers indicate p values of 0.05 ⩽ p ⩽ 0.1 in (b) and (c).

    Techniques Used: Activation Assay, Negative Control, Transwell Assay, Electroporation, Cell Culture, Incubation, Marker, Flow Cytometry, Cytometry

    NK cells stimulated with caIKKβ-DCs can kill K562 cells. Cytokine-matured DCs were electroporated either with caIKKβ-RNA or, as a control, were mock electroporated. (a) Transfected dendritic cells (DCs) were co-cultured with fresh autologous peripheral blood mononuclear cells (PBMCs) at a ratio of 1:2 (final concentrations: 1 × 10 6 DCs/ml and 2 × 10 6 PBMCs/ml) or 1:10 (final concentrations: 2 × 10 5 DCs/ml and 2 × 10 6 PBMCs/ml) and incubated for 1 week. The cytolytic capacity of the resulting cell population was determined in a 51 chromium release assay. The K562 cell line was used as target at the indicated effector to target ratios. Average values ± SEM of three independent donors, each analyzed in triplicates, are shown; for original data see Supplemental Table S9 . (b) Transfected DCs were co-cultured with fresh autologous NK cells at a ratio of 5:1 (final concentrations: 1 × 10 6 DCs/ml and 2 × 10 5 NK cells/ml) and 1:1 (final concentrations: 1 × 10 6 DCs/ml and 1 × 10 6 NK cells/ml) and incubated for 1 week. The lytic capacity of the resulting NK cells was determined as depicted in (a). Average values ± SEM of three independent donors, each analyzed in triplicates, are shown; for original data, see Supplemental Table S10 . p values were calculated to the respective mock condition using the paired Student’s t test, ** p ⩽ 0.01, * p ⩽ 0.05, numbers indicate p values of 0.05 ⩽ p ⩽ 0.1.
    Figure Legend Snippet: NK cells stimulated with caIKKβ-DCs can kill K562 cells. Cytokine-matured DCs were electroporated either with caIKKβ-RNA or, as a control, were mock electroporated. (a) Transfected dendritic cells (DCs) were co-cultured with fresh autologous peripheral blood mononuclear cells (PBMCs) at a ratio of 1:2 (final concentrations: 1 × 10 6 DCs/ml and 2 × 10 6 PBMCs/ml) or 1:10 (final concentrations: 2 × 10 5 DCs/ml and 2 × 10 6 PBMCs/ml) and incubated for 1 week. The cytolytic capacity of the resulting cell population was determined in a 51 chromium release assay. The K562 cell line was used as target at the indicated effector to target ratios. Average values ± SEM of three independent donors, each analyzed in triplicates, are shown; for original data see Supplemental Table S9 . (b) Transfected DCs were co-cultured with fresh autologous NK cells at a ratio of 5:1 (final concentrations: 1 × 10 6 DCs/ml and 2 × 10 5 NK cells/ml) and 1:1 (final concentrations: 1 × 10 6 DCs/ml and 1 × 10 6 NK cells/ml) and incubated for 1 week. The lytic capacity of the resulting NK cells was determined as depicted in (a). Average values ± SEM of three independent donors, each analyzed in triplicates, are shown; for original data, see Supplemental Table S10 . p values were calculated to the respective mock condition using the paired Student’s t test, ** p ⩽ 0.01, * p ⩽ 0.05, numbers indicate p values of 0.05 ⩽ p ⩽ 0.1.

    Techniques Used: Transfection, Cell Culture, Incubation, Release Assay

    Stimulation with caIKKβ-transfected mature dendritic cells (DCs) results in the upregulation of activation markers on NK cells. Cytokine-matured DCs were electroporated either with caIKKβ-RNA or as a control were mock electroporated. (a) Transfected DCs were co-cultured with fresh autologous peripheral blood mononuclear cells (PBMCs) 2–4 h after electroporation at a ratio of 1:2 (final concentrations: 1 × 10 6 DCs/ml and 2 × 10 6 PBMCs/ml) or 1:10 (final concentrations: 2 × 10 5 DCs/ml and 2 × 10 6 PBMCs/ml). To determine background levels, PBMCs were cultured alone. Cells were harvested after 24 h or 48 h and the expression of the surface markers CD54, CD69, and CD25 was determined via flow cytometry (using the gating strategy shown in Supplemental Figure S2 ). All values show the upregulation of each surface marker, calculated relative to the mean fluorescence intensity (MFI) of PBMCs alone. The average fold induction of four different donors with the SEM is shown; for original data, see Supplemental Table S1 . Each donor was analyzed in independent experiments. (b) DCs were co-cultured with fresh autologous NK cells at a ratio of 5:1 (final concentrations: 1 × 10 6 DCs/ml and 2 × 10 5 NK cells/ml) or 1:1 (final concentrations: 1 × 10 6 DCs/ml and 1 × 10 6 NK cells/ml). To determine background levels, NK cells were cultured alone. Cells were analyzed as described in (a). Average fold induction (relative to MFI of NK cells alone) is shown from four different donors with SEM; for original data see Supplemental Table S2 . p values were calculated to the respective mock condition with the paired Student’s t test using the specific MFI values, ** p ⩽ 0.01, * p ⩽ 0.05, numbers indicate p value of 0.05 ⩽ p ⩽ 0.1.
    Figure Legend Snippet: Stimulation with caIKKβ-transfected mature dendritic cells (DCs) results in the upregulation of activation markers on NK cells. Cytokine-matured DCs were electroporated either with caIKKβ-RNA or as a control were mock electroporated. (a) Transfected DCs were co-cultured with fresh autologous peripheral blood mononuclear cells (PBMCs) 2–4 h after electroporation at a ratio of 1:2 (final concentrations: 1 × 10 6 DCs/ml and 2 × 10 6 PBMCs/ml) or 1:10 (final concentrations: 2 × 10 5 DCs/ml and 2 × 10 6 PBMCs/ml). To determine background levels, PBMCs were cultured alone. Cells were harvested after 24 h or 48 h and the expression of the surface markers CD54, CD69, and CD25 was determined via flow cytometry (using the gating strategy shown in Supplemental Figure S2 ). All values show the upregulation of each surface marker, calculated relative to the mean fluorescence intensity (MFI) of PBMCs alone. The average fold induction of four different donors with the SEM is shown; for original data, see Supplemental Table S1 . Each donor was analyzed in independent experiments. (b) DCs were co-cultured with fresh autologous NK cells at a ratio of 5:1 (final concentrations: 1 × 10 6 DCs/ml and 2 × 10 5 NK cells/ml) or 1:1 (final concentrations: 1 × 10 6 DCs/ml and 1 × 10 6 NK cells/ml). To determine background levels, NK cells were cultured alone. Cells were analyzed as described in (a). Average fold induction (relative to MFI of NK cells alone) is shown from four different donors with SEM; for original data see Supplemental Table S2 . p values were calculated to the respective mock condition with the paired Student’s t test using the specific MFI values, ** p ⩽ 0.01, * p ⩽ 0.05, numbers indicate p value of 0.05 ⩽ p ⩽ 0.1.

    Techniques Used: Transfection, Activation Assay, Cell Culture, Electroporation, Expressing, Flow Cytometry, Cytometry, Marker, Fluorescence

    9) Product Images from "Both Sphingomyelin and Cholesterol in the Host Cell Membrane Are Essential for Rubella Virus Entry"

    Article Title: Both Sphingomyelin and Cholesterol in the Host Cell Membrane Are Essential for Rubella Virus Entry

    Journal: Journal of Virology

    doi: 10.1128/JVI.01130-17

    Effects of SMase, myriocin, HPA-12, and IFN on RuV replicon systems. (A) Structure of the subgenomic replicon RuV-Luc genome (HS-Rep-C-P2R). P2R, a reporter fusion protein composed of puromycin N -acetyl-transferase (Puro), the foot-and-mouth disease virus 2A self-cleavage domain, and Renilla luciferase (RLuc); IRES, internal ribosome entry site sequence of encephalomyocarditis virus; P150 and P90, RuV nonstructural proteins; C, RuV capsid protein; AG1, monomeric Aami-Green1. (B) The replicon RuV-Luc genome was synthesized in vitro , and Vero cells were transfected with the in vitro -synthesized replicon RuV-Luc genome. At 4 h posttransfection, the cells were left untreated or treated with SMase (150 mU/ml) and incubated for 72 h at 35°C. The luciferase activity in the cells was then measured. (C) RuV-RNA replicon cells (Vero-HS-Rep-C-P2R cells) were left untreated or treated with SMase (150 mU/ml), myriocin (100 nM), HPA-12 (5 μM), or IFN (100 units/ml) and incubated for 2 days at 35°C. The Renilla luciferase activity in the cells was then measured. For panels B and C, the average luciferase activity in untreated cells (−) was set to 100%. The asterisk indicates a significant difference based on a t test ( P
    Figure Legend Snippet: Effects of SMase, myriocin, HPA-12, and IFN on RuV replicon systems. (A) Structure of the subgenomic replicon RuV-Luc genome (HS-Rep-C-P2R). P2R, a reporter fusion protein composed of puromycin N -acetyl-transferase (Puro), the foot-and-mouth disease virus 2A self-cleavage domain, and Renilla luciferase (RLuc); IRES, internal ribosome entry site sequence of encephalomyocarditis virus; P150 and P90, RuV nonstructural proteins; C, RuV capsid protein; AG1, monomeric Aami-Green1. (B) The replicon RuV-Luc genome was synthesized in vitro , and Vero cells were transfected with the in vitro -synthesized replicon RuV-Luc genome. At 4 h posttransfection, the cells were left untreated or treated with SMase (150 mU/ml) and incubated for 72 h at 35°C. The luciferase activity in the cells was then measured. (C) RuV-RNA replicon cells (Vero-HS-Rep-C-P2R cells) were left untreated or treated with SMase (150 mU/ml), myriocin (100 nM), HPA-12 (5 μM), or IFN (100 units/ml) and incubated for 2 days at 35°C. The Renilla luciferase activity in the cells was then measured. For panels B and C, the average luciferase activity in untreated cells (−) was set to 100%. The asterisk indicates a significant difference based on a t test ( P

    Techniques Used: Luciferase, Sequencing, Synthesized, In Vitro, Transfection, Incubation, Activity Assay

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    Article Snippet: .. In vitro RNA transcription from XhoI-linearized WT or mutant EAV full-length cDNA clones was performed using the mMESSAGE mMACHINE T7 Kit (Ambion). .. Five micrograms of full-length EAV RNA was electroporated into 5.0 × 106 BHK-21 cells using the Amaxa Cell Line Nucleofector Kit T and the program T-020 of the Amaxa Nucleofector (Lonza) according to the manufacturer’s instructions.

    Article Title: NF-κB activation triggers NK-cell stimulation by monocyte-derived dendritic cells
    Article Snippet: .. In vitro RNA transcription and electroporation of DCs In vitro transcription of mRNA was carried out using the mMESSAGE mMACHINE™ T7 ULTRA Transcription Kit (Life Technologies, Carlsbad, CA, USA) and purified with an RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturers’ protocols. .. The RNA used for electroporation encoded a constitutively active mutant of IKKβ, which activates the classical NF-κB pathway.

    Article Title: Arterivirus Nsp1 Modulates the Accumulation of Minus-Strand Templates to Control the Relative Abundance of Viral mRNAs
    Article Snippet: .. In vitro RNA transcription from Xho I-linearized wt or mutant EAV full-length cDNA clones was performed using the mMESSAGE mMACHINE T7 Kit (Ambion). .. Seven µg of in vitro-synthesized EAV RNA were electroporated into 3.5×106 BHK-21 cells using the Amaxa Cell Line Nucleofector Kit T and the program T-020 of the Amaxa Nucleofector (Lonza) according to the manufacturer's instructions.

    Synthesized:

    Article Title: Both Sphingomyelin and Cholesterol in the Host Cell Membrane Are Essential for Rubella Virus Entry
    Article Snippet: .. The RNA encoding the full-length C protein was synthesized from the plasmid encoding the C protein of the RuV HS strain (C1–300 ) by in vitro RNA transcription with a mMESSAGE mMACHINE T7 transcription kit (Life Technologies). .. The quality of the synthesized RNAs was confirmed by electrophoresis, and the amounts of RNAs were calculated spectrophotometrically.

    Article Title: SmD3 Regulates Intronic Noncoding RNA Biogenesis
    Article Snippet: .. Double-stranded RNA (dsRNA) templates were generated for probes for each rpL13a snoRNA by PCR amplification of cloned hamster rpL13a genomic sequence templates using primers containing the T7 RNA polymerase promoter and used for in vitro RNA transcription of 32 P-labeled snoRNA probes. miR-16 probes were synthesized using templates from the mirVana miRNA detection kit (Ambion). .. RNA was isolated from cells using a mirVana miRNA isolation kit (Ambion) and hybridized to 32 P-labeled RNA probes (mirVana miRNA detection kit) overnight at 42 to 52°C, followed by RNase digestion and ethanol precipitation.

    Mutagenesis:

    Article Title: Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells
    Article Snippet: .. In vitro RNA transcription from XhoI-linearized WT or mutant EAV full-length cDNA clones was performed using the mMESSAGE mMACHINE T7 Kit (Ambion). .. Five micrograms of full-length EAV RNA was electroporated into 5.0 × 106 BHK-21 cells using the Amaxa Cell Line Nucleofector Kit T and the program T-020 of the Amaxa Nucleofector (Lonza) according to the manufacturer’s instructions.

    Article Title: Arterivirus Nsp1 Modulates the Accumulation of Minus-Strand Templates to Control the Relative Abundance of Viral mRNAs
    Article Snippet: .. In vitro RNA transcription from Xho I-linearized wt or mutant EAV full-length cDNA clones was performed using the mMESSAGE mMACHINE T7 Kit (Ambion). .. Seven µg of in vitro-synthesized EAV RNA were electroporated into 3.5×106 BHK-21 cells using the Amaxa Cell Line Nucleofector Kit T and the program T-020 of the Amaxa Nucleofector (Lonza) according to the manufacturer's instructions.

    Purification:

    Article Title: Cis-acting structural element in 5′ UTR is essential for infectivity of porcine reproductive and respiratory syndrome virus
    Article Snippet: .. 1 μg of purified products were used as templates for in vitro RNA transcription by T7 promoter, with T7 mMESSAGE mMachine (Ambion Inc., Austin, TX). .. After removal of the template from reaction mixture using DNAse I, the synthetic RNAs were verified by 1.2% native RNA agarose gel electrophoretic, and stored at −80 °C until use ( ).

    Article Title: NF-κB activation triggers NK-cell stimulation by monocyte-derived dendritic cells
    Article Snippet: .. In vitro RNA transcription and electroporation of DCs In vitro transcription of mRNA was carried out using the mMESSAGE mMACHINE™ T7 ULTRA Transcription Kit (Life Technologies, Carlsbad, CA, USA) and purified with an RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturers’ protocols. .. The RNA used for electroporation encoded a constitutively active mutant of IKKβ, which activates the classical NF-κB pathway.

    Polymerase Chain Reaction:

    Article Title: SmD3 Regulates Intronic Noncoding RNA Biogenesis
    Article Snippet: .. Double-stranded RNA (dsRNA) templates were generated for probes for each rpL13a snoRNA by PCR amplification of cloned hamster rpL13a genomic sequence templates using primers containing the T7 RNA polymerase promoter and used for in vitro RNA transcription of 32 P-labeled snoRNA probes. miR-16 probes were synthesized using templates from the mirVana miRNA detection kit (Ambion). .. RNA was isolated from cells using a mirVana miRNA isolation kit (Ambion) and hybridized to 32 P-labeled RNA probes (mirVana miRNA detection kit) overnight at 42 to 52°C, followed by RNase digestion and ethanol precipitation.

    Generated:

    Article Title: SmD3 Regulates Intronic Noncoding RNA Biogenesis
    Article Snippet: .. Double-stranded RNA (dsRNA) templates were generated for probes for each rpL13a snoRNA by PCR amplification of cloned hamster rpL13a genomic sequence templates using primers containing the T7 RNA polymerase promoter and used for in vitro RNA transcription of 32 P-labeled snoRNA probes. miR-16 probes were synthesized using templates from the mirVana miRNA detection kit (Ambion). .. RNA was isolated from cells using a mirVana miRNA isolation kit (Ambion) and hybridized to 32 P-labeled RNA probes (mirVana miRNA detection kit) overnight at 42 to 52°C, followed by RNase digestion and ethanol precipitation.

    Sequencing:

    Article Title: SmD3 Regulates Intronic Noncoding RNA Biogenesis
    Article Snippet: .. Double-stranded RNA (dsRNA) templates were generated for probes for each rpL13a snoRNA by PCR amplification of cloned hamster rpL13a genomic sequence templates using primers containing the T7 RNA polymerase promoter and used for in vitro RNA transcription of 32 P-labeled snoRNA probes. miR-16 probes were synthesized using templates from the mirVana miRNA detection kit (Ambion). .. RNA was isolated from cells using a mirVana miRNA isolation kit (Ambion) and hybridized to 32 P-labeled RNA probes (mirVana miRNA detection kit) overnight at 42 to 52°C, followed by RNase digestion and ethanol precipitation.

    Electroporation:

    Article Title: NF-κB activation triggers NK-cell stimulation by monocyte-derived dendritic cells
    Article Snippet: .. In vitro RNA transcription and electroporation of DCs In vitro transcription of mRNA was carried out using the mMESSAGE mMACHINE™ T7 ULTRA Transcription Kit (Life Technologies, Carlsbad, CA, USA) and purified with an RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturers’ protocols. .. The RNA used for electroporation encoded a constitutively active mutant of IKKβ, which activates the classical NF-κB pathway.

    Plasmid Preparation:

    Article Title: Both Sphingomyelin and Cholesterol in the Host Cell Membrane Are Essential for Rubella Virus Entry
    Article Snippet: .. The RNA encoding the full-length C protein was synthesized from the plasmid encoding the C protein of the RuV HS strain (C1–300 ) by in vitro RNA transcription with a mMESSAGE mMACHINE T7 transcription kit (Life Technologies). .. The quality of the synthesized RNAs was confirmed by electrophoresis, and the amounts of RNAs were calculated spectrophotometrically.

    Article Title: Improved Molecular Diagnosis of COVID-19 by the Novel, Highly Sensitive and Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-PCR Assay Validated In Vitro and with Clinical Specimens
    Article Snippet: .. Linearized pCR2.1-TOPO plasmid (Invitrogen, Carlsbad, CA, USA) with a T7 promoter and a cloned target region (RdRp/Hel, S, or N) of SARS-CoV-2 were used for in vitro RNA transcription using MEGAscript T7 transcription kit (Ambion, Austin, TX, USA) for the standards and limit of detection (LOD) as previously described ( , ). .. Each linearized plasmid template was mixed with 2 μl each of ATP, GTP, CTP, and UTP, 10× reaction buffer, and enzyme mix in a standard 20-μl reaction mixture.

    Article Title: A twist in the tail: SHAPE mapping of long-range interactions and structural rearrangements of RNA elements involved in HCV replication
    Article Snippet: .. In vitro RNA transcription One microgram of either J6/JFH-1 plasmid cDNA, which includes a 3′ cis -acting ribozyme, or ScaI-linearized Con1b–luc–rep cDNA was used as template for the production of RNA in vitro using a T7 MEGAscript kit (Ambion), according to the manufacturers’ instructions. .. After transcription, the DNA template was removed by DNase 1 (Ambion) treatment and the RNA purified with an RNeasy mini-kit column (Qiagen).

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    Thermo Fisher sertoli cell tj basal es barrier
    Knockdown of Spire 1 perturbs MT organization through disruptive changes in detyrosinated α-tubulin and EB1 distribution. a Knockdown of Spire 1 in <t>Sertoli</t> cells by RNAi perturbed the organization of MTs since α-tubulin (note: α- and β-tubulins are blocking blocks of MTs 12 ) no longer stretched across the Sertoli cell cytosol as noted in control cells. Instead, MTs appeared to be largely truncated and wrapped around the cell nuclei, retracting from cell peripheries. On the other hand, detyrosinated α-tubulin (rendering MTs to become more stabilized) was found to wrap around the Sertoli cell nucleus loosely, displaying a pattern similar to α-tubulin following Spire 1 knockdown instead of stretching across the Sertoli cell cytosols as noted in control cells. Furthermore, the +TIP protein EB1 known to stabilize MTs and involved in promoting MT growth from the plus (+)-end also retracted from the Sertoli cell cytosol, and localized closer to the Sertoli cell nucleus following Spire 1 knockdown, dissimilar from control cells wherein EB1 scattered along the MTs that stretched across the long axis of the entire Sertoli cell. Co-transfection of rhodamine-siGLO indicator (red fluorescence) indicated successful transfection. Scale bar, 40 µm, which applies to other micrographs. b Microtubule spin-down assay was used to quantify the relative amount of polymerized microtubules (in pellet) vs. non-polymerized and free-tubulins (in supernatant) in Sertoli cell cytosol after knockdown of Spire 1. Knockdown of Spire 1, as confirmed by IB using Sertoli cell lysates, was found to perturb MT dynamics by reducing the level of polymerized MTs quantified in Sertoli cells. The presence of Taxol (20 µM) and CaCl 2 (2 mM) in Sertoli cell lysates in this assay served as the corresponding positive (+ve) and negative (−ve) control, respectively. GAPDH served as the protein loading control. Each bar in the histogram is a mean ± s.d. of at n = 3 independent experiments. ** P
    Sertoli Cell Tj Basal Es Barrier, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher transcriptome analysis
    RNA-seq and connectivity Map (CMAP) analysis of differentially expressed genes (DEG) in Fmr1 knockout (KO) hippocampal neuron. ( a ) Gene expression differences between wild type (WT) and Fmr1 KO samples. There are 587 up-regulated and 724 down-regulated genes in Fmr1 KO hippocampal neuron. ( b ) Top 15 GO biology processes that are associated with DEG between WT and Fmr1 KO samples. ( c ) KEGG pathways that are associated with DEG between WT and Fmr1 KO samples. ( d ) Top-ranked 10 CMAP compounds/drugs that induce <t>transcriptome</t> alterations oppositional to (indicated by negative similarity mean) or overlapping with (indicated by positive similarity mean) that caused by Fmr1 deficiency. Rank is determined by P value and enrichment score. Drug name and cell line indicate the name of compound used for treatment with specific cell lines in CMAP database. Full information of P value, enrichment score, and similarity mean is shown in Supplementary Table 4.
    Transcriptome Analysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher ercc rna spike in mix
    Nanopore DRS with cap-dependent ligation of 5′ adapter <t>RNA.</t> ( A ) Histogram showing the distribution of 5′ adapter RNA length in the nanopore raw current signal, as inferred from alignment of the mRNA sequence to the signal using nanopolish eventalign. The median signal length was 1441 points and 96% of adapter signals were 3000 points or less. ( B ) Out-of-bag receiver operator characteristic curve showing the performance of the trained convolutional neural network at detecting 5′ adapter RNA using 3000 points of signal. The curve was generated using five-fold cross validation. ( C ) Out-of-bag precision recall curve showing the performance of trained neural network, generated using five-fold cross validation. ( D ) Alternative transcription start sites were identified using nanopore DRS with cap-dependent ligation of a 5′ end adapter at the AT1G17050 and AT5G18650 genes. Orange, 5’ coverage for capped nanoPARE reads; blue track, nanopore DRS coverage with cap-dependent ligation of 5′ adapter RNA; blue, isoforms detected by nanopore DRS with cap-dependent ligation of 5′ adapter RNA; black, Araport11 annotation. ( E ) Reads mapping to <t>ERCC</t> RNA spike-ins lack approximately 11 nt of sequence at the 5′ end. Histogram showing the distance to the 5′ end for ERCC RNA spike-in reads (each spike-in is shown in a different colour; only those with > 1000 supporting reads are shown). ( F ) Reads mapping to in vitro transcribed mGFP lack approximately 11 nt of sequence at the 5′ end. Histogram showing the distance to the 5′ end for in vitro transcribed mGFP. ( G ) Araport11 annotation overestimates the length of 5′ UTRs. The cumulative distribution function shows the distance to the nearest TSS identified from full-length transcripts cloned as part of the RIKEN RAFL project (blue) and Araport11 annotation (orange). ( H ) Nanopore DRS detects miR170/miR171 cleavage products of Hairy Meristem 1 (HAM1, AT2G45160) transcripts. Orange, 5’ coverage from capped nanoPARE reads; purple, 5’ coverage from uncapped nanoPARE reads; blue, nanopore DRS 5’ coverage; grey, miRNA target site alignment is shown; black, Araport11 annotation. microRNA cleavage site predictions supported by enrichment of nanopore 5’ ends – Figure 3—figure supplement 1H .
    Ercc Rna Spike In Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Knockdown of Spire 1 perturbs MT organization through disruptive changes in detyrosinated α-tubulin and EB1 distribution. a Knockdown of Spire 1 in Sertoli cells by RNAi perturbed the organization of MTs since α-tubulin (note: α- and β-tubulins are blocking blocks of MTs 12 ) no longer stretched across the Sertoli cell cytosol as noted in control cells. Instead, MTs appeared to be largely truncated and wrapped around the cell nuclei, retracting from cell peripheries. On the other hand, detyrosinated α-tubulin (rendering MTs to become more stabilized) was found to wrap around the Sertoli cell nucleus loosely, displaying a pattern similar to α-tubulin following Spire 1 knockdown instead of stretching across the Sertoli cell cytosols as noted in control cells. Furthermore, the +TIP protein EB1 known to stabilize MTs and involved in promoting MT growth from the plus (+)-end also retracted from the Sertoli cell cytosol, and localized closer to the Sertoli cell nucleus following Spire 1 knockdown, dissimilar from control cells wherein EB1 scattered along the MTs that stretched across the long axis of the entire Sertoli cell. Co-transfection of rhodamine-siGLO indicator (red fluorescence) indicated successful transfection. Scale bar, 40 µm, which applies to other micrographs. b Microtubule spin-down assay was used to quantify the relative amount of polymerized microtubules (in pellet) vs. non-polymerized and free-tubulins (in supernatant) in Sertoli cell cytosol after knockdown of Spire 1. Knockdown of Spire 1, as confirmed by IB using Sertoli cell lysates, was found to perturb MT dynamics by reducing the level of polymerized MTs quantified in Sertoli cells. The presence of Taxol (20 µM) and CaCl 2 (2 mM) in Sertoli cell lysates in this assay served as the corresponding positive (+ve) and negative (−ve) control, respectively. GAPDH served as the protein loading control. Each bar in the histogram is a mean ± s.d. of at n = 3 independent experiments. ** P

    Journal: Cell Death & Disease

    Article Title: Actin nucleator Spire 1 is a regulator of ectoplasmic specialization in the testis

    doi: 10.1038/s41419-017-0201-6

    Figure Lengend Snippet: Knockdown of Spire 1 perturbs MT organization through disruptive changes in detyrosinated α-tubulin and EB1 distribution. a Knockdown of Spire 1 in Sertoli cells by RNAi perturbed the organization of MTs since α-tubulin (note: α- and β-tubulins are blocking blocks of MTs 12 ) no longer stretched across the Sertoli cell cytosol as noted in control cells. Instead, MTs appeared to be largely truncated and wrapped around the cell nuclei, retracting from cell peripheries. On the other hand, detyrosinated α-tubulin (rendering MTs to become more stabilized) was found to wrap around the Sertoli cell nucleus loosely, displaying a pattern similar to α-tubulin following Spire 1 knockdown instead of stretching across the Sertoli cell cytosols as noted in control cells. Furthermore, the +TIP protein EB1 known to stabilize MTs and involved in promoting MT growth from the plus (+)-end also retracted from the Sertoli cell cytosol, and localized closer to the Sertoli cell nucleus following Spire 1 knockdown, dissimilar from control cells wherein EB1 scattered along the MTs that stretched across the long axis of the entire Sertoli cell. Co-transfection of rhodamine-siGLO indicator (red fluorescence) indicated successful transfection. Scale bar, 40 µm, which applies to other micrographs. b Microtubule spin-down assay was used to quantify the relative amount of polymerized microtubules (in pellet) vs. non-polymerized and free-tubulins (in supernatant) in Sertoli cell cytosol after knockdown of Spire 1. Knockdown of Spire 1, as confirmed by IB using Sertoli cell lysates, was found to perturb MT dynamics by reducing the level of polymerized MTs quantified in Sertoli cells. The presence of Taxol (20 µM) and CaCl 2 (2 mM) in Sertoli cell lysates in this assay served as the corresponding positive (+ve) and negative (−ve) control, respectively. GAPDH served as the protein loading control. Each bar in the histogram is a mean ± s.d. of at n = 3 independent experiments. ** P

    Article Snippet: BTB integrity assay in vivo The Sertoli cell BTB integrity in vivo was assessed by the ability of the Sertoli cell TJ/basal ES-barrier to block the diffusion of a membrane impermeable biotinylation reagent EZ-Link Sulfo-NHS-LC-biotin (Thermo Fisher Scientific, Waltham, MA) across the barrier as earlier described .

    Techniques: Blocking Assay, Cotransfection, Fluorescence, Transfection, Spin Down Assay

    Knockdown of Spire 1 in the testis in vivo perturbs the Sertoli cell BTB. ( a ) In normal testes and testes transfected with non-targeting negative control siRNA duplexes (Ctrl RNAi), the BTB localized adjacent to the basement membrane (annotated by the white dash line) effectively blocked the diffusion of biotin (green fluorescence) from the basal (annotated by the white bracket in control groups) to the adluminal compartment. However, biotin freely diffused into the adluminal compartment in tubules from testes following treatment of rats with cadmium chloride (via i.m.) which is known to irreversibly disrupt the BTB function (positive control group) as annotated by the yellow bracket. Similarly, biotin was detected deep inside the adluminal compartment in testes following Spire 1 knockdown. Scale bar, 350 µm, first and third panel; 80 µm in second and fourth panel. The white boxed area was magnified and shown in the lower panel. b Semi-quantitative data illustrating the BTB was grossly disrupted by cadmium chloride, but a knockdown of Spire 1 RNAi also perturbed the BTB integrity considerably. Each bar is a mean ± s.d. of n = 3 rats. ** P

    Journal: Cell Death & Disease

    Article Title: Actin nucleator Spire 1 is a regulator of ectoplasmic specialization in the testis

    doi: 10.1038/s41419-017-0201-6

    Figure Lengend Snippet: Knockdown of Spire 1 in the testis in vivo perturbs the Sertoli cell BTB. ( a ) In normal testes and testes transfected with non-targeting negative control siRNA duplexes (Ctrl RNAi), the BTB localized adjacent to the basement membrane (annotated by the white dash line) effectively blocked the diffusion of biotin (green fluorescence) from the basal (annotated by the white bracket in control groups) to the adluminal compartment. However, biotin freely diffused into the adluminal compartment in tubules from testes following treatment of rats with cadmium chloride (via i.m.) which is known to irreversibly disrupt the BTB function (positive control group) as annotated by the yellow bracket. Similarly, biotin was detected deep inside the adluminal compartment in testes following Spire 1 knockdown. Scale bar, 350 µm, first and third panel; 80 µm in second and fourth panel. The white boxed area was magnified and shown in the lower panel. b Semi-quantitative data illustrating the BTB was grossly disrupted by cadmium chloride, but a knockdown of Spire 1 RNAi also perturbed the BTB integrity considerably. Each bar is a mean ± s.d. of n = 3 rats. ** P

    Article Snippet: BTB integrity assay in vivo The Sertoli cell BTB integrity in vivo was assessed by the ability of the Sertoli cell TJ/basal ES-barrier to block the diffusion of a membrane impermeable biotinylation reagent EZ-Link Sulfo-NHS-LC-biotin (Thermo Fisher Scientific, Waltham, MA) across the barrier as earlier described .

    Techniques: In Vivo, Transfection, Negative Control, Diffusion-based Assay, Fluorescence, Positive Control

    Knockdown of Spire1 in the testis in vivo perturbs F-actin organization in the seminiferous epithelium. a As noted in control testes transfected with non-targeting negative control siRNA duplexes, F-actin was prominently organized around the Sertoli–spermatid interface (yellow boxed areas) and also at the Sertoli cell–cell interface near the basement membrane (green boxed areas) to support the apical ES and basal ES/BTB function, respectively, as earlier reported 14 , 73 , 74 , such as in stage VII, VIII, and IX tubules. Furthermore, F-actin also formed the track-like structures (annotated by white arrowheads), mostly notably detected in late stage VIII tubules, to support phagosome and spermatid transport. After Spire 1 knockdown, F-actin no longer tightly localized at the basal ES to support the BTB, instead, F-actin appeared as truncated/branched network, diffusely localized at the site (see yellow brackets in Spire 1 RNAi testes vs. white brackets in control testes) and no obvious actin-conferred track-like structures were noted. Also, F-actin that appeared as bulb-like structures prominently found at the concave side of spermatid heads in control testes were re-distributed, appeared as dis-organized structures by wrapping around spermatid heads after Spire 1 knockdown. Moreover, F-actin virtually was non-detected or considerably diminished in spermatids that had loss their polarity (i.e., no longer have the heads pointing toward the basement membrane, annotated by yellow arrowheads in Spire 1 RNAi group) or still embedded deep inside the seminiferous epithelium (annotated by green arrowheads) when they should have been released into the tubule lumen at spermiation. These defects of F-actin organization was detected even in tubules that appeared as ‘normal’ staged tubules following Spire 1 knockdown. Thus, when defects of F-actin organization was taken into consideration, the percentage of defective tubules were considerably higher (see b below). Co-transfection of rhodamine-siGLO indicator (red fluorescence) illustrated successful transfection in the tubules shown herein. Scale bar, 20 µm; inset, 10 µm. b When the % of defective tubules were scored based on disruptive organization of F-actin, the percentage of disorganized tubules was considerably increased as noted in this bar graph when compared to defective tubules noted based on histological analysis as noted in Fig. 6c . Each bar is a mean ± s.d. of n = 4 rats, ~2000 tubules were randomly scored from each testis to assess disruptive changes in F-actin organization in the Spire 1 knockdown vs. control group. ** P

    Journal: Cell Death & Disease

    Article Title: Actin nucleator Spire 1 is a regulator of ectoplasmic specialization in the testis

    doi: 10.1038/s41419-017-0201-6

    Figure Lengend Snippet: Knockdown of Spire1 in the testis in vivo perturbs F-actin organization in the seminiferous epithelium. a As noted in control testes transfected with non-targeting negative control siRNA duplexes, F-actin was prominently organized around the Sertoli–spermatid interface (yellow boxed areas) and also at the Sertoli cell–cell interface near the basement membrane (green boxed areas) to support the apical ES and basal ES/BTB function, respectively, as earlier reported 14 , 73 , 74 , such as in stage VII, VIII, and IX tubules. Furthermore, F-actin also formed the track-like structures (annotated by white arrowheads), mostly notably detected in late stage VIII tubules, to support phagosome and spermatid transport. After Spire 1 knockdown, F-actin no longer tightly localized at the basal ES to support the BTB, instead, F-actin appeared as truncated/branched network, diffusely localized at the site (see yellow brackets in Spire 1 RNAi testes vs. white brackets in control testes) and no obvious actin-conferred track-like structures were noted. Also, F-actin that appeared as bulb-like structures prominently found at the concave side of spermatid heads in control testes were re-distributed, appeared as dis-organized structures by wrapping around spermatid heads after Spire 1 knockdown. Moreover, F-actin virtually was non-detected or considerably diminished in spermatids that had loss their polarity (i.e., no longer have the heads pointing toward the basement membrane, annotated by yellow arrowheads in Spire 1 RNAi group) or still embedded deep inside the seminiferous epithelium (annotated by green arrowheads) when they should have been released into the tubule lumen at spermiation. These defects of F-actin organization was detected even in tubules that appeared as ‘normal’ staged tubules following Spire 1 knockdown. Thus, when defects of F-actin organization was taken into consideration, the percentage of defective tubules were considerably higher (see b below). Co-transfection of rhodamine-siGLO indicator (red fluorescence) illustrated successful transfection in the tubules shown herein. Scale bar, 20 µm; inset, 10 µm. b When the % of defective tubules were scored based on disruptive organization of F-actin, the percentage of disorganized tubules was considerably increased as noted in this bar graph when compared to defective tubules noted based on histological analysis as noted in Fig. 6c . Each bar is a mean ± s.d. of n = 4 rats, ~2000 tubules were randomly scored from each testis to assess disruptive changes in F-actin organization in the Spire 1 knockdown vs. control group. ** P

    Article Snippet: BTB integrity assay in vivo The Sertoli cell BTB integrity in vivo was assessed by the ability of the Sertoli cell TJ/basal ES-barrier to block the diffusion of a membrane impermeable biotinylation reagent EZ-Link Sulfo-NHS-LC-biotin (Thermo Fisher Scientific, Waltham, MA) across the barrier as earlier described .

    Techniques: In Vivo, Transfection, Negative Control, Cotransfection, Fluorescence

    Expression, and cellular and stage-specific localization of Spire1 in the rat testis. a A study by RT-PCR using corresponding primer pairs specific to different target genes (Table 3 ) was performed to illustrate the expression of Spire 1 in adult rat testes (T), Sertoli cells (SC), and germ cells (GC) vs. brain (BR; positive control) with S16 served as a loading control. Spire 2 was also expressed in T, GC and BR, but not SC under the conditions used in our experiments. M, DNA markers in base pairs (bp). The identity of the PCR products was confirmed by direct nucleotide sequencing. b Specificity of the anti-Spire 1 antibody (Table 1 ) was assessed by immunoblotting using lysates of adult rat testes (T) at 80 µg protein. c Changes in the relative steady-state protein level of Spire1 expressed by Sertoli cells (left panel) were detected during the assembly of the Sertoli cell TJ-permeability barrier (right panel) when cultured in vitro for 6 days in serum-free F12/DMEM using Sertoli cell lysates (40 µg protein per lane) with β-actin as a protein loading control. Assembly of the TJ-barrier was assessed by a rise of transepithelial electrical resistance (TER) across the Sertoli cell epithelium which blocked the conductivity of current across the barrier. A considerable rise of actin nucleation protein Spire 1 expression was noted during the initial phase of TJ-barrier assembly which became plateau when the barrier was established by ~day 3. d Staining of Sertoli cells with anti-Spire1 antibody (red fluorescence) to illustrate cellular localization of Spire 1 which was shown to partially co-localize with F-actin (visualized by Alexa Fluor 488 phalloidin, green fluorescence) in Sertoli cell cytosol. Sertoli cell nuclei were visualized by DAPI (blue). Scale bar, 40 µm. e Stage-specific expression of Spire1 (red fluorescence) and its co-localization with F-actin (green fluorescence) in the seminiferous epithelium of adult rat testes. Cell nuclei were visualized by DAPI (blue). Spire1, mostly expressed at the convex (dorsal) side of spermatid heads, was found to partially co-localize with F-actin at the apical ES (yellow boxes) and also at the basal ES/BTB (green boxes) near the basement membrane (annotated by dashed white line). Scale bar, 80 µm; inset, 40 µm

    Journal: Cell Death & Disease

    Article Title: Actin nucleator Spire 1 is a regulator of ectoplasmic specialization in the testis

    doi: 10.1038/s41419-017-0201-6

    Figure Lengend Snippet: Expression, and cellular and stage-specific localization of Spire1 in the rat testis. a A study by RT-PCR using corresponding primer pairs specific to different target genes (Table 3 ) was performed to illustrate the expression of Spire 1 in adult rat testes (T), Sertoli cells (SC), and germ cells (GC) vs. brain (BR; positive control) with S16 served as a loading control. Spire 2 was also expressed in T, GC and BR, but not SC under the conditions used in our experiments. M, DNA markers in base pairs (bp). The identity of the PCR products was confirmed by direct nucleotide sequencing. b Specificity of the anti-Spire 1 antibody (Table 1 ) was assessed by immunoblotting using lysates of adult rat testes (T) at 80 µg protein. c Changes in the relative steady-state protein level of Spire1 expressed by Sertoli cells (left panel) were detected during the assembly of the Sertoli cell TJ-permeability barrier (right panel) when cultured in vitro for 6 days in serum-free F12/DMEM using Sertoli cell lysates (40 µg protein per lane) with β-actin as a protein loading control. Assembly of the TJ-barrier was assessed by a rise of transepithelial electrical resistance (TER) across the Sertoli cell epithelium which blocked the conductivity of current across the barrier. A considerable rise of actin nucleation protein Spire 1 expression was noted during the initial phase of TJ-barrier assembly which became plateau when the barrier was established by ~day 3. d Staining of Sertoli cells with anti-Spire1 antibody (red fluorescence) to illustrate cellular localization of Spire 1 which was shown to partially co-localize with F-actin (visualized by Alexa Fluor 488 phalloidin, green fluorescence) in Sertoli cell cytosol. Sertoli cell nuclei were visualized by DAPI (blue). Scale bar, 40 µm. e Stage-specific expression of Spire1 (red fluorescence) and its co-localization with F-actin (green fluorescence) in the seminiferous epithelium of adult rat testes. Cell nuclei were visualized by DAPI (blue). Spire1, mostly expressed at the convex (dorsal) side of spermatid heads, was found to partially co-localize with F-actin at the apical ES (yellow boxes) and also at the basal ES/BTB (green boxes) near the basement membrane (annotated by dashed white line). Scale bar, 80 µm; inset, 40 µm

    Article Snippet: BTB integrity assay in vivo The Sertoli cell BTB integrity in vivo was assessed by the ability of the Sertoli cell TJ/basal ES-barrier to block the diffusion of a membrane impermeable biotinylation reagent EZ-Link Sulfo-NHS-LC-biotin (Thermo Fisher Scientific, Waltham, MA) across the barrier as earlier described .

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction, Sequencing, Permeability, Cell Culture, In Vitro, Staining, Fluorescence

    Spire1 is a component of apical and basal ES in the seminiferous epithelium of adult rat testes. a Dual-labeled immunofluorescence (IF) analysis of Spire1 (red fluorescence) with two ES regulatory proteins Arp3 (green fluorescence) and Eps8 (green fluorescence) at stage VII to early VIII tubules. Since Arp3 and Eps8 were mostly expressed at the concave side of spermatid heads as bulb-like structures, only partial localization with Spire 1 were detected because Spire 1 only weakly expressed at this site but robustly expressed at the convex side of spermatid heads. Scale bar, 20 µm, applies to other micrographs. b Dual-labeled IF analysis showed almost superimposable co-localization of Spire 1 (red fluorescence) with apical ES proteins (green fluorescence) β1-integrin (Sertoli cell-specific), nectin 2 (expressed by both Sertoli cells and spermatids) and nectin 3 (elongated spermatid-specific) in stage VII to early VIII tubules since these apical ES proteins also expressed predominantly at the convex side of spermatid heads. Scale bar, 20 µm, applies to other micrographs. c Dual-labeled IF analysis illustrated partial co-localization of Spire 1 (red fluorescence) with basal ES/BTB proteins (green fluorescence) N-cadherin and γ-catenin at the BTB site. Scale bar, 20 µm, applies to other micrographs

    Journal: Cell Death & Disease

    Article Title: Actin nucleator Spire 1 is a regulator of ectoplasmic specialization in the testis

    doi: 10.1038/s41419-017-0201-6

    Figure Lengend Snippet: Spire1 is a component of apical and basal ES in the seminiferous epithelium of adult rat testes. a Dual-labeled immunofluorescence (IF) analysis of Spire1 (red fluorescence) with two ES regulatory proteins Arp3 (green fluorescence) and Eps8 (green fluorescence) at stage VII to early VIII tubules. Since Arp3 and Eps8 were mostly expressed at the concave side of spermatid heads as bulb-like structures, only partial localization with Spire 1 were detected because Spire 1 only weakly expressed at this site but robustly expressed at the convex side of spermatid heads. Scale bar, 20 µm, applies to other micrographs. b Dual-labeled IF analysis showed almost superimposable co-localization of Spire 1 (red fluorescence) with apical ES proteins (green fluorescence) β1-integrin (Sertoli cell-specific), nectin 2 (expressed by both Sertoli cells and spermatids) and nectin 3 (elongated spermatid-specific) in stage VII to early VIII tubules since these apical ES proteins also expressed predominantly at the convex side of spermatid heads. Scale bar, 20 µm, applies to other micrographs. c Dual-labeled IF analysis illustrated partial co-localization of Spire 1 (red fluorescence) with basal ES/BTB proteins (green fluorescence) N-cadherin and γ-catenin at the BTB site. Scale bar, 20 µm, applies to other micrographs

    Article Snippet: BTB integrity assay in vivo The Sertoli cell BTB integrity in vivo was assessed by the ability of the Sertoli cell TJ/basal ES-barrier to block the diffusion of a membrane impermeable biotinylation reagent EZ-Link Sulfo-NHS-LC-biotin (Thermo Fisher Scientific, Waltham, MA) across the barrier as earlier described .

    Techniques: Labeling, Immunofluorescence, Fluorescence

    Knockdown of Spire 1 in Sertoli cells perturbs F-actin organization through disruptive changes in actin polymerization and bundling activity. a Knockdown of Spire 1 in Sertoli cells by RNAi was found to induce disruptive changes in the organization of actin microfilaments across Sertoli cell cytosol. These included truncation of actin filaments so that they no longer stretched across the cell cytosol as noted in control cells to support the Sertoli cell TJ-permeability barrier function as noted in Fig. 3d . These changes are supported by disruptive changes in spatial organization of actin bundling protein palladin and linear actin polymerization protein formin 1, which no longer stretched across the entire cell cytosol following Spire 1 knockdown as found in control cells, but retracted to localize closer to the cell nuclei (yellow arrowheads). Branched actin polymerization protein Arp3 and actin barbed-end capping and bundling protein Eps8 also no longer prominently expressed at the Sertoli cell–cell interface. Instead, Apr3 and Eps8 were internalized into cell cytosol, disrupting the ability to confer plasticity to the actin microfilaments near the cell cortical zone to support basal ES function to tighten the TJ-barrier. Co-transfection of rhodamine-siGLO indicator (red fluorescence) indicated successful transfection. Scale bar, 40 µm, which applies to other micrographs. b Actin spin-down assay was performed as detailed in Materials and Methods, which separated filamentous (F, in pellet) actin from globular (G, in supernatant (S/N)) actin in cell lysates from Sertoli cell cultures after Spire1 knockdown. As noted herein (bar graph in lower panel), Spire1 knockdown by ~ 70% reduced the level of F-actin in Sertoli cells considerably. GAPDH served as a protein loading control. Phalloidin (0.1 µM) and urea (80 mM) included in the Sertoli cell lysates for the assay served as the corresponding positive (+ve) and negative (−ve) control. Each bar in the histogram is a mean ± s.d. of n = 3 independent experiments. ** P

    Journal: Cell Death & Disease

    Article Title: Actin nucleator Spire 1 is a regulator of ectoplasmic specialization in the testis

    doi: 10.1038/s41419-017-0201-6

    Figure Lengend Snippet: Knockdown of Spire 1 in Sertoli cells perturbs F-actin organization through disruptive changes in actin polymerization and bundling activity. a Knockdown of Spire 1 in Sertoli cells by RNAi was found to induce disruptive changes in the organization of actin microfilaments across Sertoli cell cytosol. These included truncation of actin filaments so that they no longer stretched across the cell cytosol as noted in control cells to support the Sertoli cell TJ-permeability barrier function as noted in Fig. 3d . These changes are supported by disruptive changes in spatial organization of actin bundling protein palladin and linear actin polymerization protein formin 1, which no longer stretched across the entire cell cytosol following Spire 1 knockdown as found in control cells, but retracted to localize closer to the cell nuclei (yellow arrowheads). Branched actin polymerization protein Arp3 and actin barbed-end capping and bundling protein Eps8 also no longer prominently expressed at the Sertoli cell–cell interface. Instead, Apr3 and Eps8 were internalized into cell cytosol, disrupting the ability to confer plasticity to the actin microfilaments near the cell cortical zone to support basal ES function to tighten the TJ-barrier. Co-transfection of rhodamine-siGLO indicator (red fluorescence) indicated successful transfection. Scale bar, 40 µm, which applies to other micrographs. b Actin spin-down assay was performed as detailed in Materials and Methods, which separated filamentous (F, in pellet) actin from globular (G, in supernatant (S/N)) actin in cell lysates from Sertoli cell cultures after Spire1 knockdown. As noted herein (bar graph in lower panel), Spire1 knockdown by ~ 70% reduced the level of F-actin in Sertoli cells considerably. GAPDH served as a protein loading control. Phalloidin (0.1 µM) and urea (80 mM) included in the Sertoli cell lysates for the assay served as the corresponding positive (+ve) and negative (−ve) control. Each bar in the histogram is a mean ± s.d. of n = 3 independent experiments. ** P

    Article Snippet: BTB integrity assay in vivo The Sertoli cell BTB integrity in vivo was assessed by the ability of the Sertoli cell TJ/basal ES-barrier to block the diffusion of a membrane impermeable biotinylation reagent EZ-Link Sulfo-NHS-LC-biotin (Thermo Fisher Scientific, Waltham, MA) across the barrier as earlier described .

    Techniques: Activity Assay, Permeability, Cotransfection, Fluorescence, Transfection, Spin Down Assay

    Knockdown of Spire 1 in Sertoli cell epithelium perturbs the TJ-permeability barrier function through disruptive distribution of TJ- and basal ES-proteins at the Sertoli cell–cell interface. a Illustration of the treatment regimen to obtain Sertoli cells for qPCR, IB or IF and TER analysis. b Sertoli cells harvested on day 5 were used to obtain cell lysates for IB (20 µg protein per lane) with corresponding specific antibodies listed in Table 1 , illustrating Spire1 expression was considerably reduced. However, virtually all other BTB-associated proteins were not affected following Spire 1 knockdown, illustrating that there were no apparent off-target effects by using the Spire 1-specific siRNA duplexes vs. the non-targeting negative control siRNA duplexes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a protein loading control. c qPCR (left panel) and IB (right panel) was performed using RNAs or protein lysates obtained from Sertoli cells harvested on day 4 or 5, respectively, illustrating the expression of Spire 1 was reduced by ~70% when examined by qPCR or IB. Each bar in the histogram is a mean ± s.d. of n = 3 independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: Actin nucleator Spire 1 is a regulator of ectoplasmic specialization in the testis

    doi: 10.1038/s41419-017-0201-6

    Figure Lengend Snippet: Knockdown of Spire 1 in Sertoli cell epithelium perturbs the TJ-permeability barrier function through disruptive distribution of TJ- and basal ES-proteins at the Sertoli cell–cell interface. a Illustration of the treatment regimen to obtain Sertoli cells for qPCR, IB or IF and TER analysis. b Sertoli cells harvested on day 5 were used to obtain cell lysates for IB (20 µg protein per lane) with corresponding specific antibodies listed in Table 1 , illustrating Spire1 expression was considerably reduced. However, virtually all other BTB-associated proteins were not affected following Spire 1 knockdown, illustrating that there were no apparent off-target effects by using the Spire 1-specific siRNA duplexes vs. the non-targeting negative control siRNA duplexes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a protein loading control. c qPCR (left panel) and IB (right panel) was performed using RNAs or protein lysates obtained from Sertoli cells harvested on day 4 or 5, respectively, illustrating the expression of Spire 1 was reduced by ~70% when examined by qPCR or IB. Each bar in the histogram is a mean ± s.d. of n = 3 independent experiments. * P

    Article Snippet: BTB integrity assay in vivo The Sertoli cell BTB integrity in vivo was assessed by the ability of the Sertoli cell TJ/basal ES-barrier to block the diffusion of a membrane impermeable biotinylation reagent EZ-Link Sulfo-NHS-LC-biotin (Thermo Fisher Scientific, Waltham, MA) across the barrier as earlier described .

    Techniques: Permeability, Real-time Polymerase Chain Reaction, Expressing, Negative Control

    RNA-seq and connectivity Map (CMAP) analysis of differentially expressed genes (DEG) in Fmr1 knockout (KO) hippocampal neuron. ( a ) Gene expression differences between wild type (WT) and Fmr1 KO samples. There are 587 up-regulated and 724 down-regulated genes in Fmr1 KO hippocampal neuron. ( b ) Top 15 GO biology processes that are associated with DEG between WT and Fmr1 KO samples. ( c ) KEGG pathways that are associated with DEG between WT and Fmr1 KO samples. ( d ) Top-ranked 10 CMAP compounds/drugs that induce transcriptome alterations oppositional to (indicated by negative similarity mean) or overlapping with (indicated by positive similarity mean) that caused by Fmr1 deficiency. Rank is determined by P value and enrichment score. Drug name and cell line indicate the name of compound used for treatment with specific cell lines in CMAP database. Full information of P value, enrichment score, and similarity mean is shown in Supplementary Table 4.

    Journal: bioRxiv

    Article Title: Computational analysis of transcriptome signature repurposes low dose trifluoperazine for the treatment of fragile X syndrome in mouse model

    doi: 10.1101/683169

    Figure Lengend Snippet: RNA-seq and connectivity Map (CMAP) analysis of differentially expressed genes (DEG) in Fmr1 knockout (KO) hippocampal neuron. ( a ) Gene expression differences between wild type (WT) and Fmr1 KO samples. There are 587 up-regulated and 724 down-regulated genes in Fmr1 KO hippocampal neuron. ( b ) Top 15 GO biology processes that are associated with DEG between WT and Fmr1 KO samples. ( c ) KEGG pathways that are associated with DEG between WT and Fmr1 KO samples. ( d ) Top-ranked 10 CMAP compounds/drugs that induce transcriptome alterations oppositional to (indicated by negative similarity mean) or overlapping with (indicated by positive similarity mean) that caused by Fmr1 deficiency. Rank is determined by P value and enrichment score. Drug name and cell line indicate the name of compound used for treatment with specific cell lines in CMAP database. Full information of P value, enrichment score, and similarity mean is shown in Supplementary Table 4.

    Article Snippet: Transcriptome analysis of gene expression in Fmr1 KO neurons Whole genome transcript changes were determined by RNA-seq with total RNA extracted from primary hippocampal cultures at DIV (days in vitro) 14.

    Techniques: RNA Sequencing Assay, Knock-Out, Expressing

    Nanopore DRS with cap-dependent ligation of 5′ adapter RNA. ( A ) Histogram showing the distribution of 5′ adapter RNA length in the nanopore raw current signal, as inferred from alignment of the mRNA sequence to the signal using nanopolish eventalign. The median signal length was 1441 points and 96% of adapter signals were 3000 points or less. ( B ) Out-of-bag receiver operator characteristic curve showing the performance of the trained convolutional neural network at detecting 5′ adapter RNA using 3000 points of signal. The curve was generated using five-fold cross validation. ( C ) Out-of-bag precision recall curve showing the performance of trained neural network, generated using five-fold cross validation. ( D ) Alternative transcription start sites were identified using nanopore DRS with cap-dependent ligation of a 5′ end adapter at the AT1G17050 and AT5G18650 genes. Orange, 5’ coverage for capped nanoPARE reads; blue track, nanopore DRS coverage with cap-dependent ligation of 5′ adapter RNA; blue, isoforms detected by nanopore DRS with cap-dependent ligation of 5′ adapter RNA; black, Araport11 annotation. ( E ) Reads mapping to ERCC RNA spike-ins lack approximately 11 nt of sequence at the 5′ end. Histogram showing the distance to the 5′ end for ERCC RNA spike-in reads (each spike-in is shown in a different colour; only those with > 1000 supporting reads are shown). ( F ) Reads mapping to in vitro transcribed mGFP lack approximately 11 nt of sequence at the 5′ end. Histogram showing the distance to the 5′ end for in vitro transcribed mGFP. ( G ) Araport11 annotation overestimates the length of 5′ UTRs. The cumulative distribution function shows the distance to the nearest TSS identified from full-length transcripts cloned as part of the RIKEN RAFL project (blue) and Araport11 annotation (orange). ( H ) Nanopore DRS detects miR170/miR171 cleavage products of Hairy Meristem 1 (HAM1, AT2G45160) transcripts. Orange, 5’ coverage from capped nanoPARE reads; purple, 5’ coverage from uncapped nanoPARE reads; blue, nanopore DRS 5’ coverage; grey, miRNA target site alignment is shown; black, Araport11 annotation. microRNA cleavage site predictions supported by enrichment of nanopore 5’ ends – Figure 3—figure supplement 1H .

    Journal: eLife

    Article Title: Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m6A modification

    doi: 10.7554/eLife.49658

    Figure Lengend Snippet: Nanopore DRS with cap-dependent ligation of 5′ adapter RNA. ( A ) Histogram showing the distribution of 5′ adapter RNA length in the nanopore raw current signal, as inferred from alignment of the mRNA sequence to the signal using nanopolish eventalign. The median signal length was 1441 points and 96% of adapter signals were 3000 points or less. ( B ) Out-of-bag receiver operator characteristic curve showing the performance of the trained convolutional neural network at detecting 5′ adapter RNA using 3000 points of signal. The curve was generated using five-fold cross validation. ( C ) Out-of-bag precision recall curve showing the performance of trained neural network, generated using five-fold cross validation. ( D ) Alternative transcription start sites were identified using nanopore DRS with cap-dependent ligation of a 5′ end adapter at the AT1G17050 and AT5G18650 genes. Orange, 5’ coverage for capped nanoPARE reads; blue track, nanopore DRS coverage with cap-dependent ligation of 5′ adapter RNA; blue, isoforms detected by nanopore DRS with cap-dependent ligation of 5′ adapter RNA; black, Araport11 annotation. ( E ) Reads mapping to ERCC RNA spike-ins lack approximately 11 nt of sequence at the 5′ end. Histogram showing the distance to the 5′ end for ERCC RNA spike-in reads (each spike-in is shown in a different colour; only those with > 1000 supporting reads are shown). ( F ) Reads mapping to in vitro transcribed mGFP lack approximately 11 nt of sequence at the 5′ end. Histogram showing the distance to the 5′ end for in vitro transcribed mGFP. ( G ) Araport11 annotation overestimates the length of 5′ UTRs. The cumulative distribution function shows the distance to the nearest TSS identified from full-length transcripts cloned as part of the RIKEN RAFL project (blue) and Araport11 annotation (orange). ( H ) Nanopore DRS detects miR170/miR171 cleavage products of Hairy Meristem 1 (HAM1, AT2G45160) transcripts. Orange, 5’ coverage from capped nanoPARE reads; purple, 5’ coverage from uncapped nanoPARE reads; blue, nanopore DRS 5’ coverage; grey, miRNA target site alignment is shown; black, Araport11 annotation. microRNA cleavage site predictions supported by enrichment of nanopore 5’ ends – Figure 3—figure supplement 1H .

    Article Snippet: Nanopore libraries were prepared from 1 μg poly(A)+ RNA combined with 1 μl undiluted ERCC RNA Spike-In mix (Thermo Fisher Scientific) using the nanopore DRS Kit (SQK-RNA001, Oxford Nanopore Technologies) according to manufacturer’s instructions.

    Techniques: Ligation, Sequencing, Generated, In Vitro, Clone Assay

    Properties of nanopore DRS sequencing data. ( A ) Nanopore DRS identified a 12.8 kb transcript generated from the AT1G67120 gene that includes 58 exons. Blue, nanopore DRS isoform; black, Araport11 annotation. ( B ) Synthetic ERCC RNA spike-in mixes are detected in a quantitative manner. Absolute concentrations of spike-ins are plotted against counts per million (CPM) reads in log 10 scale. Blue, ERCC RNA spike-in mix 1; orange, ERCC RNA spike-in mix 2. ( C ) Overview of the sequencing and alignment characteristics of nanopore DRS data for ERCC RNA spike-ins. Left, distribution of the length fraction of each sequenced read that aligns to the ERCC RNA spike-in reference; centre, distribution of fraction of identity that matches between the sequence of the read and the ERCC RNA spike-in reference for the aligned portion of each read; right, distributions of the occurrence of insertions (black), substitutions (orange) and deletions (blue) as a proportion of the number of aligned bases in each read. ( D ) Substitution preference for each nucleotide (left to right: adenine [A], uracil [U], guanine [G], cytosine [C]). When substituted, G is replaced with A in more than 63% of its substitutions, while C is replaced by U in 73%. Conversely, U is rarely replaced with G (12%) and A is rarely substituted with C (16%). ( E ) Nucleotide representation within the ERCC RNA Spike-In reference sequences (black dots) compared with nucleotide representation within four categories from the nanopore DRS reads. Identity matches between the sequence of the read and the ERCC RNA spike-in reference (green crosses), insertions (blue pentagons), deletions (yellow stars) and substitutions (purple diamonds).G is under-represented and U is over-represented for all three categories of error (insertion, deletion and substitution) relative to the reference nucleotide distribution. C is over-represented in deletions and substitutions. A is over-represented in insertions and deletions and under-represented in substitutions. ( F ) Signals originating from the RH3 transcripts are susceptible to systematic over-splitting around exons 7–9 (highlighted using a purple dashed box), resulting in reads with apparently novel 5′ or 3′ positions. This appears only to occur at high frequency in datasets collected after May 2018 ( Supplementary file 1 ) and may result from an update to the MinKNOW software. ( G ) PIN7 antisense RNAs detected using nanopore DRS. Blue, Col-0 PIN7 sense Illumina RNAseq coverage and nanopore PIN7 sense read alignments; orange, Col-0 PIN7 antisense Illumina RNAseq coverage and nanopore PIN7 antisense read alignments; green, hen2–two mutant PIN7 sense Illumina RNAseq coverage; purple, hen2–two mutant PIN7 antisense Illumina RNAseq coverage; black, PIN7 sense RNA isoforms found in Araport11 annotation; grey, PIN7 antisense differentially expressed regions detected with DERfinder.

    Journal: eLife

    Article Title: Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m6A modification

    doi: 10.7554/eLife.49658

    Figure Lengend Snippet: Properties of nanopore DRS sequencing data. ( A ) Nanopore DRS identified a 12.8 kb transcript generated from the AT1G67120 gene that includes 58 exons. Blue, nanopore DRS isoform; black, Araport11 annotation. ( B ) Synthetic ERCC RNA spike-in mixes are detected in a quantitative manner. Absolute concentrations of spike-ins are plotted against counts per million (CPM) reads in log 10 scale. Blue, ERCC RNA spike-in mix 1; orange, ERCC RNA spike-in mix 2. ( C ) Overview of the sequencing and alignment characteristics of nanopore DRS data for ERCC RNA spike-ins. Left, distribution of the length fraction of each sequenced read that aligns to the ERCC RNA spike-in reference; centre, distribution of fraction of identity that matches between the sequence of the read and the ERCC RNA spike-in reference for the aligned portion of each read; right, distributions of the occurrence of insertions (black), substitutions (orange) and deletions (blue) as a proportion of the number of aligned bases in each read. ( D ) Substitution preference for each nucleotide (left to right: adenine [A], uracil [U], guanine [G], cytosine [C]). When substituted, G is replaced with A in more than 63% of its substitutions, while C is replaced by U in 73%. Conversely, U is rarely replaced with G (12%) and A is rarely substituted with C (16%). ( E ) Nucleotide representation within the ERCC RNA Spike-In reference sequences (black dots) compared with nucleotide representation within four categories from the nanopore DRS reads. Identity matches between the sequence of the read and the ERCC RNA spike-in reference (green crosses), insertions (blue pentagons), deletions (yellow stars) and substitutions (purple diamonds).G is under-represented and U is over-represented for all three categories of error (insertion, deletion and substitution) relative to the reference nucleotide distribution. C is over-represented in deletions and substitutions. A is over-represented in insertions and deletions and under-represented in substitutions. ( F ) Signals originating from the RH3 transcripts are susceptible to systematic over-splitting around exons 7–9 (highlighted using a purple dashed box), resulting in reads with apparently novel 5′ or 3′ positions. This appears only to occur at high frequency in datasets collected after May 2018 ( Supplementary file 1 ) and may result from an update to the MinKNOW software. ( G ) PIN7 antisense RNAs detected using nanopore DRS. Blue, Col-0 PIN7 sense Illumina RNAseq coverage and nanopore PIN7 sense read alignments; orange, Col-0 PIN7 antisense Illumina RNAseq coverage and nanopore PIN7 antisense read alignments; green, hen2–two mutant PIN7 sense Illumina RNAseq coverage; purple, hen2–two mutant PIN7 antisense Illumina RNAseq coverage; black, PIN7 sense RNA isoforms found in Araport11 annotation; grey, PIN7 antisense differentially expressed regions detected with DERfinder.

    Article Snippet: Nanopore libraries were prepared from 1 μg poly(A)+ RNA combined with 1 μl undiluted ERCC RNA Spike-In mix (Thermo Fisher Scientific) using the nanopore DRS Kit (SQK-RNA001, Oxford Nanopore Technologies) according to manufacturer’s instructions.

    Techniques: Sequencing, Generated, Software, Mutagenesis

    3′ end processing is revealed by nanopore DRS. ( A ) Poly(A) tail length estimates for ERCC spike-in controls. Boxplots showing distribution of poly(A) length estimates for ERCC spike-in controls with more than 100 mapped reads for which tail length could be successfully estimated. Expected poly(A) tail lengths are shown as orange points. ( B ) The RNA poly(A) tail length negatively correlates with the gene expression level. Expression in log 2 scale of counts per million (CPM) obtained from nanopore DRS data is plotted against the median poly(A) tail length. ρ, Spearman’s correlation coefficient; black line, locally weighted scatterplot smoothing (LOWESS) regression fit. ( C ) Nanopore DRS identified known 3′ polyadenylation sites in RNAs transcribed from the IBM1 (AT3G07610) locus. Blue track, nanopore DRS coverage; blue, isoforms detected by nanopore DRS; black, Araport11 annotation; PAS, proximal polyadenylation site. ( D ) Nanopore DRS identified novel 3’ polyadenylation sites in RNAs transcribed from the PTM (AT5G35210) locus. Black (on the top), Helicos DRS 3’ coverage; blue track, nanopore DRS coverage; blue, isoforms detected by nanopore DRS; black (on the bottom), Araport11 annotation; green, five annotated transmembrane domain regions from Uniprot entry F4JYC8 (PTM_ARATH) that mapped to exons; pPAS, proximal polyadenylation site; dPAS, distal polyadenylation sites. Poly(A) tail length estimations generated from ERCC spike-in reads – Figure 2—figure supplement 1A . Per gene poly(A) tail length estimate distributions generated from Col-0 reads - Figure 2—figure supplement 1B .

    Journal: eLife

    Article Title: Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m6A modification

    doi: 10.7554/eLife.49658

    Figure Lengend Snippet: 3′ end processing is revealed by nanopore DRS. ( A ) Poly(A) tail length estimates for ERCC spike-in controls. Boxplots showing distribution of poly(A) length estimates for ERCC spike-in controls with more than 100 mapped reads for which tail length could be successfully estimated. Expected poly(A) tail lengths are shown as orange points. ( B ) The RNA poly(A) tail length negatively correlates with the gene expression level. Expression in log 2 scale of counts per million (CPM) obtained from nanopore DRS data is plotted against the median poly(A) tail length. ρ, Spearman’s correlation coefficient; black line, locally weighted scatterplot smoothing (LOWESS) regression fit. ( C ) Nanopore DRS identified known 3′ polyadenylation sites in RNAs transcribed from the IBM1 (AT3G07610) locus. Blue track, nanopore DRS coverage; blue, isoforms detected by nanopore DRS; black, Araport11 annotation; PAS, proximal polyadenylation site. ( D ) Nanopore DRS identified novel 3’ polyadenylation sites in RNAs transcribed from the PTM (AT5G35210) locus. Black (on the top), Helicos DRS 3’ coverage; blue track, nanopore DRS coverage; blue, isoforms detected by nanopore DRS; black (on the bottom), Araport11 annotation; green, five annotated transmembrane domain regions from Uniprot entry F4JYC8 (PTM_ARATH) that mapped to exons; pPAS, proximal polyadenylation site; dPAS, distal polyadenylation sites. Poly(A) tail length estimations generated from ERCC spike-in reads – Figure 2—figure supplement 1A . Per gene poly(A) tail length estimate distributions generated from Col-0 reads - Figure 2—figure supplement 1B .

    Article Snippet: Nanopore libraries were prepared from 1 μg poly(A)+ RNA combined with 1 μl undiluted ERCC RNA Spike-In mix (Thermo Fisher Scientific) using the nanopore DRS Kit (SQK-RNA001, Oxford Nanopore Technologies) according to manufacturer’s instructions.

    Techniques: Expressing, Generated