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

    1) Product Images from "Hemodynamic forces sculpt developing heart valves through a KLF2-WNT9B paracrine signaling axis"

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

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2017.09.023

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

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

    2) Product Images from "Programmable RNA recognition and cleavage by CRISPR/Cas9"

    Article Title: Programmable RNA recognition and cleavage by CRISPR/Cas9

    Journal: Nature

    doi: 10.1038/nature13769

    RNA-guided Cas9 can target non-PAM sites on ssRNA and isolate GAPDH mRNA from HeLa cells in a tagless manner a ,Schematic of the approach designed to avoid cleavage of template DNA by targeting non-PAM sites in the ssRNA target. b , The panel of nucleic acid substrates tested in ( c ). c , Cas9–gRNA cleaves ssRNA targets with equal efficiency when the 5’-NGG-3’ of the PAMmer is mismatched with the ssRNA. This strategy enables selective cleavage of ssRNA in the presence of non-PAM target dsDNA. d , Schematic of the dCas9 RNA pull-down experiment. e , GAPDH mRNA transcript isoform 3 shown schematically, with exons common to all GAPDH protein-coding transcripts in red and gRNA/PAMmer targets G 1- G 4 indicated. f , Northern blot showing that gRNAs and 5’-extended PAMmers enable tagless isolation of GAPDH mRNA from HeLa total RNA; β-actin mRNA is shown as a control. g , Northern blot showing tagless isolation of GAPDH mRNA from HeLa cell lysate with varying 2’-OMe-modified PAMmers. RNase H cleavage is abrogated with v4 and v5 PAMmers; β-actin mRNA is shown as a control. h , Sequences of unmodified and modified GAPDH PAMmers used in (g) ; 2’-OMe-modified nucleotides are shown in red.
    Figure Legend Snippet: RNA-guided Cas9 can target non-PAM sites on ssRNA and isolate GAPDH mRNA from HeLa cells in a tagless manner a ,Schematic of the approach designed to avoid cleavage of template DNA by targeting non-PAM sites in the ssRNA target. b , The panel of nucleic acid substrates tested in ( c ). c , Cas9–gRNA cleaves ssRNA targets with equal efficiency when the 5’-NGG-3’ of the PAMmer is mismatched with the ssRNA. This strategy enables selective cleavage of ssRNA in the presence of non-PAM target dsDNA. d , Schematic of the dCas9 RNA pull-down experiment. e , GAPDH mRNA transcript isoform 3 shown schematically, with exons common to all GAPDH protein-coding transcripts in red and gRNA/PAMmer targets G 1- G 4 indicated. f , Northern blot showing that gRNAs and 5’-extended PAMmers enable tagless isolation of GAPDH mRNA from HeLa total RNA; β-actin mRNA is shown as a control. g , Northern blot showing tagless isolation of GAPDH mRNA from HeLa cell lysate with varying 2’-OMe-modified PAMmers. RNase H cleavage is abrogated with v4 and v5 PAMmers; β-actin mRNA is shown as a control. h , Sequences of unmodified and modified GAPDH PAMmers used in (g) ; 2’-OMe-modified nucleotides are shown in red.

    Techniques Used: Northern Blot, Isolation, Modification

    Cas9 programmed with GAPDH-specific gRNAs can pull-down GAPDH mRNA in the absence of PAMmer a , Northern blot showing that, in some cases, Cas9-gRNA is able to pull down detectable amounts of GAPDH mRNA from total RNA without requiring a PAMmer. a , Northern blot showing that Cas9-gRNA 1 is also able to pull-down quantitative amounts of GAPDH mRNA from HeLa cell lysate without requiring a PAMmer. s: standard; v: 2’-OMe-modified PAMmers.
    Figure Legend Snippet: Cas9 programmed with GAPDH-specific gRNAs can pull-down GAPDH mRNA in the absence of PAMmer a , Northern blot showing that, in some cases, Cas9-gRNA is able to pull down detectable amounts of GAPDH mRNA from total RNA without requiring a PAMmer. a , Northern blot showing that Cas9-gRNA 1 is also able to pull-down quantitative amounts of GAPDH mRNA from HeLa cell lysate without requiring a PAMmer. s: standard; v: 2’-OMe-modified PAMmers.

    Techniques Used: Northern Blot, Modification

    3) Product Images from "Molecular Analysis of the Multiple GroEL Proteins of Chlamydiae"

    Article Title: Molecular Analysis of the Multiple GroEL Proteins of Chlamydiae

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.185.6.1958-1966.2003

    (A) The top panel shows control PCR amplification of groEL1 , groEL2 , and groEL3 with serovar D DNA as the template as a measure to compare primer efficiency. The bottom panel shows an RT-PCR analysis of total RNA from HeLa cells infected with C. trachomatis serovar D isolated at 12 h after infection. (B) The top panel shows a Western blot analysis of C. trachomatis serovar D GroEL1, GroEL2, and GroEL3. Boiled samples of purified serovar D C. trachomatis elementary bodies were subjected to SDS-7.5% polyacrylamide gel electrophoresis, and the blotted nitrocellulose membranes were incubated with polyclonal anti-GroEL1 (first lane), anti-GroEL2 (middle lane), or anti-GroEL3 (third lane) antibodies, followed by incubation with alkaline phosphatase-conjugated secondary antibodies and color detection. In the bottom panel is shown a quantification of GroEL1, GroEL2, and GroEL3 by using the procedure described above except that 1-, 10-, and 100-ng portions of recombinant proteins were loaded per lane.
    Figure Legend Snippet: (A) The top panel shows control PCR amplification of groEL1 , groEL2 , and groEL3 with serovar D DNA as the template as a measure to compare primer efficiency. The bottom panel shows an RT-PCR analysis of total RNA from HeLa cells infected with C. trachomatis serovar D isolated at 12 h after infection. (B) The top panel shows a Western blot analysis of C. trachomatis serovar D GroEL1, GroEL2, and GroEL3. Boiled samples of purified serovar D C. trachomatis elementary bodies were subjected to SDS-7.5% polyacrylamide gel electrophoresis, and the blotted nitrocellulose membranes were incubated with polyclonal anti-GroEL1 (first lane), anti-GroEL2 (middle lane), or anti-GroEL3 (third lane) antibodies, followed by incubation with alkaline phosphatase-conjugated secondary antibodies and color detection. In the bottom panel is shown a quantification of GroEL1, GroEL2, and GroEL3 by using the procedure described above except that 1-, 10-, and 100-ng portions of recombinant proteins were loaded per lane.

    Techniques Used: Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, Infection, Isolation, Western Blot, Purification, Polyacrylamide Gel Electrophoresis, Incubation, Recombinant

    Quantification of C. trachomatis serovar D groEL1 , groEL2 , and groEL3 expression after heat shock by using microarray. ( groES and dnaK genes were included for comparison purposes). Heat-shocked and non-heat-shocked RNA samples were reverse transcribed, labeled with Cy3 or Cy5, and hybridized to the microarray. The fluorescence intensity was measured for each spot and then normalized to the average fluorescence intensity for the entire microarray. Data analysis was performed with the GeneSpring software (see Materials and Methods for more details).
    Figure Legend Snippet: Quantification of C. trachomatis serovar D groEL1 , groEL2 , and groEL3 expression after heat shock by using microarray. ( groES and dnaK genes were included for comparison purposes). Heat-shocked and non-heat-shocked RNA samples were reverse transcribed, labeled with Cy3 or Cy5, and hybridized to the microarray. The fluorescence intensity was measured for each spot and then normalized to the average fluorescence intensity for the entire microarray. Data analysis was performed with the GeneSpring software (see Materials and Methods for more details).

    Techniques Used: Expressing, Microarray, Labeling, Fluorescence, Software

    4) Product Images from "Expression profiling of cell-intrinsic regulators in the process of differentiation of human iPSCs into retinal lineages"

    Article Title: Expression profiling of cell-intrinsic regulators in the process of differentiation of human iPSCs into retinal lineages

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-018-0848-7

    Differentially expressed components of epigenetic complexes. a Hierarchical clustering of genes encoding components of PcG complexes. b Hierarchical clustering of genes encoding components of COMPASS/MLL complexes. c Hierarchical clustering of genes encoding components of SWI/SNF complexes. d qRT-PCR validation of expression patterns of ACTL6B , PCGF5 and CECR2 genes using same RNA as used for microarray (left) and RNA obtained from independent set of cells (right) that included two stages of differentiation of RGCs (9 and 17 days post OV), hiPSC-derived RPE cells (RPE) and RPE cell line ARPE-19. FC fold-change, OV optic vesicle, RGC retinal ganglion cell, RPE retinal pigment epithelium
    Figure Legend Snippet: Differentially expressed components of epigenetic complexes. a Hierarchical clustering of genes encoding components of PcG complexes. b Hierarchical clustering of genes encoding components of COMPASS/MLL complexes. c Hierarchical clustering of genes encoding components of SWI/SNF complexes. d qRT-PCR validation of expression patterns of ACTL6B , PCGF5 and CECR2 genes using same RNA as used for microarray (left) and RNA obtained from independent set of cells (right) that included two stages of differentiation of RGCs (9 and 17 days post OV), hiPSC-derived RPE cells (RPE) and RPE cell line ARPE-19. FC fold-change, OV optic vesicle, RGC retinal ganglion cell, RPE retinal pigment epithelium

    Techniques Used: Quantitative RT-PCR, Expressing, Microarray, Derivative Assay

    Validation of microarray results by qRT-PCR and western blot analysis. a qRT-PCR testing expression of selected TF-encoding mRNAs using the same total RNA as analyzed by microarray. Data for OVs, RGCs and RPE cells presented as fold-changes relative to mRNA level in hiPSC, standard deviation between replicates shown as error bars. b qRT-PCR testing expression of selected TF-encoding mRNAs using RNA obtained from independent set of cells: hiPSCs, RGCs after 9 days of differentiation from OVs (RGC 9), RGCs after 17 days of differentiation from OVs (RGC 17), hiPSC-derived RPE cells (RPE) and RPE cell line ARPE-19 (ARPE19). Data presented as fold-changes relative to hiPSC. c Western blot analysis demonstrating overexpression of EBF1 and EBF3 TFs in RGCs, but not in RPE cells and hiPSCs, similarly to well-known RGC markers SNCG and ISL1. iPSC induced pluripotent stem cell, RGC retinal ganglion cell, RPE retinal pigment epithelium
    Figure Legend Snippet: Validation of microarray results by qRT-PCR and western blot analysis. a qRT-PCR testing expression of selected TF-encoding mRNAs using the same total RNA as analyzed by microarray. Data for OVs, RGCs and RPE cells presented as fold-changes relative to mRNA level in hiPSC, standard deviation between replicates shown as error bars. b qRT-PCR testing expression of selected TF-encoding mRNAs using RNA obtained from independent set of cells: hiPSCs, RGCs after 9 days of differentiation from OVs (RGC 9), RGCs after 17 days of differentiation from OVs (RGC 17), hiPSC-derived RPE cells (RPE) and RPE cell line ARPE-19 (ARPE19). Data presented as fold-changes relative to hiPSC. c Western blot analysis demonstrating overexpression of EBF1 and EBF3 TFs in RGCs, but not in RPE cells and hiPSCs, similarly to well-known RGC markers SNCG and ISL1. iPSC induced pluripotent stem cell, RGC retinal ganglion cell, RPE retinal pigment epithelium

    Techniques Used: Microarray, Quantitative RT-PCR, Western Blot, Expressing, Standard Deviation, Derivative Assay, Over Expression

    5) Product Images from "Enriched Bone Marrow Derived Disseminated Neuroblastoma Cells Can Be a Reliable Source for Gene Expression Studies—A Validation Study"

    Article Title: Enriched Bone Marrow Derived Disseminated Neuroblastoma Cells Can Be a Reliable Source for Gene Expression Studies—A Validation Study

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0137995

    Experimental design. (a) We spiked LAN-1 NB cells into fresh PB and kept the samples for 0, 24, 48 and 72h at room temperature and, for the same time periods, at 4°C prior to density gradient separation. The LAN-1 cells were enriched from the MNC fraction with magnetic beads to a 99% purity of the tumor cell fractions prior to homogenization in TRIzol. RNA was isolated from all seven samples simultaneously and used for the qPCR array. ( b) LAN-1 cells were spiked into PB and tumor-free BM, and density gradient separation was immediately performed. The MNCs were frozen in 20% DMSO for seven days at -80°C. After thawing, the LAN-1 cells were either directly enriched by magnetic bead-based separation, or an additional density gradient separation (*) was performed prior to magnetic bead-based separation. The samples were homogenized in TRIzol and the isolated RNA was used for qPCR (in case of PB) and microarrays (in case of BM). (c) LAN-1 cells were spiked into PB and density gradient separation of MNCs was performed without delay, following two enrichment steps in a row. The > 99% LAN-1 cell fractions were homogenized in TRIzol and RNA was isolated from all samples simultaneously. qPCR arrays were performed in order to analyze the effect of enrichment on selected genes.
    Figure Legend Snippet: Experimental design. (a) We spiked LAN-1 NB cells into fresh PB and kept the samples for 0, 24, 48 and 72h at room temperature and, for the same time periods, at 4°C prior to density gradient separation. The LAN-1 cells were enriched from the MNC fraction with magnetic beads to a 99% purity of the tumor cell fractions prior to homogenization in TRIzol. RNA was isolated from all seven samples simultaneously and used for the qPCR array. ( b) LAN-1 cells were spiked into PB and tumor-free BM, and density gradient separation was immediately performed. The MNCs were frozen in 20% DMSO for seven days at -80°C. After thawing, the LAN-1 cells were either directly enriched by magnetic bead-based separation, or an additional density gradient separation (*) was performed prior to magnetic bead-based separation. The samples were homogenized in TRIzol and the isolated RNA was used for qPCR (in case of PB) and microarrays (in case of BM). (c) LAN-1 cells were spiked into PB and density gradient separation of MNCs was performed without delay, following two enrichment steps in a row. The > 99% LAN-1 cell fractions were homogenized in TRIzol and RNA was isolated from all samples simultaneously. qPCR arrays were performed in order to analyze the effect of enrichment on selected genes.

    Techniques Used: Magnetic Beads, Homogenization, Isolation, Real-time Polymerase Chain Reaction

    6) Product Images from "An alternative splicing switch in FLNB promotes the mesenchymal cell state in human breast cancer"

    Article Title: An alternative splicing switch in FLNB promotes the mesenchymal cell state in human breast cancer

    Journal: eLife

    doi: 10.7554/eLife.37184

    Identification of splicing targets regulated by QKI and RBFOX1. ( A ) Quantification of the different types of alternative splicing events regulated by QKI or RBFOX1 overexpression as determined using rMATS. Exclusion or inclusion are relative to control cells overexpressing EGFP. ( B ) RT-PCR validation of individual splicing events regulated by QKI or RBFOX1. The cDNA from cells expressing the indicated ORFs were subjected to PCR amplification using primers flanking the alternative exon. The ratios of the intensity of the upper (inclusion) and lower (exclusion) PCR product bands were quantified and the relative intensity of the upper band is indicated. Below are shown RNA-sequencing based quantification of the % inclusion of the alternative exon. n = 3 (EGFP, HcRed and RBFOX1) or n = 2 (QKI and SNAI1).
    Figure Legend Snippet: Identification of splicing targets regulated by QKI and RBFOX1. ( A ) Quantification of the different types of alternative splicing events regulated by QKI or RBFOX1 overexpression as determined using rMATS. Exclusion or inclusion are relative to control cells overexpressing EGFP. ( B ) RT-PCR validation of individual splicing events regulated by QKI or RBFOX1. The cDNA from cells expressing the indicated ORFs were subjected to PCR amplification using primers flanking the alternative exon. The ratios of the intensity of the upper (inclusion) and lower (exclusion) PCR product bands were quantified and the relative intensity of the upper band is indicated. Below are shown RNA-sequencing based quantification of the % inclusion of the alternative exon. n = 3 (EGFP, HcRed and RBFOX1) or n = 2 (QKI and SNAI1).

    Techniques Used: Over Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction, Amplification, RNA Sequencing Assay

    7) Product Images from "Determination of the Human Cardiomyocyte mRNA and miRNA Differentiation Network by Fine-Scale Profiling"

    Article Title: Determination of the Human Cardiomyocyte mRNA and miRNA Differentiation Network by Fine-Scale Profiling

    Journal: Stem Cells and Development

    doi: 10.1089/scd.2011.0357

    The differentiation time course from human-induced pluripotent stem cells (hiPSCs) to cardiomyocytes. Three independent differentiations were performed (Run 1, 2, and 3), and RNA was sampled at days 0, 3, 7, 10, 14, 20, 25, 35, 45, 60, 90, and 120 days.
    Figure Legend Snippet: The differentiation time course from human-induced pluripotent stem cells (hiPSCs) to cardiomyocytes. Three independent differentiations were performed (Run 1, 2, and 3), and RNA was sampled at days 0, 3, 7, 10, 14, 20, 25, 35, 45, 60, 90, and 120 days.

    Techniques Used:

    8) Product Images from "RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control in pluripotent stem cells"

    Article Title: RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control in pluripotent stem cells

    Journal: Nature genetics

    doi: 10.1038/s41588-018-0060-9

    PSPC1 mediates TET2 recruitment to chromatin through RNA a , (Top) Depiction of the wild-type (PSPC1WT) and RNA binding mutant (PSPC1Mut) PSPC1 protein structures. (Bottom) The maintained interactions of 3xFL TET2 with both Myc PSPC1WT and Myc PSPC1Mut by coimmunoprecipitation (CoIP). HEK293T cells were transiently transfected with constructs expressing 3xFL TET2 and Myc PSPC1 or Myc PSPC1Mut followed by IP with anti-FLAG and western blotting with the indicated antibodies. b , Reduction of chromatin-bound TET2 in Pspc1 KO lines relative to WT control. The relative levels of TET2, normalized to Histone 3 (H3) and WT controls, are indicated. c , Chromatin-bound 3xFL PSPC1WT and 3xFL PSPC1Mut proteins in the Pspc1 KO rescued cell lines. OCT4 is shown as a negative control. d , (Top) Depiction of the in vitro RNA-immunoprecipitation assay (iv-RIP) protocol. (Bottom) Agarose gel analysis of RNA bound by PSPC1 (IP: PSPC1) or TET2 (IP: TET2) protein complexes in Pspc1 WT and KO ESCs. Total RNA used for the IP was incubated with DNase I to ensure the complete absence of DNA contamination (0.1% input is shown on the right panel). e , TET2 chromatin binding is reduced upon the inhibition of transcription by α-Amanitin treatment. TET2 levels in chromatin were evaluated by nucleosome pulldown (IP: Histone H3) compared to total TET2 protein in whole cell extract (WCE) after transcriptional inhibition with α-Amanitin for 2 and 4 hours, compared to untreated cells (0 hours). Histone H3 was used as a loading control in b, c, and e. All images are representative of at least 2 independent experiments.
    Figure Legend Snippet: PSPC1 mediates TET2 recruitment to chromatin through RNA a , (Top) Depiction of the wild-type (PSPC1WT) and RNA binding mutant (PSPC1Mut) PSPC1 protein structures. (Bottom) The maintained interactions of 3xFL TET2 with both Myc PSPC1WT and Myc PSPC1Mut by coimmunoprecipitation (CoIP). HEK293T cells were transiently transfected with constructs expressing 3xFL TET2 and Myc PSPC1 or Myc PSPC1Mut followed by IP with anti-FLAG and western blotting with the indicated antibodies. b , Reduction of chromatin-bound TET2 in Pspc1 KO lines relative to WT control. The relative levels of TET2, normalized to Histone 3 (H3) and WT controls, are indicated. c , Chromatin-bound 3xFL PSPC1WT and 3xFL PSPC1Mut proteins in the Pspc1 KO rescued cell lines. OCT4 is shown as a negative control. d , (Top) Depiction of the in vitro RNA-immunoprecipitation assay (iv-RIP) protocol. (Bottom) Agarose gel analysis of RNA bound by PSPC1 (IP: PSPC1) or TET2 (IP: TET2) protein complexes in Pspc1 WT and KO ESCs. Total RNA used for the IP was incubated with DNase I to ensure the complete absence of DNA contamination (0.1% input is shown on the right panel). e , TET2 chromatin binding is reduced upon the inhibition of transcription by α-Amanitin treatment. TET2 levels in chromatin were evaluated by nucleosome pulldown (IP: Histone H3) compared to total TET2 protein in whole cell extract (WCE) after transcriptional inhibition with α-Amanitin for 2 and 4 hours, compared to untreated cells (0 hours). Histone H3 was used as a loading control in b, c, and e. All images are representative of at least 2 independent experiments.

    Techniques Used: RNA Binding Assay, Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Construct, Expressing, Western Blot, Negative Control, In Vitro, Immunoprecipitation, Agarose Gel Electrophoresis, Incubation, Binding Assay, Inhibition

    TET2 is recruited to chromatin by the RNA-binding protein PSPC1 a , Illustration of the two complementary techniques (Rep1 and Rep2) employed to identify TET2- interacting proteins in mouse ESCs. (Left) The experimental scheme for FLAG immunoprecipitation (IP) followed by mass spectrometry (MS) of 3xFL Tet2 knock-in and wild-type (WT) control ESC lines. (Right) Scheme of the SILAC-based in vivo labeling approach used to determine TET2 partners by IP with an anti-FLAG antibody using the nuclear extracts from 3xFL Tet2 knock-in ESCs and wild-type (WT) ESCs followed by MS analysis. (Center) Ratios of TET2-interacting peptides versus non-specific peptides detected by AP-MS in both IP-MS experiments. b , Relative RNA expression levels of Pspc1 , Tet2 and the ESC marker Oct4 in differentiated (MEF) versus pluripotent (iPSC, ESC) cell lines. Data are from one representative experiment (n=3 technical replicates) and presented as mean ± s.e.m. c , Validation of the interaction between endogenous PSPC1 and TET2 by coimmunoprecipitation followed by western blotting analysis. IgG was used as a negative control for the IP. The percentage of input (15%) is shown. d , Reduced TET2 chromatin occupancy upon Pspc1 depletion. (Top) Western blot analysis of total (whole cell extract, WCE) and chromatin-bound (Chromatin) PSPC1 and TET2 in control (shEV) and PSPC1 knock-down (shPspc1) ESCs. Histone H3 was used as a loading control. (Bottom) Quantitation of the relative levels of PSPC1 and TET2 in WCE and chromatin (Chr.) compared to H3 and shEV. In c and d, images are representative of immunoblots from 2 independent experiments.
    Figure Legend Snippet: TET2 is recruited to chromatin by the RNA-binding protein PSPC1 a , Illustration of the two complementary techniques (Rep1 and Rep2) employed to identify TET2- interacting proteins in mouse ESCs. (Left) The experimental scheme for FLAG immunoprecipitation (IP) followed by mass spectrometry (MS) of 3xFL Tet2 knock-in and wild-type (WT) control ESC lines. (Right) Scheme of the SILAC-based in vivo labeling approach used to determine TET2 partners by IP with an anti-FLAG antibody using the nuclear extracts from 3xFL Tet2 knock-in ESCs and wild-type (WT) ESCs followed by MS analysis. (Center) Ratios of TET2-interacting peptides versus non-specific peptides detected by AP-MS in both IP-MS experiments. b , Relative RNA expression levels of Pspc1 , Tet2 and the ESC marker Oct4 in differentiated (MEF) versus pluripotent (iPSC, ESC) cell lines. Data are from one representative experiment (n=3 technical replicates) and presented as mean ± s.e.m. c , Validation of the interaction between endogenous PSPC1 and TET2 by coimmunoprecipitation followed by western blotting analysis. IgG was used as a negative control for the IP. The percentage of input (15%) is shown. d , Reduced TET2 chromatin occupancy upon Pspc1 depletion. (Top) Western blot analysis of total (whole cell extract, WCE) and chromatin-bound (Chromatin) PSPC1 and TET2 in control (shEV) and PSPC1 knock-down (shPspc1) ESCs. Histone H3 was used as a loading control. (Bottom) Quantitation of the relative levels of PSPC1 and TET2 in WCE and chromatin (Chr.) compared to H3 and shEV. In c and d, images are representative of immunoblots from 2 independent experiments.

    Techniques Used: RNA Binding Assay, Immunoprecipitation, Mass Spectrometry, Knock-In, In Vivo, Labeling, RNA Expression, Marker, Western Blot, Negative Control, Quantitation Assay

    PSPC1 and TET2 silence MERVL transcriptionally and post-transcriptionally a , MERVL expression in Tet1/2/3 triple knock-out ( Tet TKO) ESCs rescued with an empty vector (+EV), a wild-type (+TET2WT), or a catalytic mutant (+TET2Mut) TET2. Center line, median; box and whisker plots: ± 10th–90th percentile range. Data are from 5 independent experiments (n=14 total technical replicates for each rescue). Two-tailed Student’s t -test was applied. ns, not significant. b–c , MERVL and IAP enrichment, compared to U6 negative control, among anti-5hmC immunoprecipitated RNAs in Tet TKO (b) and Pspc1 KO (c) ESCs rescued with an empty vector (+EV), a wild-type, or a mutant TET2/PSPC1. Data are presented as mean ± s.e.m. (n=3 independent experiments). Two-tailed Student’s t -test was applied. ns, not significant. d , (Top) Schematic of the protocol used for inhibition of transcription with α-Amanitin for RNA stability assay. (Bottom) Relative abundance of MERVL RNA in Pspc1 WT an d KO ESCs after transcriptional inhibition for 1, 2, or 4 hours with α-Amanitin. Data are normalized to untreated cells at time 0 h (Vehicle without treatment). Error bars indicate s.e.m. (n=3). Two-tailed Student’s t -test was applied. ns, not significant. e , A model of MERVL regulation by PSPC1/TET2 and HDAC1/2 in ESCs. PSPC1 binding to actively transcribed MERVL RNAs recruits TET2 and HDAC1/2 to chromatin. TET2 catalyzes 5hmC modification of MERVL RNAs resulting in their destabilization, and HDAC1/2 deacetylate histones at the chromatin level leading to transcriptional repression of the MERVL loci. Transcriptional and posttranscriptional repression of MERVL leads to the release of the PSPC1-TET2-HDAC1/2 complex from chromatin. Sporadic reactivation of MERVL , via a yet-to-be defined mechanism, leads to the recruitment PSPC1-TET2-HDAC1/2 for transcriptional and posttranscriptional control of MERVL and coordinated gene expression. Illustration by Jill Gregory. Printed with permission of ©Mount Sinai Health System.
    Figure Legend Snippet: PSPC1 and TET2 silence MERVL transcriptionally and post-transcriptionally a , MERVL expression in Tet1/2/3 triple knock-out ( Tet TKO) ESCs rescued with an empty vector (+EV), a wild-type (+TET2WT), or a catalytic mutant (+TET2Mut) TET2. Center line, median; box and whisker plots: ± 10th–90th percentile range. Data are from 5 independent experiments (n=14 total technical replicates for each rescue). Two-tailed Student’s t -test was applied. ns, not significant. b–c , MERVL and IAP enrichment, compared to U6 negative control, among anti-5hmC immunoprecipitated RNAs in Tet TKO (b) and Pspc1 KO (c) ESCs rescued with an empty vector (+EV), a wild-type, or a mutant TET2/PSPC1. Data are presented as mean ± s.e.m. (n=3 independent experiments). Two-tailed Student’s t -test was applied. ns, not significant. d , (Top) Schematic of the protocol used for inhibition of transcription with α-Amanitin for RNA stability assay. (Bottom) Relative abundance of MERVL RNA in Pspc1 WT an d KO ESCs after transcriptional inhibition for 1, 2, or 4 hours with α-Amanitin. Data are normalized to untreated cells at time 0 h (Vehicle without treatment). Error bars indicate s.e.m. (n=3). Two-tailed Student’s t -test was applied. ns, not significant. e , A model of MERVL regulation by PSPC1/TET2 and HDAC1/2 in ESCs. PSPC1 binding to actively transcribed MERVL RNAs recruits TET2 and HDAC1/2 to chromatin. TET2 catalyzes 5hmC modification of MERVL RNAs resulting in their destabilization, and HDAC1/2 deacetylate histones at the chromatin level leading to transcriptional repression of the MERVL loci. Transcriptional and posttranscriptional repression of MERVL leads to the release of the PSPC1-TET2-HDAC1/2 complex from chromatin. Sporadic reactivation of MERVL , via a yet-to-be defined mechanism, leads to the recruitment PSPC1-TET2-HDAC1/2 for transcriptional and posttranscriptional control of MERVL and coordinated gene expression. Illustration by Jill Gregory. Printed with permission of ©Mount Sinai Health System.

    Techniques Used: Expressing, Knock-Out, Plasmid Preparation, Mutagenesis, Whisker Assay, Two Tailed Test, Negative Control, Immunoprecipitation, Inhibition, Stability Assay, Binding Assay, Modification

    9) Product Images from "Virus relatedness predicts susceptibility in novel host species"

    Article Title: Virus relatedness predicts susceptibility in novel host species

    Journal: bioRxiv

    doi: 10.1101/2021.02.16.431403

    Change in viral load across a diverse panel of Drosophila host species for different virus isolates. Bar height and colour show the mean change in RNA viral load by 2 dpi on a log2 scale, with error bars representing the standard error of the mean. The phylogeny of Drosophila hosts is presented on the left. The scale bar represents number of nucleotide substitutions per site and the scale axis represents the approximate age since divergence in millions of years (my) based on estimates from [ 79 , 80 ]. The virus cladogram is based on the evolutionary relationships shown in Figure 1 .
    Figure Legend Snippet: Change in viral load across a diverse panel of Drosophila host species for different virus isolates. Bar height and colour show the mean change in RNA viral load by 2 dpi on a log2 scale, with error bars representing the standard error of the mean. The phylogeny of Drosophila hosts is presented on the left. The scale bar represents number of nucleotide substitutions per site and the scale axis represents the approximate age since divergence in millions of years (my) based on estimates from [ 79 , 80 ]. The virus cladogram is based on the evolutionary relationships shown in Figure 1 .

    Techniques Used:

    10) Product Images from "Age and Race related changes in Gene Expression during Human PBMC aging"

    Article Title: Age and Race related changes in Gene Expression during Human PBMC aging

    Journal: bioRxiv

    doi: 10.1101/2021.02.22.432179

    The characteristic gene expression of PBMC aging in Asian. (A) principal component analyses (PCA) for 19 Chinese PBMC RNA-seq data. Young and old individuals were largely separated according to the principal component1 scores (PC1). (B) Cluster dendrogram. Each color represents one specific co-expression module. In the colored rows below the dendrogram, the two colored rows represent the original modules and merged modules; (C) Supervised hierarchical cluster of each row correspond to a module eigengene (n = 40), column to a trait. Each cell contained the corresponding correlation. High correlations was colored in orange, low correlation in blue. (D) Hierarchical cluster analysis of four interested modules, based on the module-trait’s correlation and p value (absolute r > 0.5, P
    Figure Legend Snippet: The characteristic gene expression of PBMC aging in Asian. (A) principal component analyses (PCA) for 19 Chinese PBMC RNA-seq data. Young and old individuals were largely separated according to the principal component1 scores (PC1). (B) Cluster dendrogram. Each color represents one specific co-expression module. In the colored rows below the dendrogram, the two colored rows represent the original modules and merged modules; (C) Supervised hierarchical cluster of each row correspond to a module eigengene (n = 40), column to a trait. Each cell contained the corresponding correlation. High correlations was colored in orange, low correlation in blue. (D) Hierarchical cluster analysis of four interested modules, based on the module-trait’s correlation and p value (absolute r > 0.5, P

    Techniques Used: Expressing, RNA Sequencing Assay

    11) Product Images from "A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits"

    Article Title: A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA120.015434

    Environmental testing protocol and qPCR standard curve. A , environmental testing protocol (see “Environmental testing”). B , standard curves used to calculate the magnitude of environmental surface contamination and qPCR efficiencies ( E = 10 (−1/slope) − 1) using TaqPath and IDT CDC primers and probes. Left template , Quantitative Synthetic SARS-CoV-2 RNA (ATCC #VR-3276SD), N1 r 2 = 0.993, N2 r 2 = 0.994. Right template , positive control plasmid viral DNA (IDT, #10006625), N1 r 2 = 0.997, N2 r 2 = 0.992.
    Figure Legend Snippet: Environmental testing protocol and qPCR standard curve. A , environmental testing protocol (see “Environmental testing”). B , standard curves used to calculate the magnitude of environmental surface contamination and qPCR efficiencies ( E = 10 (−1/slope) − 1) using TaqPath and IDT CDC primers and probes. Left template , Quantitative Synthetic SARS-CoV-2 RNA (ATCC #VR-3276SD), N1 r 2 = 0.993, N2 r 2 = 0.994. Right template , positive control plasmid viral DNA (IDT, #10006625), N1 r 2 = 0.997, N2 r 2 = 0.992.

    Techniques Used: Real-time Polymerase Chain Reaction, Positive Control, Plasmid Preparation

    12) Product Images from "Association of large intergenic noncoding RNA expression with disease activity and organ damage in systemic lupus erythematosus"

    Article Title: Association of large intergenic noncoding RNA expression with disease activity and organ damage in systemic lupus erythematosus

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-015-0632-3

    Regulation of large intergenic noncoding RNAs in peripheral blood mononuclear cells by Pam3CSK4. (A) Expression of the large intergenic noncoding RNA linc0949 was lower in untreated and stimulated peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE) than in those of healthy controls. linc0949 was suppressed by the Toll-like receptor 2 (TLR2) ligand Pam3CK4 (20 ng/ml) in healthy controls, and it was not significantly different after treatment in patients with SLE. (B) linc0597 was upregulated by TLR2 agonist Pam3CK4 (20 ng/ml) in PBMCs of healthy controls, whereas it did not respond to the stimuli in patients with SLE. linc0597 expression was significantly decreased in PBMCs of patients with SLE compared with healthy controls both before stimulation and after treatment. The data shown are from five healthy donors and five patients with SLE after incubation with the indicated stimuli for 4 hours. Real-time quantitative PCR of the indicated RNAs is normalized to RPL13A levels (mean ± standard deviation (SD)). Data are mean ± SD. P -values were determined by performing unpaired t -tests. NS, Not significant. * P
    Figure Legend Snippet: Regulation of large intergenic noncoding RNAs in peripheral blood mononuclear cells by Pam3CSK4. (A) Expression of the large intergenic noncoding RNA linc0949 was lower in untreated and stimulated peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE) than in those of healthy controls. linc0949 was suppressed by the Toll-like receptor 2 (TLR2) ligand Pam3CK4 (20 ng/ml) in healthy controls, and it was not significantly different after treatment in patients with SLE. (B) linc0597 was upregulated by TLR2 agonist Pam3CK4 (20 ng/ml) in PBMCs of healthy controls, whereas it did not respond to the stimuli in patients with SLE. linc0597 expression was significantly decreased in PBMCs of patients with SLE compared with healthy controls both before stimulation and after treatment. The data shown are from five healthy donors and five patients with SLE after incubation with the indicated stimuli for 4 hours. Real-time quantitative PCR of the indicated RNAs is normalized to RPL13A levels (mean ± standard deviation (SD)). Data are mean ± SD. P -values were determined by performing unpaired t -tests. NS, Not significant. * P

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

    13) Product Images from "An Antiviral Defense Role of AGO2 in Plants"

    Article Title: An Antiviral Defense Role of AGO2 in Plants

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0014639

    AGO2 is induced by TCV, affects accumulation of viral RNA and coat protein and binds viral siRNA. a ) Q-RT-PCR of TCV accumulation in WT and ago2-1 plants. b ) Western blots of AGO2 and TCV CP accumulation in mock- and TCV-infected WT (Col-0) and ago2-1 plants, as indicated at 7 and 14dpi. Ponceau S-stained RUBISCO large subunit serves as a loading control. c ) TCV siRNAs bound to AGO2, sequenced by Illumina, derived from along the TCV genome. Those above the x-axis match the TCV positive strand, while those below the x-axis match the negative strand.
    Figure Legend Snippet: AGO2 is induced by TCV, affects accumulation of viral RNA and coat protein and binds viral siRNA. a ) Q-RT-PCR of TCV accumulation in WT and ago2-1 plants. b ) Western blots of AGO2 and TCV CP accumulation in mock- and TCV-infected WT (Col-0) and ago2-1 plants, as indicated at 7 and 14dpi. Ponceau S-stained RUBISCO large subunit serves as a loading control. c ) TCV siRNAs bound to AGO2, sequenced by Illumina, derived from along the TCV genome. Those above the x-axis match the TCV positive strand, while those below the x-axis match the negative strand.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Infection, Staining, Derivative Assay

    14) Product Images from "Long noncoding RNA TP73-AS1 accelerates the epithelial ovarian cancer via epigenetically repressing p21"

    Article Title: Long noncoding RNA TP73-AS1 accelerates the epithelial ovarian cancer via epigenetically repressing p21

    Journal: American Journal of Translational Research

    doi:

    TP73-AS1 epigenetically inhibits p21 through binding with EZH2. A. The subcellular location of TP73-AS1 in the nuclear and the cytoplasm fraction. B. The level of CDK inhibitors (CKIs), including p15, p16, p21, p27, p57. C. Western blot revealed the p21 protein in the TP73-AS1 silencing transfection. D. RNA-binding protein immunoprecipitation (RIP) assay showed the binding of TP73-AS1 with EZH2. E. Chromatin immunoprecipitation (ChIP) revealed the direct bound of EZH2 with p21 promoter region. F. Kaplan-Meier survival curve analysis based on the TCGA database suggested the prognosis of EOC patients with higher or lower group. **
    Figure Legend Snippet: TP73-AS1 epigenetically inhibits p21 through binding with EZH2. A. The subcellular location of TP73-AS1 in the nuclear and the cytoplasm fraction. B. The level of CDK inhibitors (CKIs), including p15, p16, p21, p27, p57. C. Western blot revealed the p21 protein in the TP73-AS1 silencing transfection. D. RNA-binding protein immunoprecipitation (RIP) assay showed the binding of TP73-AS1 with EZH2. E. Chromatin immunoprecipitation (ChIP) revealed the direct bound of EZH2 with p21 promoter region. F. Kaplan-Meier survival curve analysis based on the TCGA database suggested the prognosis of EOC patients with higher or lower group. **

    Techniques Used: Binding Assay, Western Blot, Transfection, RNA Binding Assay, Immunoprecipitation, Chromatin Immunoprecipitation

    15) Product Images from "Tet1 is a tumor suppressor of hematopoietic malignancy"

    Article Title: Tet1 is a tumor suppressor of hematopoietic malignancy

    Journal: Nature immunology

    doi: 10.1038/ni.3148

    TET1 is hypermethylated and transcriptionally down-regulated in B-NHL a) Methylation profiling by HELP-assay of the TET1 promoter in DLBCL and FL patients compared to normal naïve (NB) and centroblast (CB) B cells. b) mRNA expression analysis of TET1 in B-NHL patients. c) MassARRAY analysis of TET1 CpG methylation in 26 FL patients compared to normal Germinal Center B (GCB) cells and d) RNA-seq of TET1 and TET2 expression in the same 26 FL patients and normal GCB cells. Each circle indicates an individual patient in all panels; * P =
    Figure Legend Snippet: TET1 is hypermethylated and transcriptionally down-regulated in B-NHL a) Methylation profiling by HELP-assay of the TET1 promoter in DLBCL and FL patients compared to normal naïve (NB) and centroblast (CB) B cells. b) mRNA expression analysis of TET1 in B-NHL patients. c) MassARRAY analysis of TET1 CpG methylation in 26 FL patients compared to normal Germinal Center B (GCB) cells and d) RNA-seq of TET1 and TET2 expression in the same 26 FL patients and normal GCB cells. Each circle indicates an individual patient in all panels; * P =

    Techniques Used: Methylation, HELP Assay, Expressing, CpG Methylation Assay, RNA Sequencing Assay

    16) Product Images from "Novel Pituitary Actions of Epidermal Growth Factor: Receptor Specificity and Signal Transduction for UTS1, EGR1, and MMP13 Regulation by EGF"

    Article Title: Novel Pituitary Actions of Epidermal Growth Factor: Receptor Specificity and Signal Transduction for UTS1, EGR1, and MMP13 Regulation by EGF

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20205172

    EGF induced TIMP3 mRNA expression and receptor specificity in grass carp pituitary. ( A ) Time course of EGF (0.5 μM) treatment on TIMP3 mRNA expression. ( B ) Effect of EGF concentration (0.05–500 nM)-induced on TIMP3 mRNA expression in grass carp pituitary cells. ( C – E ) Effects of ErbB1 antagonist AG1478 (10 µM), ErbB2 antagonist AG879 (10 µM), and IGF receptor antagonist AG1024 (10 µM) on TIMP3 mRNA expression for 24 h, respectively. After drug treatment, total RNA was isolated for real-time PCR of MMP13 mRNA expression. In the data present (mean ± SEM), the differences between groups were considered as significant at p
    Figure Legend Snippet: EGF induced TIMP3 mRNA expression and receptor specificity in grass carp pituitary. ( A ) Time course of EGF (0.5 μM) treatment on TIMP3 mRNA expression. ( B ) Effect of EGF concentration (0.05–500 nM)-induced on TIMP3 mRNA expression in grass carp pituitary cells. ( C – E ) Effects of ErbB1 antagonist AG1478 (10 µM), ErbB2 antagonist AG879 (10 µM), and IGF receptor antagonist AG1024 (10 µM) on TIMP3 mRNA expression for 24 h, respectively. After drug treatment, total RNA was isolated for real-time PCR of MMP13 mRNA expression. In the data present (mean ± SEM), the differences between groups were considered as significant at p

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

    The functional role of in EGF-induced UTS1 and EGR1 in grass carp pituitary. ( A ) Effect of the inhibitor of MMPs BB94 (10 µM) on UTS1 mRNA expression. ( B ) Effect of the inhibitor of MMPs BB94 (10 µM) on EGR1 mRNA expression. After drug treatment, total RNA was isolated for real-time PCR of UTS1 and EGR1 mRNA expression. In the data present (mean ± SEM), the differences between groups were considered as significant at p
    Figure Legend Snippet: The functional role of in EGF-induced UTS1 and EGR1 in grass carp pituitary. ( A ) Effect of the inhibitor of MMPs BB94 (10 µM) on UTS1 mRNA expression. ( B ) Effect of the inhibitor of MMPs BB94 (10 µM) on EGR1 mRNA expression. After drug treatment, total RNA was isolated for real-time PCR of UTS1 and EGR1 mRNA expression. In the data present (mean ± SEM), the differences between groups were considered as significant at p

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

    Synergistic effects of EGF on UTS1 mRNA expression and receptor specificity and post-receptor signal pathway of EGF (0.5 μM)-induced UTS1 mRNA expression in grass carp pituitary cells. ( A ) Time course of EGF (0.5 μM) treatment on UTS1 mRNA expression. ( B ) Effect of EGF concentration (0.05–500 nM)-induced on UTS1 mRNA expression in grass carp pituitary cells. ( C – E ) Effects of ErbB1 antagonist AG1478, ErbB2 antagonist AG879, and IGF receptor antagonist AG1024 on EGF-induced UTS1 mRNA expression, respectively. ( F – H ) The effects of EGF (0.5 μM) induced UTS1 mRNA transcription with the MEK inhibitor U0126 (10 μM), ERK1/2 inhibitor LY3214996 (10 μM), and p38MAPK inhibitor SB203580, respectively. ( I – K ) Co-treatment with the PI 3 K inhibitor Wortmannin (10 μM), AKT inhibitor MK2206 (10 μM), and mTOR inhibitor Rapamycin (10 μM) on EGF (0.5 μm)-induced UTS1 mRNA expression for 24 h, respectively. After drug treatment, total RNA was isolated and used for real-time PCR of UTS1 mRNA expression. The differences between groups were considered as significant at p
    Figure Legend Snippet: Synergistic effects of EGF on UTS1 mRNA expression and receptor specificity and post-receptor signal pathway of EGF (0.5 μM)-induced UTS1 mRNA expression in grass carp pituitary cells. ( A ) Time course of EGF (0.5 μM) treatment on UTS1 mRNA expression. ( B ) Effect of EGF concentration (0.05–500 nM)-induced on UTS1 mRNA expression in grass carp pituitary cells. ( C – E ) Effects of ErbB1 antagonist AG1478, ErbB2 antagonist AG879, and IGF receptor antagonist AG1024 on EGF-induced UTS1 mRNA expression, respectively. ( F – H ) The effects of EGF (0.5 μM) induced UTS1 mRNA transcription with the MEK inhibitor U0126 (10 μM), ERK1/2 inhibitor LY3214996 (10 μM), and p38MAPK inhibitor SB203580, respectively. ( I – K ) Co-treatment with the PI 3 K inhibitor Wortmannin (10 μM), AKT inhibitor MK2206 (10 μM), and mTOR inhibitor Rapamycin (10 μM) on EGF (0.5 μm)-induced UTS1 mRNA expression for 24 h, respectively. After drug treatment, total RNA was isolated and used for real-time PCR of UTS1 mRNA expression. The differences between groups were considered as significant at p

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

    EGF induced EGR1 mRNA expression in grass carp pituitary cells, including receptor specificity and signal transduction pathways. ( A ) In the time course experiment, pituitary cells were treated with EGF (0.5 μM). ( B ) In the dose experiment, pituitary cells were cultured with EGF (0.05–500 nM). ( C – E ) Receptor specificity of EGF (0.5 µM)-induced EGR1 mRNA expression; effects of ErbB1 antagonist AG1478 (10 µM), ErbB2 antagonist AG879 (10 µM), and IGF receptor antagonist AG1024 (10 µM) on EGR1 mRNA expression for 24 h, respectively. ( F – H ) Signal transduction of EGR1 mRNA expression induced by EGF (0.5 μM) in grass carp pituitary cells. The effects of UTS1 mRNA transcription induced by EGF (0.5 μM) with EGF (0.5 μM) in the presence or absence of the MEK inhibitor U0126 (10 μM), ERK1/2 inhibitor LY3214996 (10 μM), or p38MAPK inhibitor SB203580 (10 μM), respectively. ( I – K ) The effects of EGF (0.5 μM) induced EGR1 mRNA expression with the PI 3 K inhibitor Wortmannin (10 μM), AKT inhibitor MK2206 (10 μM), or mTOR inhibitor Rapamycin (10 μM) by EGF (0.5 μM)-induced EGR1 mRNA expression for 24 h, respectively. After drug treatment, total RNA was isolated and used for real-time PCR of UTS1 mRNA expression. The differences between groups were considered as highly significant at p
    Figure Legend Snippet: EGF induced EGR1 mRNA expression in grass carp pituitary cells, including receptor specificity and signal transduction pathways. ( A ) In the time course experiment, pituitary cells were treated with EGF (0.5 μM). ( B ) In the dose experiment, pituitary cells were cultured with EGF (0.05–500 nM). ( C – E ) Receptor specificity of EGF (0.5 µM)-induced EGR1 mRNA expression; effects of ErbB1 antagonist AG1478 (10 µM), ErbB2 antagonist AG879 (10 µM), and IGF receptor antagonist AG1024 (10 µM) on EGR1 mRNA expression for 24 h, respectively. ( F – H ) Signal transduction of EGR1 mRNA expression induced by EGF (0.5 μM) in grass carp pituitary cells. The effects of UTS1 mRNA transcription induced by EGF (0.5 μM) with EGF (0.5 μM) in the presence or absence of the MEK inhibitor U0126 (10 μM), ERK1/2 inhibitor LY3214996 (10 μM), or p38MAPK inhibitor SB203580 (10 μM), respectively. ( I – K ) The effects of EGF (0.5 μM) induced EGR1 mRNA expression with the PI 3 K inhibitor Wortmannin (10 μM), AKT inhibitor MK2206 (10 μM), or mTOR inhibitor Rapamycin (10 μM) by EGF (0.5 μM)-induced EGR1 mRNA expression for 24 h, respectively. After drug treatment, total RNA was isolated and used for real-time PCR of UTS1 mRNA expression. The differences between groups were considered as highly significant at p

    Techniques Used: Expressing, Transduction, Cell Culture, Isolation, Real-time Polymerase Chain Reaction

    EGF induced MMP13 mRNA expression and receptor specificity and signal transduction mechanism in grass carp pituitary cells. ( A ) Pituitary cells were treated with EGF (0.5 μM) in a time dependent manner. ( B ) dose-dependent manner of EGF (0.05–500 nM) induced MMP13 mRNA expression, respectively. ( C – E ) Effects of ErbB1 antagonist AG1478 (10 µM), ErbB2 antagonist AG879 (10 µM), and IGF receptor antagonist AG1024 (10 µM) on MMP13 mRNA expression for 24 h, respectively. ( F – H ) Signal transduction of EGF-induced MMP13 mRNA expression in grass carp pituitary cells. Co-treatment of 24 h with the MEK blocker U0126 (10 μM), ERK1/2 inhibitor LY3214996 (10 μM), or p38MAPK inhibitor SB203580(10 μM) induced MMP13 mRNA expression was examined in grass carp pituitary cells, respectively. ( I – K ) Co-treatment of 24 h with the PI 3 K inhibitor Wortmannin (10 μM), AKT inhibitor MK2206 (10 μM), and mTOR inhibitor Rapamycin (10 μM) induced MMP13 mRNA expression was examined, respectively. After drug treatment, total RNA was isolated for real-time PCR of MMP13 mRNA expression. The differences between groups were considered as significant at p
    Figure Legend Snippet: EGF induced MMP13 mRNA expression and receptor specificity and signal transduction mechanism in grass carp pituitary cells. ( A ) Pituitary cells were treated with EGF (0.5 μM) in a time dependent manner. ( B ) dose-dependent manner of EGF (0.05–500 nM) induced MMP13 mRNA expression, respectively. ( C – E ) Effects of ErbB1 antagonist AG1478 (10 µM), ErbB2 antagonist AG879 (10 µM), and IGF receptor antagonist AG1024 (10 µM) on MMP13 mRNA expression for 24 h, respectively. ( F – H ) Signal transduction of EGF-induced MMP13 mRNA expression in grass carp pituitary cells. Co-treatment of 24 h with the MEK blocker U0126 (10 μM), ERK1/2 inhibitor LY3214996 (10 μM), or p38MAPK inhibitor SB203580(10 μM) induced MMP13 mRNA expression was examined in grass carp pituitary cells, respectively. ( I – K ) Co-treatment of 24 h with the PI 3 K inhibitor Wortmannin (10 μM), AKT inhibitor MK2206 (10 μM), and mTOR inhibitor Rapamycin (10 μM) induced MMP13 mRNA expression was examined, respectively. After drug treatment, total RNA was isolated for real-time PCR of MMP13 mRNA expression. The differences between groups were considered as significant at p

    Techniques Used: Expressing, Transduction, Isolation, Real-time Polymerase Chain Reaction

    17) Product Images from "Plasmodium falciparum: hrp3 promoter region is associated with stage-specificity and episomal recombination"

    Article Title: Plasmodium falciparum: hrp3 promoter region is associated with stage-specificity and episomal recombination

    Journal: Experimental parasitology

    doi: 10.1016/j.exppara.2007.01.020

    Deletion of the hrp 3 promoter switched stage specific expression from ring to trophozoite stage ( A ) Schematic representation of hrp 3 promoter truncations (diagonal lines) driving human DHFR fused to GFP (HDGFP, filled box) in plasmid pH1.7HG containing the 1.7-kb full length promoter as well as the plasmids pH1.1HG, pH0.6HG carrying the truncated promoters of 1.1-kb and 0.6-kb respectively. ( B ) Total RNA from stable cell lines expressing HDGFP was purified from ring (i, iii) and trophozoite (ii, iv) stages, separated on agarose gels stained with ethidium bromide (iii, iv) and analyzed by northern blot (i, ii) using 32 P-GFP antisense probe.
    Figure Legend Snippet: Deletion of the hrp 3 promoter switched stage specific expression from ring to trophozoite stage ( A ) Schematic representation of hrp 3 promoter truncations (diagonal lines) driving human DHFR fused to GFP (HDGFP, filled box) in plasmid pH1.7HG containing the 1.7-kb full length promoter as well as the plasmids pH1.1HG, pH0.6HG carrying the truncated promoters of 1.1-kb and 0.6-kb respectively. ( B ) Total RNA from stable cell lines expressing HDGFP was purified from ring (i, iii) and trophozoite (ii, iv) stages, separated on agarose gels stained with ethidium bromide (iii, iv) and analyzed by northern blot (i, ii) using 32 P-GFP antisense probe.

    Techniques Used: Expressing, Plasmid Preparation, Stable Transfection, Purification, Staining, Northern Blot

    18) Product Images from "Expression of protein kinase C gamma promotes cell migration in colon cancer"

    Article Title: Expression of protein kinase C gamma promotes cell migration in colon cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18916

    Gene expression of PKC genes in colon cancer Tissue samples measuring approximately 0.5cm in diameter were collected from 23 patients undergoing surgery in University Hospital Limerick. Normal tissue from the 23 patients was also collected approximately 10 cm away from the cancer tissue. RNA was extracted from the tissue, cDNA was synthesised and real time PCR was carried out. All data was normalized using the housekeeping genes PRGK1, GUSB, PPIA and HRPT1. ( A ) Fold change of PRKCG (PKC Gamma coding gene) in cancer tissue of each patient. Results were obtained by comparing mRNA level of PRKCG in individual's normal tissue compared to levels in that individual's cancer tissue (Statistical difference based on Pair Wise Fixed Reallocation Randomisation Test © as per REST © software). ( B ) Fold change of PRKCG in stage 2 cancer tissue. ( C ) Fold change of PRKCG in stage 3 cancer tissue (No statistical difference found between stages as determined by Mann Whitney U test).
    Figure Legend Snippet: Gene expression of PKC genes in colon cancer Tissue samples measuring approximately 0.5cm in diameter were collected from 23 patients undergoing surgery in University Hospital Limerick. Normal tissue from the 23 patients was also collected approximately 10 cm away from the cancer tissue. RNA was extracted from the tissue, cDNA was synthesised and real time PCR was carried out. All data was normalized using the housekeeping genes PRGK1, GUSB, PPIA and HRPT1. ( A ) Fold change of PRKCG (PKC Gamma coding gene) in cancer tissue of each patient. Results were obtained by comparing mRNA level of PRKCG in individual's normal tissue compared to levels in that individual's cancer tissue (Statistical difference based on Pair Wise Fixed Reallocation Randomisation Test © as per REST © software). ( B ) Fold change of PRKCG in stage 2 cancer tissue. ( C ) Fold change of PRKCG in stage 3 cancer tissue (No statistical difference found between stages as determined by Mann Whitney U test).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Software, MANN-WHITNEY

    19) Product Images from "Identification of amino acid residues important for anti-IFN activity of porcine reproductive and respiratory syndrome virus non-structural protein 1"

    Article Title: Identification of amino acid residues important for anti-IFN activity of porcine reproductive and respiratory syndrome virus non-structural protein 1

    Journal: Virology

    doi: 10.1016/j.virol.2012.08.034

    The 16-5A mutant induces higher level of type I IFN mRNAs . Porcine monocyte- derived macrophages were mock-infected/infected with 1 MOI of 16-5A mutant or wt FL12 virus. Total RNA isolated from cells was reverse transcribed and real time PCR was done for detection of porcine IFN-α (A), IFN-β (B) mRNA. The mRNA copy numbers were calculated after normalization with porcine β-actin copy number and expressed relative to mock control. Bars show average of mRNA copy numbers±SEM from three independent experiments (‘ ⁎ ’ indicates p
    Figure Legend Snippet: The 16-5A mutant induces higher level of type I IFN mRNAs . Porcine monocyte- derived macrophages were mock-infected/infected with 1 MOI of 16-5A mutant or wt FL12 virus. Total RNA isolated from cells was reverse transcribed and real time PCR was done for detection of porcine IFN-α (A), IFN-β (B) mRNA. The mRNA copy numbers were calculated after normalization with porcine β-actin copy number and expressed relative to mock control. Bars show average of mRNA copy numbers±SEM from three independent experiments (‘ ⁎ ’ indicates p

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

    Growth attenuation and instability of 16-5A virus in vivo . (A) The in vivo growth property of 16-5A virus. Wt FL12 or 16-5A mutant viruses were inoculated into growing pigs and serum was collected at indicated days post-infection. After isolation of total RNA from serum, 4 μl of RNA was used in a single step real-time PCR reaction to detect viral RNA copy numbers. The bars represent mean of the viral RNA copy number from 4 different animals in each group. Error bars indicate SEM values. ( ⁎ indicate p
    Figure Legend Snippet: Growth attenuation and instability of 16-5A virus in vivo . (A) The in vivo growth property of 16-5A virus. Wt FL12 or 16-5A mutant viruses were inoculated into growing pigs and serum was collected at indicated days post-infection. After isolation of total RNA from serum, 4 μl of RNA was used in a single step real-time PCR reaction to detect viral RNA copy numbers. The bars represent mean of the viral RNA copy number from 4 different animals in each group. Error bars indicate SEM values. ( ⁎ indicate p

    Techniques Used: In Vivo, Mutagenesis, Infection, Isolation, Real-time Polymerase Chain Reaction

    20) Product Images from "Biological characteristics of the rtA181T/sW172* mutant strain of Hepatitis B virus in animal model"

    Article Title: Biological characteristics of the rtA181T/sW172* mutant strain of Hepatitis B virus in animal model

    Journal: Virology Journal

    doi: 10.1186/1743-422X-9-280

    Time-dependent viral transcription after hydrodynamic transfection in vivo. A : HBV RNA of mice liver were detected by Northern blotting, and GAPDH transcript was used as an internal control for RNA loading per lane; B : The 3.5-kb HBV mRNA was quantitatively calculated automatically by the Quantity-One software (Bio-Rad); C or D : The total levels of HBV S-mRNA or C-mRNA in mouse liver were detected by qPCR, and the mean value of Wt of day 1 was defined as 1.
    Figure Legend Snippet: Time-dependent viral transcription after hydrodynamic transfection in vivo. A : HBV RNA of mice liver were detected by Northern blotting, and GAPDH transcript was used as an internal control for RNA loading per lane; B : The 3.5-kb HBV mRNA was quantitatively calculated automatically by the Quantity-One software (Bio-Rad); C or D : The total levels of HBV S-mRNA or C-mRNA in mouse liver were detected by qPCR, and the mean value of Wt of day 1 was defined as 1.

    Techniques Used: Transfection, In Vivo, Mouse Assay, Northern Blot, Software, Real-time Polymerase Chain Reaction

    21) Product Images from "Genomic Profiling of Messenger RNAs and MicroRNAs Reveals Potential Mechanisms of TWEAK-Induced Skeletal Muscle Wasting in Mice"

    Article Title: Genomic Profiling of Messenger RNAs and MicroRNAs Reveals Potential Mechanisms of TWEAK-Induced Skeletal Muscle Wasting in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008760

    Differential expression of miRNAs in TWEAK-treated C2C12 myotubes measured by low-density miRNA array. A) C2C12 myotubes were treated with 10ng/ml of TWEAK for 18h following isolation of total RNA enriched with small RNAs. Untreated C2C12 myotubes under exactly similar conditions served as control. The normalized expression ratios were plotted for each miRNA are mean ± SD (n = 3). Low-density miRNA array of TWEAK-treated C2C12 myotubes showed down-regulation of miR-1, miR-133a, miR-133b, miR-206, miR-27, miR-23, miR-93, miR-199, miR-107, and miR-192. The numbers above the bar represents the fold changes with TWEAK treatment against control with p-values ≤0.05. B). TWEAK increased the expression of miR-715, miR-146a, miR-455, miR-322, mir-98, and miR-470 in C2C12 myotubes. The relative expression values from the QRT-PCR analysis were plotted for each gene are mean ± SD (n = 3). The values significantly different from corresponding untreated control (p-value ≤0.01) were represented with ‘*’.
    Figure Legend Snippet: Differential expression of miRNAs in TWEAK-treated C2C12 myotubes measured by low-density miRNA array. A) C2C12 myotubes were treated with 10ng/ml of TWEAK for 18h following isolation of total RNA enriched with small RNAs. Untreated C2C12 myotubes under exactly similar conditions served as control. The normalized expression ratios were plotted for each miRNA are mean ± SD (n = 3). Low-density miRNA array of TWEAK-treated C2C12 myotubes showed down-regulation of miR-1, miR-133a, miR-133b, miR-206, miR-27, miR-23, miR-93, miR-199, miR-107, and miR-192. The numbers above the bar represents the fold changes with TWEAK treatment against control with p-values ≤0.05. B). TWEAK increased the expression of miR-715, miR-146a, miR-455, miR-322, mir-98, and miR-470 in C2C12 myotubes. The relative expression values from the QRT-PCR analysis were plotted for each gene are mean ± SD (n = 3). The values significantly different from corresponding untreated control (p-value ≤0.01) were represented with ‘*’.

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

    Expression profiles of select microRNAs and TRAF-6 proteins in skeletal muscle of TWEAK-Tg mice. A) TaqMan QRT-PCR analysis of miR-1-1, miR-133a, miR-133b, and miR-146a in skeletal muscles of TWEAK-Tg mice. Gastrocnemius muscle from 6 months old TWEAK-Tg mice and littermate control mice were taken and total RNA enriched with small RNAs was isolated for TaqMan qRT-PCR analysis. The normalized expression ratios were plotted for each miRNA are mean ± SD (n = 3). ‘*’ represents the statistical significance (p-value ≤0.01). B). Gastrocnemius muscle of 6 months old TWEAK-Tg mice and littermate control mice were taken and total protein was isolated for Western blotting analysis. Representative immunoblot presented here show that the levels of TRAF-6 are considerably reduced in skeletal muscle of TWEAK-Tg (n = 4) mice compared to control (n = 4) mice. Equal amounts of protein loading were ensured by the expression levels of β-actin.
    Figure Legend Snippet: Expression profiles of select microRNAs and TRAF-6 proteins in skeletal muscle of TWEAK-Tg mice. A) TaqMan QRT-PCR analysis of miR-1-1, miR-133a, miR-133b, and miR-146a in skeletal muscles of TWEAK-Tg mice. Gastrocnemius muscle from 6 months old TWEAK-Tg mice and littermate control mice were taken and total RNA enriched with small RNAs was isolated for TaqMan qRT-PCR analysis. The normalized expression ratios were plotted for each miRNA are mean ± SD (n = 3). ‘*’ represents the statistical significance (p-value ≤0.01). B). Gastrocnemius muscle of 6 months old TWEAK-Tg mice and littermate control mice were taken and total protein was isolated for Western blotting analysis. Representative immunoblot presented here show that the levels of TRAF-6 are considerably reduced in skeletal muscle of TWEAK-Tg (n = 4) mice compared to control (n = 4) mice. Equal amounts of protein loading were ensured by the expression levels of β-actin.

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

    22) Product Images from "An important role for Akt3 in platelet activation and thrombosis"

    Article Title: An important role for Akt3 in platelet activation and thrombosis

    Journal: Blood

    doi: 10.1182/blood-2010-12-323204

    Expression of Akt3 in platelets. (A) Human platelet RNA was isolated from washed platelets (3 × 10 8 ). RT-PCR was performed with primers specific for Akt3 or a housekeeping gene, GAPDH. (B) Mouse platelet RNA was isolated from 3 × 10 8 platelets of wild-type or Akt3 −/− platelets and RT-PCR was performed similarly. Leukocyte contamination of platelet preparation was 4 × 10 4 /mL as determined using Hemavet blood cell analyzer. RNA was isolated from 4 × 10 4 /mL of WT mouse leukocytes and was also analyzed by RT-PCR using Akt3 specific primers under the same conditions as for platelet preparations to verify that the Akt3 fragment was not from leukocyte contamination. (C) Washed human platelets, wild-type and Akt3 −/− mouse platelets were solubilized and immunoblotted with a rabbit antibody specifically recognizing Akt3, and α-tubulin is used as loading control. (D) Washed human platelets were solubilized, and immunoabsorbed with anti-Akt3 to remove Akt3 from lysates or with control rabbit IgG, and then immunoblotted with anti-Akt3 or an antibody recognizing all Akt isoforms (Total Akt). (E) Experiments in panel D were scanned and quantified using NIH Image J for uncalibrated optical density (mean ± SE, 4 experiments). The difference in percent of total Akt between IgG and Akt3 immunoabsorbed lysates is significant ( P
    Figure Legend Snippet: Expression of Akt3 in platelets. (A) Human platelet RNA was isolated from washed platelets (3 × 10 8 ). RT-PCR was performed with primers specific for Akt3 or a housekeeping gene, GAPDH. (B) Mouse platelet RNA was isolated from 3 × 10 8 platelets of wild-type or Akt3 −/− platelets and RT-PCR was performed similarly. Leukocyte contamination of platelet preparation was 4 × 10 4 /mL as determined using Hemavet blood cell analyzer. RNA was isolated from 4 × 10 4 /mL of WT mouse leukocytes and was also analyzed by RT-PCR using Akt3 specific primers under the same conditions as for platelet preparations to verify that the Akt3 fragment was not from leukocyte contamination. (C) Washed human platelets, wild-type and Akt3 −/− mouse platelets were solubilized and immunoblotted with a rabbit antibody specifically recognizing Akt3, and α-tubulin is used as loading control. (D) Washed human platelets were solubilized, and immunoabsorbed with anti-Akt3 to remove Akt3 from lysates or with control rabbit IgG, and then immunoblotted with anti-Akt3 or an antibody recognizing all Akt isoforms (Total Akt). (E) Experiments in panel D were scanned and quantified using NIH Image J for uncalibrated optical density (mean ± SE, 4 experiments). The difference in percent of total Akt between IgG and Akt3 immunoabsorbed lysates is significant ( P

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

    23) Product Images from "Exopolysaccharides from a Codonopsis pilosula endophyte activate macrophages and inhibit cancer cell proliferation and migration"

    Article Title: Exopolysaccharides from a Codonopsis pilosula endophyte activate macrophages and inhibit cancer cell proliferation and migration

    Journal: Thoracic Cancer

    doi: 10.1111/1759-7714.12630

    DSPS activates macrophages. ( a ) RAW264.7 cells were treated with various concentrations of DSPS for 24 hours, and the cellular morphology was analyzed with a phase‐contrast microscope. ( b ) Quantification of activated macrophages for experiments performed as described in ( a ). ( c ) Quantitative reverse transcriptase‐PCR analysis of the relative expression of TNF‐α messenger RNA (mRNA). ( d ) Quantification of nitric oxide (NO) production by RAW264.7 cells.
    Figure Legend Snippet: DSPS activates macrophages. ( a ) RAW264.7 cells were treated with various concentrations of DSPS for 24 hours, and the cellular morphology was analyzed with a phase‐contrast microscope. ( b ) Quantification of activated macrophages for experiments performed as described in ( a ). ( c ) Quantitative reverse transcriptase‐PCR analysis of the relative expression of TNF‐α messenger RNA (mRNA). ( d ) Quantification of nitric oxide (NO) production by RAW264.7 cells.

    Techniques Used: Microscopy, Polymerase Chain Reaction, Expressing

    24) Product Images from "Trans control of cardiac mRNA translation in a protein length-dependent fashion"

    Article Title: Trans control of cardiac mRNA translation in a protein length-dependent fashion

    Journal: bioRxiv

    doi: 10.1101/2020.06.05.133298

    Impaired ribosome assembly and half-mer formation drive the chromosome 3p teQTL, related to Figure 3. (A) Rat congenic line comparison of differences in polysomal configuration as measured by normalized area under curves (AUCs) of the polysome profiles, for SHR.BN-(3L) (grey) and SHR.BN-(3S) (green). (B) Left panel: dot plots with RIN values of HXB/BXH RI lines separated by Chr. 3p teQTL genotype. RIN values shown are calculated on the Agilent BioAnalyzer 2100 using the RNA nano assay. The middle panel contains dot plots of the HXB/BXH RI lines, with (i) 18S abundance, (ii) 28S abundance and (iii) 28S / 18S ratios, as calculated by the percentage of total RNA with an Agilent BioAnalyzer 2100 RNA nano assay. The right panel contains dot box plots comparing congenic rat line mean mRNA expression values for ribosomal protein genes involved in the structure of the 40S (SSU) and 60S (LSU) ribosomal subunits. Values are separated by local genotype at the chromosome 3p teQTL locus and only left ventricular heart tissue RNA data is shown. Error bars indicate mean values with standard deviation (SD). All these analyses indicate no imbalance between the production levels of both ribosomal subunits. (C) Meta-gene codon 3-nt periodicity bar plots displaying P-sites of 29-nt ribosome footprints to illustrate the similarity in translation elongation rates between both congenic lines. The visualized data are a merger of all replicate SHR.BN-(3L) (grey, top) and SHR.BN-(3S) (green, bottom) Ribo-seq 29-nt footprints. Plots are generated with Ribo-seQC ( Calviello et al., 2019 ) and modified to only display the following sections of genes: (i) 25nt before and 33nt after the start codon, followed by (ii) 33nt from the middle of the CDS, and finally (iii) 33nt before and 25nt after the stop codon ( Calviello et al., 2019 ). (D) Western blots and dot plots with quantification of band intensities for the ER stress markers IRE1-alha phosphorylation (normalized for IRE1-alpha and TOM20 expression; top) and XBP1s production (normalized for GAPDH expression; bottom). Error bars indicate mean values with SD. These protein level analyses show no difference in the expression or activation of typical ER stress response markers. (E) Scatter plot showing CDS length versus fold change (FC (SHR.BN-(3S) vs SHR.BN-(3L)) for Ribo-seq data, highlighting all genes with actively translated uORFs in the rat heart. The square correlation coefficient (r 2 ) based on standardized major axis (SMA) is calculated using expression values of this subset of uORF-containing genes only, and precisely matches that of the whole translatome (r 2 = 0.20), showing that there is no differential regulation of genes with uORFs as opposed to the full set of translated genes.
    Figure Legend Snippet: Impaired ribosome assembly and half-mer formation drive the chromosome 3p teQTL, related to Figure 3. (A) Rat congenic line comparison of differences in polysomal configuration as measured by normalized area under curves (AUCs) of the polysome profiles, for SHR.BN-(3L) (grey) and SHR.BN-(3S) (green). (B) Left panel: dot plots with RIN values of HXB/BXH RI lines separated by Chr. 3p teQTL genotype. RIN values shown are calculated on the Agilent BioAnalyzer 2100 using the RNA nano assay. The middle panel contains dot plots of the HXB/BXH RI lines, with (i) 18S abundance, (ii) 28S abundance and (iii) 28S / 18S ratios, as calculated by the percentage of total RNA with an Agilent BioAnalyzer 2100 RNA nano assay. The right panel contains dot box plots comparing congenic rat line mean mRNA expression values for ribosomal protein genes involved in the structure of the 40S (SSU) and 60S (LSU) ribosomal subunits. Values are separated by local genotype at the chromosome 3p teQTL locus and only left ventricular heart tissue RNA data is shown. Error bars indicate mean values with standard deviation (SD). All these analyses indicate no imbalance between the production levels of both ribosomal subunits. (C) Meta-gene codon 3-nt periodicity bar plots displaying P-sites of 29-nt ribosome footprints to illustrate the similarity in translation elongation rates between both congenic lines. The visualized data are a merger of all replicate SHR.BN-(3L) (grey, top) and SHR.BN-(3S) (green, bottom) Ribo-seq 29-nt footprints. Plots are generated with Ribo-seQC ( Calviello et al., 2019 ) and modified to only display the following sections of genes: (i) 25nt before and 33nt after the start codon, followed by (ii) 33nt from the middle of the CDS, and finally (iii) 33nt before and 25nt after the stop codon ( Calviello et al., 2019 ). (D) Western blots and dot plots with quantification of band intensities for the ER stress markers IRE1-alha phosphorylation (normalized for IRE1-alpha and TOM20 expression; top) and XBP1s production (normalized for GAPDH expression; bottom). Error bars indicate mean values with SD. These protein level analyses show no difference in the expression or activation of typical ER stress response markers. (E) Scatter plot showing CDS length versus fold change (FC (SHR.BN-(3S) vs SHR.BN-(3L)) for Ribo-seq data, highlighting all genes with actively translated uORFs in the rat heart. The square correlation coefficient (r 2 ) based on standardized major axis (SMA) is calculated using expression values of this subset of uORF-containing genes only, and precisely matches that of the whole translatome (r 2 = 0.20), showing that there is no differential regulation of genes with uORFs as opposed to the full set of translated genes.

    Techniques Used: Expressing, Standard Deviation, Generated, Modification, Western Blot, Activation Assay

    Identification of translational efficiency QTLs in the HXB/BXH panel, related to Figure 1. (A) Bar plot with RNA integrity numbers (RIN values) for total RNA isolated from rat heart (top) and liver (bottom). The dashed line indicates the average RIN of 9.11, illustrating the high integrity of the processed tissue samples. (B) Stacked bar plots with sequencing read filtering statistics for heart (top) and liver (bottom) tissue. Reads derived from ribosomal RNA (rRNA), mitochondrial RNA (mtRNA) and transfer RNA (tRNA) are removed from the mRNA-seq and Ribo-seq data prior to mapping. This results in a set of ‘cleaned reads’, which are used as input for mapping and downstream data analyses. (C) Correlation analyses and scatter plots for biological replicate Ribo-seq data for 3 replicates of two RI lines (BXH12 and BXH13), illustrating the high technical reproducibility of our Ribo-seq approach across biological replicates. (D) Correlation scatter plots for heart (left) and liver (right) tissue, showing the correlation between mean mRNA-seq and Ribo-seq based quantifications of gene expression. (E) Correlation scatter plots between mRNA-seq or Ribo-seq reads and rat liver proteomics data, as quantified using iBAQ values obtained from MaxQuant ( Cox Mann, 2008 ). Ribo-seq is a slightly better proxy for final protein levels than mRNA-seq (Pearson’s r = 0.60 vs 0.53). (F) Dot plot showing the ribosome footprint (Ribo-seq read) length distribution across the 30 lines in rat heart (left) and liver (right). (G) Venn diagrams with tissue-specific comparisons of all identified translated genes, translated lncRNAs and translated uORFs. (H) Bar plot with a meta-analysis of stacked P-sites derived from Ribo-seq reads in heart (top) and liver (bottom) tissue. Blue bars indicate the number (left) and percentage (right) of footprints that precisely match annotated protein-coding gene open reading frames (ORFs; 90%). (I) Bar plots with significance values for detected local (top) and distant (bottom) eQTLs and riboQTLs, sorted by the delta of the p-values for both quantitative traits (bottom track of each panel). This analysis illustrates that most eQTLs are prolonged during translation, though they may sometimes near-miss the significance cutoff. Concordant with the teQTL results, this analysis furthermore highlights a highly translation-specific set of QTLs. (J) Dot plots with expression values for 2 genes with a highly specific local teQTL in rat hearts. Bars indicate mean values. (K) Rtel1 is the only gene for which a local teQTL and uORF-QTL coincide, though both associations occur with similar directionality (lower translation associate with the SHR/Ola genotype). (L) Bee swarm dot plots with correlation values (Spearman’s rho ) between the translation rates of uORFs and the primary ORF TE in heart and liver. Genes with uORFs that show a strong negative correlation with primary ORF translation are highlighted in red. Overall, most uORFs seem to positively correlate with primary ORF TE, as previously reported for the human heart ( Heesch et al., 2019 ). (M) Two rare examples of genes with strongly anti-correlating translation rates for the uORF vs. the primary ORF.
    Figure Legend Snippet: Identification of translational efficiency QTLs in the HXB/BXH panel, related to Figure 1. (A) Bar plot with RNA integrity numbers (RIN values) for total RNA isolated from rat heart (top) and liver (bottom). The dashed line indicates the average RIN of 9.11, illustrating the high integrity of the processed tissue samples. (B) Stacked bar plots with sequencing read filtering statistics for heart (top) and liver (bottom) tissue. Reads derived from ribosomal RNA (rRNA), mitochondrial RNA (mtRNA) and transfer RNA (tRNA) are removed from the mRNA-seq and Ribo-seq data prior to mapping. This results in a set of ‘cleaned reads’, which are used as input for mapping and downstream data analyses. (C) Correlation analyses and scatter plots for biological replicate Ribo-seq data for 3 replicates of two RI lines (BXH12 and BXH13), illustrating the high technical reproducibility of our Ribo-seq approach across biological replicates. (D) Correlation scatter plots for heart (left) and liver (right) tissue, showing the correlation between mean mRNA-seq and Ribo-seq based quantifications of gene expression. (E) Correlation scatter plots between mRNA-seq or Ribo-seq reads and rat liver proteomics data, as quantified using iBAQ values obtained from MaxQuant ( Cox Mann, 2008 ). Ribo-seq is a slightly better proxy for final protein levels than mRNA-seq (Pearson’s r = 0.60 vs 0.53). (F) Dot plot showing the ribosome footprint (Ribo-seq read) length distribution across the 30 lines in rat heart (left) and liver (right). (G) Venn diagrams with tissue-specific comparisons of all identified translated genes, translated lncRNAs and translated uORFs. (H) Bar plot with a meta-analysis of stacked P-sites derived from Ribo-seq reads in heart (top) and liver (bottom) tissue. Blue bars indicate the number (left) and percentage (right) of footprints that precisely match annotated protein-coding gene open reading frames (ORFs; 90%). (I) Bar plots with significance values for detected local (top) and distant (bottom) eQTLs and riboQTLs, sorted by the delta of the p-values for both quantitative traits (bottom track of each panel). This analysis illustrates that most eQTLs are prolonged during translation, though they may sometimes near-miss the significance cutoff. Concordant with the teQTL results, this analysis furthermore highlights a highly translation-specific set of QTLs. (J) Dot plots with expression values for 2 genes with a highly specific local teQTL in rat hearts. Bars indicate mean values. (K) Rtel1 is the only gene for which a local teQTL and uORF-QTL coincide, though both associations occur with similar directionality (lower translation associate with the SHR/Ola genotype). (L) Bee swarm dot plots with correlation values (Spearman’s rho ) between the translation rates of uORFs and the primary ORF TE in heart and liver. Genes with uORFs that show a strong negative correlation with primary ORF translation are highlighted in red. Overall, most uORFs seem to positively correlate with primary ORF TE, as previously reported for the human heart ( Heesch et al., 2019 ). (M) Two rare examples of genes with strongly anti-correlating translation rates for the uORF vs. the primary ORF.

    Techniques Used: Isolation, Sequencing, Derivative Assay, Expressing

    25) Product Images from "ArtinM Mediates Murine T Cell Activation and Induces Cell Death in Jurkat Human Leukemic T Cells"

    Article Title: ArtinM Mediates Murine T Cell Activation and Induces Cell Death in Jurkat Human Leukemic T Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18071400

    Detection of activation markers in ArtinM-stimulated CD4 + and CD8 + T cells. CD4 + and CD8 + T cells (1.5 × 10 6 /mL) were distributed in 96-well microplates and incubated under stimulation with ArtinM (1.25 μg/mL), PMA (50 ng/mL) plus ionomycin (1 μM), IL-4 (50 ng/mL), or IL-12 (50 ng/mL) plus IFN-γ (30 ng/mL) at 37 °C for different periods of time. Medium alone was used as the negative control. ( A ) Culture supernatants of CD4 + and CD8 + T cells were used to measure IFN-γ production by ELISA after 48 h of incubation; ( B , C ) CD4 + T cells were stimulated for 8 h and the extracted RNA was used for real-time quantitative polymerase chain reaction analysis of T-bet and GATA-3 mRNA. The results are expressed as means ± SEM; * p
    Figure Legend Snippet: Detection of activation markers in ArtinM-stimulated CD4 + and CD8 + T cells. CD4 + and CD8 + T cells (1.5 × 10 6 /mL) were distributed in 96-well microplates and incubated under stimulation with ArtinM (1.25 μg/mL), PMA (50 ng/mL) plus ionomycin (1 μM), IL-4 (50 ng/mL), or IL-12 (50 ng/mL) plus IFN-γ (30 ng/mL) at 37 °C for different periods of time. Medium alone was used as the negative control. ( A ) Culture supernatants of CD4 + and CD8 + T cells were used to measure IFN-γ production by ELISA after 48 h of incubation; ( B , C ) CD4 + T cells were stimulated for 8 h and the extracted RNA was used for real-time quantitative polymerase chain reaction analysis of T-bet and GATA-3 mRNA. The results are expressed as means ± SEM; * p

    Techniques Used: Activation Assay, Incubation, Negative Control, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

    26) Product Images from "Attenuation of estrogen receptor ?-mediated transcription through estrogen-stimulated recruitment of a negative elongation factor"

    Article Title: Attenuation of estrogen receptor ?-mediated transcription through estrogen-stimulated recruitment of a negative elongation factor

    Journal: Genes & Development

    doi: 10.1101/gad.1214104

    Ectopically expressed COBRA1 represses ligand-dependent transcriptional activation by ERα. ( A ) COBRA1 represses transcription from the luciferase-linked C3 and pS2 promoters, but not the CATD promoter. T47D cells were transfected with 0.5 μg COBRA1 and 0.5 μg of various luciferase reporter plasmids in the presence or absence of 10 nM E2. ( B ) COBRA1 does not affect transcription from ERα-unresponsive promoters. GAL4-p53 and c-Jun were cotransfected with a GAL4- or AP1-responsive luciferase reporter construct, respectively, in the presence or absence of Flag-COBRA1. ( C ) Northern hybridization indicates the impact of ectopic COBRA1 on the transcription of the endogenous C3 gene in T47D cells. Stably transfected T47D clones harboring either the pcDNA3 empty vector or Flag-COBRA1-expressing plasmid were treated with either ethanol or E2 for the indicated periods of time and harvested for RNA extraction. ( D ) RT–PCR analysis of the COBRA1 effect on the transcription of various ER-responsive genes. Pools of T47D cells stably transfected with either empty or COBRA1-expressing retroviral vectors were treated with ethanol or E2 (10 nM) for 24 h. RT–PCR was conducted on the total RNA to assess the transcription level of three ERα-responsive genes. In both C and D , β-Actin was used as an internal control. Also shown on the top of the panel is an anti-COBRA1 immunoblot indicating the total level of COBRA1 in the control and Flag-COBRA1-expressing cells.
    Figure Legend Snippet: Ectopically expressed COBRA1 represses ligand-dependent transcriptional activation by ERα. ( A ) COBRA1 represses transcription from the luciferase-linked C3 and pS2 promoters, but not the CATD promoter. T47D cells were transfected with 0.5 μg COBRA1 and 0.5 μg of various luciferase reporter plasmids in the presence or absence of 10 nM E2. ( B ) COBRA1 does not affect transcription from ERα-unresponsive promoters. GAL4-p53 and c-Jun were cotransfected with a GAL4- or AP1-responsive luciferase reporter construct, respectively, in the presence or absence of Flag-COBRA1. ( C ) Northern hybridization indicates the impact of ectopic COBRA1 on the transcription of the endogenous C3 gene in T47D cells. Stably transfected T47D clones harboring either the pcDNA3 empty vector or Flag-COBRA1-expressing plasmid were treated with either ethanol or E2 for the indicated periods of time and harvested for RNA extraction. ( D ) RT–PCR analysis of the COBRA1 effect on the transcription of various ER-responsive genes. Pools of T47D cells stably transfected with either empty or COBRA1-expressing retroviral vectors were treated with ethanol or E2 (10 nM) for 24 h. RT–PCR was conducted on the total RNA to assess the transcription level of three ERα-responsive genes. In both C and D , β-Actin was used as an internal control. Also shown on the top of the panel is an anti-COBRA1 immunoblot indicating the total level of COBRA1 in the control and Flag-COBRA1-expressing cells.

    Techniques Used: Activation Assay, Luciferase, Transfection, Construct, Northern Blot, Hybridization, Stable Transfection, Clone Assay, Plasmid Preparation, Expressing, RNA Extraction, Reverse Transcription Polymerase Chain Reaction

    27) Product Images from "MicroRNA-183 family conservation and ciliated neurosensory organ expression"

    Article Title: MicroRNA-183 family conservation and ciliated neurosensory organ expression

    Journal: Evolution & development

    doi: 10.1111/j.1525-142X.2007.00217.x

    miR-183 family member expression in sea urchin tube feet. (A) ISH detecting moderate expression of spu-miR-183-like in the sucker cup rim of green sea urchin tube feet compared to negative control lacking probe. (B) Tubulin ICC demonstrates innervation of spu-miR-183-like positive regions containing cilia (arrowheads). Scale bars indicate 100 μm. (C) spu-miR-183-like-primary transcript expression is specific to tube feet. Depicted is primary transcript detection relative to U6 snRNA by RT-PCR of total RNA isolated from sea urchin digestive gland (D), gonad (G), and tube feet (T) in reactions prepared with (+) or without (−) reverse transcriptase (RT) along side 100 bp ladder (L).
    Figure Legend Snippet: miR-183 family member expression in sea urchin tube feet. (A) ISH detecting moderate expression of spu-miR-183-like in the sucker cup rim of green sea urchin tube feet compared to negative control lacking probe. (B) Tubulin ICC demonstrates innervation of spu-miR-183-like positive regions containing cilia (arrowheads). Scale bars indicate 100 μm. (C) spu-miR-183-like-primary transcript expression is specific to tube feet. Depicted is primary transcript detection relative to U6 snRNA by RT-PCR of total RNA isolated from sea urchin digestive gland (D), gonad (G), and tube feet (T) in reactions prepared with (+) or without (−) reverse transcriptase (RT) along side 100 bp ladder (L).

    Techniques Used: Expressing, In Situ Hybridization, Negative Control, Immunocytochemistry, Reverse Transcription Polymerase Chain Reaction, Isolation

    28) Product Images from "Circadian Clock Regulation of Hepatic Lipid Metabolism by Modulation of m6A mRNA Methylation"

    Article Title: Circadian Clock Regulation of Hepatic Lipid Metabolism by Modulation of m6A mRNA Methylation

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.10.068

    ROS Significantly Impacts m 6 A RNA Methylation (A) Representative pictures of ROS detection using dihydroethidium (DHE)-stained liver cryosections from WT and Bmal1 −/− mice. Scale bars, 100 μm. (B) Quantification of ROS formation was achieved by measuring mean gray values of DHE signal per cell using ImageJ software (n = 3 per genotype). (C) The relative cell viability of HepG2 cells assayed by MTT at 24 hr after H 2 O 2 treatment (n = 6). (D) The expression of YTHDF2 mRNA in HepG2 cells 6, 12, and 24 hr after H 2 O 2 treatment (n = 3). (E) Immunoblotting of HepG2 cells after H 2 O 2 treatment for YTHDF2 protein levels normalized to GAPDH (n = 3). (F) Subcellular localization of YTHDF2 in HepG2 cells 6 hr after H 2 O 2 treatment (n = 3). Scale bars, 80 μm. (G) Gene-specific m 6 A validation of m 6 A level changes within the PPaRα gene in HepG2 cells 48 hr after H 2 O 2 treatment (n = 3). (H) Expression of methyltransferase ( METTL3 ), demethylases (ALKBH5 and FTO ), and methyl-specific binding proteins ( YTHDF2 ) mRNA in the liver of WT mice determined by qRT-PCR after injection of APAP or PBS (n = 4–5 per group). (I) The m 6 A enrichment of PPaRα in the liver of mice after injection of APAP or PBS (n = 4–5 per group). . Bmal1 −/− , Bmal1 -AlbCre; WT, Bmal1 f/f ; ZT, Zeitgeber Time. All data are representative of two independent experiments. Data are represented as mean ± SEM. *p
    Figure Legend Snippet: ROS Significantly Impacts m 6 A RNA Methylation (A) Representative pictures of ROS detection using dihydroethidium (DHE)-stained liver cryosections from WT and Bmal1 −/− mice. Scale bars, 100 μm. (B) Quantification of ROS formation was achieved by measuring mean gray values of DHE signal per cell using ImageJ software (n = 3 per genotype). (C) The relative cell viability of HepG2 cells assayed by MTT at 24 hr after H 2 O 2 treatment (n = 6). (D) The expression of YTHDF2 mRNA in HepG2 cells 6, 12, and 24 hr after H 2 O 2 treatment (n = 3). (E) Immunoblotting of HepG2 cells after H 2 O 2 treatment for YTHDF2 protein levels normalized to GAPDH (n = 3). (F) Subcellular localization of YTHDF2 in HepG2 cells 6 hr after H 2 O 2 treatment (n = 3). Scale bars, 80 μm. (G) Gene-specific m 6 A validation of m 6 A level changes within the PPaRα gene in HepG2 cells 48 hr after H 2 O 2 treatment (n = 3). (H) Expression of methyltransferase ( METTL3 ), demethylases (ALKBH5 and FTO ), and methyl-specific binding proteins ( YTHDF2 ) mRNA in the liver of WT mice determined by qRT-PCR after injection of APAP or PBS (n = 4–5 per group). (I) The m 6 A enrichment of PPaRα in the liver of mice after injection of APAP or PBS (n = 4–5 per group). . Bmal1 −/− , Bmal1 -AlbCre; WT, Bmal1 f/f ; ZT, Zeitgeber Time. All data are representative of two independent experiments. Data are represented as mean ± SEM. *p

    Techniques Used: Methylation, Staining, Mouse Assay, Software, MTT Assay, Expressing, Binding Assay, Quantitative RT-PCR, Injection

    29) Product Images from "An alternative splicing switch in FLNB promotes the mesenchymal cell state in human breast cancer"

    Article Title: An alternative splicing switch in FLNB promotes the mesenchymal cell state in human breast cancer

    Journal: eLife

    doi: 10.7554/eLife.37184

    RNA-Seq and eCLIP-Seq analysis of HME cells expressing QKI and RBFOX1. ( A ) (Upper) Venn diagrams illustrating the overlap in AS events regulated by QKI and RBFOX1 for each AS event type, including alternative 5’ splice sites (A5SS), alternative 3’ splice sites (A3SS), mutually exclusive exons (MXE) and retained introns (RI) (related to Figure 4A ). The significance of overlapping events was determined by Fisher’s exact test and p values are shown. (Lower) Scatter plot of the change in ‘Percentage Spliced In’ (PSI) for the corresponding alternative splicing events shared between QKI and RBFOX1. ΔPSI values are for each ORF relative to EGFP (related to Figure 4G ). ( B ) Schematic of the eCLIP-seq experiment. Briefly, protein-RNA interactions were stabilized with UV crosslinking followed by immunoprecipitation. The RNA was then ligated to a 3’ end adapter and reverse-transcribed. An additional adapter was ligated to the 3’ end of the cDNA and the cDNA was amplified by PCR for Illumina sequencing. The crosslinked sites (‘RT stops’) correspond to the 5’ end of sequenced reads and allow the identification of specific protein-RNA interaction sites. ( C ) Interaction between QKI and RBFOX1 as determined by immunoprecipitation of endogenous QKI protein followed by immunoblotting for QKI, RBFOX1 or vinculin. The cell lysate was pretreated with the indicated concentration of RNase at room temperature for 20 min (left). Cell lysate was pretreated with 50 ng/ml of RNase before the immunoprecipitation of endogenous QKI proteins (right).
    Figure Legend Snippet: RNA-Seq and eCLIP-Seq analysis of HME cells expressing QKI and RBFOX1. ( A ) (Upper) Venn diagrams illustrating the overlap in AS events regulated by QKI and RBFOX1 for each AS event type, including alternative 5’ splice sites (A5SS), alternative 3’ splice sites (A3SS), mutually exclusive exons (MXE) and retained introns (RI) (related to Figure 4A ). The significance of overlapping events was determined by Fisher’s exact test and p values are shown. (Lower) Scatter plot of the change in ‘Percentage Spliced In’ (PSI) for the corresponding alternative splicing events shared between QKI and RBFOX1. ΔPSI values are for each ORF relative to EGFP (related to Figure 4G ). ( B ) Schematic of the eCLIP-seq experiment. Briefly, protein-RNA interactions were stabilized with UV crosslinking followed by immunoprecipitation. The RNA was then ligated to a 3’ end adapter and reverse-transcribed. An additional adapter was ligated to the 3’ end of the cDNA and the cDNA was amplified by PCR for Illumina sequencing. The crosslinked sites (‘RT stops’) correspond to the 5’ end of sequenced reads and allow the identification of specific protein-RNA interaction sites. ( C ) Interaction between QKI and RBFOX1 as determined by immunoprecipitation of endogenous QKI protein followed by immunoblotting for QKI, RBFOX1 or vinculin. The cell lysate was pretreated with the indicated concentration of RNase at room temperature for 20 min (left). Cell lysate was pretreated with 50 ng/ml of RNase before the immunoprecipitation of endogenous QKI proteins (right).

    Techniques Used: RNA Sequencing Assay, Expressing, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Sequencing, Concentration Assay

    30) Product Images from "Histone methylation-mediated silencing of miR-139 enhances invasion of non-small-cell lung cancer"

    Article Title: Histone methylation-mediated silencing of miR-139 enhances invasion of non-small-cell lung cancer

    Journal: Cancer Medicine

    doi: 10.1002/cam4.505

    Expression of miR-139 in lung cancer. (A) Expression of miR-139 in primary NSCLCs ( n = 75) and adjacent normal lung tissues ( n = 15). The patient characteristics of the 75 primary NSCLCs are described in Table 1A . Adjacent normal lung tissues were obtained from randomly selected 15 cases. The experiments were in duplicate and the expression levels are relative to the average expression of the 15 normal lung tissues. The statistical significance of the expression difference was determined using Student's t -test. (B) Association of miR-139 and PDE2A expression in 25 lung cancer cell lines and NHBE. U6 small nuclear RNA and ACTB was used as internal controls. The experiments were in triplicate and the list of cell lines is shown in Table S2 . A Pearson's correlation coefficient was calculated between miR-139 and PDE2A expression. (C) Association of miR-139 and PDE2A expression in primary NSCLCs and adjacent normal lung tissues. U6 small nuclear RNA and ACTB was used as internal controls. A Pearson's correlation coefficient was calculated between miR-139 and PDE2A expression. NSCLCs, non-small-cell lung cancer; NHBE, normal human bronchial epithelial cells.
    Figure Legend Snippet: Expression of miR-139 in lung cancer. (A) Expression of miR-139 in primary NSCLCs ( n = 75) and adjacent normal lung tissues ( n = 15). The patient characteristics of the 75 primary NSCLCs are described in Table 1A . Adjacent normal lung tissues were obtained from randomly selected 15 cases. The experiments were in duplicate and the expression levels are relative to the average expression of the 15 normal lung tissues. The statistical significance of the expression difference was determined using Student's t -test. (B) Association of miR-139 and PDE2A expression in 25 lung cancer cell lines and NHBE. U6 small nuclear RNA and ACTB was used as internal controls. The experiments were in triplicate and the list of cell lines is shown in Table S2 . A Pearson's correlation coefficient was calculated between miR-139 and PDE2A expression. (C) Association of miR-139 and PDE2A expression in primary NSCLCs and adjacent normal lung tissues. U6 small nuclear RNA and ACTB was used as internal controls. A Pearson's correlation coefficient was calculated between miR-139 and PDE2A expression. NSCLCs, non-small-cell lung cancer; NHBE, normal human bronchial epithelial cells.

    Techniques Used: Expressing

    Chromatin immunoprecipitation assay. (A) Structure of PDE2A genomic locus. The locations of miR-139 and the ChIP primers are shown. (B) Expression of PDE2A short transcript in five lung cancer cell lines and NHBE. ACTB was used as an internal control. The data are shown as mean ± SD ( n = 3). (C) Expression of miR-139 in five lung cancer cell lines and NHBE. U6 small nuclear RNA was used as an internal control. The data are shown as mean ± SD ( n = 3). (D) Histone methylation status in NHBE. The horizontal axis indicates the numbers of primers shown in Figure 2A . The experiments were in duplicate, and the results shown as the ratio of IP DNA to input DNA. IP/Input values were normalized to a control locus ( ACTB promoter). (E–G) ChIP analysis of H3K4me3, H3K27me3, and H3K9me3 in five lung cancer cell lines and NHBE. The experiments were in duplicate, and IP/Input values were normalized to a control locus ( ACTB promoter). ChIP, chromatin immunoprecipitation; NHBE, normal human bronchial epithelial cells.
    Figure Legend Snippet: Chromatin immunoprecipitation assay. (A) Structure of PDE2A genomic locus. The locations of miR-139 and the ChIP primers are shown. (B) Expression of PDE2A short transcript in five lung cancer cell lines and NHBE. ACTB was used as an internal control. The data are shown as mean ± SD ( n = 3). (C) Expression of miR-139 in five lung cancer cell lines and NHBE. U6 small nuclear RNA was used as an internal control. The data are shown as mean ± SD ( n = 3). (D) Histone methylation status in NHBE. The horizontal axis indicates the numbers of primers shown in Figure 2A . The experiments were in duplicate, and the results shown as the ratio of IP DNA to input DNA. IP/Input values were normalized to a control locus ( ACTB promoter). (E–G) ChIP analysis of H3K4me3, H3K27me3, and H3K9me3 in five lung cancer cell lines and NHBE. The experiments were in duplicate, and IP/Input values were normalized to a control locus ( ACTB promoter). ChIP, chromatin immunoprecipitation; NHBE, normal human bronchial epithelial cells.

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Methylation

    31) Product Images from "Molecular detection of drug resistant polymorphisms in Plasmodium falciparum isolates from Southwest, Nigeria"

    Article Title: Molecular detection of drug resistant polymorphisms in Plasmodium falciparum isolates from Southwest, Nigeria

    Journal: BMC Research Notes

    doi: 10.1186/s13104-020-05334-5

    Messenger RNA transcript levels for Pfk13 and PfATPase using 2 −ΔΔCt . Total RNA was isolated from whole blood preserved in RNALater, reverse transcribed to cDNA in order to measure gene expression profiles
    Figure Legend Snippet: Messenger RNA transcript levels for Pfk13 and PfATPase using 2 −ΔΔCt . Total RNA was isolated from whole blood preserved in RNALater, reverse transcribed to cDNA in order to measure gene expression profiles

    Techniques Used: Isolation, Expressing

    32) Product Images from "Characterization of a Second Rhodococcus erythropolis SQ1 3-Ketosteroid 9?-Hydroxylase Activity Comprising a Terminal Oxygenase Homologue, KshA2, Active with Oxygenase-Reductase Component KshB ▿"

    Article Title: Characterization of a Second Rhodococcus erythropolis SQ1 3-Ketosteroid 9?-Hydroxylase Activity Comprising a Terminal Oxygenase Homologue, KshA2, Active with Oxygenase-Reductase Component KshB ▿

    Journal:

    doi: 10.1128/AEM.00888-08

    Transcriptional analysis of kshA2 , kstD1 (positive control for AD induction), and 16S rRNA (control) by RT-PCR using total RNA isolated from noninduced cultures (lane 1) and AD-induced cultures of parent strain R. erythropolis SQ1 (lane 2). PCR primers
    Figure Legend Snippet: Transcriptional analysis of kshA2 , kstD1 (positive control for AD induction), and 16S rRNA (control) by RT-PCR using total RNA isolated from noninduced cultures (lane 1) and AD-induced cultures of parent strain R. erythropolis SQ1 (lane 2). PCR primers

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

    RNA isolation and RT-PCR experiments.
    Figure Legend Snippet: RNA isolation and RT-PCR experiments.

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction

    33) Product Images from "Ionic currents in intimal cultured synoviocytes from the rabbit"

    Article Title: Ionic currents in intimal cultured synoviocytes from the rabbit

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00028.2010

    Cells from passage 6 (the passage used for electrophysiological studies) were subjected to total RNA extraction using the RNeasy Micro Kit. Total RNA was also prepared from freshly microdissected synovium using the TRIzol method. A : the transcription product was amplified with primers specific for hyaluronan synthase 2 (HAS2), and the resulting DNA bands are shown. HAS2 message was evident in both the passage 6 -cultured synoviocytes (Cult Syn P6) and in intact synovium at dilutions of 1:1 and 1:5 but absent from the nontemplate control (NTC). B, bottom : fixed erythrocyte exclusion test. Under normal conditions the synoviocytes were surrounded by a clear area from which erythrocytes were excluded. This clear area disappeared after hyaluronidase addition, suggesting that it was due to hyaluronan secretion by the synoviocyte. Rab, rabbit; P4H, prolyl 4-hydroxylase. Black calibration bar represents 20 μm in each case.
    Figure Legend Snippet: Cells from passage 6 (the passage used for electrophysiological studies) were subjected to total RNA extraction using the RNeasy Micro Kit. Total RNA was also prepared from freshly microdissected synovium using the TRIzol method. A : the transcription product was amplified with primers specific for hyaluronan synthase 2 (HAS2), and the resulting DNA bands are shown. HAS2 message was evident in both the passage 6 -cultured synoviocytes (Cult Syn P6) and in intact synovium at dilutions of 1:1 and 1:5 but absent from the nontemplate control (NTC). B, bottom : fixed erythrocyte exclusion test. Under normal conditions the synoviocytes were surrounded by a clear area from which erythrocytes were excluded. This clear area disappeared after hyaluronidase addition, suggesting that it was due to hyaluronan secretion by the synoviocyte. Rab, rabbit; P4H, prolyl 4-hydroxylase. Black calibration bar represents 20 μm in each case.

    Techniques Used: RNA Extraction, Amplification, Cell Culture

    34) Product Images from "Opposing activities of oncogenic MIR17HG and tumor suppressive MIR100HG clusters and their gene targets regulate replicative senescence in human adult stem cells"

    Article Title: Opposing activities of oncogenic MIR17HG and tumor suppressive MIR100HG clusters and their gene targets regulate replicative senescence in human adult stem cells

    Journal: NPJ Aging and Mechanisms of Disease

    doi: 10.1038/s41514-017-0006-y

    MiRNA clusters and SEN-associated miRNA (SA-miRNAs) discovered via RNA-seq analysis and experimentally validated by qPCR. a Differential expression of non-coding RNA genes in SR versus SEN hADSCs revealed by RNA-seq analysis. Fold-change values (log 2 SEN/SR) are shown on the x -axis and RPKM differences (log 2 SEN-SR) are shown on the y -axis. SEN upregulated non-coding RNA genes are shown in red (upper right quadrant). b Genomic locations and locus names for SEN upregulated miRNA gene clusters revealed by RNA-seq analysis. c Graphical representation of oncogenic MIR17HG locus and qPCR analysis of mature mirRNA expression in SR ( blue bar ) and senescent (SEN, red bar ) states of hADSCs. Relative expression of either passenger strand mature miRNAs (depicted in the graphs as -3p ) or guide strand mature miRNAs (depicted in the graphs as -5p ) to U6 small RNA was measured. Data are shown as fold change (ΔΔC τ ) The mean ± SD from three independent experiments is shown. The statistical difference was evaluated by Student’s t- test and P- value ( p ) related to experimental measurements and are listed under the graphs, where *** p
    Figure Legend Snippet: MiRNA clusters and SEN-associated miRNA (SA-miRNAs) discovered via RNA-seq analysis and experimentally validated by qPCR. a Differential expression of non-coding RNA genes in SR versus SEN hADSCs revealed by RNA-seq analysis. Fold-change values (log 2 SEN/SR) are shown on the x -axis and RPKM differences (log 2 SEN-SR) are shown on the y -axis. SEN upregulated non-coding RNA genes are shown in red (upper right quadrant). b Genomic locations and locus names for SEN upregulated miRNA gene clusters revealed by RNA-seq analysis. c Graphical representation of oncogenic MIR17HG locus and qPCR analysis of mature mirRNA expression in SR ( blue bar ) and senescent (SEN, red bar ) states of hADSCs. Relative expression of either passenger strand mature miRNAs (depicted in the graphs as -3p ) or guide strand mature miRNAs (depicted in the graphs as -5p ) to U6 small RNA was measured. Data are shown as fold change (ΔΔC τ ) The mean ± SD from three independent experiments is shown. The statistical difference was evaluated by Student’s t- test and P- value ( p ) related to experimental measurements and are listed under the graphs, where *** p

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

    35) Product Images from "Serum amyloid A3 does not contribute to circulating SAA levels"

    Article Title: Serum amyloid A3 does not contribute to circulating SAA levels

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M900089-JLR200

    SAA mRNA expression is increased and apoA-I expression decreased in livers of obese mice. Mice were euthanized and the livers were removed and frozen under liquid nitrogen. Total RNA was extracted using TRIzol reagent and converted to cDNA. SAA1/2 (A),
    Figure Legend Snippet: SAA mRNA expression is increased and apoA-I expression decreased in livers of obese mice. Mice were euthanized and the livers were removed and frozen under liquid nitrogen. Total RNA was extracted using TRIzol reagent and converted to cDNA. SAA1/2 (A),

    Techniques Used: Expressing, Mouse Assay

    SAA3 mRNA expression is increased in intra-abdominal fat of obese mice. Mice were euthanized and epididymal fat pads were removed and frozen under liquid nitrogen. Total RNA was extracted using TRIzol reagent and converted to cDNA. SAA1/2 (A) and SAA3
    Figure Legend Snippet: SAA3 mRNA expression is increased in intra-abdominal fat of obese mice. Mice were euthanized and epididymal fat pads were removed and frozen under liquid nitrogen. Total RNA was extracted using TRIzol reagent and converted to cDNA. SAA1/2 (A) and SAA3

    Techniques Used: Expressing, Mouse Assay

    SAA3 mRNA expression is not increased in subcutaneous fat of obese mice. Mice were euthanized and subcutaneous fat was removed and frozen under liquid nitrogen. Total RNA was extracted using TRIzol reagent and converted to cDNA. SAA1/2 (A) and SAA3 (B)
    Figure Legend Snippet: SAA3 mRNA expression is not increased in subcutaneous fat of obese mice. Mice were euthanized and subcutaneous fat was removed and frozen under liquid nitrogen. Total RNA was extracted using TRIzol reagent and converted to cDNA. SAA1/2 (A) and SAA3 (B)

    Techniques Used: Expressing, Mouse Assay

    36) Product Images from "IgG1 memory B cells keep the memory of IgE responses"

    Article Title: IgG1 memory B cells keep the memory of IgE responses

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00723-0

    Transcriptional profile suggests readiness for activation of DP IgG1 MBC. a Inter-sample similarity between DP, SP and DN IgG1 MBC, IgG1 GC cells and naive B cells, visualised as a multidimensional scaling (MDS) plot. b Connectivity map (CMap) analysis to test the similarity of the transcriptional profile of sorted IgG1 MBC, IgG1 GC and naive B cells, with B cells activated with LPS or LPS + CD40 (GSE35998 data set). a – e The B cell populations were sorted from pooled spleen and mLN of 3 BALB/c mice at 10 weeks after infection with N.b . c – e Heatmaps derived from the RNA-seq analysis showed the clustering of DEG related to c transcriptional regulation, d cellular receptors and e kinases, among IgG1 MBC subsets (DP, SP and DN) and naive B cell samples
    Figure Legend Snippet: Transcriptional profile suggests readiness for activation of DP IgG1 MBC. a Inter-sample similarity between DP, SP and DN IgG1 MBC, IgG1 GC cells and naive B cells, visualised as a multidimensional scaling (MDS) plot. b Connectivity map (CMap) analysis to test the similarity of the transcriptional profile of sorted IgG1 MBC, IgG1 GC and naive B cells, with B cells activated with LPS or LPS + CD40 (GSE35998 data set). a – e The B cell populations were sorted from pooled spleen and mLN of 3 BALB/c mice at 10 weeks after infection with N.b . c – e Heatmaps derived from the RNA-seq analysis showed the clustering of DEG related to c transcriptional regulation, d cellular receptors and e kinases, among IgG1 MBC subsets (DP, SP and DN) and naive B cell samples

    Techniques Used: Activation Assay, Mouse Assay, Infection, Derivative Assay, RNA Sequencing Assay

    37) Product Images from "T-614 Promotes Osteoblastic Cell Differentiation by Increasing Dlx5 Expression and Regulating the Activation of p38 and NF-κB"

    Article Title: T-614 Promotes Osteoblastic Cell Differentiation by Increasing Dlx5 Expression and Regulating the Activation of p38 and NF-κB

    Journal: BioMed Research International

    doi: 10.1155/2018/4901591

    Osteogenesis markers and the related transcription factor expressions in MSCs. The mRNA levels of the osteogenesis markers Alp , Col1 , and Bgp in MSCs cultured in the indicated condition (a) and the mRNA levels of the related transcription factors ( Osterix and Dlx5 ) ( n = 3) (b) were measured by real-time PCR. The β -actin gene was used as a reference to normalize the differences in total RNA in each sample ( n = 3). Statistical analysis is expressed as the means +/− SD. The data are representative of five experiments with similar results. ∗∗ P
    Figure Legend Snippet: Osteogenesis markers and the related transcription factor expressions in MSCs. The mRNA levels of the osteogenesis markers Alp , Col1 , and Bgp in MSCs cultured in the indicated condition (a) and the mRNA levels of the related transcription factors ( Osterix and Dlx5 ) ( n = 3) (b) were measured by real-time PCR. The β -actin gene was used as a reference to normalize the differences in total RNA in each sample ( n = 3). Statistical analysis is expressed as the means +/− SD. The data are representative of five experiments with similar results. ∗∗ P

    Techniques Used: ALP Assay, Cell Culture, Real-time Polymerase Chain Reaction

    38) Product Images from "Natural and Experimental Infection of Caenorhabditis Nematodes by Novel Viruses Related to NodavirusesNematodes Go Viral"

    Article Title: Natural and Experimental Infection of Caenorhabditis Nematodes by Novel Viruses Related to NodavirusesNematodes Go Viral

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1000586

    RNAi-deficient mutants of C. elegans can be infected by the Orsay virus. (A) JU1580bl, N2, rde-1(ne219) ( n = 10 independent replicates each), rde-2(ne221) , rde-4(ne301) , and mut-7(pk204) ( n = 5 independent replicates each) were tested by qRT-PCR for infection with Orsay virus extract. RNA levels were normalized to ama-1 and shown as average fold-change relative to JU1580bl. Error bars represent SEM. Same results as displayed in Figure 5C for N2 and JU1580. (B) Scoring of symptoms in two independent replicates of infection of rde-1 mutant and wild-type N2 animals by the Orsay virus filtrate, after 4 d.
    Figure Legend Snippet: RNAi-deficient mutants of C. elegans can be infected by the Orsay virus. (A) JU1580bl, N2, rde-1(ne219) ( n = 10 independent replicates each), rde-2(ne221) , rde-4(ne301) , and mut-7(pk204) ( n = 5 independent replicates each) were tested by qRT-PCR for infection with Orsay virus extract. RNA levels were normalized to ama-1 and shown as average fold-change relative to JU1580bl. Error bars represent SEM. Same results as displayed in Figure 5C for N2 and JU1580. (B) Scoring of symptoms in two independent replicates of infection of rde-1 mutant and wild-type N2 animals by the Orsay virus filtrate, after 4 d.

    Techniques Used: Infection, Quantitative RT-PCR, Mutagenesis

    Specificity of infection by the Orsay and Santeuil viruses. (A) Specificity of infection by the Orsay virus. Each Caenorhabditis strain (name indicated below the gel) was mock-infected (−) or infected with a virus filtrate (+). RT-PCR on cultures after 7 d at 23°C. See Figure S3 for corresponding morphological symptom scoring. (B) Specificity of infection by the Santeuil virus. RT-PCR results after 4 d at 23°C. (C) Quantitative variation in viral replication N2 versus JU1580. N2 and JU1580 were tested by qRT-PCR for infection with Orsay virus extract ( n = 10 independent replicates for each strain). By conventional RT-PCR assay, Orsay virus infection of N2 yielded positive bands in 3 out of 10 replicate infections whereas 7 out of 10 replicate infections of JU1580bl were positive in these conditions. Control RNA ( n = 6) was extracted from JU1580bl animals grown in parallel without virus filtrate, and to which filtrate was added at the time of sample collection. RNA levels were normalized to ama-1 and shown as average fold-change relative to JU1580bl. Error bars represent SEM.
    Figure Legend Snippet: Specificity of infection by the Orsay and Santeuil viruses. (A) Specificity of infection by the Orsay virus. Each Caenorhabditis strain (name indicated below the gel) was mock-infected (−) or infected with a virus filtrate (+). RT-PCR on cultures after 7 d at 23°C. See Figure S3 for corresponding morphological symptom scoring. (B) Specificity of infection by the Santeuil virus. RT-PCR results after 4 d at 23°C. (C) Quantitative variation in viral replication N2 versus JU1580. N2 and JU1580 were tested by qRT-PCR for infection with Orsay virus extract ( n = 10 independent replicates for each strain). By conventional RT-PCR assay, Orsay virus infection of N2 yielded positive bands in 3 out of 10 replicate infections whereas 7 out of 10 replicate infections of JU1580bl were positive in these conditions. Control RNA ( n = 6) was extracted from JU1580bl animals grown in parallel without virus filtrate, and to which filtrate was added at the time of sample collection. RNA levels were normalized to ama-1 and shown as average fold-change relative to JU1580bl. Error bars represent SEM.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    Molecular evidence of viral infection. (A) RT-PCR detection of the Orsay virus in the original JU1580 wild isolate (I), after bleaching (bl) and after re-infection by a 0.2 µM filtrate after 7 d (RI 1 ) and 3 wk (RI 2 ) of culture at 23°C. (B) RT-PCR detection of the Santeuil virus in the original wild isolate (I), after bleaching (bl) and after re-infection by a 0.2 µM filtrate after 4 d (RI 1 ) and 4 wk (RI 2 ) at 23°C. (C) Northern blots of Santeuil virus RNA1 and RNA2 segments hybridized using a double-stranded DNA probe. (D) Northern blots of Santeuil virus RNA1 segment using + and − sense riboprobes. (E) RNA FISH with a probe targeting Orsay virus RNA1 segment. Representative JU1580bl animals following infection by Orsay virus (top and middle rows) or uninfected (bottom row). S corresponds to ovary sheath cells, OO is an oocyte, and I is an intestinal cell.
    Figure Legend Snippet: Molecular evidence of viral infection. (A) RT-PCR detection of the Orsay virus in the original JU1580 wild isolate (I), after bleaching (bl) and after re-infection by a 0.2 µM filtrate after 7 d (RI 1 ) and 3 wk (RI 2 ) of culture at 23°C. (B) RT-PCR detection of the Santeuil virus in the original wild isolate (I), after bleaching (bl) and after re-infection by a 0.2 µM filtrate after 4 d (RI 1 ) and 4 wk (RI 2 ) at 23°C. (C) Northern blots of Santeuil virus RNA1 and RNA2 segments hybridized using a double-stranded DNA probe. (D) Northern blots of Santeuil virus RNA1 segment using + and − sense riboprobes. (E) RNA FISH with a probe targeting Orsay virus RNA1 segment. Representative JU1580bl animals following infection by Orsay virus (top and middle rows) or uninfected (bottom row). S corresponds to ovary sheath cells, OO is an oocyte, and I is an intestinal cell.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Fluorescence In Situ Hybridization

    39) Product Images from "Small Regulatory RNAs of Rickettsia conorii"

    Article Title: Small Regulatory RNAs of Rickettsia conorii

    Journal: Scientific Reports

    doi: 10.1038/srep36728

    Representative graphs showing the read coverage of novel sRNAs in R. conorii during the infection of HMECs. HMECs were infected with R. conorii (MOI = 20) and total RNA was harvested at 3 and 24 h post-infection. High throughput RNA sequencing was performed on total RNA enriched for bacterial transcripts (see materials and methods). The strand specific reads were mapped onto R. conorii genome (PATRIC genome ID: 27944.4). The read coverage plots of two cis-acting sRNAs ( a ) Rc _sR19 and ( b ) Rc _sR26, and two riboswitches ( c ) Rc _sR3 and ( d ) Rc _sR15 are presented. The reads above and below the x-axis represent the forward (green) and reverse strand (blue), respectively. The identified sRNAs are indicated by red arrow. The ORFs up- and downstream, and cis-acting ORF are shown by black arrow. The orientation and genomic location coordinates correspond to the R. conorii genome annotation in PATRIC.
    Figure Legend Snippet: Representative graphs showing the read coverage of novel sRNAs in R. conorii during the infection of HMECs. HMECs were infected with R. conorii (MOI = 20) and total RNA was harvested at 3 and 24 h post-infection. High throughput RNA sequencing was performed on total RNA enriched for bacterial transcripts (see materials and methods). The strand specific reads were mapped onto R. conorii genome (PATRIC genome ID: 27944.4). The read coverage plots of two cis-acting sRNAs ( a ) Rc _sR19 and ( b ) Rc _sR26, and two riboswitches ( c ) Rc _sR3 and ( d ) Rc _sR15 are presented. The reads above and below the x-axis represent the forward (green) and reverse strand (blue), respectively. The identified sRNAs are indicated by red arrow. The ORFs up- and downstream, and cis-acting ORF are shown by black arrow. The orientation and genomic location coordinates correspond to the R. conorii genome annotation in PATRIC.

    Techniques Used: Infection, High Throughput Screening Assay, RNA Sequencing Assay

    Expression profile of R. conorii novel small RNAs, Rc _sR31, Rc _sR33, Rc _sR35 and Rc _sR42, during the infection of human endothelium and tick cells in vitro . Confluent monolayer of HMECs and AAE2 cells were infected with R. conorii (MOI = 20) for 3 and 24 h. Total RNA was extracted by Tri-reagent ® method and genomic DNA contamination was eliminated by DNase I treatment. Complementary DNA was generated and the sRNA transcript abundance was assessed by quantitative PCR using sRNA specific primers listed in Supplementary table S2 . R. conorii 16S rRNA was used as housekeeping control and HMECs or AAE2 cells infected with R. conorii for 15 minutes were used as baseline. The data from a minimum of three independent experiments were analyzed by ∆∆ CT method and presented as mean ± SEM. Significant differences in fold change of Rc _sR35 and Rc _sR42 were observed depending on the host (bottom panel), while the expression of Rc _sR31 and Rc _sR33 were similar (top panel) during human and tick cell infection in vitro . Legend: Red: Expression of R. conorii sRNAs in human cell line (HMECs); Green: Expression of R. conorii sRNAs in tick cell line (AAE2). * = p
    Figure Legend Snippet: Expression profile of R. conorii novel small RNAs, Rc _sR31, Rc _sR33, Rc _sR35 and Rc _sR42, during the infection of human endothelium and tick cells in vitro . Confluent monolayer of HMECs and AAE2 cells were infected with R. conorii (MOI = 20) for 3 and 24 h. Total RNA was extracted by Tri-reagent ® method and genomic DNA contamination was eliminated by DNase I treatment. Complementary DNA was generated and the sRNA transcript abundance was assessed by quantitative PCR using sRNA specific primers listed in Supplementary table S2 . R. conorii 16S rRNA was used as housekeeping control and HMECs or AAE2 cells infected with R. conorii for 15 minutes were used as baseline. The data from a minimum of three independent experiments were analyzed by ∆∆ CT method and presented as mean ± SEM. Significant differences in fold change of Rc _sR35 and Rc _sR42 were observed depending on the host (bottom panel), while the expression of Rc _sR31 and Rc _sR33 were similar (top panel) during human and tick cell infection in vitro . Legend: Red: Expression of R. conorii sRNAs in human cell line (HMECs); Green: Expression of R. conorii sRNAs in tick cell line (AAE2). * = p

    Techniques Used: Expressing, Infection, In Vitro, Generated, Real-time Polymerase Chain Reaction

    Northern blots showing the expression of selected R. conorii novel trans-acting and known bacterial sRNAs during the infection of HMECs. HMECs were infected with R. conorii (MOI = 20) and total RNA was extracted at 24 h post-infection. The DNase I treated total RNA was enriched for bacterial transcripts using MICROB Enrich and MICROB Express kits (Ambion). Enriched RNA was size separated on 1.5% agarose-formaldehyde gel and transferred onto nylon membranes (BioRad). The membranes were probed with [α- 32 P] UTP-labeled strand-specific RNA probes generated by in vitro transcription. The membranes were washed following the NorthernMax kit manufacturer’s protocol (Ambion) and developed by autoradiography. All sRNAs were expressed as independent transcripts in R. conorii during the infection of HMECs. The scanned images for four novel trans-acting identified in this study ( Rc _sR31, Rc _sR33, Rc _sR35 and Rc _sR42) and four well-known sRNAs (6S, a_tmRNA, RNaseP_bact_a and ffs) are shown. Two bands of varying transcript sizes were detected in Rc _sR33 and Rc _sR42 which may represent both primary and processed transcripts. Asterisk indicates a processed transcript of lower size and a non-specific band in Rc _sR33 is shown by arrow. The adjacent up and downstream genes of each sRNA are shown in green arrows. The sRNA is indicated by red arrow and the estimated size is shown above the arrow. The orientation of all sRNA and ORFs are based on R. conorii genome annotation available in PATRIC. Total RNA from uninfected HMECs was used as a control (C).
    Figure Legend Snippet: Northern blots showing the expression of selected R. conorii novel trans-acting and known bacterial sRNAs during the infection of HMECs. HMECs were infected with R. conorii (MOI = 20) and total RNA was extracted at 24 h post-infection. The DNase I treated total RNA was enriched for bacterial transcripts using MICROB Enrich and MICROB Express kits (Ambion). Enriched RNA was size separated on 1.5% agarose-formaldehyde gel and transferred onto nylon membranes (BioRad). The membranes were probed with [α- 32 P] UTP-labeled strand-specific RNA probes generated by in vitro transcription. The membranes were washed following the NorthernMax kit manufacturer’s protocol (Ambion) and developed by autoradiography. All sRNAs were expressed as independent transcripts in R. conorii during the infection of HMECs. The scanned images for four novel trans-acting identified in this study ( Rc _sR31, Rc _sR33, Rc _sR35 and Rc _sR42) and four well-known sRNAs (6S, a_tmRNA, RNaseP_bact_a and ffs) are shown. Two bands of varying transcript sizes were detected in Rc _sR33 and Rc _sR42 which may represent both primary and processed transcripts. Asterisk indicates a processed transcript of lower size and a non-specific band in Rc _sR33 is shown by arrow. The adjacent up and downstream genes of each sRNA are shown in green arrows. The sRNA is indicated by red arrow and the estimated size is shown above the arrow. The orientation of all sRNA and ORFs are based on R. conorii genome annotation available in PATRIC. Total RNA from uninfected HMECs was used as a control (C).

    Techniques Used: Northern Blot, Expressing, Infection, Labeling, Generated, In Vitro, Autoradiography

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

    Journal: Developmental cell

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

    doi: 10.1016/j.devcel.2017.09.023

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

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

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

    RNA isolation, cDNA synthesis and real-time PCR

    Journal: Molecular oral microbiology

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

    doi: 10.1111/j.2041-1014.2010.00602.x

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

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

    Techniques: Isolation, Real-time Polymerase Chain Reaction

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

    Journal:

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

    doi:

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

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

    Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction

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

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

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

    doi:

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

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

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

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

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

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

    doi:

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

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

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