rna  (Qiagen)


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    RNase Free DNase Set
    Description:
    For DNase digestion during RNA purification Kit contents Qiagen RNase free DNase Set 50 preps For DNase Digestion During RNA Purification Silica gel Membrane Spin column Technology Efficiently Removes the Majority of the DNA Without DNase Treatment The Buffer is Also Well suited for Efficient DNase Digestion in Solution Includes 1500U RNase free DNase I RNase free Buffer RDD and RNase free Water
    Catalog Number:
    79254
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    RNase Free DNase Set
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    Structured Review

    Qiagen rna
    RNase Free DNase Set
    For DNase digestion during RNA purification Kit contents Qiagen RNase free DNase Set 50 preps For DNase Digestion During RNA Purification Silica gel Membrane Spin column Technology Efficiently Removes the Majority of the DNA Without DNase Treatment The Buffer is Also Well suited for Efficient DNase Digestion in Solution Includes 1500U RNase free DNase I RNase free Buffer RDD and RNase free Water
    https://www.bioz.com/result/rna/product/Qiagen
    Average 98 stars, based on 33180 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "Depleting Components of the THO Complex Causes Increased Telomere Length by Reducing the Expression of the Telomere-Associated Protein Rif1p"

    Article Title: Depleting Components of the THO Complex Causes Increased Telomere Length by Reducing the Expression of the Telomere-Associated Protein Rif1p

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033498

    THO2 and HPR1 function in the same pathway as RIF1 to regulate telomere length. ( A ) Yeast DNA from the indicated mutations was isolated and analyzed by Southern blotting assays using the Y′ element probe. ( B ) The decreased Tel1p level does not contribute to telomere lengthening in tho2 and hpr1 cells. Total mRNA from the indicated strains was isolated and analyzed for GCN1 and TEL1 RNA levels using real time RT-PCR (left panel). Results were presented as relative levels normalized to the wild-type expression level. The bars were standard deviations determined from three independent experiments. Yeast DNA from indicated strains was analyzed for telomere length (right panel). ( C ) Overexpressing Rif1p suppresses the telomere lengthening in tho2 and hpr1 cells. Wild-type, tho2 , or hpr1 yeast cells carrying plasmids pRS426 or pRS426- RIF1 (o/e RIF1 ) were cultured at 30°C. Telomere lengths of these cells were then analyzed using Southern blotting assays (top panel). Immunoblotting analysis of the Rif1p level was also performed (bottom panel).
    Figure Legend Snippet: THO2 and HPR1 function in the same pathway as RIF1 to regulate telomere length. ( A ) Yeast DNA from the indicated mutations was isolated and analyzed by Southern blotting assays using the Y′ element probe. ( B ) The decreased Tel1p level does not contribute to telomere lengthening in tho2 and hpr1 cells. Total mRNA from the indicated strains was isolated and analyzed for GCN1 and TEL1 RNA levels using real time RT-PCR (left panel). Results were presented as relative levels normalized to the wild-type expression level. The bars were standard deviations determined from three independent experiments. Yeast DNA from indicated strains was analyzed for telomere length (right panel). ( C ) Overexpressing Rif1p suppresses the telomere lengthening in tho2 and hpr1 cells. Wild-type, tho2 , or hpr1 yeast cells carrying plasmids pRS426 or pRS426- RIF1 (o/e RIF1 ) were cultured at 30°C. Telomere lengths of these cells were then analyzed using Southern blotting assays (top panel). Immunoblotting analysis of the Rif1p level was also performed (bottom panel).

    Techniques Used: Isolation, Southern Blot, Quantitative RT-PCR, Expressing, Cell Culture

    Overexpressing SUB2 cannot restore the Rif1p level and telomere length in tho2 and hpr1 cells. ( A ) Overexpressing SUB2 suppressed the growth defect of tho2 or hpr1 cells. Strains of the indicated genotypes derived from THO2 / tho2 , or HPR1 / hpr1 diploids carrying SUB2 or sub2-5 overexpressing plasmids were grown on YC plates at 30° or 37°C. ( B ) SUB2 or sub2-5 overexpression did not restore the Rif1p level in tho2 or hpr1 cells. Immunoblotting assays were carried out as described. ( C ) SUB2 overexpression did not restore the RIF1 level in tho2 or hpr1 cells. Total mRNA from the indicated strains was isolated and analyzed for the RIF1 RNA level using real time RT-PCR. Results were presented as relative levels normalized to the wild-type expression level. The bars were standard deviations calculated using data from three independent experiments. ( D ) SUB2 or sub2-5 overexpression did not restore the telomere length in tho2 or hpr1 cells. Telomere length analyses were performed as previously described.
    Figure Legend Snippet: Overexpressing SUB2 cannot restore the Rif1p level and telomere length in tho2 and hpr1 cells. ( A ) Overexpressing SUB2 suppressed the growth defect of tho2 or hpr1 cells. Strains of the indicated genotypes derived from THO2 / tho2 , or HPR1 / hpr1 diploids carrying SUB2 or sub2-5 overexpressing plasmids were grown on YC plates at 30° or 37°C. ( B ) SUB2 or sub2-5 overexpression did not restore the Rif1p level in tho2 or hpr1 cells. Immunoblotting assays were carried out as described. ( C ) SUB2 overexpression did not restore the RIF1 level in tho2 or hpr1 cells. Total mRNA from the indicated strains was isolated and analyzed for the RIF1 RNA level using real time RT-PCR. Results were presented as relative levels normalized to the wild-type expression level. The bars were standard deviations calculated using data from three independent experiments. ( D ) SUB2 or sub2-5 overexpression did not restore the telomere length in tho2 or hpr1 cells. Telomere length analyses were performed as previously described.

    Techniques Used: Derivative Assay, Over Expression, Isolation, Quantitative RT-PCR, Expressing

    2) Product Images from "Extensive Natural Variation in Arabidopsis Seed Mucilage Structure"

    Article Title: Extensive Natural Variation in Arabidopsis Seed Mucilage Structure

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2016.00803

    Gene expression in siliques of accessions with altered Gal and Man content. (A,B) qRT-PCR analysis of RNA isolated from 7 DPA siliques. Data show means + SD of two biological replicates analyzed at least twice (only technical error shown for one Le-0 plant). Target gene transcript levels were first normalized to the geometric mean of two reference genes ( UBQ5 and elF4a ), and were then set as 1.0 for Col-0. Significant changes in gene expression relative to Col-0 ( t -test, P
    Figure Legend Snippet: Gene expression in siliques of accessions with altered Gal and Man content. (A,B) qRT-PCR analysis of RNA isolated from 7 DPA siliques. Data show means + SD of two biological replicates analyzed at least twice (only technical error shown for one Le-0 plant). Target gene transcript levels were first normalized to the geometric mean of two reference genes ( UBQ5 and elF4a ), and were then set as 1.0 for Col-0. Significant changes in gene expression relative to Col-0 ( t -test, P

    Techniques Used: Expressing, Quantitative RT-PCR, Isolation

    3) Product Images from "Transcription Profile of Aging and Cognition-Related Genes in the Medial Prefrontal Cortex"

    Article Title: Transcription Profile of Aging and Cognition-Related Genes in the Medial Prefrontal Cortex

    Journal: Frontiers in Aging Neuroscience

    doi: 10.3389/fnagi.2016.00113

    Comparison between RT-qPCR and RNA-seq . Six genes were selected for validation experiments using a subset of animals. Each panel provides the mPFC expression determined by RT-qPCR (left, ΔΔCT values) and RNA-seq (right, counts). Two-tailed t -tests confirmed increased expression of Arc, Fos, Egr1, Egr2, and Egr4 in AI, relative to AU rats. Gene expression for young animals is provided for comparison to aged animals. For two genes, Lin7b and Egr4 , age differences were confirmed ( *** p
    Figure Legend Snippet: Comparison between RT-qPCR and RNA-seq . Six genes were selected for validation experiments using a subset of animals. Each panel provides the mPFC expression determined by RT-qPCR (left, ΔΔCT values) and RNA-seq (right, counts). Two-tailed t -tests confirmed increased expression of Arc, Fos, Egr1, Egr2, and Egr4 in AI, relative to AU rats. Gene expression for young animals is provided for comparison to aged animals. For two genes, Lin7b and Egr4 , age differences were confirmed ( *** p

    Techniques Used: Quantitative RT-PCR, RNA Sequencing Assay, Expressing, Two Tailed Test

    Region of the mPFC and white matter (WM) collected for RNA-seq . The right panel provides a schematic of a coronal slice +2.7 anterior to bregma diagram as adapted from Paxinos and Watson ( 1986 ) and illustrates the region of the mPFC and white matter collected for RNA-seq. The left panel shows a coronal slice from this same region.
    Figure Legend Snippet: Region of the mPFC and white matter (WM) collected for RNA-seq . The right panel provides a schematic of a coronal slice +2.7 anterior to bregma diagram as adapted from Paxinos and Watson ( 1986 ) and illustrates the region of the mPFC and white matter collected for RNA-seq. The left panel shows a coronal slice from this same region.

    Techniques Used: RNA Sequencing Assay

    4) Product Images from "Loss of Ezh2 promotes a midbrain-to-forebrain identity switch by direct gene derepression and Wnt-dependent regulation"

    Article Title: Loss of Ezh2 promotes a midbrain-to-forebrain identity switch by direct gene derepression and Wnt-dependent regulation

    Journal: BMC Biology

    doi: 10.1186/s12915-015-0210-9

    Neural progenitor cell proliferation is controlled by Ezh2-mediated repression of cell cycle and Wnt/β-catenin signaling inhibitors. ( a ) Microarray analysis of three dissected E10.5 control and mutant midbrains identified 126 differentially expressed genes (≥1.75×, P ≤0.01), the majority of which (114) are upregulated upon Ezh2 ablation. Genes further analyzed are indicated. ( b ) qRT-PCR for Ezh2 , cell cycle regulators Cdkn2a and Cdkn2c , and Wnt signaling inhibitors Wif1 and Dkk2 on control and mutant E11.5 midbrains confirms microarray data. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, * P ≤0.05, Student’s t -test. ( c ) Chromatin immunoprecipitation confirms the presence of H3K27me3 at the transcription start site (±500 bp) of Cdkn2a , Cdkn2c , Wif1 , and Dkk2 . Intergenic region Int1 serves as unmethylated negative control. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, Student’s t -test. ( d – e ) In situ hybridization for Cdkn2a ( d ) and Wif1 ( e ) mRNA illustrates increased gene expression in Ezh2 mutants. ( f ) Immunostaining for β-galactosidase + cells on the BAT- gal Wnt/β-catenin signaling reporter line demonstrates diminished signaling in Ezh2-deficient midbrains. n ≥3 in each group, ** P ≤0.01, Student’s t -test. Cartoon insert indicates area of analysis for f and g . ( g ) Immunostaining against CyclinD1 and qRT-PCR. ( h ) Ccnd1 and Lef1 Wnt signaling downstream targets show decreased expression upon Ezh2 ablation. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, Student’s t -test. ( i ) H E staining of E12.5 sagittal midbrain sections of controls and Wnt/β-catenin signaling-ablated embryos. Mutant embryos exhibit reduced neuroepithelium thickness indicated with grey brackets in the magnifications. DAPI staining serves as nuclear marker: f , g ; Scale bars: d , e , 100 μm; f , g , 40 μm; i , 400 μm; Error bars indicate SD; ctrl, Control; dMB, Dorsal midbrain; vMB, Ventral midbrain
    Figure Legend Snippet: Neural progenitor cell proliferation is controlled by Ezh2-mediated repression of cell cycle and Wnt/β-catenin signaling inhibitors. ( a ) Microarray analysis of three dissected E10.5 control and mutant midbrains identified 126 differentially expressed genes (≥1.75×, P ≤0.01), the majority of which (114) are upregulated upon Ezh2 ablation. Genes further analyzed are indicated. ( b ) qRT-PCR for Ezh2 , cell cycle regulators Cdkn2a and Cdkn2c , and Wnt signaling inhibitors Wif1 and Dkk2 on control and mutant E11.5 midbrains confirms microarray data. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, * P ≤0.05, Student’s t -test. ( c ) Chromatin immunoprecipitation confirms the presence of H3K27me3 at the transcription start site (±500 bp) of Cdkn2a , Cdkn2c , Wif1 , and Dkk2 . Intergenic region Int1 serves as unmethylated negative control. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, Student’s t -test. ( d – e ) In situ hybridization for Cdkn2a ( d ) and Wif1 ( e ) mRNA illustrates increased gene expression in Ezh2 mutants. ( f ) Immunostaining for β-galactosidase + cells on the BAT- gal Wnt/β-catenin signaling reporter line demonstrates diminished signaling in Ezh2-deficient midbrains. n ≥3 in each group, ** P ≤0.01, Student’s t -test. Cartoon insert indicates area of analysis for f and g . ( g ) Immunostaining against CyclinD1 and qRT-PCR. ( h ) Ccnd1 and Lef1 Wnt signaling downstream targets show decreased expression upon Ezh2 ablation. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, Student’s t -test. ( i ) H E staining of E12.5 sagittal midbrain sections of controls and Wnt/β-catenin signaling-ablated embryos. Mutant embryos exhibit reduced neuroepithelium thickness indicated with grey brackets in the magnifications. DAPI staining serves as nuclear marker: f , g ; Scale bars: d , e , 100 μm; f , g , 40 μm; i , 400 μm; Error bars indicate SD; ctrl, Control; dMB, Dorsal midbrain; vMB, Ventral midbrain

    Techniques Used: Microarray, Mutagenesis, Quantitative RT-PCR, Chromatin Immunoprecipitation, Negative Control, In Situ Hybridization, Expressing, Immunostaining, Staining, Marker

    5) Product Images from "Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4"

    Article Title: Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4

    Journal: Scientific Reports

    doi: 10.1038/srep30529

    Viperin expression inhibits RABV replication. ( A ) Viperin inhibits RABV replication in viperin-eGFP-transfected BHK-21 cells. The viperin stably expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.1. Virus titres were determined at 24, 36, and 48 hpi. ( B ) RABV proteins in the infected viperin stably expressing BHK-21 cells were detected by Western blotting. ( C ) The N protein/actin, P protein/actin and M protein/actin ratios in Figure 2F were measured using Li-Cor Odyssey 3.0 analytical software version 29. ( D ) RNA expression levels of viperin. rRC-HL vRNA and N mRNA expression levels were detected by qRT-PCR at 24, 36, and 48 hpi. Viperin-expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.01. Data were normalized to β-actin expression and are presented as relative fold expression values to each control cell population infected with rRC-HL.
    Figure Legend Snippet: Viperin expression inhibits RABV replication. ( A ) Viperin inhibits RABV replication in viperin-eGFP-transfected BHK-21 cells. The viperin stably expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.1. Virus titres were determined at 24, 36, and 48 hpi. ( B ) RABV proteins in the infected viperin stably expressing BHK-21 cells were detected by Western blotting. ( C ) The N protein/actin, P protein/actin and M protein/actin ratios in Figure 2F were measured using Li-Cor Odyssey 3.0 analytical software version 29. ( D ) RNA expression levels of viperin. rRC-HL vRNA and N mRNA expression levels were detected by qRT-PCR at 24, 36, and 48 hpi. Viperin-expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.01. Data were normalized to β-actin expression and are presented as relative fold expression values to each control cell population infected with rRC-HL.

    Techniques Used: Expressing, Transfection, Stable Transfection, Infection, Western Blot, Software, RNA Expression, Quantitative RT-PCR

    6) Product Images from "FTO Is a Relevant Factor for the Development of the Metabolic Syndrome in Mice"

    Article Title: FTO Is a Relevant Factor for the Development of the Metabolic Syndrome in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105349

    Detailed analysis of adipose tissue. All data are collected from 30 weeks old mice. *indicate significant p-values between Lep ob/ob ;Fto +/+ and Lep ob/ob ; Fto −/− . a) Weights of different fat pads from female mice (n = 13, 16, 18, 16). b) Area size of epigonadal fat cells from female mice (n = 4, 4, 8, 7). c+d) Expression analysis for different marker genes of epigonadal adipose tissue (n = 6, 4, 5, 5, 5). Following p-values were calculated: between Lep ob/ob ;Fto +/− and Lep ob/ob ; Fto −/− : PPARγ2: p = 0,08, Adiponectin: p = 0,21, TNFα:p = 0,06, IL-6:p = 0,03. Data are presented as mean. Error bars indicate the SEM.
    Figure Legend Snippet: Detailed analysis of adipose tissue. All data are collected from 30 weeks old mice. *indicate significant p-values between Lep ob/ob ;Fto +/+ and Lep ob/ob ; Fto −/− . a) Weights of different fat pads from female mice (n = 13, 16, 18, 16). b) Area size of epigonadal fat cells from female mice (n = 4, 4, 8, 7). c+d) Expression analysis for different marker genes of epigonadal adipose tissue (n = 6, 4, 5, 5, 5). Following p-values were calculated: between Lep ob/ob ;Fto +/− and Lep ob/ob ; Fto −/− : PPARγ2: p = 0,08, Adiponectin: p = 0,21, TNFα:p = 0,06, IL-6:p = 0,03. Data are presented as mean. Error bars indicate the SEM.

    Techniques Used: Mouse Assay, Expressing, Marker

    7) Product Images from "A novel role for the 3′-5′ exoribonuclease Dis3L2 in controlling cell proliferation and tissue growth"

    Article Title: A novel role for the 3′-5′ exoribonuclease Dis3L2 in controlling cell proliferation and tissue growth

    Journal: RNA Biology

    doi: 10.1080/15476286.2016.1232238

    RNA-seq validation. (A) Comparison between the fold changes of the selected transcripts by RNA-seq (red dots) and qRT-PCR (gray bars). The parental control (left) and dis3L2 knockdown (right) values are presented for each transcript. n ≥ 4, error bars represent standard error. See Table 1 for p-values. (B) pyrexia ( pyx) and CG2678 show post-transcriptional changes in gene expression as the mature mRNA (dark gray) but not the pre-mRNA (light gray) show significant increases in expression. cyt-c-d and CG31808 show transcriptional changes as both the pre- (light gray) and mature (dark gray) mRNA increase in expression. The parental control (left) and dis3L2 knockdown (right) values are presented for each transcript for both the pre- and mature mRNA. n ≥ 4, error bars represent standard error. See Table 1 for p-values.
    Figure Legend Snippet: RNA-seq validation. (A) Comparison between the fold changes of the selected transcripts by RNA-seq (red dots) and qRT-PCR (gray bars). The parental control (left) and dis3L2 knockdown (right) values are presented for each transcript. n ≥ 4, error bars represent standard error. See Table 1 for p-values. (B) pyrexia ( pyx) and CG2678 show post-transcriptional changes in gene expression as the mature mRNA (dark gray) but not the pre-mRNA (light gray) show significant increases in expression. cyt-c-d and CG31808 show transcriptional changes as both the pre- (light gray) and mature (dark gray) mRNA increase in expression. The parental control (left) and dis3L2 knockdown (right) values are presented for each transcript for both the pre- and mature mRNA. n ≥ 4, error bars represent standard error. See Table 1 for p-values.

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

    8) Product Images from "Towards an understanding of the molecular basis of effective RNAi against a global insect pest, the whitefly Bemisia tabaci"

    Article Title: Towards an understanding of the molecular basis of effective RNAi against a global insect pest, the whitefly Bemisia tabaci

    Journal: Insect Biochemistry and Molecular Biology

    doi: 10.1016/j.ibmb.2017.07.005

    The dsRNase genes of Bemisia tabaci . (A) Neighbor-joining phylogenetic tree constructed using the protein sequence of the conserved DNA/RNA non-specific nuclease domain of insect dsRNase genes. The numbers at the branches indicate the %bootstrap support, based on the frequency of the clusters for 1000 bootstraps. The d sRNase sequences were: Bombyx mori 2 ( NP_001091744.1 ), Papilio machaon ( XP_014355571.1 ), Spodoptera littorallis (CAR92522.1), Spodoptera frugiperda (CAR92521.1), Aedes aegypti ( XP_001648469.1 ), Drosophila melanogaster 1 ( NM_140821.4 ), D. melanogaster 2 ( NP_649078.1 , CG3819), Tribolium castaneum 1 ( XP_973587.1 ), T. castaneum 2 ( XP_970494.1 ), Schistocerca gregaria 1 ( KJ135008 ), S. gregaria 2 ( KJ135009 ), S. gregaria 3 ( KJ135010 ), S. gregaria 4 ( KJ135011 ), Acyrthosiphon pisum (ACYPI008471), Myzus persicae (MYZPE13164_0_v1.0_000125730.4_pep) and Bemisia tabaci 1(KX390872), B. tabaci 2 (KX390873) and B. tabaci 3 (Unigene11878_BT_Q_SG_ZJU). (B) qRT-PCR analysis of the expression of BtdsRNase-1 (top) and BtdsRNase-2 (bottom) in dissected guts of B. tabaci , relative to the whole body (Wb). Mean ± s. e. from 3 replicates are shown.
    Figure Legend Snippet: The dsRNase genes of Bemisia tabaci . (A) Neighbor-joining phylogenetic tree constructed using the protein sequence of the conserved DNA/RNA non-specific nuclease domain of insect dsRNase genes. The numbers at the branches indicate the %bootstrap support, based on the frequency of the clusters for 1000 bootstraps. The d sRNase sequences were: Bombyx mori 2 ( NP_001091744.1 ), Papilio machaon ( XP_014355571.1 ), Spodoptera littorallis (CAR92522.1), Spodoptera frugiperda (CAR92521.1), Aedes aegypti ( XP_001648469.1 ), Drosophila melanogaster 1 ( NM_140821.4 ), D. melanogaster 2 ( NP_649078.1 , CG3819), Tribolium castaneum 1 ( XP_973587.1 ), T. castaneum 2 ( XP_970494.1 ), Schistocerca gregaria 1 ( KJ135008 ), S. gregaria 2 ( KJ135009 ), S. gregaria 3 ( KJ135010 ), S. gregaria 4 ( KJ135011 ), Acyrthosiphon pisum (ACYPI008471), Myzus persicae (MYZPE13164_0_v1.0_000125730.4_pep) and Bemisia tabaci 1(KX390872), B. tabaci 2 (KX390873) and B. tabaci 3 (Unigene11878_BT_Q_SG_ZJU). (B) qRT-PCR analysis of the expression of BtdsRNase-1 (top) and BtdsRNase-2 (bottom) in dissected guts of B. tabaci , relative to the whole body (Wb). Mean ± s. e. from 3 replicates are shown.

    Techniques Used: Construct, Sequencing, Quantitative RT-PCR, Expressing, Western Blot

    9) Product Images from "Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule"

    Article Title: Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule

    Journal: Journal of Medicinal Chemistry

    doi: 10.1021/acs.jmedchem.7b01781

    Differentially down-regulated genes common to both PANC-1 and MIA PaCa-2 are enriched in PQs after treatment with 400 nM CM03. (a,b) MIA PaCa-2 and PANC-1 cells were treated with 400 nM CM03 for 6 and 24 h and mRNA extracted for analysis by RNA-Seq. Genes were split into four subgroups according to their fold change upon CM03 treatment versus untreated: Down (Log 2 FC
    Figure Legend Snippet: Differentially down-regulated genes common to both PANC-1 and MIA PaCa-2 are enriched in PQs after treatment with 400 nM CM03. (a,b) MIA PaCa-2 and PANC-1 cells were treated with 400 nM CM03 for 6 and 24 h and mRNA extracted for analysis by RNA-Seq. Genes were split into four subgroups according to their fold change upon CM03 treatment versus untreated: Down (Log 2 FC

    Techniques Used: RNA Sequencing Assay

    Validation of mRNA down regulation by qRT-PCR for a subset of down-regulated genes, selected from RNA-Seq experiments. (a–d) MIA PaCa-2 and PANC-1 cells were treated (a and b) with 400 nM CM03 and (c and d) with 400 nM gemcitabine, all for 6 and 24 h. Total mRNA was extracted, reverse transcribed into cDNA, and then qRT-PCR was performed. The C t values were normalized to the genomic mean of three housekeeping genes ( ACTB , GAPDH , and TUBB ), and the relative gene expression was determined using the Livak method, 2 –ΔΔ C t . The log-fold expression changes (Log 2 FC) for each gene are shown relative to vehicle-treated controls (PBS for CM03 and DMSO for gemcitabine). Student’s t test along with 2 –Δ C t values were used to determine the statistical significance of the observed changes, which are the mean of in each case at least three determinations. Those genes with changes in expression with p
    Figure Legend Snippet: Validation of mRNA down regulation by qRT-PCR for a subset of down-regulated genes, selected from RNA-Seq experiments. (a–d) MIA PaCa-2 and PANC-1 cells were treated (a and b) with 400 nM CM03 and (c and d) with 400 nM gemcitabine, all for 6 and 24 h. Total mRNA was extracted, reverse transcribed into cDNA, and then qRT-PCR was performed. The C t values were normalized to the genomic mean of three housekeeping genes ( ACTB , GAPDH , and TUBB ), and the relative gene expression was determined using the Livak method, 2 –ΔΔ C t . The log-fold expression changes (Log 2 FC) for each gene are shown relative to vehicle-treated controls (PBS for CM03 and DMSO for gemcitabine). Student’s t test along with 2 –Δ C t values were used to determine the statistical significance of the observed changes, which are the mean of in each case at least three determinations. Those genes with changes in expression with p

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

    10) Product Images from "Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis"

    Article Title: Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky437

    No overlap in DEGs between the different LOF methods upon depletion of SLC25A25-AS1 . ( A ) Expression levels of SLC25A25-AS1 after RNAi, LNA and CRISPRi-mediated depletion. qPCR revealed only a 25% reduction in SLC25A25-AS1 transcription after siRNA-mediated knockdown relative to negative control siRNA from Dharmacon (Control Dharm), and no significant difference relative to negative control siRNA from Ambion (Control Ambion). LNA-mediated knockdown of SLC25A25-AS1 was performed using LNA oligonucleotide sequence 2 (LNA 2), and showed 90% reduction. CRISPRi-mediated repression of SLC25A25-AS1 using two guide RNAs targeting the TSS of SLC25A25-AS1 relative to the negative (non-targeting) guide RNA 2 yielded 70–90% knockdown in clonal cells. Only one guide RNA (guide 9) was efficient in depleting SLC25A25-AS1 in non-clonal cells. Statistical significance by two-tailed Student's t -test: ** P
    Figure Legend Snippet: No overlap in DEGs between the different LOF methods upon depletion of SLC25A25-AS1 . ( A ) Expression levels of SLC25A25-AS1 after RNAi, LNA and CRISPRi-mediated depletion. qPCR revealed only a 25% reduction in SLC25A25-AS1 transcription after siRNA-mediated knockdown relative to negative control siRNA from Dharmacon (Control Dharm), and no significant difference relative to negative control siRNA from Ambion (Control Ambion). LNA-mediated knockdown of SLC25A25-AS1 was performed using LNA oligonucleotide sequence 2 (LNA 2), and showed 90% reduction. CRISPRi-mediated repression of SLC25A25-AS1 using two guide RNAs targeting the TSS of SLC25A25-AS1 relative to the negative (non-targeting) guide RNA 2 yielded 70–90% knockdown in clonal cells. Only one guide RNA (guide 9) was efficient in depleting SLC25A25-AS1 in non-clonal cells. Statistical significance by two-tailed Student's t -test: ** P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Negative Control, Sequencing, Two Tailed Test

    SLC25A25-AS1 is an archetypal lncRNA expressed in the nucleus. ( A ) Schematic representation of the genomic landscape surrounding SLC25A25-AS1 (annotated in RefSeq as loc100289019; chr9:128108581-128118693, hg38), including three transcriptional start sites ( 101 ) and a polyadenylation site ( 102 ) . SLC25A25-AS1 is not occupied by ribosomes ( 64 ), shows no protein coding potential (PhyloCSF, ( 46 )), and has clear hallmarks of active transcription in HeLa cells (H3K4me3 and H3K27ac data sets obtained from ENCODE via the UCSC browser). The arrows denote the direction of transcription, and green boxes represent the five exons. Note that all PhyloCSF scores at this locus are negative. ( B ) Expression of SLC25A25-AS1 in cytosol and nuclei of ENCODE cell lines ( www.ebi.ac.uk/gxa/home ), shown as reads per kilobase of exon per million reads mapped (RPKM). ( C ) Computational analysis of the mature SLC25A25-AS1 transcript using the CPC and CPAT tools reveals SLC25A25-AS1 has low coding potential. ( D ) Nuclear localization of SLC25A25-AS1 in HeLa cells was determined using single-molecule RNA FISH with exonic probes (green). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar represents 5 μm. ( E ) SLC25A25-AS1 is enriched in chromatin of HeLa cells. RNA distribution in the cytoplasm, nucleoplasm and chromatin was quantified by qPCR, and RPS18 and MALAT1 were used as positive controls for the cytoplasmic and chromatin fraction, respectively. Error bars represent the standard error of the mean (s.e.m) values of four independent experiments.
    Figure Legend Snippet: SLC25A25-AS1 is an archetypal lncRNA expressed in the nucleus. ( A ) Schematic representation of the genomic landscape surrounding SLC25A25-AS1 (annotated in RefSeq as loc100289019; chr9:128108581-128118693, hg38), including three transcriptional start sites ( 101 ) and a polyadenylation site ( 102 ) . SLC25A25-AS1 is not occupied by ribosomes ( 64 ), shows no protein coding potential (PhyloCSF, ( 46 )), and has clear hallmarks of active transcription in HeLa cells (H3K4me3 and H3K27ac data sets obtained from ENCODE via the UCSC browser). The arrows denote the direction of transcription, and green boxes represent the five exons. Note that all PhyloCSF scores at this locus are negative. ( B ) Expression of SLC25A25-AS1 in cytosol and nuclei of ENCODE cell lines ( www.ebi.ac.uk/gxa/home ), shown as reads per kilobase of exon per million reads mapped (RPKM). ( C ) Computational analysis of the mature SLC25A25-AS1 transcript using the CPC and CPAT tools reveals SLC25A25-AS1 has low coding potential. ( D ) Nuclear localization of SLC25A25-AS1 in HeLa cells was determined using single-molecule RNA FISH with exonic probes (green). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar represents 5 μm. ( E ) SLC25A25-AS1 is enriched in chromatin of HeLa cells. RNA distribution in the cytoplasm, nucleoplasm and chromatin was quantified by qPCR, and RPS18 and MALAT1 were used as positive controls for the cytoplasmic and chromatin fraction, respectively. Error bars represent the standard error of the mean (s.e.m) values of four independent experiments.

    Techniques Used: Expressing, Fluorescence In Situ Hybridization, Staining, Real-time Polymerase Chain Reaction

    11) Product Images from "Oxidized phospholipids regulate amino acid metabolism through MTHFD2 to facilitate nucleotide release in endothelial cells"

    Article Title: Oxidized phospholipids regulate amino acid metabolism through MTHFD2 to facilitate nucleotide release in endothelial cells

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04602-0

    OxPAPC elicits ATP release and inhibition of ATP release prevents induction of MTHFD2 . a−d Nucleoside measurement in HAEC exposed to medium (1% FCS) with (oxP) or without (Ct) oxPAPC for 24 h. Cell lysates were measured by mass spectrometry ( n = 6). (* p ≤ 0.05 Student’s t test). e−h Nucleoside measurement in supernatants of HAEC exposed to medium (1% FCS) with or without oxPAPC for 24 h. Supernatants were measured by mass spectrometry ( n = 4) (* p ≤ 0.05 Student’s t -test) i Scheme of flow of serine- and glycine-derived carbons which can be incorporated into the purine backbone. j , k HAEC were treated with 13 C 3 -serine ( j ) or 13 C 2 -glycine ( k ) and oxPAPC or control for 24 h and supernatants were measured by mass spectrometry ( n = 3). Relative fractions of extracellular AMP containing no ( m ), one ( m + 1), two ( m + 2) or three ( m + 3) heavy carbons are shown. l 24 h flux analysis with 13 C 3 -serine labeling in HAEC with or without siRNA mediated knockdown of MTHFD2 ( n = 3). m ATP measurement of supernatants of HAEC exposed to medium (1% FCS) with or without oxPAPC and flufenamic acid (FFA, 50 µM) for 8 h. ATP was measured by luminescence and normalized to intracellular RNA concentration ( n = 7). n , o qRT-PCR detection of MTHFD2 and PHGDH in HAEC exposed to medium (1% FCS) with or without oxPAPC and flufenamic acid (FFA, 50 µM) for 24 h ( n = 5). p , q Spheroid outgrowth assay ( p ) and quantification ( q ) of the cumulative sprout length of HUVEC treated with combinations of oxPAPC, flufenamic acid (FFA, 50 µM) and VEGF-A165 (10 ng ml −1 ) as indicated ( n = 6). Scale bar: 50 µM. Data are represented as mean ± SEM, * p ≤ 0.05 (oxP vs Ct), # p ≤ 0.05 (inhibitor present vs absent), $ p ≤ 0.05 (VEGFA vs Ct), (ANOVA with Bonferroni post-hoc test if not otherwise indicated)
    Figure Legend Snippet: OxPAPC elicits ATP release and inhibition of ATP release prevents induction of MTHFD2 . a−d Nucleoside measurement in HAEC exposed to medium (1% FCS) with (oxP) or without (Ct) oxPAPC for 24 h. Cell lysates were measured by mass spectrometry ( n = 6). (* p ≤ 0.05 Student’s t test). e−h Nucleoside measurement in supernatants of HAEC exposed to medium (1% FCS) with or without oxPAPC for 24 h. Supernatants were measured by mass spectrometry ( n = 4) (* p ≤ 0.05 Student’s t -test) i Scheme of flow of serine- and glycine-derived carbons which can be incorporated into the purine backbone. j , k HAEC were treated with 13 C 3 -serine ( j ) or 13 C 2 -glycine ( k ) and oxPAPC or control for 24 h and supernatants were measured by mass spectrometry ( n = 3). Relative fractions of extracellular AMP containing no ( m ), one ( m + 1), two ( m + 2) or three ( m + 3) heavy carbons are shown. l 24 h flux analysis with 13 C 3 -serine labeling in HAEC with or without siRNA mediated knockdown of MTHFD2 ( n = 3). m ATP measurement of supernatants of HAEC exposed to medium (1% FCS) with or without oxPAPC and flufenamic acid (FFA, 50 µM) for 8 h. ATP was measured by luminescence and normalized to intracellular RNA concentration ( n = 7). n , o qRT-PCR detection of MTHFD2 and PHGDH in HAEC exposed to medium (1% FCS) with or without oxPAPC and flufenamic acid (FFA, 50 µM) for 24 h ( n = 5). p , q Spheroid outgrowth assay ( p ) and quantification ( q ) of the cumulative sprout length of HUVEC treated with combinations of oxPAPC, flufenamic acid (FFA, 50 µM) and VEGF-A165 (10 ng ml −1 ) as indicated ( n = 6). Scale bar: 50 µM. Data are represented as mean ± SEM, * p ≤ 0.05 (oxP vs Ct), # p ≤ 0.05 (inhibitor present vs absent), $ p ≤ 0.05 (VEGFA vs Ct), (ANOVA with Bonferroni post-hoc test if not otherwise indicated)

    Techniques Used: Inhibition, Mass Spectrometry, Flow Cytometry, Derivative Assay, Labeling, Concentration Assay, Quantitative RT-PCR

    12) Product Images from "Genome-wide transcriptional profiling for elucidating the effects of brassinosteroids on Glycine max during early vegetative development"

    Article Title: Genome-wide transcriptional profiling for elucidating the effects of brassinosteroids on Glycine max during early vegetative development

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52599-3

    Validation and comparison of eight differently expressed genes selected from RNA-seq analysis by qRT-PCR. qRT-PCR data were normalized to the stable endogenous control (Cyclin gene). The fold changes are presented as mean with standard errors (SE) of three biological replications. RNA-seq results showed in gray and qRT-PCR results showed in black.
    Figure Legend Snippet: Validation and comparison of eight differently expressed genes selected from RNA-seq analysis by qRT-PCR. qRT-PCR data were normalized to the stable endogenous control (Cyclin gene). The fold changes are presented as mean with standard errors (SE) of three biological replications. RNA-seq results showed in gray and qRT-PCR results showed in black.

    Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR

    13) Product Images from "IFI16, a nuclear innate immune DNA sensor, mediates epigenetic silencing of herpesvirus genomes by its association with H3K9 methyltransferases SUV39H1 and GLP"

    Article Title: IFI16, a nuclear innate immune DNA sensor, mediates epigenetic silencing of herpesvirus genomes by its association with H3K9 methyltransferases SUV39H1 and GLP

    Journal: eLife

    doi: 10.7554/eLife.49500

    Effect of IFI16 knockdown (KD) on H3K9me3 and RNA Pol II deposition on KSHV lytic gene promoters. ( A ) IFI16 was KD in BCBL-1 cells using shRNA lentivirus for 72 hr and KD efficiency was assessed by q-RT PCR and successful induction of lytic KSHV ORF50 gene as a result of IFI16 KD was assessed by q-RT PCR of ORF50. ( B ) WB showing IFI16 KD compared to untreated or shC treated BCBL-1 cells. ( C ) ChIP was performed after lentivirus-mediated IFI16 KD in BCBL-1 cells or shC-BCBL-1 cells. Deposition of different histone H3 lysine tri-methylation marks (H3, H3K4me3, H3K9me3, H3K27me3, H3K36me3 and H3K79me3) and RNA Pol II on four different KSHV promoters (pORF73- La, pK8- IE, pvIRF2- E, and pORF63- L) representing the four different temporal KSHV gene classes were tested by q-PCR. ChIP efficiencies normalized to input chromatin are shown as relative to shC control. ( D - H ) TIME cells were electroporated with either siC or siIFI16. After 72 hr, cells were de novo infected with KSHV (100 DNA copies/cell) for 6 or 48 hr. IFI16 KD efficiencies were assessed by q-RT PCR of the IFI16 gene ( D ) and WB of IFI16 ( E ). ( F ) ChIP was performed after 48 hr of de novo infection of IFI16 KD TIME. ( G and H ) q-RT PCR (one step TaqMan) of KSHV latent ORF73 ( G ) and lytic ORF50 ( H ) mRNA expression normalized to cellular RNaseP after 6 and 48 hr of de novo infection of TIME cells previously treated with siIFI16 of siC for 72 hr. ( I ) Lytic cycle was induced in TRExBCBL1-RTA cells using doxycycline. At 0, 1, 2, 3 and 4 days post-induction, ChIP was performed. Deposition of different H3 lysine tri-methylation marks (H3, H3K4me3, H3K9me3 and H3K27me3) and RNA Pol II on the ORF63 promoters was tested by q-PCR. ChIP with control IgG was also performed. ChIP efficiencies are represented as % input. Data shown are averages of the results of at least three experiments ± SD. *=p
    Figure Legend Snippet: Effect of IFI16 knockdown (KD) on H3K9me3 and RNA Pol II deposition on KSHV lytic gene promoters. ( A ) IFI16 was KD in BCBL-1 cells using shRNA lentivirus for 72 hr and KD efficiency was assessed by q-RT PCR and successful induction of lytic KSHV ORF50 gene as a result of IFI16 KD was assessed by q-RT PCR of ORF50. ( B ) WB showing IFI16 KD compared to untreated or shC treated BCBL-1 cells. ( C ) ChIP was performed after lentivirus-mediated IFI16 KD in BCBL-1 cells or shC-BCBL-1 cells. Deposition of different histone H3 lysine tri-methylation marks (H3, H3K4me3, H3K9me3, H3K27me3, H3K36me3 and H3K79me3) and RNA Pol II on four different KSHV promoters (pORF73- La, pK8- IE, pvIRF2- E, and pORF63- L) representing the four different temporal KSHV gene classes were tested by q-PCR. ChIP efficiencies normalized to input chromatin are shown as relative to shC control. ( D - H ) TIME cells were electroporated with either siC or siIFI16. After 72 hr, cells were de novo infected with KSHV (100 DNA copies/cell) for 6 or 48 hr. IFI16 KD efficiencies were assessed by q-RT PCR of the IFI16 gene ( D ) and WB of IFI16 ( E ). ( F ) ChIP was performed after 48 hr of de novo infection of IFI16 KD TIME. ( G and H ) q-RT PCR (one step TaqMan) of KSHV latent ORF73 ( G ) and lytic ORF50 ( H ) mRNA expression normalized to cellular RNaseP after 6 and 48 hr of de novo infection of TIME cells previously treated with siIFI16 of siC for 72 hr. ( I ) Lytic cycle was induced in TRExBCBL1-RTA cells using doxycycline. At 0, 1, 2, 3 and 4 days post-induction, ChIP was performed. Deposition of different H3 lysine tri-methylation marks (H3, H3K4me3, H3K9me3 and H3K27me3) and RNA Pol II on the ORF63 promoters was tested by q-PCR. ChIP with control IgG was also performed. ChIP efficiencies are represented as % input. Data shown are averages of the results of at least three experiments ± SD. *=p

    Techniques Used: shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Chromatin Immunoprecipitation, Methylation, Polymerase Chain Reaction, Infection, Expressing

    14) Product Images from "MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae"

    Article Title: MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1458171

    MoSNT2 is associated with the MoTor signaling pathway. (A) Vegetative growth of M. oryzae on CM agar medium supplemented with or without 1 μg/ml rapamycin (rapa.). (B) Inhibition rate of rapamycin on the mycelial growth. (C) Expression profiles of MoSNT2 and MoTOR in the wild-type Guy11 strain at different developmental processes. (D) Linear correlation between qRT-PCR-measured expression levels of MoSNT2 and MoTOR . (E) qRT-PCR analysis of MoSNT2 expression levels in the Guy11 strain in response to rapamycin. The Guy11 strain grown in liquid CM for 48 h was transferred into fresh liquid CM in the presence or absence of 1 μg/ml rapamycin for 6 h before total RNA extraction.
    Figure Legend Snippet: MoSNT2 is associated with the MoTor signaling pathway. (A) Vegetative growth of M. oryzae on CM agar medium supplemented with or without 1 μg/ml rapamycin (rapa.). (B) Inhibition rate of rapamycin on the mycelial growth. (C) Expression profiles of MoSNT2 and MoTOR in the wild-type Guy11 strain at different developmental processes. (D) Linear correlation between qRT-PCR-measured expression levels of MoSNT2 and MoTOR . (E) qRT-PCR analysis of MoSNT2 expression levels in the Guy11 strain in response to rapamycin. The Guy11 strain grown in liquid CM for 48 h was transferred into fresh liquid CM in the presence or absence of 1 μg/ml rapamycin for 6 h before total RNA extraction.

    Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, RNA Extraction

    15) Product Images from "Knockdown of LMP1-induced miR-155 sensitizes nasopharyngeal carcinoma cells to radiotherapy in vitro"

    Article Title: Knockdown of LMP1-induced miR-155 sensitizes nasopharyngeal carcinoma cells to radiotherapy in vitro

    Journal: Oncology Letters

    doi: 10.3892/ol.2016.4400

    LMP1 of Epstein-Barr virus promotes miR-155 expression in CNE-2 cells. CNE-2 cells were separated into three groups: Non-transfected CNE-2 cells (Blank), CNE-2 cells transfected with empty pcDNA3.1 as negative control (Con) and LMP1-pcDNA3.1-transfected CNE-2 cells (LMP1). (A) Relative mRNA expression levels of LMP1 in the three groups of CNE-2 cells, compared with the levels of GAPDH. (B) Percentage of LMP1 expression at the protein level in the three CNE-2 cell groups, as revealed by western blot analysis. (C) Relative mRNA levels of LMP1 vs. GAPDH in the three groups of CNE-2 cells upon a number of serial passages, as determined by reverse transcription-quantitative polymerase chain reaction. (D) Overexpression of LMP1 protein in CNE-2 cells following a number of serial passages. (E) Relative miR-155 levels in the three groups of CNE-2 cells, compared with the levels of U6 snRNA. (F) Relative miR-155 levels in CNE-2 cells transfected with various concentrations of LMP1-pcDNA3.1, compared with the levels of U6 snRNA. *P
    Figure Legend Snippet: LMP1 of Epstein-Barr virus promotes miR-155 expression in CNE-2 cells. CNE-2 cells were separated into three groups: Non-transfected CNE-2 cells (Blank), CNE-2 cells transfected with empty pcDNA3.1 as negative control (Con) and LMP1-pcDNA3.1-transfected CNE-2 cells (LMP1). (A) Relative mRNA expression levels of LMP1 in the three groups of CNE-2 cells, compared with the levels of GAPDH. (B) Percentage of LMP1 expression at the protein level in the three CNE-2 cell groups, as revealed by western blot analysis. (C) Relative mRNA levels of LMP1 vs. GAPDH in the three groups of CNE-2 cells upon a number of serial passages, as determined by reverse transcription-quantitative polymerase chain reaction. (D) Overexpression of LMP1 protein in CNE-2 cells following a number of serial passages. (E) Relative miR-155 levels in the three groups of CNE-2 cells, compared with the levels of U6 snRNA. (F) Relative miR-155 levels in CNE-2 cells transfected with various concentrations of LMP1-pcDNA3.1, compared with the levels of U6 snRNA. *P

    Techniques Used: Expressing, Transfection, Negative Control, Western Blot, Real-time Polymerase Chain Reaction, Over Expression

    16) Product Images from "Characterization of macroautophagic flux in vivo using a leupeptin-based assay"

    Article Title: Characterization of macroautophagic flux in vivo using a leupeptin-based assay

    Journal: Autophagy

    doi: 10.4161/auto.7.6.15100

    Analysis of basal macroautophagic flux in beclin 1 +/+ and beclin 1 +/− mice using the leupeptin assay. Mice were injected with PBS or 40 mg/kg leupeptin and sacrificed 0–180 min later (n = 3–4 mice per time point). (A) Western blot
    Figure Legend Snippet: Analysis of basal macroautophagic flux in beclin 1 +/+ and beclin 1 +/− mice using the leupeptin assay. Mice were injected with PBS or 40 mg/kg leupeptin and sacrificed 0–180 min later (n = 3–4 mice per time point). (A) Western blot

    Techniques Used: Mouse Assay, Injection, Western Blot

    17) Product Images from "Preclinical development of HIvax: Human survivin highly immunogenic vaccines"

    Article Title: Preclinical development of HIvax: Human survivin highly immunogenic vaccines

    Journal: Human Vaccines & Immunotherapeutics

    doi: 10.1080/21645515.2015.1050572

    HIvax1 and HIvax2 induce activation of CD8 + and CD4 + T cells, respectively, in a donor with haplotype HLA-A 01:01, 11:01; HLA-B 08:01, 42:01; HLA-DRB1 03:01. Human dendritic cells (DC) were infected with HIvax1 or HIvax2 and co-cultured for 2 wks with
    Figure Legend Snippet: HIvax1 and HIvax2 induce activation of CD8 + and CD4 + T cells, respectively, in a donor with haplotype HLA-A 01:01, 11:01; HLA-B 08:01, 42:01; HLA-DRB1 03:01. Human dendritic cells (DC) were infected with HIvax1 or HIvax2 and co-cultured for 2 wks with

    Techniques Used: Activation Assay, Infection, Cell Culture

    Survivin epitopes have been included in HIvax1 e HIvax2. ( A ) HIvax 1 and 2 diagrams. Epitopes from Hsurv 5–7 were included in HIvax1, separated by AAY spacers and targeted to the ER by including an IgK signal sequence. This strategy facilitates
    Figure Legend Snippet: Survivin epitopes have been included in HIvax1 e HIvax2. ( A ) HIvax 1 and 2 diagrams. Epitopes from Hsurv 5–7 were included in HIvax1, separated by AAY spacers and targeted to the ER by including an IgK signal sequence. This strategy facilitates

    Techniques Used: Sequencing

    Cytolytic potential of HIvax-activated (T)cells against malignant mesothelioma cells overexpressing survivin. ( A ) ELISPOT analysis of released granzyme B was performed after stimulation by DC infected with FP-ctrl or HIvax1/HIvax2 simultaneously (HIvax).
    Figure Legend Snippet: Cytolytic potential of HIvax-activated (T)cells against malignant mesothelioma cells overexpressing survivin. ( A ) ELISPOT analysis of released granzyme B was performed after stimulation by DC infected with FP-ctrl or HIvax1/HIvax2 simultaneously (HIvax).

    Techniques Used: Enzyme-linked Immunospot, Infection

    18) Product Images from "Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza"

    Article Title: Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04792-7

    The transfection efficiency and antiviral activity of TAT-P1 with DIG-3 could be increased by P1 peptide. a Transfection efficiency of TAT-P1/pLuc increased by additional ATPase inhibitor (bafilomycin A1) or P1 peptide. Before TAT-P1/pLuc (2.0 μg/0.5 μg) complex was added to 293T cells for transfection, the indicated concentrations of bafilomycin A1 (A1, nM), P1 peptide (μg per ml), or PA1 peptide (μg per ml) were added to cell culture media. Mock indicates cells treated with A1 or TAT-P1 without DNA. Data were presented as mean ± SD of three independent experiments. b Transfection efficiency of TAT-P1/pCMV-Luc increased by P1 in mouse lungs. c Representative In Vivo Imaging System image showed increased luciferase expression by P1 in mouse lungs. TAT-P1/pCMV-Luc (20 μg/5 μg) with additional P1 (20 μg, 10 μg, or 0 μg) or PA1 (20 μg) were inoculated to mouse lungs at 48 and 24 h before measuring bioluminescence signal or taking bioluminescence image. Mock indicates mouse lungs inoculated with TAT-P1 + P1 without DNA. d The RNA expression of DI-PA increased by P1 in mouse lungs. TAT-P1/DI-PA (20 μg/5 μg) with additional P1 (20 μg, 10 μg, or 0 μg) were inoculated to mouse lungs at 48 and 24 h before detecting RNA expression. Data were presented as mean ± SD of ≥3 mice. * Indicates P
    Figure Legend Snippet: The transfection efficiency and antiviral activity of TAT-P1 with DIG-3 could be increased by P1 peptide. a Transfection efficiency of TAT-P1/pLuc increased by additional ATPase inhibitor (bafilomycin A1) or P1 peptide. Before TAT-P1/pLuc (2.0 μg/0.5 μg) complex was added to 293T cells for transfection, the indicated concentrations of bafilomycin A1 (A1, nM), P1 peptide (μg per ml), or PA1 peptide (μg per ml) were added to cell culture media. Mock indicates cells treated with A1 or TAT-P1 without DNA. Data were presented as mean ± SD of three independent experiments. b Transfection efficiency of TAT-P1/pCMV-Luc increased by P1 in mouse lungs. c Representative In Vivo Imaging System image showed increased luciferase expression by P1 in mouse lungs. TAT-P1/pCMV-Luc (20 μg/5 μg) with additional P1 (20 μg, 10 μg, or 0 μg) or PA1 (20 μg) were inoculated to mouse lungs at 48 and 24 h before measuring bioluminescence signal or taking bioluminescence image. Mock indicates mouse lungs inoculated with TAT-P1 + P1 without DNA. d The RNA expression of DI-PA increased by P1 in mouse lungs. TAT-P1/DI-PA (20 μg/5 μg) with additional P1 (20 μg, 10 μg, or 0 μg) were inoculated to mouse lungs at 48 and 24 h before detecting RNA expression. Data were presented as mean ± SD of ≥3 mice. * Indicates P

    Techniques Used: Transfection, Activity Assay, Cell Culture, In Vivo Imaging, Luciferase, Expressing, RNA Expression, Mouse Assay

    Construction and antiviral activity of defective interfering genes (DIG). a The plasmid construction of DI-PB2, DI-PB1, and DI-PA. The indicated sequences of shortened viral polymerase gene PB2, PB1, and PA were inserted into phw2000, respectively. Dotted lines indicate the internal deletion of wild-type (WT) viral polymerase genes. b , c DI RNA expression in 293T and A549 cells. The plasmids of DI-PB2, DI-PB1, and DI-PA were co-transfected into cells with the indicated concentrations. At 24 h post transfection, DI RNAs were extracted from cells and digested by DNase I for RT-qPCR. Empty vector was used as a negative control for RT-qPCR. d Anti-A(H7N7) virus activity of individual plasmid of DI-PB2, DI-PB1, and DI-PA or three combined plasmid DIG (DIG-3, 0.6 μg per well). e , f Dose-dependent anti-A(H7N7) virus activity of DIG-3 in 293T and A549 cells. g Anti-A(H5N1) virus activity of DIG-3. Empty vector phw2000 and plasmids with DIG were individually transfected to cells. At 24 h post transfection, cells were infected with A(H7N7) or A(H5N1) virus at MOI = 0.005 and cell supernatants were collected at 40 h post infection. Viral titers in the supernatants were detected by plaque assay. Data were presented as mean ± SD of three independent experiments. * Indicates P
    Figure Legend Snippet: Construction and antiviral activity of defective interfering genes (DIG). a The plasmid construction of DI-PB2, DI-PB1, and DI-PA. The indicated sequences of shortened viral polymerase gene PB2, PB1, and PA were inserted into phw2000, respectively. Dotted lines indicate the internal deletion of wild-type (WT) viral polymerase genes. b , c DI RNA expression in 293T and A549 cells. The plasmids of DI-PB2, DI-PB1, and DI-PA were co-transfected into cells with the indicated concentrations. At 24 h post transfection, DI RNAs were extracted from cells and digested by DNase I for RT-qPCR. Empty vector was used as a negative control for RT-qPCR. d Anti-A(H7N7) virus activity of individual plasmid of DI-PB2, DI-PB1, and DI-PA or three combined plasmid DIG (DIG-3, 0.6 μg per well). e , f Dose-dependent anti-A(H7N7) virus activity of DIG-3 in 293T and A549 cells. g Anti-A(H5N1) virus activity of DIG-3. Empty vector phw2000 and plasmids with DIG were individually transfected to cells. At 24 h post transfection, cells were infected with A(H7N7) or A(H5N1) virus at MOI = 0.005 and cell supernatants were collected at 40 h post infection. Viral titers in the supernatants were detected by plaque assay. Data were presented as mean ± SD of three independent experiments. * Indicates P

    Techniques Used: Activity Assay, Plasmid Preparation, RNA Expression, Transfection, Quantitative RT-PCR, Negative Control, Infection, Plaque Assay

    19) Product Images from "Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells"

    Article Title: Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells

    Journal: Immunity

    doi: 10.1016/j.immuni.2018.01.013

    BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from RNA-Seq analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of DNase-Seq read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.
    Figure Legend Snippet: BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from RNA-Seq analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of DNase-Seq read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.

    Techniques Used: Binding Assay, Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation, Modification, Marker

    20) Product Images from "Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells"

    Article Title: Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells

    Journal: Immunity

    doi: 10.1016/j.immuni.2018.01.013

    BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from RNA-Seq analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of DNase-Seq read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.
    Figure Legend Snippet: BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from RNA-Seq analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of DNase-Seq read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.

    Techniques Used: Binding Assay, Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation, Modification, Marker

    21) Product Images from "Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses"

    Article Title: Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.01588

    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.
    Figure Legend Snippet: Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Techniques Used: Environmental Sampling, Purification, Real-time Polymerase Chain Reaction, Lysis

    22) Product Images from "HeT-A_pi1, a piRNA Target Sequence in the Drosophila Telomeric Retrotransposon HeT-A, Is Extremely Conserved across Copies and Species"

    Article Title: HeT-A_pi1, a piRNA Target Sequence in the Drosophila Telomeric Retrotransposon HeT-A, Is Extremely Conserved across Copies and Species

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0037405

    Alignment of 3′UTR transcripts obtained from testes and ovaries of D. melanogaster Oregon R. Nucleotide polymorphisms are indicated. piRNA target HeT-A_pi1 is labelled with a red rectangle.
    Figure Legend Snippet: Alignment of 3′UTR transcripts obtained from testes and ovaries of D. melanogaster Oregon R. Nucleotide polymorphisms are indicated. piRNA target HeT-A_pi1 is labelled with a red rectangle.

    Techniques Used:

    23) Product Images from "Cholecalciferol (Vitamin D3) Improves Myelination and Recovery after Nerve Injury"

    Article Title: Cholecalciferol (Vitamin D3) Improves Myelination and Recovery after Nerve Injury

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065034

    Analysis of the main functions altered by vitamin D supplementation using the Ingenuity Pathway Analysis Tool. A. List of functions for the genes involved in “nervous system development and function” whose expression was altered after addition of calcitriol to Schwann cells ( A ) or Schwann cells and dorsal root ganglion cells ( B ). Red arrows indicate an over-expression; green arrows, an under-expression. C. Twenty-five nervous system-related genes were used to generate a network representation. The genes shaded red are upregulated and those that are green are downregulated. The intensity of the shading shows to what degree each gene was up- or downregulated. The genes in white colour were not significantly changed in the analysis and can be considered as “missing links”. Orange solid lines represent a known direct interaction between calcitriol and the genes present in the network.
    Figure Legend Snippet: Analysis of the main functions altered by vitamin D supplementation using the Ingenuity Pathway Analysis Tool. A. List of functions for the genes involved in “nervous system development and function” whose expression was altered after addition of calcitriol to Schwann cells ( A ) or Schwann cells and dorsal root ganglion cells ( B ). Red arrows indicate an over-expression; green arrows, an under-expression. C. Twenty-five nervous system-related genes were used to generate a network representation. The genes shaded red are upregulated and those that are green are downregulated. The intensity of the shading shows to what degree each gene was up- or downregulated. The genes in white colour were not significantly changed in the analysis and can be considered as “missing links”. Orange solid lines represent a known direct interaction between calcitriol and the genes present in the network.

    Techniques Used: Expressing, Over Expression

    Main metabolic pathways associated to in vitro calcitriol supplementation. A. Venn diagram showing the functional pathways affected by the addition of calcitriol in cultures of Schwann cells or in co-cultures of DRG/Schwann cells. Five of the fifteen metabolic calcitriol-regulated pathways are affected in both cell types. B. Validation by qPCR of four selected up-regulated genes ( Prx, Tspan2, IgF1, Spp1 ) involved in axogenesis and myelination.
    Figure Legend Snippet: Main metabolic pathways associated to in vitro calcitriol supplementation. A. Venn diagram showing the functional pathways affected by the addition of calcitriol in cultures of Schwann cells or in co-cultures of DRG/Schwann cells. Five of the fifteen metabolic calcitriol-regulated pathways are affected in both cell types. B. Validation by qPCR of four selected up-regulated genes ( Prx, Tspan2, IgF1, Spp1 ) involved in axogenesis and myelination.

    Techniques Used: In Vitro, Functional Assay, Real-time Polymerase Chain Reaction

    24) Product Images from "Tracking Fungal Community Responses to Maize Plants by DNA- and RNA-Based Pyrosequencing"

    Article Title: Tracking Fungal Community Responses to Maize Plants by DNA- and RNA-Based Pyrosequencing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0069973

    Percentage of fungal OTUs shared between nucleic acid type (DNA; RNA) in different sampling times (t1 = 47 days; t2 = 104 days).
    Figure Legend Snippet: Percentage of fungal OTUs shared between nucleic acid type (DNA; RNA) in different sampling times (t1 = 47 days; t2 = 104 days).

    Techniques Used: Sampling

    NMDS analysis based on relative abundance of fungal OTUs based on environmental DNA and RNA both at sampling times t1-47 days and t2-104 days of plant age.
    Figure Legend Snippet: NMDS analysis based on relative abundance of fungal OTUs based on environmental DNA and RNA both at sampling times t1-47 days and t2-104 days of plant age.

    Techniques Used: Sampling

    Relative abundance of different fungal phyla recovered from environmental DNA and RNA in soils with two maize cultivars (M = Monumental; D = DKC 3420) and their respective genetically modified lines (M-GM = event MON810; D-GM = DKC 3421YG) at two different sampling times (t1 = 47 days; t2 = 104 days) of plant age.
    Figure Legend Snippet: Relative abundance of different fungal phyla recovered from environmental DNA and RNA in soils with two maize cultivars (M = Monumental; D = DKC 3420) and their respective genetically modified lines (M-GM = event MON810; D-GM = DKC 3421YG) at two different sampling times (t1 = 47 days; t2 = 104 days) of plant age.

    Techniques Used: Genetically Modified, Sampling

    Non-metric multidimensional scaling (NMDS) analysis of presence and absence of fungal OTUs based on Jaccard-index of similarity with 95% confidence intervals shared between sampling times (t1 = 47 days; t2 = 104 days) and nucleic acid type (DNA, RNA).
    Figure Legend Snippet: Non-metric multidimensional scaling (NMDS) analysis of presence and absence of fungal OTUs based on Jaccard-index of similarity with 95% confidence intervals shared between sampling times (t1 = 47 days; t2 = 104 days) and nucleic acid type (DNA, RNA).

    Techniques Used: Sampling

    25) Product Images from "Molecular and Cellular Features of Murine Craniofacial and Trunk Neural Crest Cells as Stem Cell-Like Cells"

    Article Title: Molecular and Cellular Features of Murine Craniofacial and Trunk Neural Crest Cells as Stem Cell-Like Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0084072

    Differential expression profiles of cNCCs and tNCCs in P0-Cre/Floxed-EGFP mouse embryos. (A) Scatter plot of Craniofacial EGFP + cells (Cp) and Trunk EGFP + cells (Tp) as assessed by microarray analysis (3D-Gene; Toray Industries). (B) Most up-regulated genes in Craniofacial EGFP + cells (blue) and Trunk EGFP + cells (red), compared with those in the EGFP + cells of trunk and craniofacial regions, respectively. (C) Biplot of principal component analysis of the eight samples revealed three sample groups. Black dots indicate all genes and red dots indicate known stem cell genes selected from GO annotations. Cp, Tp, Cn; craniofacial EGFP − cells, and Tn; trunk EGFP − cells.
    Figure Legend Snippet: Differential expression profiles of cNCCs and tNCCs in P0-Cre/Floxed-EGFP mouse embryos. (A) Scatter plot of Craniofacial EGFP + cells (Cp) and Trunk EGFP + cells (Tp) as assessed by microarray analysis (3D-Gene; Toray Industries). (B) Most up-regulated genes in Craniofacial EGFP + cells (blue) and Trunk EGFP + cells (red), compared with those in the EGFP + cells of trunk and craniofacial regions, respectively. (C) Biplot of principal component analysis of the eight samples revealed three sample groups. Black dots indicate all genes and red dots indicate known stem cell genes selected from GO annotations. Cp, Tp, Cn; craniofacial EGFP − cells, and Tn; trunk EGFP − cells.

    Techniques Used: Expressing, Microarray

    26) Product Images from "GmBZL3 acts as a major BR signaling regulator through crosstalk with multiple pathways in Glycine max"

    Article Title: GmBZL3 acts as a major BR signaling regulator through crosstalk with multiple pathways in Glycine max

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-019-1677-2

    Expression patterns of GmBZL3 target genes in response to a BR inhibitor in combination with or without epibrassinolide. a Heatmap representation of expression patterns of different GmBZL3 targets in soybean Williams 82 under following conditions (Pcz: 5 μM Pcz for 10 days. Pcz-BL: 5 μM Pcz with 10 nM BL for 10 days. Pcz-BL-1 h: 5 μM Pcz for 10 days then with 1 μM BL for 1 h. Pcz-BL-8 h: 5 μM Pcz for 10 days then with 1 μM BL for 8 h). The expression data values were median-centered and normalized for each gene before transforming to the color scale (log2-transformed ratios). The color bar at the bottom shows the range of expression values from highest expression level (red) to lowest expression level (green). 0 is the median expression level (Black). b qRT-PCR analysis of six GmBZL3 target genes was performed using total RNA isolated from Wm82 seedlings under control. Pcz. Pcz-BL. Pcz-BL-1 h and Pcz-BL-8 h treatments. Relative gene expression levels (fold change, log2) are shown following normalization with actin (Glyma.18G290800) transcript values. Error bars represent the standard error of the mean. The y-axis represents the relative gene expression level in different samples. Three independent experiments were performed. A representative result is shown. The star (*) indicates statistically significant differences among the means ( p
    Figure Legend Snippet: Expression patterns of GmBZL3 target genes in response to a BR inhibitor in combination with or without epibrassinolide. a Heatmap representation of expression patterns of different GmBZL3 targets in soybean Williams 82 under following conditions (Pcz: 5 μM Pcz for 10 days. Pcz-BL: 5 μM Pcz with 10 nM BL for 10 days. Pcz-BL-1 h: 5 μM Pcz for 10 days then with 1 μM BL for 1 h. Pcz-BL-8 h: 5 μM Pcz for 10 days then with 1 μM BL for 8 h). The expression data values were median-centered and normalized for each gene before transforming to the color scale (log2-transformed ratios). The color bar at the bottom shows the range of expression values from highest expression level (red) to lowest expression level (green). 0 is the median expression level (Black). b qRT-PCR analysis of six GmBZL3 target genes was performed using total RNA isolated from Wm82 seedlings under control. Pcz. Pcz-BL. Pcz-BL-1 h and Pcz-BL-8 h treatments. Relative gene expression levels (fold change, log2) are shown following normalization with actin (Glyma.18G290800) transcript values. Error bars represent the standard error of the mean. The y-axis represents the relative gene expression level in different samples. Three independent experiments were performed. A representative result is shown. The star (*) indicates statistically significant differences among the means ( p

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

    27) Product Images from "RNA-seq analysis of PHD and VHL inhibitors reveals differences and similarities to the hypoxia response."

    Article Title: RNA-seq analysis of PHD and VHL inhibitors reveals differences and similarities to the hypoxia response.

    Journal: Wellcome Open Research

    doi: 10.12688/wellcomeopenres.15044.1

    Analysis of protein levels of genes with increased transcription in hypoxia, IOX2 and VH032. HIF targets were increased in hypoxia, VH298 and FG-4592. 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 were introduced to ( A ) HeLa or HFF for 24 hours and ( B ) HeLa for indicated time. Protein levels were analysed by immunoblotting using antibodies against indicated proteins, with β-Actin as loading control. The blots shown are representative of three independent experiments. * indicates longer exposure.
    Figure Legend Snippet: Analysis of protein levels of genes with increased transcription in hypoxia, IOX2 and VH032. HIF targets were increased in hypoxia, VH298 and FG-4592. 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 were introduced to ( A ) HeLa or HFF for 24 hours and ( B ) HeLa for indicated time. Protein levels were analysed by immunoblotting using antibodies against indicated proteins, with β-Actin as loading control. The blots shown are representative of three independent experiments. * indicates longer exposure.

    Techniques Used:

    Validation of genes with increased transcript level in hypoxia, IOX2 and VH032. ( A ) Bar plot showing log2FC according to data obtained from RNA seq analysis of known HIF target genes in hypoxia, IOX2 and VH032. ( B ) HeLa and ( C ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05.
    Figure Legend Snippet: Validation of genes with increased transcript level in hypoxia, IOX2 and VH032. ( A ) Bar plot showing log2FC according to data obtained from RNA seq analysis of known HIF target genes in hypoxia, IOX2 and VH032. ( B ) HeLa and ( C ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05.

    Techniques Used: RNA Sequencing Assay, Two Tailed Test

    Differential gene expression analysis of RNA-seq results. ( A ) Heatmap of Pearson correlations among RNA-seq samples that have been normalised to their total counts. ( B ) Multidimensional scaling plot of RNA-seq data. The distance between two samples reflects the leading logFC of the corresponding samples. The leading logFC is the average (root mean square) of the 2000 largest absolute logFCs for genes between those two samples. ( C–E ) Heatmaps of log2 counts per million (logcpm) across all the samples using the top 100 most differentially expressed (DE) genes in ( C ) Hypoxia, ( D ) VH032, and ( E ) IOX2. The Pearson correlation was used to compute distances between genes and samples, and the clustering was performed using average linkage. Each column corresponds to a sample and each row corresponds to a specific gene. ( F ) Heatmaps of Pearson correlations between replicates of the same conditions. Each data had been normalised to their total counts. ( G ) Each dot represents a differentially expressed gene comparing the condition stated in the heading legend to DMSO vehicle control. Blue dots represent genes with increased expression (logFC > 0.58; to the right) or decreased expression (logFC
    Figure Legend Snippet: Differential gene expression analysis of RNA-seq results. ( A ) Heatmap of Pearson correlations among RNA-seq samples that have been normalised to their total counts. ( B ) Multidimensional scaling plot of RNA-seq data. The distance between two samples reflects the leading logFC of the corresponding samples. The leading logFC is the average (root mean square) of the 2000 largest absolute logFCs for genes between those two samples. ( C–E ) Heatmaps of log2 counts per million (logcpm) across all the samples using the top 100 most differentially expressed (DE) genes in ( C ) Hypoxia, ( D ) VH032, and ( E ) IOX2. The Pearson correlation was used to compute distances between genes and samples, and the clustering was performed using average linkage. Each column corresponds to a sample and each row corresponds to a specific gene. ( F ) Heatmaps of Pearson correlations between replicates of the same conditions. Each data had been normalised to their total counts. ( G ) Each dot represents a differentially expressed gene comparing the condition stated in the heading legend to DMSO vehicle control. Blue dots represent genes with increased expression (logFC > 0.58; to the right) or decreased expression (logFC

    Techniques Used: Expressing, RNA Sequencing Assay

    RNA seq validation of genes solely upregulated in hypoxia and IOX2, but not VH032. ( A ) HeLa and ( B ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05. ( C ) Table showing log2FC according to data obtained from RNA-seq analysis of known HIF target genes in hypoxia and IOX2, but not VH032. ( D ) Gene set enrichment analysis (GSEA) MsigDB showing significant enrichment of gene set signatures for genes upregulated in hypoxia and IOX2, but not found in VH032 at 5% false discovery rate (FDR). ( E ) Transcription factor enrichment analysis using TFEA.ChIP showing binding site enrichment for genes upregulated in hypoxia and IOX2, but not B032. The graph represents the adjusted p value (-log10 FDR) and the log-odds ratio (Log2.OR) for the association of ChIP datasets.
    Figure Legend Snippet: RNA seq validation of genes solely upregulated in hypoxia and IOX2, but not VH032. ( A ) HeLa and ( B ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05. ( C ) Table showing log2FC according to data obtained from RNA-seq analysis of known HIF target genes in hypoxia and IOX2, but not VH032. ( D ) Gene set enrichment analysis (GSEA) MsigDB showing significant enrichment of gene set signatures for genes upregulated in hypoxia and IOX2, but not found in VH032 at 5% false discovery rate (FDR). ( E ) Transcription factor enrichment analysis using TFEA.ChIP showing binding site enrichment for genes upregulated in hypoxia and IOX2, but not B032. The graph represents the adjusted p value (-log10 FDR) and the log-odds ratio (Log2.OR) for the association of ChIP datasets.

    Techniques Used: RNA Sequencing Assay, Two Tailed Test, Chromatin Immunoprecipitation, Binding Assay

    Transcription factor enrichment analysis. Transcription factor enrichment analysis using TFEA.ChIP showing binding site enrichment for genes upregulated in ( A ) VH032, ( B ) IOX2 and ( C ) hypoxia, or ( D ) downregulated in hypoxia. The graph represents the adjusted p-value (-log10 false discovery rate (FDR)) and the log-odds ratio (Log2.OR) for the association of ChIP datasets.
    Figure Legend Snippet: Transcription factor enrichment analysis. Transcription factor enrichment analysis using TFEA.ChIP showing binding site enrichment for genes upregulated in ( A ) VH032, ( B ) IOX2 and ( C ) hypoxia, or ( D ) downregulated in hypoxia. The graph represents the adjusted p-value (-log10 false discovery rate (FDR)) and the log-odds ratio (Log2.OR) for the association of ChIP datasets.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay

    28) Product Images from "RNA-seq analysis of PHD and VHL inhibitors reveals differences and similarities to the hypoxia response."

    Article Title: RNA-seq analysis of PHD and VHL inhibitors reveals differences and similarities to the hypoxia response.

    Journal: Wellcome Open Research

    doi: 10.12688/wellcomeopenres.15044.1

    Validation of genes with increased transcript level in hypoxia, IOX2 and VH032. ( A ) Bar plot showing log2FC according to data obtained from RNA seq analysis of known HIF target genes in hypoxia, IOX2 and VH032. ( B ) HeLa and ( C ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05.
    Figure Legend Snippet: Validation of genes with increased transcript level in hypoxia, IOX2 and VH032. ( A ) Bar plot showing log2FC according to data obtained from RNA seq analysis of known HIF target genes in hypoxia, IOX2 and VH032. ( B ) HeLa and ( C ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05.

    Techniques Used: RNA Sequencing Assay, Two Tailed Test

    Differential gene expression analysis of RNA-seq results. ( A ) Heatmap of Pearson correlations among RNA-seq samples that have been normalised to their total counts. ( B ) Multidimensional scaling plot of RNA-seq data. The distance between two samples reflects the leading logFC of the corresponding samples. The leading logFC is the average (root mean square) of the 2000 largest absolute logFCs for genes between those two samples. ( C–E ) Heatmaps of log2 counts per million (logcpm) across all the samples using the top 100 most differentially expressed (DE) genes in ( C ) Hypoxia, ( D ) VH032, and ( E ) IOX2. The Pearson correlation was used to compute distances between genes and samples, and the clustering was performed using average linkage. Each column corresponds to a sample and each row corresponds to a specific gene. ( F ) Heatmaps of Pearson correlations between replicates of the same conditions. Each data had been normalised to their total counts. ( G ) Each dot represents a differentially expressed gene comparing the condition stated in the heading legend to DMSO vehicle control. Blue dots represent genes with increased expression (logFC > 0.58; to the right) or decreased expression (logFC
    Figure Legend Snippet: Differential gene expression analysis of RNA-seq results. ( A ) Heatmap of Pearson correlations among RNA-seq samples that have been normalised to their total counts. ( B ) Multidimensional scaling plot of RNA-seq data. The distance between two samples reflects the leading logFC of the corresponding samples. The leading logFC is the average (root mean square) of the 2000 largest absolute logFCs for genes between those two samples. ( C–E ) Heatmaps of log2 counts per million (logcpm) across all the samples using the top 100 most differentially expressed (DE) genes in ( C ) Hypoxia, ( D ) VH032, and ( E ) IOX2. The Pearson correlation was used to compute distances between genes and samples, and the clustering was performed using average linkage. Each column corresponds to a sample and each row corresponds to a specific gene. ( F ) Heatmaps of Pearson correlations between replicates of the same conditions. Each data had been normalised to their total counts. ( G ) Each dot represents a differentially expressed gene comparing the condition stated in the heading legend to DMSO vehicle control. Blue dots represent genes with increased expression (logFC > 0.58; to the right) or decreased expression (logFC

    Techniques Used: Expressing, RNA Sequencing Assay

    RNA seq validation of genes solely upregulated in hypoxia and IOX2, but not VH032. ( A ) HeLa and ( B ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05. ( C ) Table showing log2FC according to data obtained from RNA-seq analysis of known HIF target genes in hypoxia and IOX2, but not VH032. ( D ) Gene set enrichment analysis (GSEA) MsigDB showing significant enrichment of gene set signatures for genes upregulated in hypoxia and IOX2, but not found in VH032 at 5% false discovery rate (FDR). ( E ) Transcription factor enrichment analysis using TFEA.ChIP showing binding site enrichment for genes upregulated in hypoxia and IOX2, but not B032. The graph represents the adjusted p value (-log10 FDR) and the log-odds ratio (Log2.OR) for the association of ChIP datasets.
    Figure Legend Snippet: RNA seq validation of genes solely upregulated in hypoxia and IOX2, but not VH032. ( A ) HeLa and ( B ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05. ( C ) Table showing log2FC according to data obtained from RNA-seq analysis of known HIF target genes in hypoxia and IOX2, but not VH032. ( D ) Gene set enrichment analysis (GSEA) MsigDB showing significant enrichment of gene set signatures for genes upregulated in hypoxia and IOX2, but not found in VH032 at 5% false discovery rate (FDR). ( E ) Transcription factor enrichment analysis using TFEA.ChIP showing binding site enrichment for genes upregulated in hypoxia and IOX2, but not B032. The graph represents the adjusted p value (-log10 FDR) and the log-odds ratio (Log2.OR) for the association of ChIP datasets.

    Techniques Used: RNA Sequencing Assay, Two Tailed Test, Chromatin Immunoprecipitation, Binding Assay

    29) Product Images from "Sex differences in gene regulation in the dorsal root ganglion after nerve injury"

    Article Title: Sex differences in gene regulation in the dorsal root ganglion after nerve injury

    Journal: BMC Genomics

    doi: 10.1186/s12864-019-5512-9

    Comparison of differential gene expression between CCI and naïve groups in male and female rats. a Schematic diagram of experiment. Male and female rats were randomly assigned to the naïve group or receive CCI. RNA-seq performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG) defined as genes expressed after CCI versus naïve with a |log 2 FC| > 0.5 and an FDR
    Figure Legend Snippet: Comparison of differential gene expression between CCI and naïve groups in male and female rats. a Schematic diagram of experiment. Male and female rats were randomly assigned to the naïve group or receive CCI. RNA-seq performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG) defined as genes expressed after CCI versus naïve with a |log 2 FC| > 0.5 and an FDR

    Techniques Used: Expressing, RNA Sequencing Assay

    Gene ontology in differentially expressed genes that are upregulated after CCI. a Schematic diagram of experiment. Male and female rats were randomly assigned to the naïve group or receive CCI. RNA-seq performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG) defined as genes expressed after CCI versus naïve with a |log 2 FC| > 0.5 and an FDR
    Figure Legend Snippet: Gene ontology in differentially expressed genes that are upregulated after CCI. a Schematic diagram of experiment. Male and female rats were randomly assigned to the naïve group or receive CCI. RNA-seq performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG) defined as genes expressed after CCI versus naïve with a |log 2 FC| > 0.5 and an FDR

    Techniques Used: RNA Sequencing Assay

    Schematic diagram of experimental procedures. Male and female rats were randomly assigned to the naïve group or receive CCI. Total RNA was isolated from the L4-L6 DRG of naïve rats and on day 14 after CCI to the sciatic nerve. Libraries were constructed after poly(A) selection and sequenced. RNA-seq was performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG), defined as genes expressed after CCI versus naïve with a |log 2 fold change (FC)| > 0.5 and an FDR
    Figure Legend Snippet: Schematic diagram of experimental procedures. Male and female rats were randomly assigned to the naïve group or receive CCI. Total RNA was isolated from the L4-L6 DRG of naïve rats and on day 14 after CCI to the sciatic nerve. Libraries were constructed after poly(A) selection and sequenced. RNA-seq was performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG), defined as genes expressed after CCI versus naïve with a |log 2 fold change (FC)| > 0.5 and an FDR

    Techniques Used: Isolation, Construct, Selection, RNA Sequencing Assay

    Sex differences of gene expression in DRGs from naïve rats. a Schematic diagram of experiment. RNA-seq performed on ipsilateral L4-L6 DRGs from naïve male and female rats. Differentially expressed genes (DEG) in one sex are identified by a |log 2 fold change (FC)| > 0.5 and an FDR
    Figure Legend Snippet: Sex differences of gene expression in DRGs from naïve rats. a Schematic diagram of experiment. RNA-seq performed on ipsilateral L4-L6 DRGs from naïve male and female rats. Differentially expressed genes (DEG) in one sex are identified by a |log 2 fold change (FC)| > 0.5 and an FDR

    Techniques Used: Expressing, RNA Sequencing Assay

    Differential gene expression between CCI and naïve groups in female rats. a Schematic diagram of experiment. Female rats were randomly assigned to the naïve group or receive CCI. RNA-seq performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG) are defined as genes expressed after CCI versus naïve with a |log 2 FC| > 0.5 and an FDR
    Figure Legend Snippet: Differential gene expression between CCI and naïve groups in female rats. a Schematic diagram of experiment. Female rats were randomly assigned to the naïve group or receive CCI. RNA-seq performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG) are defined as genes expressed after CCI versus naïve with a |log 2 FC| > 0.5 and an FDR

    Techniques Used: Expressing, RNA Sequencing Assay

    Differential gene expression between CCI and naïve groups in male rats. a Schematic diagram of experiment. Male rats were randomly assigned to the naïve group or receive CCI. RNA-seq performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG) are defined as genes expressed after CCI versus naïve with a |log 2 FC| > 0.5 and an adjusted p -value
    Figure Legend Snippet: Differential gene expression between CCI and naïve groups in male rats. a Schematic diagram of experiment. Male rats were randomly assigned to the naïve group or receive CCI. RNA-seq performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG) are defined as genes expressed after CCI versus naïve with a |log 2 FC| > 0.5 and an adjusted p -value

    Techniques Used: Expressing, RNA Sequencing Assay

    30) Product Images from "Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq"

    Article Title: Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00185

    Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p
    Figure Legend Snippet: Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p

    Techniques Used: Laser Capture Microdissection, Isolation

    31) Product Images from "Decoupling of DNA methylation and activity of intergenic LINE-1 promoters in colorectal cancer"

    Article Title: Decoupling of DNA methylation and activity of intergenic LINE-1 promoters in colorectal cancer

    Journal: Epigenetics

    doi: 10.1080/15592294.2017.1300729

    Relationship between methylation and expression of LCT14 in CRC. (A) Schematic diagram of the LCT14 genomic locus on human chromosome 5 (coordinates: 24,487,209–27,038,689) with indicated positions of the annotated genes CDH10, LOC105374693 , and CDH9 and of the intact intergenic LINE1 (L1) that drives transcription of LCT14. At the bottom is an enlargement of the region including the LINE-1 (L1PA2; chr5:25,378,639–25,384,665) from which LCT14 originates with the regions (black bars) tested by bisulfite or hydroxymethylated DNA (hMeDIP) and chromatin (ChIP) immunoprecipitations and, below these, the LCT14 transcript (chr5: 25,384,485–25,384,958) and the region amplified for expression studies. All coordinates are from hg19 annotations; scale is in kb. (B) Expression of LCT14 measured by real-time RT-PCR and expressed relatively to the geometric mean of 3 reference genes in matched normal (dark gray) and tumor (light gray) tissues from 4 colorectal cancer patients (left panel) and of 5 colorectal cancer cell lines (right panel). (C) Methylation levels measured by bisulfite sequencing in the paired normal and tumor tissues of the 4 patients (left panel) and cell lines (right panel) described in B.
    Figure Legend Snippet: Relationship between methylation and expression of LCT14 in CRC. (A) Schematic diagram of the LCT14 genomic locus on human chromosome 5 (coordinates: 24,487,209–27,038,689) with indicated positions of the annotated genes CDH10, LOC105374693 , and CDH9 and of the intact intergenic LINE1 (L1) that drives transcription of LCT14. At the bottom is an enlargement of the region including the LINE-1 (L1PA2; chr5:25,378,639–25,384,665) from which LCT14 originates with the regions (black bars) tested by bisulfite or hydroxymethylated DNA (hMeDIP) and chromatin (ChIP) immunoprecipitations and, below these, the LCT14 transcript (chr5: 25,384,485–25,384,958) and the region amplified for expression studies. All coordinates are from hg19 annotations; scale is in kb. (B) Expression of LCT14 measured by real-time RT-PCR and expressed relatively to the geometric mean of 3 reference genes in matched normal (dark gray) and tumor (light gray) tissues from 4 colorectal cancer patients (left panel) and of 5 colorectal cancer cell lines (right panel). (C) Methylation levels measured by bisulfite sequencing in the paired normal and tumor tissues of the 4 patients (left panel) and cell lines (right panel) described in B.

    Techniques Used: Methylation, Expressing, Chromatin Immunoprecipitation, Amplification, Quantitative RT-PCR, Methylation Sequencing

    32) Product Images from "Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators"

    Article Title: Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators

    Journal: Nature

    doi: 10.1038/nature25179

    MORC2, MPP8 and TASOR silence L1 transcription. a. Relative genomic copy number of newly integrated L1-GFP reporters in the indicated mutant K562 pools after dox-induction. PspGI-assisted qPCR assay used here was designed to selectively detect spliced GFP rather than the unspliced version (see Methods section). The L1-GFP copies were normalized to beta-actin DNAs; data then normalized to Ctrl. As a putative L1 activator, SLTM shows an opposite effect on the DNA copy number, compared with L1 suppressors. Center value as median. n = 3 technical replicates per gene. b. RNA-seq data in Ctrl K562 cells showing that most heterochromatin regulators in Fig. 2a are expressed, supporting the selective effect of HUSH and MORC2 in L1 regulation. c. Western blots validating the knockout (KO) effects in independent KO K562 cell clones. Ctrl samples were loaded at 4 different amounts (200%, 100%, 50%, 25% of KO clones). Three experiments repeated independently with similar results. To obtain KO clones, we sorted mutant K562 pools (cells used in Fig. 1e,f ) into 96-well plates, expanded cells and screened for KO clones through western blotting. Of note, all K562 KO clones were derived from the same starting L1-GFP reporter line, and thus do not differ in reporter transgene integrations among the clones. d. Representative images of single molecule FISH (smFISH) assays targeting ACTB mRNAs and RNA transcripts from L1-GFP reporters in Ctrl and KO K562 clones after 5 days of dox-induction. No signal was observed from L1-GFP reporters without dox-induction (data not shown). Two experiments repeated independently with similar results. See also panel e and Fig. 2b (showing L1-GFP mRNA only). e. Quantitation of the L1-GFP transcription level from the indicated number of K562 cells, determined by smFISH assays (panel d and Fig. 2b ). The number of L1-GFP mRNA transcripts is normalized to the number of beta-actin mRNAs within each K562 cell. Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. P-value, two-sided Wilcoxon test. 95% CI for median from 1,000× bootstrap: Control: 0.059-0.082; MORC2: 0.106-0.123; MPP8: 0.264-0.410; TASOR: 0.514-0.671. f. MORC2, MPP8, and TASOR KOs increase the genomic copy number of newly integrated L1-GFP reporters. PspGI-assisted qPCR assays were performed as in panel a), but using clonal KO K562 clones instead of mutant cell pools. Data normalized to Ctrl. n = 3 technical replicates, center value as median. g. MORC2 KO, MPP8 KO, and TASOR KO increase the expression of endogenous L1s. RT-qPCR experiments were performed as in ( Fig. 1f ), but using clonal KO K562 clones instead of mutant cell pools. n = 2 biological replicates × 3 technical replicates (center value as median). The primers do not target the L1-GFP reporter and the cell lines were not dox-induced, so these RT-qPCR assays will not detect L1-GFP transcripts. h. Western blots showing depletion effects of MORC2, MPP8 and TASOR in the mutant pools of K562 cells (left) and in the mutant pools of H9 hESCs without transgenic L1 reporters (right). Two experiments repeated independently with similar results. i. Northern blots showing increased transcription from the L1-GFP reporter in KO K562 clones (same cell lines as in panel c) after 5 days’ dox-induction. Two experiments repeated independently with similar results. As observed in Fig. 2b , while HUSH KO significantly increases L1-GFP transcription, MORC2 KO leads to only a modest increase. This is probably because the L1-GFP reporter does not contain the native L1 5’ UTR sequence, where MORC2 intensively binds (See Extended Data Fig. 7f,g ). The 5 kb and 1.9 kb marks on the membrane refer to the 28S rRNA and 18S rRNA bands respectively. j. Northern blots showing that disruption of MORC2, MPP8 and TASOR increases the expression level of endogenous L1Hs in hESCs, same cell lines as in panel h). Size marker indicated as in panel i). Two experiments repeated independently with similar results. k. Western blots showing protein abundance of L1_ORF1p and HSP90 in the mutant pools of K562 cells and hESCs (same cell line as shown in panel h). Two experiments repeated independently with similar results. Experiments were performed without dox-induction of the transgenic L1 reporter. Due to the strong signal of bands from the KO samples, the blots were exposed for a very short time and the band signal in the Ctrl samples were relatively very weak compared to the KO samples; same case for panels i, j).
    Figure Legend Snippet: MORC2, MPP8 and TASOR silence L1 transcription. a. Relative genomic copy number of newly integrated L1-GFP reporters in the indicated mutant K562 pools after dox-induction. PspGI-assisted qPCR assay used here was designed to selectively detect spliced GFP rather than the unspliced version (see Methods section). The L1-GFP copies were normalized to beta-actin DNAs; data then normalized to Ctrl. As a putative L1 activator, SLTM shows an opposite effect on the DNA copy number, compared with L1 suppressors. Center value as median. n = 3 technical replicates per gene. b. RNA-seq data in Ctrl K562 cells showing that most heterochromatin regulators in Fig. 2a are expressed, supporting the selective effect of HUSH and MORC2 in L1 regulation. c. Western blots validating the knockout (KO) effects in independent KO K562 cell clones. Ctrl samples were loaded at 4 different amounts (200%, 100%, 50%, 25% of KO clones). Three experiments repeated independently with similar results. To obtain KO clones, we sorted mutant K562 pools (cells used in Fig. 1e,f ) into 96-well plates, expanded cells and screened for KO clones through western blotting. Of note, all K562 KO clones were derived from the same starting L1-GFP reporter line, and thus do not differ in reporter transgene integrations among the clones. d. Representative images of single molecule FISH (smFISH) assays targeting ACTB mRNAs and RNA transcripts from L1-GFP reporters in Ctrl and KO K562 clones after 5 days of dox-induction. No signal was observed from L1-GFP reporters without dox-induction (data not shown). Two experiments repeated independently with similar results. See also panel e and Fig. 2b (showing L1-GFP mRNA only). e. Quantitation of the L1-GFP transcription level from the indicated number of K562 cells, determined by smFISH assays (panel d and Fig. 2b ). The number of L1-GFP mRNA transcripts is normalized to the number of beta-actin mRNAs within each K562 cell. Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. P-value, two-sided Wilcoxon test. 95% CI for median from 1,000× bootstrap: Control: 0.059-0.082; MORC2: 0.106-0.123; MPP8: 0.264-0.410; TASOR: 0.514-0.671. f. MORC2, MPP8, and TASOR KOs increase the genomic copy number of newly integrated L1-GFP reporters. PspGI-assisted qPCR assays were performed as in panel a), but using clonal KO K562 clones instead of mutant cell pools. Data normalized to Ctrl. n = 3 technical replicates, center value as median. g. MORC2 KO, MPP8 KO, and TASOR KO increase the expression of endogenous L1s. RT-qPCR experiments were performed as in ( Fig. 1f ), but using clonal KO K562 clones instead of mutant cell pools. n = 2 biological replicates × 3 technical replicates (center value as median). The primers do not target the L1-GFP reporter and the cell lines were not dox-induced, so these RT-qPCR assays will not detect L1-GFP transcripts. h. Western blots showing depletion effects of MORC2, MPP8 and TASOR in the mutant pools of K562 cells (left) and in the mutant pools of H9 hESCs without transgenic L1 reporters (right). Two experiments repeated independently with similar results. i. Northern blots showing increased transcription from the L1-GFP reporter in KO K562 clones (same cell lines as in panel c) after 5 days’ dox-induction. Two experiments repeated independently with similar results. As observed in Fig. 2b , while HUSH KO significantly increases L1-GFP transcription, MORC2 KO leads to only a modest increase. This is probably because the L1-GFP reporter does not contain the native L1 5’ UTR sequence, where MORC2 intensively binds (See Extended Data Fig. 7f,g ). The 5 kb and 1.9 kb marks on the membrane refer to the 28S rRNA and 18S rRNA bands respectively. j. Northern blots showing that disruption of MORC2, MPP8 and TASOR increases the expression level of endogenous L1Hs in hESCs, same cell lines as in panel h). Size marker indicated as in panel i). Two experiments repeated independently with similar results. k. Western blots showing protein abundance of L1_ORF1p and HSP90 in the mutant pools of K562 cells and hESCs (same cell line as shown in panel h). Two experiments repeated independently with similar results. Experiments were performed without dox-induction of the transgenic L1 reporter. Due to the strong signal of bands from the KO samples, the blots were exposed for a very short time and the band signal in the Ctrl samples were relatively very weak compared to the KO samples; same case for panels i, j).

    Techniques Used: Mutagenesis, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Western Blot, Knock-Out, Clone Assay, Derivative Assay, Fluorescence In Situ Hybridization, Quantitation Assay, Expressing, Quantitative RT-PCR, Transgenic Assay, Northern Blot, Sequencing, Marker

    HUSH/MORC2 binding at L1s decreases active host gene expression. a. Heatmaps showing MPP8 and H3K9me3 ChIP signal enrichment, centered on MPP8 and MORC2 summits and separated by L1 presence or absence. b. Expression change of genes with intronic full-length L1s that are bound or unbound by MORC2 or MPP8 (RNA-seq reads from KO K562 clones compared to Ctrl). Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. p-value, two-sided Mann-Whitney-Wilcoxon test. c. Genome browser tracks: HUSH/MORC2 loss causing H3K9me3 decrease at the target L1 and expression increase at both the target L1 and its host gene, independently repeated once with similar results. d. Deleting the target intronic L1 from CYP3A5 in K562 increases CYP3A5 expression, by RT-qPCR normalized to wild-type sample. n = 2 biological replicates × 3 technical replicates (center value as median). Gel image confirms L1 deletion; two experiments repeated independently with similar results. e. RT-qPCR for CYP3A5 expression in K562 clones, normalized to Ctrl. n = 2 biological replicates × 3 technical replicates (center value as median). f. Model: HUSH/MORC2 bind young full-length L1s within transcriptionally active genes, and promote H3K9me3 deposition at target L1s to silence L1 transcription. This pathway not only inhibits L1 retrotransposition, but also decreases host gene expression.
    Figure Legend Snippet: HUSH/MORC2 binding at L1s decreases active host gene expression. a. Heatmaps showing MPP8 and H3K9me3 ChIP signal enrichment, centered on MPP8 and MORC2 summits and separated by L1 presence or absence. b. Expression change of genes with intronic full-length L1s that are bound or unbound by MORC2 or MPP8 (RNA-seq reads from KO K562 clones compared to Ctrl). Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. p-value, two-sided Mann-Whitney-Wilcoxon test. c. Genome browser tracks: HUSH/MORC2 loss causing H3K9me3 decrease at the target L1 and expression increase at both the target L1 and its host gene, independently repeated once with similar results. d. Deleting the target intronic L1 from CYP3A5 in K562 increases CYP3A5 expression, by RT-qPCR normalized to wild-type sample. n = 2 biological replicates × 3 technical replicates (center value as median). Gel image confirms L1 deletion; two experiments repeated independently with similar results. e. RT-qPCR for CYP3A5 expression in K562 clones, normalized to Ctrl. n = 2 biological replicates × 3 technical replicates (center value as median). f. Model: HUSH/MORC2 bind young full-length L1s within transcriptionally active genes, and promote H3K9me3 deposition at target L1s to silence L1 transcription. This pathway not only inhibits L1 retrotransposition, but also decreases host gene expression.

    Techniques Used: Binding Assay, Expressing, Chromatin Immunoprecipitation, RNA Sequencing Assay, Clone Assay, MANN-WHITNEY, Quantitative RT-PCR

    HUSH/MORC2 preferentially bind intronic L1s within actively transcribed genes. a. Genes that contain MPP8 or MORC2 bound intronic L1s are expressed at significantly higher levels in Ctrl K562 cells, compared to genes that contain intronic full-length L1s unbound by MPP8 or MORC2. p-value, two-sided Mann-Whitney-Wilcoxon test. Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. b. The promoters of genes that contain MPP8 or MORC2 bound intronic full-length L1s are marked by transcriptionally permissive H3K27ac in wild-type K562 cells. H3K27ac ChIP-seq data are taken from K562 epigenome pilot study, accession number PRJEB8620. TSS, transcription start site. c. Genes selectively occupied by MORC2/MPP8 either in K562 or in hESC cells exhibit higher gene expression in the corresponding cell line (p-values = 4.3 × 10 −107 for MPP8 binding; p-values = 5.0 × 10 −92 for MORC2 binding, Kruskal-Wallis test). Boxplots defined as in panel a. RNA-seq datasets for hESC are from SRA entries SRR2043329 and SRR2043330. d. ChIP-qPCR assays quantifying HUSH/MORC2 binding to an inducible L1 transgene in K562 cells before or after its transcriptional induction via Dox. Transcriptional induction increases binding of MORC2 and MPP8 to the L1 transgene. n = 2 biological replicates × 3 technical replicates (center value as median).
    Figure Legend Snippet: HUSH/MORC2 preferentially bind intronic L1s within actively transcribed genes. a. Genes that contain MPP8 or MORC2 bound intronic L1s are expressed at significantly higher levels in Ctrl K562 cells, compared to genes that contain intronic full-length L1s unbound by MPP8 or MORC2. p-value, two-sided Mann-Whitney-Wilcoxon test. Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. b. The promoters of genes that contain MPP8 or MORC2 bound intronic full-length L1s are marked by transcriptionally permissive H3K27ac in wild-type K562 cells. H3K27ac ChIP-seq data are taken from K562 epigenome pilot study, accession number PRJEB8620. TSS, transcription start site. c. Genes selectively occupied by MORC2/MPP8 either in K562 or in hESC cells exhibit higher gene expression in the corresponding cell line (p-values = 4.3 × 10 −107 for MPP8 binding; p-values = 5.0 × 10 −92 for MORC2 binding, Kruskal-Wallis test). Boxplots defined as in panel a. RNA-seq datasets for hESC are from SRA entries SRR2043329 and SRR2043330. d. ChIP-qPCR assays quantifying HUSH/MORC2 binding to an inducible L1 transgene in K562 cells before or after its transcriptional induction via Dox. Transcriptional induction increases binding of MORC2 and MPP8 to the L1 transgene. n = 2 biological replicates × 3 technical replicates (center value as median).

    Techniques Used: MANN-WHITNEY, Chromatin Immunoprecipitation, Expressing, Binding Assay, RNA Sequencing Assay, Real-time Polymerase Chain Reaction

    33) Product Images from "Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators"

    Article Title: Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators

    Journal: Nature

    doi: 10.1038/nature25179

    MORC2, MPP8 and TASOR silence L1 transcription. a. Relative genomic copy number of newly integrated L1-GFP reporters in the indicated mutant K562 pools after dox-induction. PspGI-assisted qPCR assay used here was designed to selectively detect spliced GFP rather than the unspliced version (see Methods section). The L1-GFP copies were normalized to beta-actin DNAs; data then normalized to Ctrl. As a putative L1 activator, SLTM shows an opposite effect on the DNA copy number, compared with L1 suppressors. Center value as median. n = 3 technical replicates per gene. b. RNA-seq data in Ctrl K562 cells showing that most heterochromatin regulators in Fig. 2a are expressed, supporting the selective effect of HUSH and MORC2 in L1 regulation. c. Western blots validating the knockout (KO) effects in independent KO K562 cell clones. Ctrl samples were loaded at 4 different amounts (200%, 100%, 50%, 25% of KO clones). Three experiments repeated independently with similar results. To obtain KO clones, we sorted mutant K562 pools (cells used in Fig. 1e,f ) into 96-well plates, expanded cells and screened for KO clones through western blotting. Of note, all K562 KO clones were derived from the same starting L1-GFP reporter line, and thus do not differ in reporter transgene integrations among the clones. d. Representative images of single molecule FISH (smFISH) assays targeting ACTB mRNAs and RNA transcripts from L1-GFP reporters in Ctrl and KO K562 clones after 5 days of dox-induction. No signal was observed from L1-GFP reporters without dox-induction (data not shown). Two experiments repeated independently with similar results. See also panel e and Fig. 2b (showing L1-GFP mRNA only). e. Quantitation of the L1-GFP transcription level from the indicated number of K562 cells, determined by smFISH assays (panel d and Fig. 2b ). The number of L1-GFP mRNA transcripts is normalized to the number of beta-actin mRNAs within each K562 cell. Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. P-value, two-sided Wilcoxon test. 95% CI for median from 1,000× bootstrap: Control: 0.059-0.082; MORC2: 0.106-0.123; MPP8: 0.264-0.410; TASOR: 0.514-0.671. f. MORC2, MPP8, and TASOR KOs increase the genomic copy number of newly integrated L1-GFP reporters. PspGI-assisted qPCR assays were performed as in panel a), but using clonal KO K562 clones instead of mutant cell pools. Data normalized to Ctrl. n = 3 technical replicates, center value as median. g. MORC2 KO, MPP8 KO, and TASOR KO increase the expression of endogenous L1s. RT-qPCR experiments were performed as in ( Fig. 1f ), but using clonal KO K562 clones instead of mutant cell pools. n = 2 biological replicates × 3 technical replicates (center value as median). The primers do not target the L1-GFP reporter and the cell lines were not dox-induced, so these RT-qPCR assays will not detect L1-GFP transcripts. h. Western blots showing depletion effects of MORC2, MPP8 and TASOR in the mutant pools of K562 cells (left) and in the mutant pools of H9 hESCs without transgenic L1 reporters (right). Two experiments repeated independently with similar results. i. Northern blots showing increased transcription from the L1-GFP reporter in KO K562 clones (same cell lines as in panel c) after 5 days’ dox-induction. Two experiments repeated independently with similar results. As observed in Fig. 2b , while HUSH KO significantly increases L1-GFP transcription, MORC2 KO leads to only a modest increase. This is probably because the L1-GFP reporter does not contain the native L1 5’ UTR sequence, where MORC2 intensively binds (See Extended Data Fig. 7f,g ). The 5 kb and 1.9 kb marks on the membrane refer to the 28S rRNA and 18S rRNA bands respectively. j. Northern blots showing that disruption of MORC2, MPP8 and TASOR increases the expression level of endogenous L1Hs in hESCs, same cell lines as in panel h). Size marker indicated as in panel i). Two experiments repeated independently with similar results. k. Western blots showing protein abundance of L1_ORF1p and HSP90 in the mutant pools of K562 cells and hESCs (same cell line as shown in panel h). Two experiments repeated independently with similar results. Experiments were performed without dox-induction of the transgenic L1 reporter. Due to the strong signal of bands from the KO samples, the blots were exposed for a very short time and the band signal in the Ctrl samples were relatively very weak compared to the KO samples; same case for panels i, j).
    Figure Legend Snippet: MORC2, MPP8 and TASOR silence L1 transcription. a. Relative genomic copy number of newly integrated L1-GFP reporters in the indicated mutant K562 pools after dox-induction. PspGI-assisted qPCR assay used here was designed to selectively detect spliced GFP rather than the unspliced version (see Methods section). The L1-GFP copies were normalized to beta-actin DNAs; data then normalized to Ctrl. As a putative L1 activator, SLTM shows an opposite effect on the DNA copy number, compared with L1 suppressors. Center value as median. n = 3 technical replicates per gene. b. RNA-seq data in Ctrl K562 cells showing that most heterochromatin regulators in Fig. 2a are expressed, supporting the selective effect of HUSH and MORC2 in L1 regulation. c. Western blots validating the knockout (KO) effects in independent KO K562 cell clones. Ctrl samples were loaded at 4 different amounts (200%, 100%, 50%, 25% of KO clones). Three experiments repeated independently with similar results. To obtain KO clones, we sorted mutant K562 pools (cells used in Fig. 1e,f ) into 96-well plates, expanded cells and screened for KO clones through western blotting. Of note, all K562 KO clones were derived from the same starting L1-GFP reporter line, and thus do not differ in reporter transgene integrations among the clones. d. Representative images of single molecule FISH (smFISH) assays targeting ACTB mRNAs and RNA transcripts from L1-GFP reporters in Ctrl and KO K562 clones after 5 days of dox-induction. No signal was observed from L1-GFP reporters without dox-induction (data not shown). Two experiments repeated independently with similar results. See also panel e and Fig. 2b (showing L1-GFP mRNA only). e. Quantitation of the L1-GFP transcription level from the indicated number of K562 cells, determined by smFISH assays (panel d and Fig. 2b ). The number of L1-GFP mRNA transcripts is normalized to the number of beta-actin mRNAs within each K562 cell. Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. P-value, two-sided Wilcoxon test. 95% CI for median from 1,000× bootstrap: Control: 0.059-0.082; MORC2: 0.106-0.123; MPP8: 0.264-0.410; TASOR: 0.514-0.671. f. MORC2, MPP8, and TASOR KOs increase the genomic copy number of newly integrated L1-GFP reporters. PspGI-assisted qPCR assays were performed as in panel a), but using clonal KO K562 clones instead of mutant cell pools. Data normalized to Ctrl. n = 3 technical replicates, center value as median. g. MORC2 KO, MPP8 KO, and TASOR KO increase the expression of endogenous L1s. RT-qPCR experiments were performed as in ( Fig. 1f ), but using clonal KO K562 clones instead of mutant cell pools. n = 2 biological replicates × 3 technical replicates (center value as median). The primers do not target the L1-GFP reporter and the cell lines were not dox-induced, so these RT-qPCR assays will not detect L1-GFP transcripts. h. Western blots showing depletion effects of MORC2, MPP8 and TASOR in the mutant pools of K562 cells (left) and in the mutant pools of H9 hESCs without transgenic L1 reporters (right). Two experiments repeated independently with similar results. i. Northern blots showing increased transcription from the L1-GFP reporter in KO K562 clones (same cell lines as in panel c) after 5 days’ dox-induction. Two experiments repeated independently with similar results. As observed in Fig. 2b , while HUSH KO significantly increases L1-GFP transcription, MORC2 KO leads to only a modest increase. This is probably because the L1-GFP reporter does not contain the native L1 5’ UTR sequence, where MORC2 intensively binds (See Extended Data Fig. 7f,g ). The 5 kb and 1.9 kb marks on the membrane refer to the 28S rRNA and 18S rRNA bands respectively. j. Northern blots showing that disruption of MORC2, MPP8 and TASOR increases the expression level of endogenous L1Hs in hESCs, same cell lines as in panel h). Size marker indicated as in panel i). Two experiments repeated independently with similar results. k. Western blots showing protein abundance of L1_ORF1p and HSP90 in the mutant pools of K562 cells and hESCs (same cell line as shown in panel h). Two experiments repeated independently with similar results. Experiments were performed without dox-induction of the transgenic L1 reporter. Due to the strong signal of bands from the KO samples, the blots were exposed for a very short time and the band signal in the Ctrl samples were relatively very weak compared to the KO samples; same case for panels i, j).

    Techniques Used: Mutagenesis, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Western Blot, Knock-Out, Clone Assay, Derivative Assay, Fluorescence In Situ Hybridization, Quantitation Assay, Expressing, Quantitative RT-PCR, Transgenic Assay, Northern Blot, Sequencing, Marker

    34) Product Images from "A nested parallel experiment demonstrates differences in intensity-dependence between RNA-seq and microarrays"

    Article Title: A nested parallel experiment demonstrates differences in intensity-dependence between RNA-seq and microarrays

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv636

    Comparison of the log fold change estimates measured with qPCR compared to estimates from the microarray or RNA-Seq, for 13 selected low-intensity genes that disagreed between RNA-Seq and microarray.
    Figure Legend Snippet: Comparison of the log fold change estimates measured with qPCR compared to estimates from the microarray or RNA-Seq, for 13 selected low-intensity genes that disagreed between RNA-Seq and microarray.

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

    Boxplots comparing the normalized and centered expression values of microarrays, RNA-Seq and qPCR of the six genes for which microarray and qPCR most disagreed. This shows that in many cases, microarray measurements were very consistent between biological, technical and chip replicates. This suggests that the problem is not variation at low-intensity microarray measurements, but rather bias.
    Figure Legend Snippet: Boxplots comparing the normalized and centered expression values of microarrays, RNA-Seq and qPCR of the six genes for which microarray and qPCR most disagreed. This shows that in many cases, microarray measurements were very consistent between biological, technical and chip replicates. This suggests that the problem is not variation at low-intensity microarray measurements, but rather bias.

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

    35) Product Images from "Global identification and analysis of long non-coding RNAs in diploid strawberry Fragaria vesca during flower and fruit development"

    Article Title: Global identification and analysis of long non-coding RNAs in diploid strawberry Fragaria vesca during flower and fruit development

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-2014-2

    Validation of anther/pollen specific expression of lncRNAs by qRT-PCR. a Gel image of RT-PCR products of ten randomly selected anther/pollen specific lncRNAs. b to g The expression of six lncRNAs quantified by qRT-PCR (black bar and Y-axis on the left). Error bar indicates standard deviation (SD) of two biological replicates with three technical replicates each. The relative FPKM of the same six lncRNAs based on RNA-seq data was also shown (red line and Y-axis on the right). Gene11892 was used as the internal control for both RNA-seq (red line) and qRT-PCR (black bar). RNAs were from anthers at stage7/8, stage9, stage10, stage11, and stage12 as well as mature pollen
    Figure Legend Snippet: Validation of anther/pollen specific expression of lncRNAs by qRT-PCR. a Gel image of RT-PCR products of ten randomly selected anther/pollen specific lncRNAs. b to g The expression of six lncRNAs quantified by qRT-PCR (black bar and Y-axis on the left). Error bar indicates standard deviation (SD) of two biological replicates with three technical replicates each. The relative FPKM of the same six lncRNAs based on RNA-seq data was also shown (red line and Y-axis on the right). Gene11892 was used as the internal control for both RNA-seq (red line) and qRT-PCR (black bar). RNAs were from anthers at stage7/8, stage9, stage10, stage11, and stage12 as well as mature pollen

    Techniques Used: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, RNA Sequencing Assay

    36) Product Images from "Simvastatin induces cell cycle arrest and inhibits proliferation of bladder cancer cells via PPARγ signalling pathway"

    Article Title: Simvastatin induces cell cycle arrest and inhibits proliferation of bladder cancer cells via PPARγ signalling pathway

    Journal: Scientific Reports

    doi: 10.1038/srep35783

    Transcriptome profiling of bladder cancer compared to normal bladder tissues pointed out the PPAR family. ( a ) Heat map of the differentially expressed genes in three bladder cancer tissues compared with three normal bladder tissues. Red color indicated upregulated genes and green color indicated downregulated genes. ( b ) GO-map network analysis by GCBI platform revealed fatty acid biosynthesis and glycerolipid metabolism were linked with bladder cancer via PPAR and ErbB signalling pathways, as well as a close correlation between bladder cancer and cell cycle. ( c ) Semiquantitative RT-PCR analysis for alterations of PPAR family ( PPARα , PPARβ and PPAR γ) using pooled total RNA isolated from the three bladder cancer tissues versus three normal bladder tissues. The expression of the GAPDH mRNA was used as a loading control. ( d ) ELISA analysis revealed the relative PPARγ DNA-binding activity in the BCa tissues was significantly decreased comparing with the normal bladder tissues (n = 3). *p
    Figure Legend Snippet: Transcriptome profiling of bladder cancer compared to normal bladder tissues pointed out the PPAR family. ( a ) Heat map of the differentially expressed genes in three bladder cancer tissues compared with three normal bladder tissues. Red color indicated upregulated genes and green color indicated downregulated genes. ( b ) GO-map network analysis by GCBI platform revealed fatty acid biosynthesis and glycerolipid metabolism were linked with bladder cancer via PPAR and ErbB signalling pathways, as well as a close correlation between bladder cancer and cell cycle. ( c ) Semiquantitative RT-PCR analysis for alterations of PPAR family ( PPARα , PPARβ and PPAR γ) using pooled total RNA isolated from the three bladder cancer tissues versus three normal bladder tissues. The expression of the GAPDH mRNA was used as a loading control. ( d ) ELISA analysis revealed the relative PPARγ DNA-binding activity in the BCa tissues was significantly decreased comparing with the normal bladder tissues (n = 3). *p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay, Activity Assay, BIA-KA

    37) Product Images from "MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae"

    Article Title: MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1458171

    MoSNT2 is associated with the MoTor signaling pathway. (A) Vegetative growth of M. oryzae on CM agar medium supplemented with or without 1 μg/ml rapamycin (rapa.). (B) Inhibition rate of rapamycin on the mycelial growth. (C) Expression profiles of MoSNT2 and MoTOR in the wild-type Guy11 strain at different developmental processes. (D) Linear correlation between qRT-PCR-measured expression levels of MoSNT2 and MoTOR . (E) qRT-PCR analysis of MoSNT2 expression levels in the Guy11 strain in response to rapamycin. The Guy11 strain grown in liquid CM for 48 h was transferred into fresh liquid CM in the presence or absence of 1 μg/ml rapamycin for 6 h before total RNA extraction.
    Figure Legend Snippet: MoSNT2 is associated with the MoTor signaling pathway. (A) Vegetative growth of M. oryzae on CM agar medium supplemented with or without 1 μg/ml rapamycin (rapa.). (B) Inhibition rate of rapamycin on the mycelial growth. (C) Expression profiles of MoSNT2 and MoTOR in the wild-type Guy11 strain at different developmental processes. (D) Linear correlation between qRT-PCR-measured expression levels of MoSNT2 and MoTOR . (E) qRT-PCR analysis of MoSNT2 expression levels in the Guy11 strain in response to rapamycin. The Guy11 strain grown in liquid CM for 48 h was transferred into fresh liquid CM in the presence or absence of 1 μg/ml rapamycin for 6 h before total RNA extraction.

    Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, RNA Extraction

    38) Product Images from "Candida albicans gains azole resistance by altering sphingolipid composition"

    Article Title: Candida albicans gains azole resistance by altering sphingolipid composition

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06944-1

    PB transposition in haploid C. albicans . a Schematic description of the CaPBase construct inserted at the ADH1 locus. Ptet, Tet-On promoter; 3XSV40, SV40 nuclear localization signal; and P ADH1 , ADH1 promoter. Hyperactive mutations are indicated along the top. b Selection of transformants with PB[URA3] integration on plates. YW01 cells were grown in YPD for 48 h in the presence (+) or absence (−) of Dox before transformation with pPB[URA3]. Transformation products were spread onto GMM plates (90 mm Petri dishes). c Schematic description of the PB[URA3] cassette integrated within ARG4 . Arrows indicate the PCR primers used to detect PB excision. d Confirmation of the auxotrophic phenotypes of transposition products of YW02. Strains grown on a GMM+Uri+Arg+His (GMM+) plate were replica-transferred onto a GMM+Uri+His (-Arg) or GMM+Arg+His (-Uri) plate and then incubated at 30 °C overnight. YW01 and YW02 were included as controls. e Precise excision of PB from the ARG4 locus. Representative gel electrophoresis results of PCR products amplified from YW02 transposition products using primers shown in c . f Schematic description of the identification of PB insertion sites by inverse PCR. Arrows indicate the PCR (PBLf and PBLr) and sequencing (Seq1) primers used. g Distribution of PB insertion sites in and around ORFs in YW02 transposition products. Positions in the ORF were determined by treating the entire length of an ORF as 100 and then determining the relative position of an insertion in the ORF. Positions in the 5′ or 3′ region were determined by whether the insertion was closest to the 5′ or 3′ end of an ORF
    Figure Legend Snippet: PB transposition in haploid C. albicans . a Schematic description of the CaPBase construct inserted at the ADH1 locus. Ptet, Tet-On promoter; 3XSV40, SV40 nuclear localization signal; and P ADH1 , ADH1 promoter. Hyperactive mutations are indicated along the top. b Selection of transformants with PB[URA3] integration on plates. YW01 cells were grown in YPD for 48 h in the presence (+) or absence (−) of Dox before transformation with pPB[URA3]. Transformation products were spread onto GMM plates (90 mm Petri dishes). c Schematic description of the PB[URA3] cassette integrated within ARG4 . Arrows indicate the PCR primers used to detect PB excision. d Confirmation of the auxotrophic phenotypes of transposition products of YW02. Strains grown on a GMM+Uri+Arg+His (GMM+) plate were replica-transferred onto a GMM+Uri+His (-Arg) or GMM+Arg+His (-Uri) plate and then incubated at 30 °C overnight. YW01 and YW02 were included as controls. e Precise excision of PB from the ARG4 locus. Representative gel electrophoresis results of PCR products amplified from YW02 transposition products using primers shown in c . f Schematic description of the identification of PB insertion sites by inverse PCR. Arrows indicate the PCR (PBLf and PBLr) and sequencing (Seq1) primers used. g Distribution of PB insertion sites in and around ORFs in YW02 transposition products. Positions in the ORF were determined by treating the entire length of an ORF as 100 and then determining the relative position of an insertion in the ORF. Positions in the 5′ or 3′ region were determined by whether the insertion was closest to the 5′ or 3′ end of an ORF

    Techniques Used: Construct, Selection, Transformation Assay, Polymerase Chain Reaction, Incubation, Nucleic Acid Electrophoresis, Amplification, Inverse PCR, Sequencing

    PB transposition and insert distribution in C. albicans genome. a qPCR analysis of CaPBase expression during transposition induction. CDC28 mRNA level was used for normalization. Error bars represent standard deviation (s.d.) from the mean of triplicate samples. b Ploidy analysis during transposition induction by flow cytometry. The DNA content of induced cells at each time point was compared with that of haploid and diploid control strains. c Estimation of transposition efficiency in YW02. See the text for description. Error bars represent s.d. from the mean of three independent experiments. d Analysis of PB copy number in transposition products by comparing the levels of URA3 and CDC28 DNA using qPCR. YW01 and YW02 were included as controls. Error bars represent s.d. from the mean of triplicate samples. e Genome-wide NGS analysis of PB distribution. PB -specific reads were plotted in 10-kb sliding windows. f Histogram of PB -specific read counts and PB -specific site counts per 10-kb window. g Motif analysis of PB -insertion sites
    Figure Legend Snippet: PB transposition and insert distribution in C. albicans genome. a qPCR analysis of CaPBase expression during transposition induction. CDC28 mRNA level was used for normalization. Error bars represent standard deviation (s.d.) from the mean of triplicate samples. b Ploidy analysis during transposition induction by flow cytometry. The DNA content of induced cells at each time point was compared with that of haploid and diploid control strains. c Estimation of transposition efficiency in YW02. See the text for description. Error bars represent s.d. from the mean of three independent experiments. d Analysis of PB copy number in transposition products by comparing the levels of URA3 and CDC28 DNA using qPCR. YW01 and YW02 were included as controls. Error bars represent s.d. from the mean of triplicate samples. e Genome-wide NGS analysis of PB distribution. PB -specific reads were plotted in 10-kb sliding windows. f Histogram of PB -specific read counts and PB -specific site counts per 10-kb window. g Motif analysis of PB -insertion sites

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Standard Deviation, Flow Cytometry, Cytometry, Genome Wide, Next-Generation Sequencing

    39) Product Images from "Chronic stress enhances progression of acute lymphoblastic leukemia via ?-adrenergic signaling"

    Article Title: Chronic stress enhances progression of acute lymphoblastic leukemia via ?-adrenergic signaling

    Journal: Brain, Behavior, and Immunity

    doi: 10.1016/j.bbi.2012.01.013

    Effect of chronic restraint stress on ALL progression. (A) 21-day longitudinal growth curves for total body Nalm-6 tumor burden in control and restraint-stressed animals. (B) Nalm-6 tumor burden in separate body areas at Day 14 in control and restraint-stressed
    Figure Legend Snippet: Effect of chronic restraint stress on ALL progression. (A) 21-day longitudinal growth curves for total body Nalm-6 tumor burden in control and restraint-stressed animals. (B) Nalm-6 tumor burden in separate body areas at Day 14 in control and restraint-stressed

    Techniques Used:

    Nalm-6 ALL Model. (A) Nalm-6 pre-B ALL cells were localized and quantified by periodic imaging of leukemia-specific bioluminescence signal from ventral and dorsal sides of SCID mice. (B) Femoral bone marrow white blood cells (WBC) and CD10+ Nalm-6 ALL
    Figure Legend Snippet: Nalm-6 ALL Model. (A) Nalm-6 pre-B ALL cells were localized and quantified by periodic imaging of leukemia-specific bioluminescence signal from ventral and dorsal sides of SCID mice. (B) Femoral bone marrow white blood cells (WBC) and CD10+ Nalm-6 ALL

    Techniques Used: Imaging, Mouse Assay

    β-adrenergic signaling in stress-enhanced ALL progression. (A) Nalm-6 ALL was quantified in control mice vs. stress mice that were treated with or without the β-blocker, propranolol; data representative of 3 independent experiments. (B)
    Figure Legend Snippet: β-adrenergic signaling in stress-enhanced ALL progression. (A) Nalm-6 ALL was quantified in control mice vs. stress mice that were treated with or without the β-blocker, propranolol; data representative of 3 independent experiments. (B)

    Techniques Used: Mouse Assay

    40) Product Images from "Differential Roles of Two Homologous Cyclin-Dependent Kinase Inhibitor Genes in Regulating Cell Cycle and Innate Immunity in Arabidopsis 1Differential Roles of Two Homologous Cyclin-Dependent Kinase Inhibitor Genes in Regulating Cell Cycle and Innate Immunity in Arabidopsis 1 [OPEN]"

    Article Title: Differential Roles of Two Homologous Cyclin-Dependent Kinase Inhibitor Genes in Regulating Cell Cycle and Innate Immunity in Arabidopsis 1Differential Roles of Two Homologous Cyclin-Dependent Kinase Inhibitor Genes in Regulating Cell Cycle and Innate Immunity in Arabidopsis 1 [OPEN]

    Journal: Plant Physiology

    doi: 10.1104/pp.15.01466

    smr1-1 accumulation, and cell death in acd6-1 ; 2, acd6-1 ; 3, acd6-1 sim-1 ; 4, acd6-1 smr1-1 ; 5, acd6-1 sim-1 smr1-1 . B, Plant size comparison. Rosette diameters
    Figure Legend Snippet: smr1-1 accumulation, and cell death in acd6-1 ; 2, acd6-1 ; 3, acd6-1 sim-1 ; 4, acd6-1 smr1-1 ; 5, acd6-1 sim-1 smr1-1 . B, Plant size comparison. Rosette diameters

    Techniques Used:

    41) Product Images from "Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq"

    Article Title: Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00185

    Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p
    Figure Legend Snippet: Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p

    Techniques Used: Laser Capture Microdissection, Isolation

    42) Product Images from "Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza"

    Article Title: Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04792-7

    Construction and antiviral activity of defective interfering genes (DIG). a The plasmid construction of DI-PB2, DI-PB1, and DI-PA. The indicated sequences of shortened viral polymerase gene PB2, PB1, and PA were inserted into phw2000, respectively. Dotted lines indicate the internal deletion of wild-type (WT) viral polymerase genes. b , c DI RNA expression in 293T and A549 cells. The plasmids of DI-PB2, DI-PB1, and DI-PA were co-transfected into cells with the indicated concentrations. At 24 h post transfection, DI RNAs were extracted from cells and digested by DNase I for RT-qPCR. Empty vector was used as a negative control for RT-qPCR. d Anti-A(H7N7) virus activity of individual plasmid of DI-PB2, DI-PB1, and DI-PA or three combined plasmid DIG (DIG-3, 0.6 μg per well). e , f Dose-dependent anti-A(H7N7) virus activity of DIG-3 in 293T and A549 cells. g Anti-A(H5N1) virus activity of DIG-3. Empty vector phw2000 and plasmids with DIG were individually transfected to cells. At 24 h post transfection, cells were infected with A(H7N7) or A(H5N1) virus at MOI = 0.005 and cell supernatants were collected at 40 h post infection. Viral titers in the supernatants were detected by plaque assay. Data were presented as mean ± SD of three independent experiments. * Indicates P
    Figure Legend Snippet: Construction and antiviral activity of defective interfering genes (DIG). a The plasmid construction of DI-PB2, DI-PB1, and DI-PA. The indicated sequences of shortened viral polymerase gene PB2, PB1, and PA were inserted into phw2000, respectively. Dotted lines indicate the internal deletion of wild-type (WT) viral polymerase genes. b , c DI RNA expression in 293T and A549 cells. The plasmids of DI-PB2, DI-PB1, and DI-PA were co-transfected into cells with the indicated concentrations. At 24 h post transfection, DI RNAs were extracted from cells and digested by DNase I for RT-qPCR. Empty vector was used as a negative control for RT-qPCR. d Anti-A(H7N7) virus activity of individual plasmid of DI-PB2, DI-PB1, and DI-PA or three combined plasmid DIG (DIG-3, 0.6 μg per well). e , f Dose-dependent anti-A(H7N7) virus activity of DIG-3 in 293T and A549 cells. g Anti-A(H5N1) virus activity of DIG-3. Empty vector phw2000 and plasmids with DIG were individually transfected to cells. At 24 h post transfection, cells were infected with A(H7N7) or A(H5N1) virus at MOI = 0.005 and cell supernatants were collected at 40 h post infection. Viral titers in the supernatants were detected by plaque assay. Data were presented as mean ± SD of three independent experiments. * Indicates P

    Techniques Used: Activity Assay, Plasmid Preparation, RNA Expression, Transfection, Quantitative RT-PCR, Negative Control, Infection, Plaque Assay

    43) Product Images from "Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning"

    Article Title: Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning

    Journal: Neural Plasticity

    doi: 10.1155/2016/1752176

    Distribution of cannula placements and RAG1 antisense oligonucleotide diffusion within the amygdala. After behavioral treatments with RAG1 antisense or random oligonucleotides, animals were microinfused the next day with thionine to verify cannulae injectors' placement. Another set of animals was used to observe FITC-labeled RAG1 antisense diffusion. (a) Schematic representation of the amygdala at different rostrocaudal planes illustrating the position of cannulae injectors determined by thionine microinfusion. Injector tips for each cannula are represented by dark spots. (b) FITC- RAG1 antisense diffusion within the amygdalar complex; arrow indicates the injector's tip. (c) Schemes of coronal sections showing the diffusion of FITC- RAG1 antisense diffusion into the amygdala of animals decapitated 3 h after fluorescent oligonucleotide infusion. FITC- RAG1 antisense diffusion is represented by green shading from anterior to posterior areas of the amygdalar complex. The numbers in (a) and (c) indicate the distance from bregma in millimeters. A total of 4 mice were used in these studies. (d) Photomicrograph at higher magnification of FITC- RAG1 antisense diffusion showed clearly incorporation into the cells (depicted by the arrows) within amygdalar regions.
    Figure Legend Snippet: Distribution of cannula placements and RAG1 antisense oligonucleotide diffusion within the amygdala. After behavioral treatments with RAG1 antisense or random oligonucleotides, animals were microinfused the next day with thionine to verify cannulae injectors' placement. Another set of animals was used to observe FITC-labeled RAG1 antisense diffusion. (a) Schematic representation of the amygdala at different rostrocaudal planes illustrating the position of cannulae injectors determined by thionine microinfusion. Injector tips for each cannula are represented by dark spots. (b) FITC- RAG1 antisense diffusion within the amygdalar complex; arrow indicates the injector's tip. (c) Schemes of coronal sections showing the diffusion of FITC- RAG1 antisense diffusion into the amygdala of animals decapitated 3 h after fluorescent oligonucleotide infusion. FITC- RAG1 antisense diffusion is represented by green shading from anterior to posterior areas of the amygdalar complex. The numbers in (a) and (c) indicate the distance from bregma in millimeters. A total of 4 mice were used in these studies. (d) Photomicrograph at higher magnification of FITC- RAG1 antisense diffusion showed clearly incorporation into the cells (depicted by the arrows) within amygdalar regions.

    Techniques Used: Diffusion-based Assay, Labeling, Mouse Assay

    RAG1 antisense amygdalar treatment impaired consolidation of context fear conditioning. Top panel: diagram depicting the experimental design of these experiments for pretraining or posttraining amygdalar antisense or random oligonucleotide microinfusion experiments. In the pretraining microinfusion experiments, mice received RAG1 antisense or random bilateral oligonucleotide microinfusions directed at the amygdala 1 h before conditioning followed by either LTM testing or molecular evaluation. LTM was tested 24 h after conditioning. For molecular evaluation of antisense treatment effectiveness, another group of mice was sacrificed 30 min after conditioning and amygdalar RNA was used for real-time PCR. In the posttraining microinfusion experiments, mice were conditioned, returned to their home cages, and received microinfusions of antisense or random oligonucleotides 5 h after training and returned to their home cages until next day. Nineteen (19) hours later (24 h after conditioning), mice were reexposed to the conditioning chamber without any shocks in order to test LTM. (a) Mice receiving either RAG1 antisense or random oligonucleotide treatment displayed no significant differences during memory acquisition measured as the progressive enhancement of freezing behavior (Two-Way ANOVA, Treatment Factor: F (1,0.8457) = 0.01015, P > 0.9; Training Factor F (3,7863) = 94.37, ∗∗∗ P
    Figure Legend Snippet: RAG1 antisense amygdalar treatment impaired consolidation of context fear conditioning. Top panel: diagram depicting the experimental design of these experiments for pretraining or posttraining amygdalar antisense or random oligonucleotide microinfusion experiments. In the pretraining microinfusion experiments, mice received RAG1 antisense or random bilateral oligonucleotide microinfusions directed at the amygdala 1 h before conditioning followed by either LTM testing or molecular evaluation. LTM was tested 24 h after conditioning. For molecular evaluation of antisense treatment effectiveness, another group of mice was sacrificed 30 min after conditioning and amygdalar RNA was used for real-time PCR. In the posttraining microinfusion experiments, mice were conditioned, returned to their home cages, and received microinfusions of antisense or random oligonucleotides 5 h after training and returned to their home cages until next day. Nineteen (19) hours later (24 h after conditioning), mice were reexposed to the conditioning chamber without any shocks in order to test LTM. (a) Mice receiving either RAG1 antisense or random oligonucleotide treatment displayed no significant differences during memory acquisition measured as the progressive enhancement of freezing behavior (Two-Way ANOVA, Treatment Factor: F (1,0.8457) = 0.01015, P > 0.9; Training Factor F (3,7863) = 94.37, ∗∗∗ P

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

    RAG1 antisense amygdalar treatment does not interfere with reconsolidation of context fear conditioning. To test the effects of RAG1 gapmer antisense treatment on memory reconsolidation of context fear conditioning, another set of animals was bilaterally implanted with cannulas to target the amygdala. Top panel: diagram depicting the experimental design. On day 1, mice were trained in context fear conditioning and immediately returned to their home cages. Antisense or random oligonucleotides were microinfused into the amygdala 1 h before memory reactivation on day 2. The effect of antisense or random oligonucleotide treatment on LTM reconsolidation was assessed on day 3, 48 h after conditioning. (a) On day 1, mice were microinfused with saline 1 h before training and returned to their home cages immediately after conditioning. Two-Way RM ANOVA and Bonferroni posttesting demonstrated that the infusions did not impair the animals' response in developing and expressing fear during the conditioning experience (Treatment Assignment Factor: F (1,8.194) = 3.979, P > 0.05; Training Factor F (3,3134) = 1725, ∗∗∗ P
    Figure Legend Snippet: RAG1 antisense amygdalar treatment does not interfere with reconsolidation of context fear conditioning. To test the effects of RAG1 gapmer antisense treatment on memory reconsolidation of context fear conditioning, another set of animals was bilaterally implanted with cannulas to target the amygdala. Top panel: diagram depicting the experimental design. On day 1, mice were trained in context fear conditioning and immediately returned to their home cages. Antisense or random oligonucleotides were microinfused into the amygdala 1 h before memory reactivation on day 2. The effect of antisense or random oligonucleotide treatment on LTM reconsolidation was assessed on day 3, 48 h after conditioning. (a) On day 1, mice were microinfused with saline 1 h before training and returned to their home cages immediately after conditioning. Two-Way RM ANOVA and Bonferroni posttesting demonstrated that the infusions did not impair the animals' response in developing and expressing fear during the conditioning experience (Treatment Assignment Factor: F (1,8.194) = 3.979, P > 0.05; Training Factor F (3,3134) = 1725, ∗∗∗ P

    Techniques Used: Mouse Assay, Expressing

    RAG1 protein expression in amygdalar neuronal cells. Amygdalar coronal sections of context fear conditioning-trained mice, perfused 1 h after conditioning, were used for immunofluorescence and analyzed by confocal microscopy. Antibodies from immunofluorescence were validated by Western blot analysis. (a) Amygdalar area representative images of a double immunostaining using RAG1 antibody labeled with Alexa Fluor 488, green channel signal, and NeuN antibody labeled with Alexa Fluor 568, red channel signal. The left panel shows the NeuN positive neuronal nuclei, while the middle panel depicts RAG1 immunopositive cells. The right panel is the merge image showing colocalization of the NeuN neuronal nuclei marker and RAG1. Arrows point to some of the RAG1 immunopositive neurons. These immunofluorescent images revealed colocalization of RAG1 protein expressing cells with those expressing NeuN, suggesting the presence of RAG1 in neurons, although not all neurons expressed RAG1. (b) Tissue punches from amygdala (Amy) were obtained 1 h after context fear conditioning and analyzed in Western blot by comparative comigration with a standard molecular weight (MW) marker and protein extracts from bone marrow (BM) ((b)-1) and thymus (Thy) ((b)-2). Both sets of experiments consistently showed comigration between the tissues with a band corresponding to ~120 KD of RAG1 protein (green channel corresponding to RAG1 and red channel corresponding to beta-actin, ~42 KD); prestained molecular weight (MW) marker (ladder) was included in all the Western blots. ((b)-3) Additionally, tissue protein extracts from leg muscle (Mus) (negative control) were analyzed compared to amygdalar extracts with respect to RAG1 expression. As expected, RAG1 was not expressed in muscle compared to amygdala ((b)-3), bone marrow ((b)-1), and thymus ((b)-2). ((b)-4) RAG1 antibody preabsorption assays, either with muscle or with bone marrow extracts, showed that only bone marrow extracts, which express RAG1 as opposed to muscle, were able to block the ~120 KD band from amygdalar protein extracts in the Western blots, indicating that RAG1 antibody was preabsorbed (blocked) only by RAG1 protein expressing tissue (bone marrow).
    Figure Legend Snippet: RAG1 protein expression in amygdalar neuronal cells. Amygdalar coronal sections of context fear conditioning-trained mice, perfused 1 h after conditioning, were used for immunofluorescence and analyzed by confocal microscopy. Antibodies from immunofluorescence were validated by Western blot analysis. (a) Amygdalar area representative images of a double immunostaining using RAG1 antibody labeled with Alexa Fluor 488, green channel signal, and NeuN antibody labeled with Alexa Fluor 568, red channel signal. The left panel shows the NeuN positive neuronal nuclei, while the middle panel depicts RAG1 immunopositive cells. The right panel is the merge image showing colocalization of the NeuN neuronal nuclei marker and RAG1. Arrows point to some of the RAG1 immunopositive neurons. These immunofluorescent images revealed colocalization of RAG1 protein expressing cells with those expressing NeuN, suggesting the presence of RAG1 in neurons, although not all neurons expressed RAG1. (b) Tissue punches from amygdala (Amy) were obtained 1 h after context fear conditioning and analyzed in Western blot by comparative comigration with a standard molecular weight (MW) marker and protein extracts from bone marrow (BM) ((b)-1) and thymus (Thy) ((b)-2). Both sets of experiments consistently showed comigration between the tissues with a band corresponding to ~120 KD of RAG1 protein (green channel corresponding to RAG1 and red channel corresponding to beta-actin, ~42 KD); prestained molecular weight (MW) marker (ladder) was included in all the Western blots. ((b)-3) Additionally, tissue protein extracts from leg muscle (Mus) (negative control) were analyzed compared to amygdalar extracts with respect to RAG1 expression. As expected, RAG1 was not expressed in muscle compared to amygdala ((b)-3), bone marrow ((b)-1), and thymus ((b)-2). ((b)-4) RAG1 antibody preabsorption assays, either with muscle or with bone marrow extracts, showed that only bone marrow extracts, which express RAG1 as opposed to muscle, were able to block the ~120 KD band from amygdalar protein extracts in the Western blots, indicating that RAG1 antibody was preabsorbed (blocked) only by RAG1 protein expressing tissue (bone marrow).

    Techniques Used: Expressing, Mouse Assay, Immunofluorescence, Confocal Microscopy, Western Blot, Double Immunostaining, Labeling, Marker, Molecular Weight, Negative Control, Blocking Assay

    44) Product Images from "Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning"

    Article Title: Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning

    Journal: Neural Plasticity

    doi: 10.1155/2016/1752176

    RAG1 antisense amygdalar treatment impaired consolidation of context fear conditioning. Top panel: diagram depicting the experimental design of these experiments for pretraining or posttraining amygdalar antisense or random oligonucleotide microinfusion experiments. In the pretraining microinfusion experiments, mice received RAG1 antisense or random bilateral oligonucleotide microinfusions directed at the amygdala 1 h before conditioning followed by either LTM testing or molecular evaluation. LTM was tested 24 h after conditioning. For molecular evaluation of antisense treatment effectiveness, another group of mice was sacrificed 30 min after conditioning and amygdalar RNA was used for real-time PCR. In the posttraining microinfusion experiments, mice were conditioned, returned to their home cages, and received microinfusions of antisense or random oligonucleotides 5 h after training and returned to their home cages until next day. Nineteen (19) hours later (24 h after conditioning), mice were reexposed to the conditioning chamber without any shocks in order to test LTM. (a) Mice receiving either RAG1 antisense or random oligonucleotide treatment displayed no significant differences during memory acquisition measured as the progressive enhancement of freezing behavior (Two-Way ANOVA, Treatment Factor: F (1,0.8457) = 0.01015, P > 0.9; Training Factor F (3,7863) = 94.37, ∗∗∗ P
    Figure Legend Snippet: RAG1 antisense amygdalar treatment impaired consolidation of context fear conditioning. Top panel: diagram depicting the experimental design of these experiments for pretraining or posttraining amygdalar antisense or random oligonucleotide microinfusion experiments. In the pretraining microinfusion experiments, mice received RAG1 antisense or random bilateral oligonucleotide microinfusions directed at the amygdala 1 h before conditioning followed by either LTM testing or molecular evaluation. LTM was tested 24 h after conditioning. For molecular evaluation of antisense treatment effectiveness, another group of mice was sacrificed 30 min after conditioning and amygdalar RNA was used for real-time PCR. In the posttraining microinfusion experiments, mice were conditioned, returned to their home cages, and received microinfusions of antisense or random oligonucleotides 5 h after training and returned to their home cages until next day. Nineteen (19) hours later (24 h after conditioning), mice were reexposed to the conditioning chamber without any shocks in order to test LTM. (a) Mice receiving either RAG1 antisense or random oligonucleotide treatment displayed no significant differences during memory acquisition measured as the progressive enhancement of freezing behavior (Two-Way ANOVA, Treatment Factor: F (1,0.8457) = 0.01015, P > 0.9; Training Factor F (3,7863) = 94.37, ∗∗∗ P

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

    45) Product Images from "FTY720/Fingolimod Reduces Synucleinopathy and Improves Gut Motility in A53T Mice"

    Article Title: FTY720/Fingolimod Reduces Synucleinopathy and Improves Gut Motility in A53T Mice

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.744029

    FTY720 stimulates long term increases in BDNF protein in aging Tg mice in association with significantly lower levels of miR206–3p. A , BDNF protein normalized to β-actin on immunoblots confirmed that BDNF was increased in colons of FTY720-treated 21-month-old mice. B , the expression of the regulatory microRNA, miR206–3p, was significantly lower in response to FTY720 treatment of aged Tg mice as compared with vehicle Tg mice. The decrease in miR206–3p was further validated in a control experiment with dopaminergic MN9D cells treated with 160 n m FTY720 for 24 h. ( n = 8 mice/treatment group); *, p
    Figure Legend Snippet: FTY720 stimulates long term increases in BDNF protein in aging Tg mice in association with significantly lower levels of miR206–3p. A , BDNF protein normalized to β-actin on immunoblots confirmed that BDNF was increased in colons of FTY720-treated 21-month-old mice. B , the expression of the regulatory microRNA, miR206–3p, was significantly lower in response to FTY720 treatment of aged Tg mice as compared with vehicle Tg mice. The decrease in miR206–3p was further validated in a control experiment with dopaminergic MN9D cells treated with 160 n m FTY720 for 24 h. ( n = 8 mice/treatment group); *, p

    Techniques Used: Mouse Assay, Western Blot, Expressing

    46) Product Images from "Decreased TRPM7 inhibits activities and induces apoptosis of bladder cancer cells via ERK1/2 pathway"

    Article Title: Decreased TRPM7 inhibits activities and induces apoptosis of bladder cancer cells via ERK1/2 pathway

    Journal: Oncotarget

    doi: 10.18632/oncotarget.12146

    TRPM7 is upregulated in the BCa tissues and correlated with EMT markers ( A ) qRT-PCR analysis of relative gene expression of TRPM7 in total RNA isolated from ten BCa tissues at stage II, comparing with ten normal bladder tissues. Significance of TRPM7 expression difference was analyzed using T-test . * p
    Figure Legend Snippet: TRPM7 is upregulated in the BCa tissues and correlated with EMT markers ( A ) qRT-PCR analysis of relative gene expression of TRPM7 in total RNA isolated from ten BCa tissues at stage II, comparing with ten normal bladder tissues. Significance of TRPM7 expression difference was analyzed using T-test . * p

    Techniques Used: BIA-KA, Quantitative RT-PCR, Expressing, Isolation

    47) Product Images from "Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines"

    Article Title: Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines

    Journal: BMC Research Notes

    doi: 10.1186/s13104-017-2959-0

    Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1 , b brm , c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS - RNAi constructs with Act - Gal4 . Expression levels were normalized to the reference genes, eIF2Bγ and βCOP . Shown here, are relative expression values compared to the UAS - mCherry - RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p
    Figure Legend Snippet: Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1 , b brm , c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS - RNAi constructs with Act - Gal4 . Expression levels were normalized to the reference genes, eIF2Bγ and βCOP . Shown here, are relative expression values compared to the UAS - mCherry - RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Synthesized, Expressing, Construct, Activated Clotting Time Assay

    Schematic representation of the experimental setup. siRNAs direct site-specific cleavage of mRNAs, resulting in a 5′ and 3′ mRNA cleavage fragments. After RNA isolation total RNA samples consist of uncleaved mRNA transcripts and non-coding RNA, as well as undegraded 5′ and 3′ mRNA cleavage fragments. Purification of mRNA using poly-T beads excludes 5′ mRNA cleavage fragments and non-coding RNAs that are not polyadenylated. As indicated by the boxes, 5′ and 3′ primer sets could detect different species of RNA depending on the isolation method
    Figure Legend Snippet: Schematic representation of the experimental setup. siRNAs direct site-specific cleavage of mRNAs, resulting in a 5′ and 3′ mRNA cleavage fragments. After RNA isolation total RNA samples consist of uncleaved mRNA transcripts and non-coding RNA, as well as undegraded 5′ and 3′ mRNA cleavage fragments. Purification of mRNA using poly-T beads excludes 5′ mRNA cleavage fragments and non-coding RNAs that are not polyadenylated. As indicated by the boxes, 5′ and 3′ primer sets could detect different species of RNA depending on the isolation method

    Techniques Used: Isolation, Purification

    48) Product Images from "Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule"

    Article Title: Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule

    Journal: Journal of Medicinal Chemistry

    doi: 10.1021/acs.jmedchem.7b01781

    CM03 treatment reduces tumor volume in a MIA PaCa-2 xenograft model of PDAC. (a) Plot showing the tumor volume of MIA PaCa-2 xenografts treated with CM03, MM41, gemcitabine, or saline (control) over 62 days. There are eight CD-1 mice per condition and dosing for all cohorts was stopped on day 28, shown by the red arrow. Standard error of the mean (SEM) is indicated for all growth curves for each tumor volume. * p
    Figure Legend Snippet: CM03 treatment reduces tumor volume in a MIA PaCa-2 xenograft model of PDAC. (a) Plot showing the tumor volume of MIA PaCa-2 xenografts treated with CM03, MM41, gemcitabine, or saline (control) over 62 days. There are eight CD-1 mice per condition and dosing for all cohorts was stopped on day 28, shown by the red arrow. Standard error of the mean (SEM) is indicated for all growth curves for each tumor volume. * p

    Techniques Used: Mouse Assay

    Validation of mRNA down regulation by qRT-PCR for a subset of down-regulated genes, selected from RNA-Seq experiments. (a–d) MIA PaCa-2 and PANC-1 cells were treated (a and b) with 400 nM CM03 and (c and d) with 400 nM gemcitabine, all for 6 and 24 h. Total mRNA was extracted, reverse transcribed into cDNA, and then qRT-PCR was performed. The C t values were normalized to the genomic mean of three housekeeping genes ( ACTB , GAPDH , and TUBB ), and the relative gene expression was determined using the Livak method, 2 –ΔΔ C t . The log-fold expression changes (Log 2 FC) for each gene are shown relative to vehicle-treated controls (PBS for CM03 and DMSO for gemcitabine). Student’s t test along with 2 –Δ C t values were used to determine the statistical significance of the observed changes, which are the mean of in each case at least three determinations. Those genes with changes in expression with p
    Figure Legend Snippet: Validation of mRNA down regulation by qRT-PCR for a subset of down-regulated genes, selected from RNA-Seq experiments. (a–d) MIA PaCa-2 and PANC-1 cells were treated (a and b) with 400 nM CM03 and (c and d) with 400 nM gemcitabine, all for 6 and 24 h. Total mRNA was extracted, reverse transcribed into cDNA, and then qRT-PCR was performed. The C t values were normalized to the genomic mean of three housekeeping genes ( ACTB , GAPDH , and TUBB ), and the relative gene expression was determined using the Livak method, 2 –ΔΔ C t . The log-fold expression changes (Log 2 FC) for each gene are shown relative to vehicle-treated controls (PBS for CM03 and DMSO for gemcitabine). Student’s t test along with 2 –Δ C t values were used to determine the statistical significance of the observed changes, which are the mean of in each case at least three determinations. Those genes with changes in expression with p

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

    49) Product Images from "NADPH oxidase 5 (NOX5)—induced reactive oxygen signaling modulates normoxic HIF‐1α and p27Kip1 expression in malignant melanoma and other human tumors, et al. NADPH oxidase 5 (NOX5)—induced reactive oxygen signaling modulates normoxic HIF‐1α and p27Kip1 expression in malignant melanoma and other human tumors"

    Article Title: NADPH oxidase 5 (NOX5)—induced reactive oxygen signaling modulates normoxic HIF‐1α and p27Kip1 expression in malignant melanoma and other human tumors, et al. NADPH oxidase 5 (NOX5)—induced reactive oxygen signaling modulates normoxic HIF‐1α and p27Kip1 expression in malignant melanoma and other human tumors

    Journal: Molecular Carcinogenesis

    doi: 10.1002/mc.22708

    NOX5 expression levels in human UACC‐257 melanoma cells affect normoxic HIF‐1α and p 27Kip1 expression. (A) Whole cell lysates from log‐phase UACC‐257‐vector control clones (scrambled‐sh and pcDNA3.1), UACC257‐shNOX5 clones (1‐3), and UACC257‐NOX5‐overexpressing clones (2 and 3) were prepared and analyzed by Western blotting. NOX5 overexpression in UACC‐257 cells results in decreased p 27Kip1 expression as well as increased normoxic HIF‐1α expression. Conversely, decreased normoxic HIF‐1α and corresponding increased p 27Kip1 expression was observed in NOX5 knockdown cells. (B and C) Transient knockdown with NOX5‐specific siRNA primers targeting different domains of human NOX5 in the stably overexpressing NOX5 clone (B) and parental UACC‐257 cells (C) results in decreased normoxic HIF‐1α and corresponding increased p 27Kip1 expression. (D) Increased normoxic HIF‐1α and decreased p 27Kip1 expression observed in NOX5‐overexpressing cells is ROS‐dependent. The NOX5‐overexpressing UACC257 clone was treated with NAC or PEG‐Catalase or PEG‐SOD and examined by Western analysis. Densitometric analysis of western blot analyses (Panels A‐D) of the expression of the various proteins relative to that of β‐Actin are represented in Supplementary Figure S3. (E) Total RNA was isolated from log phase parental UACC‐257 cells, UACC‐257 cells that were transiently transfected with either scrambled control or two different NOX5‐specific siRNAs, and UACC‐257 clones that stably overexpress either NOX5 or the vector. Real‐time PCR analysis of p 27Kip1 mRNA expression levels are expressed relative to β‐Actin. (F) Total RNA from parental UACC‐257 cells and stable NOX5‐overexpressing clone 3 cells transiently transfected with either a scrambled siRNA or NOX5‐specific siRNAs, stable vector clones and two additional NOX5 overexpressing clones were isolated. Real‐time PCR analysis of HIF‐1α mRNA levels are expressed relative to β‐Actin. **, P
    Figure Legend Snippet: NOX5 expression levels in human UACC‐257 melanoma cells affect normoxic HIF‐1α and p 27Kip1 expression. (A) Whole cell lysates from log‐phase UACC‐257‐vector control clones (scrambled‐sh and pcDNA3.1), UACC257‐shNOX5 clones (1‐3), and UACC257‐NOX5‐overexpressing clones (2 and 3) were prepared and analyzed by Western blotting. NOX5 overexpression in UACC‐257 cells results in decreased p 27Kip1 expression as well as increased normoxic HIF‐1α expression. Conversely, decreased normoxic HIF‐1α and corresponding increased p 27Kip1 expression was observed in NOX5 knockdown cells. (B and C) Transient knockdown with NOX5‐specific siRNA primers targeting different domains of human NOX5 in the stably overexpressing NOX5 clone (B) and parental UACC‐257 cells (C) results in decreased normoxic HIF‐1α and corresponding increased p 27Kip1 expression. (D) Increased normoxic HIF‐1α and decreased p 27Kip1 expression observed in NOX5‐overexpressing cells is ROS‐dependent. The NOX5‐overexpressing UACC257 clone was treated with NAC or PEG‐Catalase or PEG‐SOD and examined by Western analysis. Densitometric analysis of western blot analyses (Panels A‐D) of the expression of the various proteins relative to that of β‐Actin are represented in Supplementary Figure S3. (E) Total RNA was isolated from log phase parental UACC‐257 cells, UACC‐257 cells that were transiently transfected with either scrambled control or two different NOX5‐specific siRNAs, and UACC‐257 clones that stably overexpress either NOX5 or the vector. Real‐time PCR analysis of p 27Kip1 mRNA expression levels are expressed relative to β‐Actin. (F) Total RNA from parental UACC‐257 cells and stable NOX5‐overexpressing clone 3 cells transiently transfected with either a scrambled siRNA or NOX5‐specific siRNAs, stable vector clones and two additional NOX5 overexpressing clones were isolated. Real‐time PCR analysis of HIF‐1α mRNA levels are expressed relative to β‐Actin. **, P

    Techniques Used: Expressing, Plasmid Preparation, Clone Assay, Western Blot, Over Expression, Stable Transfection, Isolation, Transfection, Real-time Polymerase Chain Reaction

    NOX5 expression in human melanoma cell lines. Total RNA was extracted from human melanoma cell lines. (A) Quantitative real‐time PCR was carried out to detect endogenous NOX5 expression. Values are expressed relative to β‐Actin. (B and C) Stable overexpression (B) and stable knockdown (C) of NOX5 in UACC‐257 cells. UACC‐257 cells were transfected with either the vector pcDNA 3.1 or pcDNA3‐HA‐NOX5β plasmid for stable NOX5 overexpression (B); and with either scrambled shVector or shNOX5 plasmids for stable NOX5 knockdown (C). Several clones were isolated after G418 selection, of which two clones (Clone 1 and 2) for each of the vector plasmids (pcDNA 3.1 and scrambled sh) and three clones (Clone 1, 2, and 3) for both the NOX5‐overexpressing and NOX5‐knockdown were evaluated for further studies. NOX5 expression was confirmed at the mRNA level (left panel; B and C) and at the protein level (right panel; B and C). To visualize endogenous expression of NOX5 relative to the NOX5 overexpressors and the decrease of NOX5 expression in the NOX5 knockdowns relative to the endogenous NOX5, additional NOX5 (long exposure [2‐5 min] and very long exposure [5‐10 min] of film) panels are represented. The HA‐tag expression confirmed the specificity of the antibody for NOX5 expression. Tables on the right (panels B and C) represent the densitometric expression of NOX5 protein expression (long exposure) relative to that of β‐Actin
    Figure Legend Snippet: NOX5 expression in human melanoma cell lines. Total RNA was extracted from human melanoma cell lines. (A) Quantitative real‐time PCR was carried out to detect endogenous NOX5 expression. Values are expressed relative to β‐Actin. (B and C) Stable overexpression (B) and stable knockdown (C) of NOX5 in UACC‐257 cells. UACC‐257 cells were transfected with either the vector pcDNA 3.1 or pcDNA3‐HA‐NOX5β plasmid for stable NOX5 overexpression (B); and with either scrambled shVector or shNOX5 plasmids for stable NOX5 knockdown (C). Several clones were isolated after G418 selection, of which two clones (Clone 1 and 2) for each of the vector plasmids (pcDNA 3.1 and scrambled sh) and three clones (Clone 1, 2, and 3) for both the NOX5‐overexpressing and NOX5‐knockdown were evaluated for further studies. NOX5 expression was confirmed at the mRNA level (left panel; B and C) and at the protein level (right panel; B and C). To visualize endogenous expression of NOX5 relative to the NOX5 overexpressors and the decrease of NOX5 expression in the NOX5 knockdowns relative to the endogenous NOX5, additional NOX5 (long exposure [2‐5 min] and very long exposure [5‐10 min] of film) panels are represented. The HA‐tag expression confirmed the specificity of the antibody for NOX5 expression. Tables on the right (panels B and C) represent the densitometric expression of NOX5 protein expression (long exposure) relative to that of β‐Actin

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Over Expression, Transfection, Plasmid Preparation, Clone Assay, Isolation, Selection

    50) Product Images from "GmBZL3 acts as a major BR signaling regulator through crosstalk with multiple pathways in Glycine max"

    Article Title: GmBZL3 acts as a major BR signaling regulator through crosstalk with multiple pathways in Glycine max

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-019-1677-2

    Multiple amino acid sequence alignment of the GmBZL in Soybean and Arabidopsis. The marked features are the N-terminal DNA binding domain (blue underline), putative 14–3-3 binding site (blue letters and box), putative sites of phosphorylation by GSK-3 kinase (red star), and the PEST domain (green letter and over line). The conserved amino acid (proline) between Arabidopsis and soybean in the PEST region is indicated by a red box, and was mutated to the amino acid leucine for the validation of conserved functional
    Figure Legend Snippet: Multiple amino acid sequence alignment of the GmBZL in Soybean and Arabidopsis. The marked features are the N-terminal DNA binding domain (blue underline), putative 14–3-3 binding site (blue letters and box), putative sites of phosphorylation by GSK-3 kinase (red star), and the PEST domain (green letter and over line). The conserved amino acid (proline) between Arabidopsis and soybean in the PEST region is indicated by a red box, and was mutated to the amino acid leucine for the validation of conserved functional

    Techniques Used: Sequencing, Binding Assay, Functional Assay

    51) Product Images from "RNA-seq analysis of PHD and VHL inhibitors reveals differences and similarities to the hypoxia response."

    Article Title: RNA-seq analysis of PHD and VHL inhibitors reveals differences and similarities to the hypoxia response.

    Journal: Wellcome Open Research

    doi: 10.12688/wellcomeopenres.15044.1

    Validation of genes with increased transcript level in hypoxia, IOX2 and VH032. ( A ) Bar plot showing log2FC according to data obtained from RNA seq analysis of known HIF target genes in hypoxia, IOX2 and VH032. ( B ) HeLa and ( C ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05.
    Figure Legend Snippet: Validation of genes with increased transcript level in hypoxia, IOX2 and VH032. ( A ) Bar plot showing log2FC according to data obtained from RNA seq analysis of known HIF target genes in hypoxia, IOX2 and VH032. ( B ) HeLa and ( C ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05.

    Techniques Used: RNA Sequencing Assay, Two Tailed Test

    RNA seq validation of genes solely upregulated in hypoxia and IOX2, but not VH032. ( A ) HeLa and ( B ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05. ( C ) Table showing log2FC according to data obtained from RNA-seq analysis of known HIF target genes in hypoxia and IOX2, but not VH032. ( D ) Gene set enrichment analysis (GSEA) MsigDB showing significant enrichment of gene set signatures for genes upregulated in hypoxia and IOX2, but not found in VH032 at 5% false discovery rate (FDR). ( E ) Transcription factor enrichment analysis using TFEA.ChIP showing binding site enrichment for genes upregulated in hypoxia and IOX2, but not B032. The graph represents the adjusted p value (-log10 FDR) and the log-odds ratio (Log2.OR) for the association of ChIP datasets.
    Figure Legend Snippet: RNA seq validation of genes solely upregulated in hypoxia and IOX2, but not VH032. ( A ) HeLa and ( B ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05. ( C ) Table showing log2FC according to data obtained from RNA-seq analysis of known HIF target genes in hypoxia and IOX2, but not VH032. ( D ) Gene set enrichment analysis (GSEA) MsigDB showing significant enrichment of gene set signatures for genes upregulated in hypoxia and IOX2, but not found in VH032 at 5% false discovery rate (FDR). ( E ) Transcription factor enrichment analysis using TFEA.ChIP showing binding site enrichment for genes upregulated in hypoxia and IOX2, but not B032. The graph represents the adjusted p value (-log10 FDR) and the log-odds ratio (Log2.OR) for the association of ChIP datasets.

    Techniques Used: RNA Sequencing Assay, Two Tailed Test, Chromatin Immunoprecipitation, Binding Assay

    52) Product Images from "Characterization of the porcine testis-expressed gene 11 (Tex11)"

    Article Title: Characterization of the porcine testis-expressed gene 11 (Tex11)

    Journal: Spermatogenesis

    doi: 10.4161/spmg.1.2.16680

    Cloning of the porcine Tex11 from adult pig testis. A partial sequence (1,028 bp) of the porcine Tex11 (slightly over 1 Kb) was amplified from adult pig testis by RT-PCR and resolved on a 1.2% agarose gel. Lane a: DNA ladder. Lane b: Tex11 . Lane c: Gapdh (983 bp).
    Figure Legend Snippet: Cloning of the porcine Tex11 from adult pig testis. A partial sequence (1,028 bp) of the porcine Tex11 (slightly over 1 Kb) was amplified from adult pig testis by RT-PCR and resolved on a 1.2% agarose gel. Lane a: DNA ladder. Lane b: Tex11 . Lane c: Gapdh (983 bp).

    Techniques Used: Clone Assay, Sequencing, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

    53) Product Images from "Testing Potential Effects of Maize Expressing the Bacillus thuringiensis Cry1Ab Endotoxin (Bt Maize) on Mycorrhizal Fungal Communities via DNA- and RNA-Based Pyrosequencing and Molecular Fingerprinting"

    Article Title: Testing Potential Effects of Maize Expressing the Bacillus thuringiensis Cry1Ab Endotoxin (Bt Maize) on Mycorrhizal Fungal Communities via DNA- and RNA-Based Pyrosequencing and Molecular Fingerprinting

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01372-12

    (a) The total number of sequences derived from AM fungi is shown as a percentage of total fungal reads (mean ± SE). At both samplings, AM fungi were highly overrepresented in RNA compared to in DNA, and both increased from the first to the second
    Figure Legend Snippet: (a) The total number of sequences derived from AM fungi is shown as a percentage of total fungal reads (mean ± SE). At both samplings, AM fungi were highly overrepresented in RNA compared to in DNA, and both increased from the first to the second

    Techniques Used: Derivative Assay

    NMDS biplots of Bray-Curtis similarities among communities found in the experimental treatments using pyrosequencing for RNA (stress = 0.14; n = 20 total samples) (a) and T-RFLP for RNA (stress = 0.16; n = 22) (b) and DNA (stress = 0.14; n = 22) (c).
    Figure Legend Snippet: NMDS biplots of Bray-Curtis similarities among communities found in the experimental treatments using pyrosequencing for RNA (stress = 0.14; n = 20 total samples) (a) and T-RFLP for RNA (stress = 0.16; n = 22) (b) and DNA (stress = 0.14; n = 22) (c).

    Techniques Used:

    54) Product Images from "Expression of diabetes-associated genes by dendritic cells and CD4 T-cells drives the loss of tolerance in nonobese diabetic mice *"

    Article Title: Expression of diabetes-associated genes by dendritic cells and CD4 T-cells drives the loss of tolerance in nonobese diabetic mice *

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0900428

    Differential expression of SLC11a1 and IL-2 protein and ACADL mRNA by NOD and Idd3/5 DCs
    Figure Legend Snippet: Differential expression of SLC11a1 and IL-2 protein and ACADL mRNA by NOD and Idd3/5 DCs

    Techniques Used: Expressing

    55) Product Images from "Knockdown of LMP1-induced miR-155 sensitizes nasopharyngeal carcinoma cells to radiotherapy in vitro"

    Article Title: Knockdown of LMP1-induced miR-155 sensitizes nasopharyngeal carcinoma cells to radiotherapy in vitro

    Journal: Oncology Letters

    doi: 10.3892/ol.2016.4400

    Blockage of LMP1-induced miR-155 sensitizes CNE-2 cells to radiotherapy in vitro . (A) miR-155 inhibitor enhanced the radiation-induced viability reduction of CNE-2 (LMP1) cells. (B) miR-155 inhibitor had no influence on the sensitivity of normal CNE-2 cells to radiation. (C and D) Difference in cell growth subsequent to radiation treatment in CNE-2 cells transfected with miR-Con vs. CNE-2 cells transfected with miR-155 inhibitor, as detected by colony formation assay for (C) CNE-2 (LMP1) cells and (D) normal CNE-2 cells. # P
    Figure Legend Snippet: Blockage of LMP1-induced miR-155 sensitizes CNE-2 cells to radiotherapy in vitro . (A) miR-155 inhibitor enhanced the radiation-induced viability reduction of CNE-2 (LMP1) cells. (B) miR-155 inhibitor had no influence on the sensitivity of normal CNE-2 cells to radiation. (C and D) Difference in cell growth subsequent to radiation treatment in CNE-2 cells transfected with miR-Con vs. CNE-2 cells transfected with miR-155 inhibitor, as detected by colony formation assay for (C) CNE-2 (LMP1) cells and (D) normal CNE-2 cells. # P

    Techniques Used: In Vitro, Transfection, Colony Assay

    LMP1 promotes the viability of CNE-2 cells by inducing miR-155. (A) CNE-2 cell viability following transfection with various concentrations of empty pcDNA3.1 or LMP1-pcDNA3.1 vectors, as revealed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (B) Growth curve of cell proliferation following transfection with empty pcDNA3.1 or LMP1-pcDNA3.1 vectors. (C) miR-155 levels in CNE-2 cells transfected with miR-Con or miR-155 mimic. (D) Growth curve of cell proliferation following transfection with miR-Con or miR-155 mimic (20 or 40 nM) by Cell Counting Kit-8 assay. *P
    Figure Legend Snippet: LMP1 promotes the viability of CNE-2 cells by inducing miR-155. (A) CNE-2 cell viability following transfection with various concentrations of empty pcDNA3.1 or LMP1-pcDNA3.1 vectors, as revealed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (B) Growth curve of cell proliferation following transfection with empty pcDNA3.1 or LMP1-pcDNA3.1 vectors. (C) miR-155 levels in CNE-2 cells transfected with miR-Con or miR-155 mimic. (D) Growth curve of cell proliferation following transfection with miR-Con or miR-155 mimic (20 or 40 nM) by Cell Counting Kit-8 assay. *P

    Techniques Used: Transfection, Cell Counting

    miR-155 inhibitor suppresses LMP1-promoted CNE-2 cell proliferation. (A) miR-155 expression was inhibited significantly in CNE-2 cells transfected with miR-155 inhibitor. Cell proliferation was suppressed following transfection with miR-155 inhibitor, as revealed by (B) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and (C) Cell Counting Kit-8 assays. *P
    Figure Legend Snippet: miR-155 inhibitor suppresses LMP1-promoted CNE-2 cell proliferation. (A) miR-155 expression was inhibited significantly in CNE-2 cells transfected with miR-155 inhibitor. Cell proliferation was suppressed following transfection with miR-155 inhibitor, as revealed by (B) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and (C) Cell Counting Kit-8 assays. *P

    Techniques Used: Expressing, Transfection, Cell Counting

    LMP1 of Epstein-Barr virus promotes miR-155 expression in CNE-2 cells. CNE-2 cells were separated into three groups: Non-transfected CNE-2 cells (Blank), CNE-2 cells transfected with empty pcDNA3.1 as negative control (Con) and LMP1-pcDNA3.1-transfected CNE-2 cells (LMP1). (A) Relative mRNA expression levels of LMP1 in the three groups of CNE-2 cells, compared with the levels of GAPDH. (B) Percentage of LMP1 expression at the protein level in the three CNE-2 cell groups, as revealed by western blot analysis. (C) Relative mRNA levels of LMP1 vs. GAPDH in the three groups of CNE-2 cells upon a number of serial passages, as determined by reverse transcription-quantitative polymerase chain reaction. (D) Overexpression of LMP1 protein in CNE-2 cells following a number of serial passages. (E) Relative miR-155 levels in the three groups of CNE-2 cells, compared with the levels of U6 snRNA. (F) Relative miR-155 levels in CNE-2 cells transfected with various concentrations of LMP1-pcDNA3.1, compared with the levels of U6 snRNA. *P
    Figure Legend Snippet: LMP1 of Epstein-Barr virus promotes miR-155 expression in CNE-2 cells. CNE-2 cells were separated into three groups: Non-transfected CNE-2 cells (Blank), CNE-2 cells transfected with empty pcDNA3.1 as negative control (Con) and LMP1-pcDNA3.1-transfected CNE-2 cells (LMP1). (A) Relative mRNA expression levels of LMP1 in the three groups of CNE-2 cells, compared with the levels of GAPDH. (B) Percentage of LMP1 expression at the protein level in the three CNE-2 cell groups, as revealed by western blot analysis. (C) Relative mRNA levels of LMP1 vs. GAPDH in the three groups of CNE-2 cells upon a number of serial passages, as determined by reverse transcription-quantitative polymerase chain reaction. (D) Overexpression of LMP1 protein in CNE-2 cells following a number of serial passages. (E) Relative miR-155 levels in the three groups of CNE-2 cells, compared with the levels of U6 snRNA. (F) Relative miR-155 levels in CNE-2 cells transfected with various concentrations of LMP1-pcDNA3.1, compared with the levels of U6 snRNA. *P

    Techniques Used: Expressing, Transfection, Negative Control, Western Blot, Real-time Polymerase Chain Reaction, Over Expression

    56) Product Images from "DDX5 Regulates DNA Replication And Is Required For Cell Proliferation In A Subset Of Breast Cancer Cells"

    Article Title: DDX5 Regulates DNA Replication And Is Required For Cell Proliferation In A Subset Of Breast Cancer Cells

    Journal: Cancer discovery

    doi: 10.1158/2159-8290.CD-12-0116

    DDX5 is required for G1-S phase progression. (A) Quantitative western blot analysis of DDX5 and Beta-actin (loading control) in whole cell extracts obtained from cultures 48hrs after transfection with the indicated siRNAs. (B) Flow cytometry analysis of cell cycle in 48hr post-transfection cells with either DDX5si2008 (left panel) or EBNA1si1666 (right panel). (C) Progression into S-phase following serum addition to serum starved cultures previously transfected with either DDX5si2008 (circles) or EBNA1si1666 (squares) siRNAs. The fraction of cells in S-phase at each time point after serum addition was determined using flow cytometry analysis of propidium iodide incorporation and gating cells with greater than 2C but less than 4C DNA content. (D) Western blot analysis of RB, G1/S cyclin expression, and DNA replication factor expression in G1 and early S-phase whole cell extracts obtained from cells at increasing time following addition of serum to serum-starved cells previously transfected with the indicated siRNAs. (E) Western blot analysis of replication factors in chromatin fractions obtained from siRNA transfected S-phase cells over time after serum addition. Note the upper band detected on the CDC45 blots and marked with an asterisk in (D) and (E) is a non-specific cross-hybridizing protein recognized by the antibody.
    Figure Legend Snippet: DDX5 is required for G1-S phase progression. (A) Quantitative western blot analysis of DDX5 and Beta-actin (loading control) in whole cell extracts obtained from cultures 48hrs after transfection with the indicated siRNAs. (B) Flow cytometry analysis of cell cycle in 48hr post-transfection cells with either DDX5si2008 (left panel) or EBNA1si1666 (right panel). (C) Progression into S-phase following serum addition to serum starved cultures previously transfected with either DDX5si2008 (circles) or EBNA1si1666 (squares) siRNAs. The fraction of cells in S-phase at each time point after serum addition was determined using flow cytometry analysis of propidium iodide incorporation and gating cells with greater than 2C but less than 4C DNA content. (D) Western blot analysis of RB, G1/S cyclin expression, and DNA replication factor expression in G1 and early S-phase whole cell extracts obtained from cells at increasing time following addition of serum to serum-starved cells previously transfected with the indicated siRNAs. (E) Western blot analysis of replication factors in chromatin fractions obtained from siRNA transfected S-phase cells over time after serum addition. Note the upper band detected on the CDC45 blots and marked with an asterisk in (D) and (E) is a non-specific cross-hybridizing protein recognized by the antibody.

    Techniques Used: Western Blot, Transfection, Flow Cytometry, Cytometry, Expressing

    DDX5 is required for expression of DNA replication genes. (A) Quantitative western blot analysis of DNA replication factors in whole cell extracts obtained from asynchronous cell cultures 48hrs post-siRNA transfection with the indicated siRNAs. The asterisk shown in the MCM2, MCM10, and CDC45 blots indicate the position of non-specific proteins detected by these antibodies. (B) QPCR analysis of DNA replication factor transcript abundance in cells either 24hrs or 48hrs after transfection with either DDX5si2008 (red bar), EBNA1si1666 (blue bar) or mock transfected (no siRNA – green bar). Results for each transcript are normalized to the abundance of the indicated transcript in cells transfected with the EBNA1si1666 siRNA. Error bars indicate standard deviations calculated from 3 independent experiments. (C) Heat map showing row-wise standardized expression level for DNA replication genes 24hrs after transfection of cells with the indicated siRNAs.
    Figure Legend Snippet: DDX5 is required for expression of DNA replication genes. (A) Quantitative western blot analysis of DNA replication factors in whole cell extracts obtained from asynchronous cell cultures 48hrs post-siRNA transfection with the indicated siRNAs. The asterisk shown in the MCM2, MCM10, and CDC45 blots indicate the position of non-specific proteins detected by these antibodies. (B) QPCR analysis of DNA replication factor transcript abundance in cells either 24hrs or 48hrs after transfection with either DDX5si2008 (red bar), EBNA1si1666 (blue bar) or mock transfected (no siRNA – green bar). Results for each transcript are normalized to the abundance of the indicated transcript in cells transfected with the EBNA1si1666 siRNA. Error bars indicate standard deviations calculated from 3 independent experiments. (C) Heat map showing row-wise standardized expression level for DNA replication genes 24hrs after transfection of cells with the indicated siRNAs.

    Techniques Used: Expressing, Western Blot, Transfection, Real-time Polymerase Chain Reaction

    57) Product Images from "Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule"

    Article Title: Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule

    Journal: Journal of Medicinal Chemistry

    doi: 10.1021/acs.jmedchem.7b01781

    Differentially down-regulated genes common to both PANC-1 and MIA PaCa-2 are enriched in PQs after treatment with 400 nM CM03. (a,b) MIA PaCa-2 and PANC-1 cells were treated with 400 nM CM03 for 6 and 24 h and mRNA extracted for analysis by RNA-Seq. Genes were split into four subgroups according to their fold change upon CM03 treatment versus untreated: Down (Log 2 FC
    Figure Legend Snippet: Differentially down-regulated genes common to both PANC-1 and MIA PaCa-2 are enriched in PQs after treatment with 400 nM CM03. (a,b) MIA PaCa-2 and PANC-1 cells were treated with 400 nM CM03 for 6 and 24 h and mRNA extracted for analysis by RNA-Seq. Genes were split into four subgroups according to their fold change upon CM03 treatment versus untreated: Down (Log 2 FC

    Techniques Used: RNA Sequencing Assay

    58) Product Images from "Antioxidant metabolism regulates CD8+ T memory stem cell formation and antitumor immunity"

    Article Title: Antioxidant metabolism regulates CD8+ T memory stem cell formation and antitumor immunity

    Journal: JCI Insight

    doi: 10.1172/jci.insight.122299

    Modulating ROS levels regulates effector and memory CD8 + T cell differentiation in vitro. ( A ) Representative FACS analysis of CCR7 and CD45RO expression in circulating CD8 + Tn cells activated with anti-CD3/28, IL-2, and IL-12 in the presence of N-acetylcysteine (NAC), reduced glutathione (GSH), vitamin C (vitC), or apocynin (Apo) for 8 days. Treatments were supplemented daily. Additional DMSO control for Apo is shown. Similar data were obtained from n = 8 HD in n = 4 experiments (exp.) (NAC) and n = 3 HD in n = 1 exp. (GSH, vitC, Apo). ( B ) Representative histogram of CFSE dilution of cells cultured, as in A . NS, CFSE-stained, nonproliferating control cells. ( C ) Fold change (mean ± SEM) in cell counts compared with baseline ( n = 11 HD, n = 6 exp.) and ( D ) MFI (mean ± SEM) of CellROX, MitoSOX, and TMRM ( n = 9 HD, n = 4 exp.) indicative of total ROS levels, O 2 •– , and ΔΨ m , respectively, in CTRL and NAC-treated Tn cells at day 8 of culture, treated as in A . NAC was replaced every 3 days. ( E ) Representative CCR7 and CD45RO expression, as detected by FACS, of Tn cells activated as in A . NAC and menadione (MD) were replaced every 3 days. ( F ) FACS analysis of CD45RA, CD27, CD95, and CXCR3 by cells cultured, as in E . PBMCs from a HD are depicted as additional staining control. ( G ) Proportion (mean ± SEM) of CD8 + T cells with the Tscm, Tcm, Tem, and Tte phenotypes (gated as in Methods) after culture in the indicated conditions (CTRL, n = 13; NAC, n = 13, MD, n = 5 from n = 7 [NAC] and n = 2 [MD] exp.). ( H ) Percentage (mean ± SEM) of cytokine production in response to PMA/ionomycin stimulation by cells cultured, as in G (CTRL and NAC, n = 18; MD, n = 5 from n = 9 [NAC] and n = 2 [MD] exp.). ( I ) Pies depicting combinations of cytokine production obtained after stimulation, as in H . In all figures showing FACS dot plots, numbers indicate the percentage of cells identified by the gate. Statistical analyses were performed with nonparametric paired Wilcoxon ( C , D , G , and H ) and permutation ( I ) tests. * P
    Figure Legend Snippet: Modulating ROS levels regulates effector and memory CD8 + T cell differentiation in vitro. ( A ) Representative FACS analysis of CCR7 and CD45RO expression in circulating CD8 + Tn cells activated with anti-CD3/28, IL-2, and IL-12 in the presence of N-acetylcysteine (NAC), reduced glutathione (GSH), vitamin C (vitC), or apocynin (Apo) for 8 days. Treatments were supplemented daily. Additional DMSO control for Apo is shown. Similar data were obtained from n = 8 HD in n = 4 experiments (exp.) (NAC) and n = 3 HD in n = 1 exp. (GSH, vitC, Apo). ( B ) Representative histogram of CFSE dilution of cells cultured, as in A . NS, CFSE-stained, nonproliferating control cells. ( C ) Fold change (mean ± SEM) in cell counts compared with baseline ( n = 11 HD, n = 6 exp.) and ( D ) MFI (mean ± SEM) of CellROX, MitoSOX, and TMRM ( n = 9 HD, n = 4 exp.) indicative of total ROS levels, O 2 •– , and ΔΨ m , respectively, in CTRL and NAC-treated Tn cells at day 8 of culture, treated as in A . NAC was replaced every 3 days. ( E ) Representative CCR7 and CD45RO expression, as detected by FACS, of Tn cells activated as in A . NAC and menadione (MD) were replaced every 3 days. ( F ) FACS analysis of CD45RA, CD27, CD95, and CXCR3 by cells cultured, as in E . PBMCs from a HD are depicted as additional staining control. ( G ) Proportion (mean ± SEM) of CD8 + T cells with the Tscm, Tcm, Tem, and Tte phenotypes (gated as in Methods) after culture in the indicated conditions (CTRL, n = 13; NAC, n = 13, MD, n = 5 from n = 7 [NAC] and n = 2 [MD] exp.). ( H ) Percentage (mean ± SEM) of cytokine production in response to PMA/ionomycin stimulation by cells cultured, as in G (CTRL and NAC, n = 18; MD, n = 5 from n = 9 [NAC] and n = 2 [MD] exp.). ( I ) Pies depicting combinations of cytokine production obtained after stimulation, as in H . In all figures showing FACS dot plots, numbers indicate the percentage of cells identified by the gate. Statistical analyses were performed with nonparametric paired Wilcoxon ( C , D , G , and H ) and permutation ( I ) tests. * P

    Techniques Used: Cell Differentiation, In Vitro, FACS, Expressing, Cell Culture, Staining, Transmission Electron Microscopy

    Early differentiated human CD8 + T cells display a substantial antioxidant phenotype. ( A ) Gene set enrichment analysis (GSEA) of glutathione-derived metabolic process signature (gene ontology c5.pb.v6.1) in Tn versus Tem and Tscm versus Tem CD8 + T cells. Net enrichment score (NES) values are shown. ( B ) Relative gene expression level of transcripts involved in the antioxidant response in Tn, Tscm, Tcm, and Tem CD8 + T cell subsets from n = 3 HD. ( C ) Representative FACS analysis of GSH levels (mBCI staining) in gated CD8 + T cell subsets from the peripheral blood of a healthy individual. ( D ) Mean ± SEM of mean fluorescence intensity (MFI) data, obtained as in C ( n = 10). HD, healthy donor; mBCI, monochlorobimane; FMO, fluorescence minus one control. In D statistical analysis was performed with parametric 1-way ANOVA test with Bonferroni post test. ** P
    Figure Legend Snippet: Early differentiated human CD8 + T cells display a substantial antioxidant phenotype. ( A ) Gene set enrichment analysis (GSEA) of glutathione-derived metabolic process signature (gene ontology c5.pb.v6.1) in Tn versus Tem and Tscm versus Tem CD8 + T cells. Net enrichment score (NES) values are shown. ( B ) Relative gene expression level of transcripts involved in the antioxidant response in Tn, Tscm, Tcm, and Tem CD8 + T cell subsets from n = 3 HD. ( C ) Representative FACS analysis of GSH levels (mBCI staining) in gated CD8 + T cell subsets from the peripheral blood of a healthy individual. ( D ) Mean ± SEM of mean fluorescence intensity (MFI) data, obtained as in C ( n = 10). HD, healthy donor; mBCI, monochlorobimane; FMO, fluorescence minus one control. In D statistical analysis was performed with parametric 1-way ANOVA test with Bonferroni post test. ** P

    Techniques Used: Derivative Assay, Transmission Electron Microscopy, Expressing, FACS, Staining, Fluorescence

    59) Product Images from "Mice harboring the human SLC30A8 R138X loss-of-function mutation have increased insulin secretory capacity"

    Article Title: Mice harboring the human SLC30A8 R138X loss-of-function mutation have increased insulin secretory capacity

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

    doi: 10.1073/pnas.1721418115

    Analysis of Slc30a8 RNA and protein in islets from male R138X mice on chow diet. ( A ) Slc30a8 RNA in situ hybridization of pancreatic islets isolated from wild-type, knockout, and R138X mice. KO islets were used as negative control. Red, glucagon RNA; green, insulin RNA; white, Slc30a8 RNA. ( B ) Quantification of islet Slc30a8 RNA levels using qPCR analysis. n.d., not detected. ( C ) Western blot of islets isolated from chow-fed WT, KO, and R138X mice. KO islets were used as negative control. The arrow indicates SLC30A8 protein; asterisks denote unspecific bands. ( D ) Dithizone staining of pancreatic islets isolated from WT, KO, and R138X mice.
    Figure Legend Snippet: Analysis of Slc30a8 RNA and protein in islets from male R138X mice on chow diet. ( A ) Slc30a8 RNA in situ hybridization of pancreatic islets isolated from wild-type, knockout, and R138X mice. KO islets were used as negative control. Red, glucagon RNA; green, insulin RNA; white, Slc30a8 RNA. ( B ) Quantification of islet Slc30a8 RNA levels using qPCR analysis. n.d., not detected. ( C ) Western blot of islets isolated from chow-fed WT, KO, and R138X mice. KO islets were used as negative control. The arrow indicates SLC30A8 protein; asterisks denote unspecific bands. ( D ) Dithizone staining of pancreatic islets isolated from WT, KO, and R138X mice.

    Techniques Used: Mouse Assay, RNA In Situ Hybridization, Isolation, Knock-Out, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, Staining

    60) Product Images from "MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae"

    Article Title: MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1458171

    MoSNT2 is associated with the MoTor signaling pathway. (A) Vegetative growth of M. oryzae on CM agar medium supplemented with or without 1 μg/ml rapamycin (rapa.). (B) Inhibition rate of rapamycin on the mycelial growth. (C) Expression profiles of MoSNT2 and MoTOR in the wild-type Guy11 strain at different developmental processes. (D) Linear correlation between qRT-PCR-measured expression levels of MoSNT2 and MoTOR . (E) qRT-PCR analysis of MoSNT2 expression levels in the Guy11 strain in response to rapamycin. The Guy11 strain grown in liquid CM for 48 h was transferred into fresh liquid CM in the presence or absence of 1 μg/ml rapamycin for 6 h before total RNA extraction.
    Figure Legend Snippet: MoSNT2 is associated with the MoTor signaling pathway. (A) Vegetative growth of M. oryzae on CM agar medium supplemented with or without 1 μg/ml rapamycin (rapa.). (B) Inhibition rate of rapamycin on the mycelial growth. (C) Expression profiles of MoSNT2 and MoTOR in the wild-type Guy11 strain at different developmental processes. (D) Linear correlation between qRT-PCR-measured expression levels of MoSNT2 and MoTOR . (E) qRT-PCR analysis of MoSNT2 expression levels in the Guy11 strain in response to rapamycin. The Guy11 strain grown in liquid CM for 48 h was transferred into fresh liquid CM in the presence or absence of 1 μg/ml rapamycin for 6 h before total RNA extraction.

    Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, RNA Extraction

    61) Product Images from "Matrix-bound nanovesicles within ECM bioscaffolds"

    Article Title: Matrix-bound nanovesicles within ECM bioscaffolds

    Journal: Science Advances

    doi: 10.1126/sciadv.1600502

    Enzymatic digestion of decellularized ECM scaffolds releases small RNA molecules. ( A ) Nucleic acid extracted from untreated UBM (no digest) and pepsin-, proteinase K–, or collagenase-treated UBM was exposed to RNase A, DNase I, or no-nuclease treatment (control). ( B ) Electropherogram depicting the small RNA pattern of nucleic acid in fluorescence units (FU) before (top panel) and after (bottom panel) DNase I treatment. ( C ) Electropherogram depicting small RNA pattern from the indicated samples in FU. ( D ) A subset of nucleic molecules in biologic scaffolds is protected from nuclease degradation.
    Figure Legend Snippet: Enzymatic digestion of decellularized ECM scaffolds releases small RNA molecules. ( A ) Nucleic acid extracted from untreated UBM (no digest) and pepsin-, proteinase K–, or collagenase-treated UBM was exposed to RNase A, DNase I, or no-nuclease treatment (control). ( B ) Electropherogram depicting the small RNA pattern of nucleic acid in fluorescence units (FU) before (top panel) and after (bottom panel) DNase I treatment. ( C ) Electropherogram depicting small RNA pattern from the indicated samples in FU. ( D ) A subset of nucleic molecules in biologic scaffolds is protected from nuclease degradation.

    Techniques Used: Fluorescence

    62) Product Images from "An overview of gene expression dynamics during early ovarian folliculogenesis: specificity of follicular compartments and bi-directional dialog"

    Article Title: An overview of gene expression dynamics during early ovarian folliculogenesis: specificity of follicular compartments and bi-directional dialog

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-904

    Summary of bioinformatics processes. The RNA-seq experiment produced a collection of 382,933 fragments that aggregated 47.5% of the LCM-aRNA reads. The annotation strategy assigned 73% of the mapped reads. A total of 221,716 genomic fragments remain unannotated. A total of 86.8% of the annotated reads are located in stop codon or 3′UTR regions, whereas only 5.5% are located in exons, 1.2% in start codon/ 5′UTR regions and 6.5% in introns. The final dataset conserved a single fragment per gene corresponding to the nearest 3′UTR region with the highest number of reads. This dataset aggregated 89.4% of the annotated LCM-aRNA reads (86.8% were located in 3′ UTR regions and 2.6% were located in exons).
    Figure Legend Snippet: Summary of bioinformatics processes. The RNA-seq experiment produced a collection of 382,933 fragments that aggregated 47.5% of the LCM-aRNA reads. The annotation strategy assigned 73% of the mapped reads. A total of 221,716 genomic fragments remain unannotated. A total of 86.8% of the annotated reads are located in stop codon or 3′UTR regions, whereas only 5.5% are located in exons, 1.2% in start codon/ 5′UTR regions and 6.5% in introns. The final dataset conserved a single fragment per gene corresponding to the nearest 3′UTR region with the highest number of reads. This dataset aggregated 89.4% of the annotated LCM-aRNA reads (86.8% were located in 3′ UTR regions and 2.6% were located in exons).

    Techniques Used: RNA Sequencing Assay, Produced, Laser Capture Microdissection

    63) Product Images from "Cholecalciferol (Vitamin D3) Improves Myelination and Recovery after Nerve Injury"

    Article Title: Cholecalciferol (Vitamin D3) Improves Myelination and Recovery after Nerve Injury

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065034

    Analysis of the main functions altered by vitamin D supplementation using the Ingenuity Pathway Analysis Tool. A. List of functions for the genes involved in “nervous system development and function” whose expression was altered after addition of calcitriol to Schwann cells ( A ) or Schwann cells and dorsal root ganglion cells ( B ). Red arrows indicate an over-expression; green arrows, an under-expression. C. Twenty-five nervous system-related genes were used to generate a network representation. The genes shaded red are upregulated and those that are green are downregulated. The intensity of the shading shows to what degree each gene was up- or downregulated. The genes in white colour were not significantly changed in the analysis and can be considered as “missing links”. Orange solid lines represent a known direct interaction between calcitriol and the genes present in the network.
    Figure Legend Snippet: Analysis of the main functions altered by vitamin D supplementation using the Ingenuity Pathway Analysis Tool. A. List of functions for the genes involved in “nervous system development and function” whose expression was altered after addition of calcitriol to Schwann cells ( A ) or Schwann cells and dorsal root ganglion cells ( B ). Red arrows indicate an over-expression; green arrows, an under-expression. C. Twenty-five nervous system-related genes were used to generate a network representation. The genes shaded red are upregulated and those that are green are downregulated. The intensity of the shading shows to what degree each gene was up- or downregulated. The genes in white colour were not significantly changed in the analysis and can be considered as “missing links”. Orange solid lines represent a known direct interaction between calcitriol and the genes present in the network.

    Techniques Used: Expressing, Over Expression

    Main metabolic pathways associated to in vitro calcitriol supplementation. A. Venn diagram showing the functional pathways affected by the addition of calcitriol in cultures of Schwann cells or in co-cultures of DRG/Schwann cells. Five of the fifteen metabolic calcitriol-regulated pathways are affected in both cell types. B. Validation by qPCR of four selected up-regulated genes ( Prx, Tspan2, IgF1, Spp1 ) involved in axogenesis and myelination.
    Figure Legend Snippet: Main metabolic pathways associated to in vitro calcitriol supplementation. A. Venn diagram showing the functional pathways affected by the addition of calcitriol in cultures of Schwann cells or in co-cultures of DRG/Schwann cells. Five of the fifteen metabolic calcitriol-regulated pathways are affected in both cell types. B. Validation by qPCR of four selected up-regulated genes ( Prx, Tspan2, IgF1, Spp1 ) involved in axogenesis and myelination.

    Techniques Used: In Vitro, Functional Assay, Real-time Polymerase Chain Reaction

    64) Product Images from "The 3'-5' exoribonuclease Dis3 regulates the expression of specific microRNAs in Drosophila wing imaginal discs"

    Article Title: The 3'-5' exoribonuclease Dis3 regulates the expression of specific microRNAs in Drosophila wing imaginal discs

    Journal: RNA Biology

    doi: 10.1080/15476286.2015.1040978

    qRT-PCR validation of missexpressed miRNAs from the miRNA sequencing data. ( A ) Grouping of RPKM of selected miRNAs for all replicates is highly consistent between controls (black dots) and knockdown replicates (red dots). ( B ) Validation of miRNA-seq fold changes (red dots) using qRT-PCR. All selected miRNAs that change in expression upon Dis3-depletion in the miRNA-seq data also significantly change in expression when using qRT-PCR (except miR-7–3p ). Dotted lines show +/−2-fold changes. Stars represent levels of significance calculated using a 2 sample t-test. (**** p
    Figure Legend Snippet: qRT-PCR validation of missexpressed miRNAs from the miRNA sequencing data. ( A ) Grouping of RPKM of selected miRNAs for all replicates is highly consistent between controls (black dots) and knockdown replicates (red dots). ( B ) Validation of miRNA-seq fold changes (red dots) using qRT-PCR. All selected miRNAs that change in expression upon Dis3-depletion in the miRNA-seq data also significantly change in expression when using qRT-PCR (except miR-7–3p ). Dotted lines show +/−2-fold changes. Stars represent levels of significance calculated using a 2 sample t-test. (**** p

    Techniques Used: Quantitative RT-PCR, Sequencing, Expressing

    Identification of a novel mature miRNA in the wing imaginal disc. Using miRDeep2 we identified a novel miRNA of moderate expression in the wing imaginal discs. The predicted pre-miRNA forms a hairpin loop secondary structure with the novel miRNA being situated at the 3' end (shown in pink). The novel miRNA was detected at moderate levels using miRNA-seq in all but one sample. Validation of the miRNA using qRT-PCR shows that this novel miRNA is expressed at similar levels to miR-277–3p in all samples.
    Figure Legend Snippet: Identification of a novel mature miRNA in the wing imaginal disc. Using miRDeep2 we identified a novel miRNA of moderate expression in the wing imaginal discs. The predicted pre-miRNA forms a hairpin loop secondary structure with the novel miRNA being situated at the 3' end (shown in pink). The novel miRNA was detected at moderate levels using miRNA-seq in all but one sample. Validation of the miRNA using qRT-PCR shows that this novel miRNA is expressed at similar levels to miR-277–3p in all samples.

    Techniques Used: Expressing, Quantitative RT-PCR

    65) Product Images from "Loss of Ezh2 promotes a midbrain-to-forebrain identity switch by direct gene derepression and Wnt-dependent regulation"

    Article Title: Loss of Ezh2 promotes a midbrain-to-forebrain identity switch by direct gene derepression and Wnt-dependent regulation

    Journal: BMC Biology

    doi: 10.1186/s12915-015-0210-9

    Neural progenitor cell proliferation is controlled by Ezh2-mediated repression of cell cycle and Wnt/β-catenin signaling inhibitors. ( a ) Microarray analysis of three dissected E10.5 control and mutant midbrains identified 126 differentially expressed genes (≥1.75×, P ≤0.01), the majority of which (114) are upregulated upon Ezh2 ablation. Genes further analyzed are indicated. ( b ) qRT-PCR for Ezh2 , cell cycle regulators Cdkn2a and Cdkn2c , and Wnt signaling inhibitors Wif1 and Dkk2 on control and mutant E11.5 midbrains confirms microarray data. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, * P ≤0.05, Student’s t -test. ( c ) Chromatin immunoprecipitation confirms the presence of H3K27me3 at the transcription start site (±500 bp) of Cdkn2a , Cdkn2c , Wif1 , and Dkk2 . Intergenic region Int1 serves as unmethylated negative control. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, Student’s t -test. ( d – e ) In situ hybridization for Cdkn2a ( d ) and Wif1 ( e ) mRNA illustrates increased gene expression in Ezh2 mutants. ( f ) Immunostaining for β-galactosidase + cells on the BAT- gal Wnt/β-catenin signaling reporter line demonstrates diminished signaling in Ezh2-deficient midbrains. n ≥3 in each group, ** P ≤0.01, Student’s t -test. Cartoon insert indicates area of analysis for f and g . ( g ) Immunostaining against CyclinD1 and qRT-PCR. ( h ) Ccnd1 and Lef1 Wnt signaling downstream targets show decreased expression upon Ezh2 ablation. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, Student’s t -test. ( i ) H E staining of E12.5 sagittal midbrain sections of controls and Wnt/β-catenin signaling-ablated embryos. Mutant embryos exhibit reduced neuroepithelium thickness indicated with grey brackets in the magnifications. DAPI staining serves as nuclear marker: f , g ; Scale bars: d , e , 100 μm; f , g , 40 μm; i , 400 μm; Error bars indicate SD; ctrl, Control; dMB, Dorsal midbrain; vMB, Ventral midbrain
    Figure Legend Snippet: Neural progenitor cell proliferation is controlled by Ezh2-mediated repression of cell cycle and Wnt/β-catenin signaling inhibitors. ( a ) Microarray analysis of three dissected E10.5 control and mutant midbrains identified 126 differentially expressed genes (≥1.75×, P ≤0.01), the majority of which (114) are upregulated upon Ezh2 ablation. Genes further analyzed are indicated. ( b ) qRT-PCR for Ezh2 , cell cycle regulators Cdkn2a and Cdkn2c , and Wnt signaling inhibitors Wif1 and Dkk2 on control and mutant E11.5 midbrains confirms microarray data. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, * P ≤0.05, Student’s t -test. ( c ) Chromatin immunoprecipitation confirms the presence of H3K27me3 at the transcription start site (±500 bp) of Cdkn2a , Cdkn2c , Wif1 , and Dkk2 . Intergenic region Int1 serves as unmethylated negative control. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, Student’s t -test. ( d – e ) In situ hybridization for Cdkn2a ( d ) and Wif1 ( e ) mRNA illustrates increased gene expression in Ezh2 mutants. ( f ) Immunostaining for β-galactosidase + cells on the BAT- gal Wnt/β-catenin signaling reporter line demonstrates diminished signaling in Ezh2-deficient midbrains. n ≥3 in each group, ** P ≤0.01, Student’s t -test. Cartoon insert indicates area of analysis for f and g . ( g ) Immunostaining against CyclinD1 and qRT-PCR. ( h ) Ccnd1 and Lef1 Wnt signaling downstream targets show decreased expression upon Ezh2 ablation. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, Student’s t -test. ( i ) H E staining of E12.5 sagittal midbrain sections of controls and Wnt/β-catenin signaling-ablated embryos. Mutant embryos exhibit reduced neuroepithelium thickness indicated with grey brackets in the magnifications. DAPI staining serves as nuclear marker: f , g ; Scale bars: d , e , 100 μm; f , g , 40 μm; i , 400 μm; Error bars indicate SD; ctrl, Control; dMB, Dorsal midbrain; vMB, Ventral midbrain

    Techniques Used: Microarray, Mutagenesis, Quantitative RT-PCR, Chromatin Immunoprecipitation, Negative Control, In Situ Hybridization, Expressing, Immunostaining, Staining, Marker

    66) Product Images from "Transcription Profile of Aging and Cognition-Related Genes in the Medial Prefrontal Cortex"

    Article Title: Transcription Profile of Aging and Cognition-Related Genes in the Medial Prefrontal Cortex

    Journal: Frontiers in Aging Neuroscience

    doi: 10.3389/fnagi.2016.00113

    Comparison between RT-qPCR and RNA-seq . Six genes were selected for validation experiments using a subset of animals. Each panel provides the mPFC expression determined by RT-qPCR (left, ΔΔCT values) and RNA-seq (right, counts). Two-tailed t -tests confirmed increased expression of Arc, Fos, Egr1, Egr2, and Egr4 in AI, relative to AU rats. Gene expression for young animals is provided for comparison to aged animals. For two genes, Lin7b and Egr4 , age differences were confirmed ( *** p
    Figure Legend Snippet: Comparison between RT-qPCR and RNA-seq . Six genes were selected for validation experiments using a subset of animals. Each panel provides the mPFC expression determined by RT-qPCR (left, ΔΔCT values) and RNA-seq (right, counts). Two-tailed t -tests confirmed increased expression of Arc, Fos, Egr1, Egr2, and Egr4 in AI, relative to AU rats. Gene expression for young animals is provided for comparison to aged animals. For two genes, Lin7b and Egr4 , age differences were confirmed ( *** p

    Techniques Used: Quantitative RT-PCR, RNA Sequencing Assay, Expressing, Two Tailed Test

    Region of the mPFC and white matter (WM) collected for RNA-seq . The right panel provides a schematic of a coronal slice +2.7 anterior to bregma diagram as adapted from Paxinos and Watson ( 1986 ) and illustrates the region of the mPFC and white matter collected for RNA-seq. The left panel shows a coronal slice from this same region.
    Figure Legend Snippet: Region of the mPFC and white matter (WM) collected for RNA-seq . The right panel provides a schematic of a coronal slice +2.7 anterior to bregma diagram as adapted from Paxinos and Watson ( 1986 ) and illustrates the region of the mPFC and white matter collected for RNA-seq. The left panel shows a coronal slice from this same region.

    Techniques Used: RNA Sequencing Assay

    67) Product Images from "MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae"

    Article Title: MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1458171

    MoSNT2 is associated with the MoTor signaling pathway. (A) Vegetative growth of M. oryzae on CM agar medium supplemented with or without 1 μg/ml rapamycin (rapa.). (B) Inhibition rate of rapamycin on the mycelial growth. (C) Expression profiles of MoSNT2 and MoTOR in the wild-type Guy11 strain at different developmental processes. (D) Linear correlation between qRT-PCR-measured expression levels of MoSNT2 and MoTOR . (E) qRT-PCR analysis of MoSNT2 expression levels in the Guy11 strain in response to rapamycin. The Guy11 strain grown in liquid CM for 48 h was transferred into fresh liquid CM in the presence or absence of 1 μg/ml rapamycin for 6 h before total RNA extraction.
    Figure Legend Snippet: MoSNT2 is associated with the MoTor signaling pathway. (A) Vegetative growth of M. oryzae on CM agar medium supplemented with or without 1 μg/ml rapamycin (rapa.). (B) Inhibition rate of rapamycin on the mycelial growth. (C) Expression profiles of MoSNT2 and MoTOR in the wild-type Guy11 strain at different developmental processes. (D) Linear correlation between qRT-PCR-measured expression levels of MoSNT2 and MoTOR . (E) qRT-PCR analysis of MoSNT2 expression levels in the Guy11 strain in response to rapamycin. The Guy11 strain grown in liquid CM for 48 h was transferred into fresh liquid CM in the presence or absence of 1 μg/ml rapamycin for 6 h before total RNA extraction.

    Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, RNA Extraction

    68) Product Images from "Delftibactin-A, a Non-ribosomal Peptide With Broad Antimicrobial Activity"

    Article Title: Delftibactin-A, a Non-ribosomal Peptide With Broad Antimicrobial Activity

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.02377

    The inhibitory effect of delftibactin A with and without gallium on Delftia strain D-2189. Delftibactin A has no inhibitory activity against D-2189 (A) . Dose dependent inhibition of gallium against D-2189 (B) . Addition of isomolar concentration of gallium and delftibacin A induces significant inhibition of Delftia strain D-2189 in a dose dependent manner (C) . (D) Illustrates the combined effect of delftibactin A- gallium, by displaying the activities of: 200 μM delfibactin A only, gallium only and isomolar mixture of delftibactin A-gallium in the same graph.
    Figure Legend Snippet: The inhibitory effect of delftibactin A with and without gallium on Delftia strain D-2189. Delftibactin A has no inhibitory activity against D-2189 (A) . Dose dependent inhibition of gallium against D-2189 (B) . Addition of isomolar concentration of gallium and delftibacin A induces significant inhibition of Delftia strain D-2189 in a dose dependent manner (C) . (D) Illustrates the combined effect of delftibactin A- gallium, by displaying the activities of: 200 μM delfibactin A only, gallium only and isomolar mixture of delftibactin A-gallium in the same graph.

    Techniques Used: Activity Assay, Inhibition, Concentration Assay

    HPLC chromatograms of Delftia strain D-2189 supernatants grown in Davis minimal medium with 0.5% glycerol after 7 days incubation at 30°C (black line) and 37°C (red line). Significant differences are apparent at 220 nm (A) , with dominant peaks at 11.5 and 12.3 min in the active supernatant (30°C). In contrast, chromatograms of the same samples at 280 nm were very similar (B) .
    Figure Legend Snippet: HPLC chromatograms of Delftia strain D-2189 supernatants grown in Davis minimal medium with 0.5% glycerol after 7 days incubation at 30°C (black line) and 37°C (red line). Significant differences are apparent at 220 nm (A) , with dominant peaks at 11.5 and 12.3 min in the active supernatant (30°C). In contrast, chromatograms of the same samples at 280 nm were very similar (B) .

    Techniques Used: High Performance Liquid Chromatography, Incubation

    69) Product Images from "Preclinical development of HIvax: Human survivin highly immunogenic vaccines"

    Article Title: Preclinical development of HIvax: Human survivin highly immunogenic vaccines

    Journal: Human Vaccines & Immunotherapeutics

    doi: 10.1080/21645515.2015.1050572

    HIvax1 and HIvax2 induce activation of CD8 + and CD4 + T cells, respectively, in a donor with haplotype HLA-A 01:01, 11:01; HLA-B 08:01, 42:01; HLA-DRB1 03:01. Human dendritic cells (DC) were infected with HIvax1 or HIvax2 and co-cultured for 2 wks with
    Figure Legend Snippet: HIvax1 and HIvax2 induce activation of CD8 + and CD4 + T cells, respectively, in a donor with haplotype HLA-A 01:01, 11:01; HLA-B 08:01, 42:01; HLA-DRB1 03:01. Human dendritic cells (DC) were infected with HIvax1 or HIvax2 and co-cultured for 2 wks with

    Techniques Used: Activation Assay, Infection, Cell Culture

    Survivin epitopes have been included in HIvax1 e HIvax2. ( A ) HIvax 1 and 2 diagrams. Epitopes from Hsurv 5–7 were included in HIvax1, separated by AAY spacers and targeted to the ER by including an IgK signal sequence. This strategy facilitates
    Figure Legend Snippet: Survivin epitopes have been included in HIvax1 e HIvax2. ( A ) HIvax 1 and 2 diagrams. Epitopes from Hsurv 5–7 were included in HIvax1, separated by AAY spacers and targeted to the ER by including an IgK signal sequence. This strategy facilitates

    Techniques Used: Sequencing

    Cytolytic potential of HIvax-activated (T)cells against malignant mesothelioma cells overexpressing survivin. ( A ) ELISPOT analysis of released granzyme B was performed after stimulation by DC infected with FP-ctrl or HIvax1/HIvax2 simultaneously (HIvax).
    Figure Legend Snippet: Cytolytic potential of HIvax-activated (T)cells against malignant mesothelioma cells overexpressing survivin. ( A ) ELISPOT analysis of released granzyme B was performed after stimulation by DC infected with FP-ctrl or HIvax1/HIvax2 simultaneously (HIvax).

    Techniques Used: Enzyme-linked Immunospot, Infection

    70) Product Images from "FGF10 and the Mystery of Duodenal Atresia in Humans"

    Article Title: FGF10 and the Mystery of Duodenal Atresia in Humans

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2018.00530

    Genotype and general phenotype characterization. (A) Product from standard and RT-qPCR shows genotyping and RNA expression for both tm1 and tm2 strains. (B) General phenotype of a representative Fgf10 null embryo (left embryo) with the expected lack of limbs and smaller than body-size when compared with wild type litter mate (right embryo). Fgf10 null embryos also demonstrated: abnormal facies (C) , lung agenesis (D–F) , anomalous tracheal rings (F) , and caecal atresia (G) . Arrows highlight features of interest. Gestational ages for panels B–G are as stated. # p -value
    Figure Legend Snippet: Genotype and general phenotype characterization. (A) Product from standard and RT-qPCR shows genotyping and RNA expression for both tm1 and tm2 strains. (B) General phenotype of a representative Fgf10 null embryo (left embryo) with the expected lack of limbs and smaller than body-size when compared with wild type litter mate (right embryo). Fgf10 null embryos also demonstrated: abnormal facies (C) , lung agenesis (D–F) , anomalous tracheal rings (F) , and caecal atresia (G) . Arrows highlight features of interest. Gestational ages for panels B–G are as stated. # p -value

    Techniques Used: Quantitative RT-PCR, RNA Expression

    71) Product Images from "Early IL-10 producing B-cells and coinciding Th/Tr17 shifts during three year grass-pollen AIT"

    Article Title: Early IL-10 producing B-cells and coinciding Th/Tr17 shifts during three year grass-pollen AIT

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2018.09.016

    Local gene expression changes indicate shifts in regulatory hierarchies in the nasal mucosa. Gene expression changes of nasal scrapings were taken from healthy control subjects during off season (HC off; n = 3), in grass pollen season (HC in; n = 3), treated patients throughout course of therapy at time points T0 ( n = 6), T4 ( n = 5), and T9 ( n = 9), untreated allergic rhinitis patients in grass pollen season (AR in; n = 5) and subjected to RNA whole transcriptome microarray analysis. (A) Volcano plot of statistically significant entities (p
    Figure Legend Snippet: Local gene expression changes indicate shifts in regulatory hierarchies in the nasal mucosa. Gene expression changes of nasal scrapings were taken from healthy control subjects during off season (HC off; n = 3), in grass pollen season (HC in; n = 3), treated patients throughout course of therapy at time points T0 ( n = 6), T4 ( n = 5), and T9 ( n = 9), untreated allergic rhinitis patients in grass pollen season (AR in; n = 5) and subjected to RNA whole transcriptome microarray analysis. (A) Volcano plot of statistically significant entities (p

    Techniques Used: Expressing, Microarray

    72) Product Images from "Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines"

    Article Title: Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines

    Journal: BMC Research Notes

    doi: 10.1186/s13104-017-2959-0

    Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1 , b brm , c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS - RNAi constructs with Act - Gal4 . Expression levels were normalized to the reference genes, eIF2Bγ and βCOP . Shown here, are relative expression values compared to the UAS - mCherry - RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p
    Figure Legend Snippet: Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1 , b brm , c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS - RNAi constructs with Act - Gal4 . Expression levels were normalized to the reference genes, eIF2Bγ and βCOP . Shown here, are relative expression values compared to the UAS - mCherry - RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Synthesized, Expressing, Construct, Activated Clotting Time Assay

    Schematic representation of the experimental setup. siRNAs direct site-specific cleavage of mRNAs, resulting in a 5′ and 3′ mRNA cleavage fragments. After RNA isolation total RNA samples consist of uncleaved mRNA transcripts and non-coding RNA, as well as undegraded 5′ and 3′ mRNA cleavage fragments. Purification of mRNA using poly-T beads excludes 5′ mRNA cleavage fragments and non-coding RNAs that are not polyadenylated. As indicated by the boxes, 5′ and 3′ primer sets could detect different species of RNA depending on the isolation method
    Figure Legend Snippet: Schematic representation of the experimental setup. siRNAs direct site-specific cleavage of mRNAs, resulting in a 5′ and 3′ mRNA cleavage fragments. After RNA isolation total RNA samples consist of uncleaved mRNA transcripts and non-coding RNA, as well as undegraded 5′ and 3′ mRNA cleavage fragments. Purification of mRNA using poly-T beads excludes 5′ mRNA cleavage fragments and non-coding RNAs that are not polyadenylated. As indicated by the boxes, 5′ and 3′ primer sets could detect different species of RNA depending on the isolation method

    Techniques Used: Isolation, Purification

    73) Product Images from "Decoupling of DNA methylation and activity of intergenic LINE-1 promoters in colorectal cancer"

    Article Title: Decoupling of DNA methylation and activity of intergenic LINE-1 promoters in colorectal cancer

    Journal: Epigenetics

    doi: 10.1080/15592294.2017.1300729

    Relationship between methylation and expression of LCT13 L1ASP in CRC. (A) Top: Schematic diagram of the LCT13 genomic locus on human chromosome 7 (chr7:93,204,042–93,540,485; center) with indicated the positions of the CALCR, TFPI-2 , and GNGT1 genes and of the 2 intact intergenic LINE1s (L1) present in this region. Middle: enlargement of the LINE-1 (L1PA2: chr7:93,213,393–93,221,079, with an SVA_D spanning the interval 93,214,544–93,216,214) from which LCT13 originates with the regions (black bars) tested by bisulfite or hMeDIP and ChIP. Bottom: enlargement of the LCT13 spliced transcript with indicated its exon structure [LINE-1 5′UTR fragment in light gray (chr7:93,220,882–93,221,083) and, in dark gray, the 2 GNGT1 exons (93,536,051–93,536,154 and 93,540,102–93,540,235), part of the LCT13 transcript]. Also indicated are the positions of the Taqman assay used for LCT13 expression studies located at the splice junction (black bar) and of the primers used for 5′RACE (arrows). All coordinates are from hg19 annotations; scale is in kilobase pairs (kb). (B) Bar charts showing the expression of LCT13 measured by real time RT-PCR and expressed relatively to the geometric mean of 3 reference genes in matched normal (dark gray, N) and tumor (light gray, T) tissues from 6 colorectal cancer patients (left panel) and 6 cell lines (right panel). NC: normal colon, commercially sourced total RNA from 7 healthy donors pooled together. (C) Bar charts of the methylation levels measured by bisulfite sequencing in the tissues of the 6 patients and cell lines presented in B.
    Figure Legend Snippet: Relationship between methylation and expression of LCT13 L1ASP in CRC. (A) Top: Schematic diagram of the LCT13 genomic locus on human chromosome 7 (chr7:93,204,042–93,540,485; center) with indicated the positions of the CALCR, TFPI-2 , and GNGT1 genes and of the 2 intact intergenic LINE1s (L1) present in this region. Middle: enlargement of the LINE-1 (L1PA2: chr7:93,213,393–93,221,079, with an SVA_D spanning the interval 93,214,544–93,216,214) from which LCT13 originates with the regions (black bars) tested by bisulfite or hMeDIP and ChIP. Bottom: enlargement of the LCT13 spliced transcript with indicated its exon structure [LINE-1 5′UTR fragment in light gray (chr7:93,220,882–93,221,083) and, in dark gray, the 2 GNGT1 exons (93,536,051–93,536,154 and 93,540,102–93,540,235), part of the LCT13 transcript]. Also indicated are the positions of the Taqman assay used for LCT13 expression studies located at the splice junction (black bar) and of the primers used for 5′RACE (arrows). All coordinates are from hg19 annotations; scale is in kilobase pairs (kb). (B) Bar charts showing the expression of LCT13 measured by real time RT-PCR and expressed relatively to the geometric mean of 3 reference genes in matched normal (dark gray, N) and tumor (light gray, T) tissues from 6 colorectal cancer patients (left panel) and 6 cell lines (right panel). NC: normal colon, commercially sourced total RNA from 7 healthy donors pooled together. (C) Bar charts of the methylation levels measured by bisulfite sequencing in the tissues of the 6 patients and cell lines presented in B.

    Techniques Used: Methylation, Expressing, Chromatin Immunoprecipitation, TaqMan Assay, Quantitative RT-PCR, Methylation Sequencing

    74) Product Images from "Sodium-myoinositol cotransporter-1, SMIT1, mediates the production of reactive oxygen species induced by hyperglycemia in the heart"

    Article Title: Sodium-myoinositol cotransporter-1, SMIT1, mediates the production of reactive oxygen species induced by hyperglycemia in the heart

    Journal: Scientific Reports

    doi: 10.1038/srep41166

    Detection of SGLT isoforms in human hearts. ( A ) SGLT1, SGLT2, SGLT3, SGLT4, SGLT5, SGLT6 and SMIT1 detection by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from non-failing human hearts (n = 4). Positive controls were intestine for SGLT1, kidney for SGLT2, SGLT3, SGLT4 and SGLT5, and brain for SGLT6 and SMIT1. mRNA copy numbers/μg of SGLT1 ( B ) and SMIT1 ( C ) RNA were measured in non-failing human hearts (n = 7) and compared to a positive control (n = 3). The clinical characteristics of patients are presented in Supplementary Table 1 . ( D ) Comparison of SGLT1 and SMIT1 mRNA copy numbers/μg of RNA in human hearts (n = 7). Data were normalized to RPL32 and expressed as Log10 copy numbers/μg RNA. Data are means ± SEM. Statistical analysis was by Student’s t-test. *Indicates values statistically different from ( C ) corresponding control tissue ( D ) hSGLT1 mRNA expression, p ≤ 0.05.
    Figure Legend Snippet: Detection of SGLT isoforms in human hearts. ( A ) SGLT1, SGLT2, SGLT3, SGLT4, SGLT5, SGLT6 and SMIT1 detection by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from non-failing human hearts (n = 4). Positive controls were intestine for SGLT1, kidney for SGLT2, SGLT3, SGLT4 and SGLT5, and brain for SGLT6 and SMIT1. mRNA copy numbers/μg of SGLT1 ( B ) and SMIT1 ( C ) RNA were measured in non-failing human hearts (n = 7) and compared to a positive control (n = 3). The clinical characteristics of patients are presented in Supplementary Table 1 . ( D ) Comparison of SGLT1 and SMIT1 mRNA copy numbers/μg of RNA in human hearts (n = 7). Data were normalized to RPL32 and expressed as Log10 copy numbers/μg RNA. Data are means ± SEM. Statistical analysis was by Student’s t-test. *Indicates values statistically different from ( C ) corresponding control tissue ( D ) hSGLT1 mRNA expression, p ≤ 0.05.

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

    Detection of SGLT isoforms in mouse heart and cardiomyocytes. ( A ) SGLT1, SGLT2, SGLT3b, SGLT4, SGLT5, SGLT6 and SMIT1 detection by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from hearts (n = 4) and isolated cardiomyocytes (cardio. n = 4) of mice. Positive controls were intestine for SGLT1, kidney for SGLT2, SGLT3, SGLT4 and SGLT5 and brain for SGLT6 and SMIT1. mRNA copy number per μg of RNA of SGLT1 ( B ) and SMIT1 ( C ) were measured in mice hearts (n = 4) and cardiomyocytes (n = 4) and compared to a positive control (n = 3). Data are means ± SEM. Statistical analysis was by one-way ANOVA. *Indicates values statistically different from corresponding control tissue, p ≤ 0.05. ( D ) Comparison of SGLT1 and SMIT1 mRNA copy numbers/μg of RNA between hearts (n = 4) and cardiomyocytes (n = 4). Data were normalized to ribosomal protein L32 (RPL32) and expressed as Log10 copy numbers/μg RNA. ( E ) SMIT1 protein expression in murine heart compared to murine brain. eEF-2 detection is used as loading control. Full-length blots are presented in Supplementary Figure 5 .
    Figure Legend Snippet: Detection of SGLT isoforms in mouse heart and cardiomyocytes. ( A ) SGLT1, SGLT2, SGLT3b, SGLT4, SGLT5, SGLT6 and SMIT1 detection by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from hearts (n = 4) and isolated cardiomyocytes (cardio. n = 4) of mice. Positive controls were intestine for SGLT1, kidney for SGLT2, SGLT3, SGLT4 and SGLT5 and brain for SGLT6 and SMIT1. mRNA copy number per μg of RNA of SGLT1 ( B ) and SMIT1 ( C ) were measured in mice hearts (n = 4) and cardiomyocytes (n = 4) and compared to a positive control (n = 3). Data are means ± SEM. Statistical analysis was by one-way ANOVA. *Indicates values statistically different from corresponding control tissue, p ≤ 0.05. ( D ) Comparison of SGLT1 and SMIT1 mRNA copy numbers/μg of RNA between hearts (n = 4) and cardiomyocytes (n = 4). Data were normalized to ribosomal protein L32 (RPL32) and expressed as Log10 copy numbers/μg RNA. ( E ) SMIT1 protein expression in murine heart compared to murine brain. eEF-2 detection is used as loading control. Full-length blots are presented in Supplementary Figure 5 .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Staining, Isolation, Mouse Assay, Positive Control, Expressing

    Impact of SGLT1 deletion on HG-induced NOX2 activation and ROS production. ( A ) LVEDV, ( B ) EF and ( C ) LV mass were measured by echocardiography of SGLT1 WT under usual diet (n = 10), of SGLT1 WT submitted to glucose and galactose free diet (glu/gal free diet, n = 10) and of SGLT1 KO (n = 10) mice. Echocardiographic data in M-mode and 2D parasternal long axis are presented in Supplementary Table 4 . ( D ) Detection of SGLT1, SGLT2, SGLT3b, SGLT4, SGLT5, SGLT6 and SMIT1 by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from the hearts of SGLT1 KO mice (n = 3) compared to WT mice (n = 3). Positive controls were intestine for SGLT1, kidneys for SGLT2, SGLT3, SGLT4 and SGLT5, brain for SGLT6 and SMIT1. ( E ) Quantification of HG-induced p47phox translocation close to cav3 in SGLT1 WT mice (with and without glu/gal free diet) compared to SGLT1 KO mice. Adult mouse cardiomyocytes were isolated from SGLT1 WT (n = 7), SGLT1 WT submitted to glu/gal free diet (n = 7) and SGLT1 KO (n = 7) hearts. PLA was performed 90 min after stimulation with HG and compared to LG. White lines correspond to 20 μm. ( F ) ROS production induced by 3 h of incubation with HG in cardiomyocytes isolated from SGLT1 WT (n = 6), SGLT1 WT submitted to glu/gal free diet (n = 6) and SGLT1 KO (n = 6) mice. Data are means ± SEM. Statistical analysis was by two-way ANOVA. $ Indicates values statistically different from LG, p ≤ 0.05.
    Figure Legend Snippet: Impact of SGLT1 deletion on HG-induced NOX2 activation and ROS production. ( A ) LVEDV, ( B ) EF and ( C ) LV mass were measured by echocardiography of SGLT1 WT under usual diet (n = 10), of SGLT1 WT submitted to glucose and galactose free diet (glu/gal free diet, n = 10) and of SGLT1 KO (n = 10) mice. Echocardiographic data in M-mode and 2D parasternal long axis are presented in Supplementary Table 4 . ( D ) Detection of SGLT1, SGLT2, SGLT3b, SGLT4, SGLT5, SGLT6 and SMIT1 by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from the hearts of SGLT1 KO mice (n = 3) compared to WT mice (n = 3). Positive controls were intestine for SGLT1, kidneys for SGLT2, SGLT3, SGLT4 and SGLT5, brain for SGLT6 and SMIT1. ( E ) Quantification of HG-induced p47phox translocation close to cav3 in SGLT1 WT mice (with and without glu/gal free diet) compared to SGLT1 KO mice. Adult mouse cardiomyocytes were isolated from SGLT1 WT (n = 7), SGLT1 WT submitted to glu/gal free diet (n = 7) and SGLT1 KO (n = 7) hearts. PLA was performed 90 min after stimulation with HG and compared to LG. White lines correspond to 20 μm. ( F ) ROS production induced by 3 h of incubation with HG in cardiomyocytes isolated from SGLT1 WT (n = 6), SGLT1 WT submitted to glu/gal free diet (n = 6) and SGLT1 KO (n = 6) mice. Data are means ± SEM. Statistical analysis was by two-way ANOVA. $ Indicates values statistically different from LG, p ≤ 0.05.

    Techniques Used: Activation Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Staining, Translocation Assay, Isolation, Proximity Ligation Assay, Incubation

    Impact of SMIT1 overexpression on NOX2 activation and ROS production. Adult rat cardiomyocytes were infected with adenoviruses (24 h, 200 MOI) expressing SMIT1 (Ad-SMIT1) or β-galactosidase (Ad-Ctl). ( A ) SMIT1 mRNA level measured by qRT-PCR (n = 3). Data were normalized to HPRT1 and expressed as relative expression vs Ad-Ctl. ( B ) SMIT1 protein expression in plasma membrane fractions obtained after cellular fractionation. Full-length blots are presented in Supplementary Figure 6 . ( C ) Quantification of picomoles myo-[3 H]inositol uptake per min and mg of proteins (n = 4). ( D ) p47phox translocation close to cav3 (n = 6) and ( E ) ROS production (n = 7) in response to increased glucose concentration (5–10 and 21 mM of glucose). ( F ) Gp91dstat and scrambled peptide were added 15 min prior to glucose (5 or 10 mM glucose). ROS production was quantified 2 h after change in glucose concentration (n = 3). Data are means ± SEM. Statistical analysis was by ( A–C ) Student’s t-test or ( D,E,F ) two-way ANOVA. $ Indicates values statistically different from LG, p ≤ 0.05. *Indicates values statistically different from ( A–E ) Ad-Ctl, and ( F ) scr, p ≤ 0.05.
    Figure Legend Snippet: Impact of SMIT1 overexpression on NOX2 activation and ROS production. Adult rat cardiomyocytes were infected with adenoviruses (24 h, 200 MOI) expressing SMIT1 (Ad-SMIT1) or β-galactosidase (Ad-Ctl). ( A ) SMIT1 mRNA level measured by qRT-PCR (n = 3). Data were normalized to HPRT1 and expressed as relative expression vs Ad-Ctl. ( B ) SMIT1 protein expression in plasma membrane fractions obtained after cellular fractionation. Full-length blots are presented in Supplementary Figure 6 . ( C ) Quantification of picomoles myo-[3 H]inositol uptake per min and mg of proteins (n = 4). ( D ) p47phox translocation close to cav3 (n = 6) and ( E ) ROS production (n = 7) in response to increased glucose concentration (5–10 and 21 mM of glucose). ( F ) Gp91dstat and scrambled peptide were added 15 min prior to glucose (5 or 10 mM glucose). ROS production was quantified 2 h after change in glucose concentration (n = 3). Data are means ± SEM. Statistical analysis was by ( A–C ) Student’s t-test or ( D,E,F ) two-way ANOVA. $ Indicates values statistically different from LG, p ≤ 0.05. *Indicates values statistically different from ( A–E ) Ad-Ctl, and ( F ) scr, p ≤ 0.05.

    Techniques Used: Over Expression, Activation Assay, Infection, Expressing, CTL Assay, Quantitative RT-PCR, Cell Fractionation, Translocation Assay, Concentration Assay

    Detection of SGLT isoforms in rat heart and cardiomyocytes. ( A ) SGLT1, SGLT2, SGLT3b, SGLT4, SGLT5, SGLT6 and SMIT1 detection by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from hearts (n = 4) and isolated cardiomyocytes (cardio. n = 4) of rats. Positive controls were intestine for SGLT1, kidney for SGLT2, SGLT3, SGLT4 and SGLT5 and brain for SGLT6 and SMIT1. mRNA copy number per μg of RNA of SGLT1 ( B ) and SMIT1 ( C ) were measured in rat hearts (n = 4) and cardiomyocytes (n = 4) and compared to a positive control (n = 3). Data are means ± SEM. Statistical analysis was by one-way ANOVA. *Indicates values statistically different from corresponding control tissue, p ≤ 0.05. ( D ) Comparison of SGLT1 and SMIT1 mRNA copy numbers/μg of RNA between hearts (n = 4) and cardiomyocytes (n = 4). Data were normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT1) and expressed as Log10 copy numbers/μg RNA. ( E ) SMIT1 protein expression in rat heart compared to rat brain and in isolated rat cardiomyocytes in culture compared to total heart extract. eEF-2 detection is used as loading control. Full-length blots are presented in Supplementary Figure 4 .
    Figure Legend Snippet: Detection of SGLT isoforms in rat heart and cardiomyocytes. ( A ) SGLT1, SGLT2, SGLT3b, SGLT4, SGLT5, SGLT6 and SMIT1 detection by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from hearts (n = 4) and isolated cardiomyocytes (cardio. n = 4) of rats. Positive controls were intestine for SGLT1, kidney for SGLT2, SGLT3, SGLT4 and SGLT5 and brain for SGLT6 and SMIT1. mRNA copy number per μg of RNA of SGLT1 ( B ) and SMIT1 ( C ) were measured in rat hearts (n = 4) and cardiomyocytes (n = 4) and compared to a positive control (n = 3). Data are means ± SEM. Statistical analysis was by one-way ANOVA. *Indicates values statistically different from corresponding control tissue, p ≤ 0.05. ( D ) Comparison of SGLT1 and SMIT1 mRNA copy numbers/μg of RNA between hearts (n = 4) and cardiomyocytes (n = 4). Data were normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT1) and expressed as Log10 copy numbers/μg RNA. ( E ) SMIT1 protein expression in rat heart compared to rat brain and in isolated rat cardiomyocytes in culture compared to total heart extract. eEF-2 detection is used as loading control. Full-length blots are presented in Supplementary Figure 4 .

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

    Impact of SMIT1 deletion on HG-induced NOX2 activation and ROS production. ( A ) LVEDV, ( B ) EF and ( C ) LV mass were measured by echocardiography of SMIT1 WT (n = 10) and KO (n = 10) mice. Echocardiographic data in M-mode and 2D parasternal long axis are presented in Supplementary Table 5 . ( D ) Detection of SGLT1, SGLT2, SGLT3b, SGLT4, SGLT5 and SGLT6 by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from the hearts of SMIT1 KO mice (n = 3) compared to WT mice (n = 3). Positive controls were intestine for SGLT1, kidneys for SGLT2, SGLT3, SGLT4 and SGLT5, brain for SGLT6 and SMIT1. ( E ) Quantification of HG-induced p47phox translocation close to cav3 in SMIT1 WT mice compared to SMIT1 KO mice. Adult mouse cardiomyocytes were isolated from SMIT1 WT (n = 4) or SMIT1 KO (n = 4) hearts. PLA was performed 90 min after stimulation with HG and compared to 5 mM of glucose. White lines correspond to 20 μm. ( F ) ROS production induced by 3 h of incubation with HG in cardiomyocytes isolated from SMIT1 WT (n = 6) or SMIT1 KO (n = 6) mice. ( G ) Cardiac glucose uptake in SMIT1 WT (n = 6) vs KO (n = 6) mice was measured under LG, HG and after insulin (3.10 −9 M insulin 30 min). Data are means ± SEM. Statistical analysis was by two-way ANOVA ( E–F ). $ Indicates values statistically different from LG, p ≤ 0.05.
    Figure Legend Snippet: Impact of SMIT1 deletion on HG-induced NOX2 activation and ROS production. ( A ) LVEDV, ( B ) EF and ( C ) LV mass were measured by echocardiography of SMIT1 WT (n = 10) and KO (n = 10) mice. Echocardiographic data in M-mode and 2D parasternal long axis are presented in Supplementary Table 5 . ( D ) Detection of SGLT1, SGLT2, SGLT3b, SGLT4, SGLT5 and SGLT6 by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from the hearts of SMIT1 KO mice (n = 3) compared to WT mice (n = 3). Positive controls were intestine for SGLT1, kidneys for SGLT2, SGLT3, SGLT4 and SGLT5, brain for SGLT6 and SMIT1. ( E ) Quantification of HG-induced p47phox translocation close to cav3 in SMIT1 WT mice compared to SMIT1 KO mice. Adult mouse cardiomyocytes were isolated from SMIT1 WT (n = 4) or SMIT1 KO (n = 4) hearts. PLA was performed 90 min after stimulation with HG and compared to 5 mM of glucose. White lines correspond to 20 μm. ( F ) ROS production induced by 3 h of incubation with HG in cardiomyocytes isolated from SMIT1 WT (n = 6) or SMIT1 KO (n = 6) mice. ( G ) Cardiac glucose uptake in SMIT1 WT (n = 6) vs KO (n = 6) mice was measured under LG, HG and after insulin (3.10 −9 M insulin 30 min). Data are means ± SEM. Statistical analysis was by two-way ANOVA ( E–F ). $ Indicates values statistically different from LG, p ≤ 0.05.

    Techniques Used: Activation Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Staining, Translocation Assay, Isolation, Proximity Ligation Assay, Incubation

    75) Product Images from "Repeat-length variation in a wheat cellulose synthase-like gene is associated with altered tiller number and stem cell wall composition"

    Article Title: Repeat-length variation in a wheat cellulose synthase-like gene is associated with altered tiller number and stem cell wall composition

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erx051

    Schematic representation of the cellulose synthase-like gene including intron/exon structure and the 5′UTR region that contains repeat-length polymorphisms between the free-tillering cultivar Banks and the reduced-tillering NILs. Exons are shown in yellow.
    Figure Legend Snippet: Schematic representation of the cellulose synthase-like gene including intron/exon structure and the 5′UTR region that contains repeat-length polymorphisms between the free-tillering cultivar Banks and the reduced-tillering NILs. Exons are shown in yellow.

    Techniques Used:

    Genetic and physical map of the tin region located on chromosome 1AS. Genetic distances (cM) based on the recombination rate in F 2 mapping family derived from Banks × NIL76. Physical maps of the tin region constructed with BAC clones from cultivar Chinese Spring and reduced-tillering donor line 492. Annotation of the DNA sequence spanning the region identified two predicted open-reading frames: Csl , cellulose synthase-like, and NB-LRR , nucleotide-binding leucine-rich repeat genes.
    Figure Legend Snippet: Genetic and physical map of the tin region located on chromosome 1AS. Genetic distances (cM) based on the recombination rate in F 2 mapping family derived from Banks × NIL76. Physical maps of the tin region constructed with BAC clones from cultivar Chinese Spring and reduced-tillering donor line 492. Annotation of the DNA sequence spanning the region identified two predicted open-reading frames: Csl , cellulose synthase-like, and NB-LRR , nucleotide-binding leucine-rich repeat genes.

    Techniques Used: Derivative Assay, Construct, BAC Assay, Clone Assay, Sequencing, Binding Assay

    Mean relative expression (±SE) of the cellulose synthase-like gene in elongating internodes of primary stems of cultivar Banks and NIL76. Three biological replicates from each line were sampled at four time points, which corresponded to double-ridge (26 d after planting, DAP), terminal spikelet (28 DAP), early stem elongation (32 DAP), and late stem elongation (41 DAP).
    Figure Legend Snippet: Mean relative expression (±SE) of the cellulose synthase-like gene in elongating internodes of primary stems of cultivar Banks and NIL76. Three biological replicates from each line were sampled at four time points, which corresponded to double-ridge (26 d after planting, DAP), terminal spikelet (28 DAP), early stem elongation (32 DAP), and late stem elongation (41 DAP).

    Techniques Used: Expressing

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    Article Snippet: Isolated RNA was measured for quality using a Nano drop ND/2000 spectrophotometer and analyzed by 2% gel electrophoresis. .. DNA contamination was removed using an RNase-Free DNase Set (Qiagen). cDNA was synthesized using equal amounts of RNA samples (800 ng), according to the AMV First Strand cDNA Synthesis Kit instructions (NEB Biolabs). β-actin and GAPDH were used as a housekeeping gene controls for mRNA analysis , .

    Article Title: Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins
    Article Snippet: Isolated RNA was measured for quality using a Nano drop ND/2000 spectrophotometer and analyzed by 2% gel electrophoresis. .. DNA contamination was removed using an RNase-Free DNase Set (Qiagen). cDNA was synthesized using equal amounts of RNA samples (800 ng), according to the AMV First Strand cDNA Synthesis Kit instructions (NEB Biolabs). β-actin and GAPDH were used as a housekeeping gene controls for mRNA analysis , .

    Article Title: Peripheral mononuclear blood cells contribute to the obesity-associated inflammatory state independently of glycemic status: involvement of the novel proinflammatory adipokines chemerin, chitinase-3-like protein 1, lipocalin-2 and osteopontin
    Article Snippet: .. Human peripheral blood mononuclear cells were isolated by density gradient centrifugation on Ficoll (GE Healthcare Life Sciences, Pittsburgh, PA), and RNA extraction was performed using the RNeasy Tissue Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s directions and treated with DNase I (RNase-free DNase Set, Qiagen) in order to remove any trace of genomic DNA. .. For first-strand cDNA synthesis, constant amounts of 1 μg of total RNA were reverse-transcribed in a 20-μL final volume using random hexamers (Roche) as primers and 200 units of M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA) (Gómez-Ambrosi et al. ).

    Labeling:

    Article Title: S-Bacillithiolation Protects Against Hypochlorite Stress in Bacillus subtilis as Revealed by Transcriptomics and Redox Proteomics *
    Article Snippet: For transcriptome analysis, 35 μg RNA were DNase-treated using the RNase-Free DNase Set (Qiagen) and purified using the RNA Clean-Up and Concentration Micro Kit (Norgen). .. Synthesis and purification of fluorescently labeled cDNA were carried out as descibed ( ) with minor modifications.

    Purification:

    Article Title: Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells
    Article Snippet: Total RNA was extracted and on-column digestion with DNase (QIAGEN, Cat#79254) was performed, followed by elution with 10μl of RNase-free water. .. PCR products were purified by Ampure XP beads (Beckman Coulter, Cat# ) and eluted with 17.5μl Nuclease-free water as described for Smart-seq2 method ( ).

    Article Title: Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses
    Article Snippet: The following DNases were tested for their ability to remove amplifiable DNA from TNA samples: DNase I (Sigma), RNase-Free DNase Set (QIAGEN), RNase-Free DNase I (Epicentre Biotechnologies) and TURBO DNA-free DNase Kit (Ambion, Life Technologies). .. All DNases were used according to manufacturers’ instructions, with the exception of incubation time, which we varied from 15 min to 2 h. The efficiency of each DNase treatment was determined by comparing the purified DNA fractions (Extract III in Figure ) with the non-reverse transcribed RNA (Extract V in Figure ), via quantitative PCR (qPCR) amplification of the 16S rRNA or the nosZ genes (details below).

    Article Title: S-Bacillithiolation Protects Against Hypochlorite Stress in Bacillus subtilis as Revealed by Transcriptomics and Redox Proteomics *
    Article Snippet: .. For transcriptome analysis, 35 μg RNA were DNase-treated using the RNase-Free DNase Set (Qiagen) and purified using the RNA Clean-Up and Concentration Micro Kit (Norgen). .. The quality of the RNA preparations was assessed by means of the Agilent 2100 Bioanalyzer according to the manufacturer's instructions.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Adipogenic differentiation of adipose tissue-derived human mesenchymal stem cells: effect of gastric bypass surgery
    Article Snippet: Paragraph title: RNA extraction and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) ... The Rnase-Free Dnase Set (Qiagen, Valencia, CA) was used to remove the genomic DNA, and cDNA synthesis was carried out by SuperScript III Reverse Transcriptase (Invitrogen) and analyzed by quantitative realtime PCR (MyIQ Single Color System, Bio-Rad, Hercules, CA).

    Article Title: Construction and Characterization of a Full-Length Infectious Simian T-Cell Lymphotropic Virus Type 3 Molecular Clone ▿
    Article Snippet: Paragraph title: RT-PCR. ... Twenty-four hours posttransfection of 293T cells, total RNA was extracted with an RNeasy mini kit (QIAGEN) and treated twice with the DNase I RNase-free DNA set (QIAGEN) to avoid any carryover of the proviral plasmid ( ).

    Article Title: The mTORC1/4E-BP pathway coordinates hemoglobin production with L-leucine availability
    Article Snippet: Paragraph title: Quantitative and semi-quantitative RT-PCR ... Total RNA was isolated from cells or 72 hpf zebrafish embryos using the Qiagen (Hilden, Germany) RNeasy Plus Mini Kit with on-column DNase digestion with the RNase-free DNase set (Qiagen) according to the manufacturer’s instructions.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells
    Article Snippet: .. Total RNA was extracted and on-column digestion with DNase (QIAGEN, Cat79254) was performed, followed by elution with 10μl of RNase-free water. .. Total RNA from 1K cells was reverse transcribed by SuperScript II (Invitrogen, Cat#18064-014) with oligo-dT and LNA-containing TSO primers in a final reaction volume of 10μl using the condition: 42°C for 90min, 10 cycles of 50°C 2min to 42°C 2min, 70°C for 15min and hold at 4°C. cDNA was pre-amplified by PCR using KAPA HiFi HotStart ReadyMix (KAPABIOSYSTEMS Cat#KK2602) with IS PCR for 12 cycles in 25μl.

    Plasmid Preparation:

    Article Title: Construction and Characterization of a Full-Length Infectious Simian T-Cell Lymphotropic Virus Type 3 Molecular Clone ▿
    Article Snippet: .. Twenty-four hours posttransfection of 293T cells, total RNA was extracted with an RNeasy mini kit (QIAGEN) and treated twice with the DNase I RNase-free DNA set (QIAGEN) to avoid any carryover of the proviral plasmid ( ). .. Total RNA (0.5 μg) was used as a matrix for RT-PCR with a one-step RT-PCR kit (QIAGEN).

    Software:

    Article Title: Adipogenic differentiation of adipose tissue-derived human mesenchymal stem cells: effect of gastric bypass surgery
    Article Snippet: The Rnase-Free Dnase Set (Qiagen, Valencia, CA) was used to remove the genomic DNA, and cDNA synthesis was carried out by SuperScript III Reverse Transcriptase (Invitrogen) and analyzed by quantitative realtime PCR (MyIQ Single Color System, Bio-Rad, Hercules, CA). .. The expression level was normalized to GAPDH, and the data were processed by Gene Expression Analysis Software (Bio-Rad Life Science, Hercules, CA, USA).

    Article Title: Peripheral mononuclear blood cells contribute to the obesity-associated inflammatory state independently of glycemic status: involvement of the novel proinflammatory adipokines chemerin, chitinase-3-like protein 1, lipocalin-2 and osteopontin
    Article Snippet: Human peripheral blood mononuclear cells were isolated by density gradient centrifugation on Ficoll (GE Healthcare Life Sciences, Pittsburgh, PA), and RNA extraction was performed using the RNeasy Tissue Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s directions and treated with DNase I (RNase-free DNase Set, Qiagen) in order to remove any trace of genomic DNA. .. Primers and probes (Supplemental Table 1) were designed using the software Primer Express 2.0 (Applied Biosystems) and purchased from Genosys (Sigma-Aldrich, Madrid, Spain).

    Real-time Polymerase Chain Reaction:

    Article Title: Adipogenic differentiation of adipose tissue-derived human mesenchymal stem cells: effect of gastric bypass surgery
    Article Snippet: The Rnase-Free Dnase Set (Qiagen, Valencia, CA) was used to remove the genomic DNA, and cDNA synthesis was carried out by SuperScript III Reverse Transcriptase (Invitrogen) and analyzed by quantitative realtime PCR (MyIQ Single Color System, Bio-Rad, Hercules, CA). .. Human RAS-related gene quantitative PCR primers were adopted from Janke et al. [ ] for AGT, renin, and ACE.

    Article Title: Insulin-like growth factor-1 mRNA isoforms and insulin-like growth factor-1 receptor mRNA expression in chronic hepatitis C
    Article Snippet: Paragraph title: Technique of quantitative real-time PCR ... Every RNA sample was subjected to additional digestion with DNase using RNase-Free DNase Set (QIAGEN) in order to avoid contamination with genomic DNA.

    Article Title: Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses
    Article Snippet: The following DNases were tested for their ability to remove amplifiable DNA from TNA samples: DNase I (Sigma), RNase-Free DNase Set (QIAGEN), RNase-Free DNase I (Epicentre Biotechnologies) and TURBO DNA-free DNase Kit (Ambion, Life Technologies). .. All DNases were used according to manufacturers’ instructions, with the exception of incubation time, which we varied from 15 min to 2 h. The efficiency of each DNase treatment was determined by comparing the purified DNA fractions (Extract III in Figure ) with the non-reverse transcribed RNA (Extract V in Figure ), via quantitative PCR (qPCR) amplification of the 16S rRNA or the nosZ genes (details below).

    Article Title: Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins
    Article Snippet: Paragraph title: RNA extraction and quantitative real-time PCR ... DNA contamination was removed using an RNase-Free DNase Set (Qiagen). cDNA was synthesized using equal amounts of RNA samples (800 ng), according to the AMV First Strand cDNA Synthesis Kit instructions (NEB Biolabs). β-actin and GAPDH were used as a housekeeping gene controls for mRNA analysis , .

    Article Title: Peripheral mononuclear blood cells contribute to the obesity-associated inflammatory state independently of glycemic status: involvement of the novel proinflammatory adipokines chemerin, chitinase-3-like protein 1, lipocalin-2 and osteopontin
    Article Snippet: Paragraph title: RNA extraction and Real-Time PCR ... Human peripheral blood mononuclear cells were isolated by density gradient centrifugation on Ficoll (GE Healthcare Life Sciences, Pittsburgh, PA), and RNA extraction was performed using the RNeasy Tissue Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s directions and treated with DNase I (RNase-free DNase Set, Qiagen) in order to remove any trace of genomic DNA.

    RNA Extraction:

    Article Title: Adipogenic differentiation of adipose tissue-derived human mesenchymal stem cells: effect of gastric bypass surgery
    Article Snippet: Paragraph title: RNA extraction and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) ... The Rnase-Free Dnase Set (Qiagen, Valencia, CA) was used to remove the genomic DNA, and cDNA synthesis was carried out by SuperScript III Reverse Transcriptase (Invitrogen) and analyzed by quantitative realtime PCR (MyIQ Single Color System, Bio-Rad, Hercules, CA).

    Article Title: Isolation of Exosomes from Semen for in vitro Uptake and HIV-1 Infection Assays
    Article Snippet: Examination of RNA integrity and content of SE Use a starting concentration of at least 12 μg of SE for RNA extraction. .. Extract SE RNA using the Qiagen RNeasy kit per the manufacturer's instructions and complete the optional DNase treatment using Qiagen RNase-free DNase set.

    Article Title: Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins
    Article Snippet: Paragraph title: RNA extraction and quantitative real-time PCR ... DNA contamination was removed using an RNase-Free DNase Set (Qiagen). cDNA was synthesized using equal amounts of RNA samples (800 ng), according to the AMV First Strand cDNA Synthesis Kit instructions (NEB Biolabs). β-actin and GAPDH were used as a housekeeping gene controls for mRNA analysis , .

    Article Title: Peripheral mononuclear blood cells contribute to the obesity-associated inflammatory state independently of glycemic status: involvement of the novel proinflammatory adipokines chemerin, chitinase-3-like protein 1, lipocalin-2 and osteopontin
    Article Snippet: .. Human peripheral blood mononuclear cells were isolated by density gradient centrifugation on Ficoll (GE Healthcare Life Sciences, Pittsburgh, PA), and RNA extraction was performed using the RNeasy Tissue Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s directions and treated with DNase I (RNase-free DNase Set, Qiagen) in order to remove any trace of genomic DNA. .. For first-strand cDNA synthesis, constant amounts of 1 μg of total RNA were reverse-transcribed in a 20-μL final volume using random hexamers (Roche) as primers and 200 units of M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA) (Gómez-Ambrosi et al. ).

    T-Test:

    Article Title: Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins
    Article Snippet: DNA contamination was removed using an RNase-Free DNase Set (Qiagen). cDNA was synthesized using equal amounts of RNA samples (800 ng), according to the AMV First Strand cDNA Synthesis Kit instructions (NEB Biolabs). β-actin and GAPDH were used as a housekeeping gene controls for mRNA analysis , . .. Significance was calculated using Student’s t-test with three independent experiments.

    Article Title: Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins
    Article Snippet: DNA contamination was removed using an RNase-Free DNase Set (Qiagen). cDNA was synthesized using equal amounts of RNA samples (800 ng), according to the AMV First Strand cDNA Synthesis Kit instructions (NEB Biolabs). β-actin and GAPDH were used as a housekeeping gene controls for mRNA analysis , . .. Significance was calculated using Student’s t-test with three independent experiments.

    Spectrophotometry:

    Article Title: Insulin-like growth factor-1 mRNA isoforms and insulin-like growth factor-1 receptor mRNA expression in chronic hepatitis C
    Article Snippet: RNA content was quantitated by spectrophotometry. .. Every RNA sample was subjected to additional digestion with DNase using RNase-Free DNase Set (QIAGEN) in order to avoid contamination with genomic DNA.

    Article Title: Isolation of Exosomes from Semen for in vitro Uptake and HIV-1 Infection Assays
    Article Snippet: Extract SE RNA using the Qiagen RNeasy kit per the manufacturer's instructions and complete the optional DNase treatment using Qiagen RNase-free DNase set. .. Determine RNA concentration by NanoDrop spectrophotometer To evaluate RNA integrity of SE, RNA can be analyzed by an Agilent BioAnalysis run using RNA 6000 Pico chips if the RNA concentration is < 50 ng/μl.

    Article Title: Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins
    Article Snippet: Isolated RNA was measured for quality using a Nano drop ND/2000 spectrophotometer and analyzed by 2% gel electrophoresis. .. DNA contamination was removed using an RNase-Free DNase Set (Qiagen). cDNA was synthesized using equal amounts of RNA samples (800 ng), according to the AMV First Strand cDNA Synthesis Kit instructions (NEB Biolabs). β-actin and GAPDH were used as a housekeeping gene controls for mRNA analysis , .

    Article Title: Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins
    Article Snippet: Isolated RNA was measured for quality using a Nano drop ND/2000 spectrophotometer and analyzed by 2% gel electrophoresis. .. DNA contamination was removed using an RNase-Free DNase Set (Qiagen). cDNA was synthesized using equal amounts of RNA samples (800 ng), according to the AMV First Strand cDNA Synthesis Kit instructions (NEB Biolabs). β-actin and GAPDH were used as a housekeeping gene controls for mRNA analysis , .

    Concentration Assay:

    Article Title: Isolation of Exosomes from Semen for in vitro Uptake and HIV-1 Infection Assays
    Article Snippet: Examination of RNA integrity and content of SE Use a starting concentration of at least 12 μg of SE for RNA extraction. .. Extract SE RNA using the Qiagen RNeasy kit per the manufacturer's instructions and complete the optional DNase treatment using Qiagen RNase-free DNase set.

    Article Title: S-Bacillithiolation Protects Against Hypochlorite Stress in Bacillus subtilis as Revealed by Transcriptomics and Redox Proteomics *
    Article Snippet: .. For transcriptome analysis, 35 μg RNA were DNase-treated using the RNase-Free DNase Set (Qiagen) and purified using the RNA Clean-Up and Concentration Micro Kit (Norgen). .. The quality of the RNA preparations was assessed by means of the Agilent 2100 Bioanalyzer according to the manufacturer's instructions.

    Lysis:

    Article Title: Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells
    Article Snippet: Three thousand cells were sorted into 700μl of QIAzol Lysis Reagent (miRNAeasy Micro Kit (QIAGEN, Cat#217084). .. Total RNA was extracted and on-column digestion with DNase (QIAGEN, Cat#79254) was performed, followed by elution with 10μl of RNase-free water.

    Gradient Centrifugation:

    Article Title: Peripheral mononuclear blood cells contribute to the obesity-associated inflammatory state independently of glycemic status: involvement of the novel proinflammatory adipokines chemerin, chitinase-3-like protein 1, lipocalin-2 and osteopontin
    Article Snippet: .. Human peripheral blood mononuclear cells were isolated by density gradient centrifugation on Ficoll (GE Healthcare Life Sciences, Pittsburgh, PA), and RNA extraction was performed using the RNeasy Tissue Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s directions and treated with DNase I (RNase-free DNase Set, Qiagen) in order to remove any trace of genomic DNA. .. For first-strand cDNA synthesis, constant amounts of 1 μg of total RNA were reverse-transcribed in a 20-μL final volume using random hexamers (Roche) as primers and 200 units of M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA) (Gómez-Ambrosi et al. ).

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    Qiagen dnase
    BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from <t>RNA-Seq</t> analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of <t>DNase-Seq</t> read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.
    Dnase, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1968 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from RNA-Seq analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of DNase-Seq read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.

    Journal: Immunity

    Article Title: Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells

    doi: 10.1016/j.immuni.2018.01.013

    Figure Lengend Snippet: BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from RNA-Seq analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of DNase-Seq read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.

    Article Snippet: Total RNA was extracted and on-column digestion with DNase (QIAGEN, Cat#79254) was performed, followed by elution with 10μl of RNase-free water.

    Techniques: Binding Assay, Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation, Modification, Marker

    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Journal: Frontiers in Microbiology

    Article Title: Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

    doi: 10.3389/fmicb.2016.01588

    Figure Lengend Snippet: Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Article Snippet: The following DNases were tested for their ability to remove amplifiable DNA from TNA samples: DNase I (Sigma), RNase-Free DNase Set (QIAGEN), RNase-Free DNase I (Epicentre Biotechnologies) and TURBO DNA-free DNase Kit (Ambion, Life Technologies).

    Techniques: Environmental Sampling, Purification, Real-time Polymerase Chain Reaction, Lysis

    Enzymatic digestion of decellularized ECM scaffolds releases small RNA molecules. ( A ) Nucleic acid extracted from untreated UBM (no digest) and pepsin-, proteinase K–, or collagenase-treated UBM was exposed to RNase A, DNase I, or no-nuclease treatment (control). ( B ) Electropherogram depicting the small RNA pattern of nucleic acid in fluorescence units (FU) before (top panel) and after (bottom panel) DNase I treatment. ( C ) Electropherogram depicting small RNA pattern from the indicated samples in FU. ( D ) A subset of nucleic molecules in biologic scaffolds is protected from nuclease degradation.

    Journal: Science Advances

    Article Title: Matrix-bound nanovesicles within ECM bioscaffolds

    doi: 10.1126/sciadv.1600502

    Figure Lengend Snippet: Enzymatic digestion of decellularized ECM scaffolds releases small RNA molecules. ( A ) Nucleic acid extracted from untreated UBM (no digest) and pepsin-, proteinase K–, or collagenase-treated UBM was exposed to RNase A, DNase I, or no-nuclease treatment (control). ( B ) Electropherogram depicting the small RNA pattern of nucleic acid in fluorescence units (FU) before (top panel) and after (bottom panel) DNase I treatment. ( C ) Electropherogram depicting small RNA pattern from the indicated samples in FU. ( D ) A subset of nucleic molecules in biologic scaffolds is protected from nuclease degradation.

    Article Snippet: RNase-free DNase was obtained from Qiagen.

    Techniques: Fluorescence

    RT-PCR analysis of viral SV2neo PPA-F3 RNAs. Total RNA was extracted from transiently transfected 293T cells, treated with DNase I, and subjected to RT-PCR to search for the presence of (A) nonspliced gag (498-bp) or (B, C) doubly spliced tax/rex (424-bp) viral messengers. (A) Lane 1, 100-bp DNA ladder; lane 2, mock-transfected cells; lane 3, RNA from SV2neo-transfected cells; lanes 4 to 6, RNA from cells transfected with SV2neo PPA-F3 clones 6, 7, and 26, respectively (these three recombinant plasmids contain the wild-type SV2neo PPA-F3 sequence). For lane 7, RT was not added to the PCR mix containing RNA extracted from clone 26-transfected cells. (B) RT-PCR strategy for amplifying the rex ). (C) RNAs were tested for the presence of a band corresponding to the spliced tax / rex mRNA transcript. Lane 1, 100-bp DNA ladder; lane 2, mock-transfected cells; lane 3, RNA from SV2neo-transfected cells; lanes 4 to 6, RNA from cells transfected with STLV-3 PPA-F3 clones 6, 7, and 26, respectively. NS, nonspecific band. (D) Sequence analysis of the 424-bp-long RT-PCR product. The gag and tax / rex gels are representative of at least three different experiments performed on different RNA preparations. Numbers to the left of the blots are in base pairs.

    Journal: Journal of Virology

    Article Title: Construction and Characterization of a Full-Length Infectious Simian T-Cell Lymphotropic Virus Type 3 Molecular Clone ▿

    doi: 10.1128/JVI.02538-06

    Figure Lengend Snippet: RT-PCR analysis of viral SV2neo PPA-F3 RNAs. Total RNA was extracted from transiently transfected 293T cells, treated with DNase I, and subjected to RT-PCR to search for the presence of (A) nonspliced gag (498-bp) or (B, C) doubly spliced tax/rex (424-bp) viral messengers. (A) Lane 1, 100-bp DNA ladder; lane 2, mock-transfected cells; lane 3, RNA from SV2neo-transfected cells; lanes 4 to 6, RNA from cells transfected with SV2neo PPA-F3 clones 6, 7, and 26, respectively (these three recombinant plasmids contain the wild-type SV2neo PPA-F3 sequence). For lane 7, RT was not added to the PCR mix containing RNA extracted from clone 26-transfected cells. (B) RT-PCR strategy for amplifying the rex ). (C) RNAs were tested for the presence of a band corresponding to the spliced tax / rex mRNA transcript. Lane 1, 100-bp DNA ladder; lane 2, mock-transfected cells; lane 3, RNA from SV2neo-transfected cells; lanes 4 to 6, RNA from cells transfected with STLV-3 PPA-F3 clones 6, 7, and 26, respectively. NS, nonspecific band. (D) Sequence analysis of the 424-bp-long RT-PCR product. The gag and tax / rex gels are representative of at least three different experiments performed on different RNA preparations. Numbers to the left of the blots are in base pairs.

    Article Snippet: Twenty-four hours posttransfection of 293T cells, total RNA was extracted with an RNeasy mini kit (QIAGEN) and treated twice with the DNase I RNase-free DNA set (QIAGEN) to avoid any carryover of the proviral plasmid ( ).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Clone Assay, Recombinant, Sequencing, Polymerase Chain Reaction