total rna  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99

    Structured Review

    Thermo Fisher total rna
    Expression patterns of stress/ABA responsive genes regulated by GmWRKY16 . (A) Expression patterns of genes responsive to salt stress. (B) Expression patterns of ABA and/or stress-responsive genes under drought stress. (C) Expression patterns of ABA-responsive genes under drought stress. Arabidopsis seeds of WT and GmWRKY16 transgenic line #12 of T 3 generation were sown in mixed soil (vermiculite and flower nutrient soil, 1:1) and cultured in the chamber room. The 2-week-old seedlings were subjected to drought treatment (withholding water) and 200 mM <t>NaCl</t> treatment for 10 days. The samples from the aerial parts of Arabidopsis plants were taken for total <t>RNA</t> extraction. The relative expression of drought- and/or salt stress-responsive genes was quantified by qRT-PCR using ACT3 as the reference gene to normalize the data ( ∗∗ P = 0.01). The 2 -ΔΔCt method was used to evaluate the quantitative variation between the examined replicates ( Lü et al., 2015 ). The details for the specific primers of the GmWRKY16 , ACT3 and stress-responsive genes are listed in Supplementary Table S1 .
    Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 277 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/total rna/product/Thermo Fisher
    Average 99 stars, based on 277 article reviews
    Price from $9.99 to $1999.99
    total rna - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) Product Images from "GmWRKY16 Enhances Drought and Salt Tolerance Through an ABA-Mediated Pathway in Arabidopsis thaliana"

    Article Title: GmWRKY16 Enhances Drought and Salt Tolerance Through an ABA-Mediated Pathway in Arabidopsis thaliana

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.01979

    Expression patterns of stress/ABA responsive genes regulated by GmWRKY16 . (A) Expression patterns of genes responsive to salt stress. (B) Expression patterns of ABA and/or stress-responsive genes under drought stress. (C) Expression patterns of ABA-responsive genes under drought stress. Arabidopsis seeds of WT and GmWRKY16 transgenic line #12 of T 3 generation were sown in mixed soil (vermiculite and flower nutrient soil, 1:1) and cultured in the chamber room. The 2-week-old seedlings were subjected to drought treatment (withholding water) and 200 mM NaCl treatment for 10 days. The samples from the aerial parts of Arabidopsis plants were taken for total RNA extraction. The relative expression of drought- and/or salt stress-responsive genes was quantified by qRT-PCR using ACT3 as the reference gene to normalize the data ( ∗∗ P = 0.01). The 2 -ΔΔCt method was used to evaluate the quantitative variation between the examined replicates ( Lü et al., 2015 ). The details for the specific primers of the GmWRKY16 , ACT3 and stress-responsive genes are listed in Supplementary Table S1 .
    Figure Legend Snippet: Expression patterns of stress/ABA responsive genes regulated by GmWRKY16 . (A) Expression patterns of genes responsive to salt stress. (B) Expression patterns of ABA and/or stress-responsive genes under drought stress. (C) Expression patterns of ABA-responsive genes under drought stress. Arabidopsis seeds of WT and GmWRKY16 transgenic line #12 of T 3 generation were sown in mixed soil (vermiculite and flower nutrient soil, 1:1) and cultured in the chamber room. The 2-week-old seedlings were subjected to drought treatment (withholding water) and 200 mM NaCl treatment for 10 days. The samples from the aerial parts of Arabidopsis plants were taken for total RNA extraction. The relative expression of drought- and/or salt stress-responsive genes was quantified by qRT-PCR using ACT3 as the reference gene to normalize the data ( ∗∗ P = 0.01). The 2 -ΔΔCt method was used to evaluate the quantitative variation between the examined replicates ( Lü et al., 2015 ). The details for the specific primers of the GmWRKY16 , ACT3 and stress-responsive genes are listed in Supplementary Table S1 .

    Techniques Used: Expressing, Transgenic Assay, Cell Culture, RNA Extraction, Quantitative RT-PCR

    2) Product Images from "Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells"

    Article Title: Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky159

    Polyadenylated mitochondrial tRNAs are rapidly degraded after EtBr removal. Northern blots probed for mitochondrial tRNA Ser(UCN) or tRNA Leu(UUR) as indicated, in wild-type 143B cybrid cells treated with 2.5 or 5 μg/ml EtBr for the indicated times, then allowed to recover in the absence of the drug as shown. Where shown, 5S rRNA was used as a loading control. RNA sizes (in nt) were extrapolated from the migration of mature tRNA Ser(UCN) (72 nt), 5S rRNA (121 nt) and ND3 mRNA ( > 345 nt). See also Supplementary Figure S3A .
    Figure Legend Snippet: Polyadenylated mitochondrial tRNAs are rapidly degraded after EtBr removal. Northern blots probed for mitochondrial tRNA Ser(UCN) or tRNA Leu(UUR) as indicated, in wild-type 143B cybrid cells treated with 2.5 or 5 μg/ml EtBr for the indicated times, then allowed to recover in the absence of the drug as shown. Where shown, 5S rRNA was used as a loading control. RNA sizes (in nt) were extrapolated from the migration of mature tRNA Ser(UCN) (72 nt), 5S rRNA (121 nt) and ND3 mRNA ( > 345 nt). See also Supplementary Figure S3A .

    Techniques Used: Northern Blot, Migration

    3) Product Images from "Involvement of Up-Regulation of DR5 Expression and Down-Regulation of c-FLIP in Niclosamide-Mediated TRAIL Sensitization in Human Renal Carcinoma Caki Cells"

    Article Title: Involvement of Up-Regulation of DR5 Expression and Down-Regulation of c-FLIP in Niclosamide-Mediated TRAIL Sensitization in Human Renal Carcinoma Caki Cells

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules23092264

    The down-regulation of c-FLIP is associated with the reduction of TRAIL resistance by niclosamide. ( A ) The messenger RNA (mRNA) expression was determined by reverse transcription polymerase chain reaction (RT-PCR) (upper panel) and quantitative polymerase chain reaction (qPCR) (lower panel) in Caki cells after treatment with niclosamide (50–200 nM) for 24 h. ( B ) The protein expression was determined by Western blotting in Caki cells after treatment with 200 nM niclosamide in the presence or absence of 20 μg/mL cycloheximide (CHX) for 3–18 h. The band intensity was measured using ImageJ. ( C ) Apoptosis levels and protein expression were determined by flow cytometry and Western blotting in Vector cells (Caki/Vec) and in c-FLIP-overexpressed cells (Caki/c-FLIP) after treatment with 200 nM niclosamide, and/or 50 ng/mL TRAIL for 24 h. The values in graph ( A , C ) represent the mean ± SEM of three independent samples. * p
    Figure Legend Snippet: The down-regulation of c-FLIP is associated with the reduction of TRAIL resistance by niclosamide. ( A ) The messenger RNA (mRNA) expression was determined by reverse transcription polymerase chain reaction (RT-PCR) (upper panel) and quantitative polymerase chain reaction (qPCR) (lower panel) in Caki cells after treatment with niclosamide (50–200 nM) for 24 h. ( B ) The protein expression was determined by Western blotting in Caki cells after treatment with 200 nM niclosamide in the presence or absence of 20 μg/mL cycloheximide (CHX) for 3–18 h. The band intensity was measured using ImageJ. ( C ) Apoptosis levels and protein expression were determined by flow cytometry and Western blotting in Vector cells (Caki/Vec) and in c-FLIP-overexpressed cells (Caki/c-FLIP) after treatment with 200 nM niclosamide, and/or 50 ng/mL TRAIL for 24 h. The values in graph ( A , C ) represent the mean ± SEM of three independent samples. * p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry, Cytometry, Plasmid Preparation

    4) Product Images from "Hemi-methylated CpG sites connect Dnmt1-knockdown-induced and Tet1-induced DNA demethylation during somatic cell reprogramming"

    Article Title: Hemi-methylated CpG sites connect Dnmt1-knockdown-induced and Tet1-induced DNA demethylation during somatic cell reprogramming

    Journal: Cell Discovery

    doi: 10.1038/s41421-018-0074-6

    Demethylation of hemi-methylated CpG sites up-regulates pluripotent genes. a – c Approximately 20,000 protein-coding genes were divided into different groups based on their methylation levels and CpG densities near TSS (−1.5 ~ + 2.0 kb). The number of genes in the different groups were summarized in a . Genes with 42–83 CpG sites near their TSS (−1.5 ~ + 2.0 kb) were highlighted as in Zone I in a . Of the genes in Zone I, the genes with methylation levels between 40 and 60% were highlighted in Zone II. The changes in methylation of approximately 14,500 genes detected in the current RRBS were summarized in b , while the changes in the expression of approximately 11,000 genes detected in the current RNA-seq were summarized in c . Groups with less than 50 genes detected in RRBS or RNA-seq were not listed in b and c . d – f Demethylation of genes highlighted in Zone I and Zone II in a were compared to the average demethylation of all genes in d , e . The expression changes of 698 genes in Zone II were compared to the expression changes of all genes ( f ). g – i GO analysis results of the 698 genes in Zone II ( g ). The 22 genes that undergo significant up-regulation were selected and their demethylation was listed in h . Of these 22 genes, the expression changes of 6 pluripotent genes were determined by qPCR ( i )
    Figure Legend Snippet: Demethylation of hemi-methylated CpG sites up-regulates pluripotent genes. a – c Approximately 20,000 protein-coding genes were divided into different groups based on their methylation levels and CpG densities near TSS (−1.5 ~ + 2.0 kb). The number of genes in the different groups were summarized in a . Genes with 42–83 CpG sites near their TSS (−1.5 ~ + 2.0 kb) were highlighted as in Zone I in a . Of the genes in Zone I, the genes with methylation levels between 40 and 60% were highlighted in Zone II. The changes in methylation of approximately 14,500 genes detected in the current RRBS were summarized in b , while the changes in the expression of approximately 11,000 genes detected in the current RNA-seq were summarized in c . Groups with less than 50 genes detected in RRBS or RNA-seq were not listed in b and c . d – f Demethylation of genes highlighted in Zone I and Zone II in a were compared to the average demethylation of all genes in d , e . The expression changes of 698 genes in Zone II were compared to the expression changes of all genes ( f ). g – i GO analysis results of the 698 genes in Zone II ( g ). The 22 genes that undergo significant up-regulation were selected and their demethylation was listed in h . Of these 22 genes, the expression changes of 6 pluripotent genes were determined by qPCR ( i )

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

    Two types of DNA demethylation differentially influence gene expression. The results obtained from the WGBS, ATAC-seq, and RNA-seq in assays of MEFs were analyzed together in a – g . The results obtained from WGBS (MEFs) and RRBS/RNA-seq (control, Tet1, and sh-Dnmt1 groups in MEFs) were analyzed in h , i . a First, genes were divided into 102 groups based their methylation levels (0%, 0–1%, …, 99–100%, and 100%). Then, the genes in each group were equally separated into two sub-groups, i.e., high enrichment and low enrichment, based on their AMDs. The genes from all high/low enrichment sub-groups were combined and sorted based on their methylation levels, and were presented along with their chromatin accessibility. The chromatin accessibilities generated from ATAC-seq were listed near TSS (−2.0 ~ + 2.0 kb). Gene methylation was plotted on the left. b First, the genes were divided into 20 groups (0–5%, …, and 95–100%). Then, the averages of gene methylation and chromatin accessibility were plotted. c – g Six sets of genes with particular methylation levels and enrichment of hemi-methylation were selected ( c ). The average methylation levels ( d ), enrichment of hemi-methylation ( e ), chromatin accessibilities ( f ), and gene expression ( g ) in MEFs were listed. The number of genes in each group was provided. h – i Of the 300 selected genes, the expression of 197 genes was detected in the current RNA-seq. The expression changes induced by Tet1 and sh-Dnmt1 were plotted in h . In total, 20 genes were randomly selected from these 197 genes, and the chromatin accessibilities of the genes in the Tet1 and sh-Dnmt1 groups were determined by ATAC-qPCR, compared, and plotted in i
    Figure Legend Snippet: Two types of DNA demethylation differentially influence gene expression. The results obtained from the WGBS, ATAC-seq, and RNA-seq in assays of MEFs were analyzed together in a – g . The results obtained from WGBS (MEFs) and RRBS/RNA-seq (control, Tet1, and sh-Dnmt1 groups in MEFs) were analyzed in h , i . a First, genes were divided into 102 groups based their methylation levels (0%, 0–1%, …, 99–100%, and 100%). Then, the genes in each group were equally separated into two sub-groups, i.e., high enrichment and low enrichment, based on their AMDs. The genes from all high/low enrichment sub-groups were combined and sorted based on their methylation levels, and were presented along with their chromatin accessibility. The chromatin accessibilities generated from ATAC-seq were listed near TSS (−2.0 ~ + 2.0 kb). Gene methylation was plotted on the left. b First, the genes were divided into 20 groups (0–5%, …, and 95–100%). Then, the averages of gene methylation and chromatin accessibility were plotted. c – g Six sets of genes with particular methylation levels and enrichment of hemi-methylation were selected ( c ). The average methylation levels ( d ), enrichment of hemi-methylation ( e ), chromatin accessibilities ( f ), and gene expression ( g ) in MEFs were listed. The number of genes in each group was provided. h – i Of the 300 selected genes, the expression of 197 genes was detected in the current RNA-seq. The expression changes induced by Tet1 and sh-Dnmt1 were plotted in h . In total, 20 genes were randomly selected from these 197 genes, and the chromatin accessibilities of the genes in the Tet1 and sh-Dnmt1 groups were determined by ATAC-qPCR, compared, and plotted in i

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

    5) Product Images from "Bioinformatic analysis of next-generation sequencing data to identify dysregulated genes in fibroblasts of idiopathic pulmonary fibrosis"

    Article Title: Bioinformatic analysis of next-generation sequencing data to identify dysregulated genes in fibroblasts of idiopathic pulmonary fibrosis

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2019.4086

    Schematic illustration of the study design. Following culture of the IPF fibroblasts and healthy lung fibroblasts, the total RNA were extracted and deep sequenced using a next-generation sequencing platform. The differentially expressed genes ( > 2 fold-change) were identified, and analyzed with IPA and DAVID for pathway analysis and functional interpretation. The GEO IPF databases were analyzed to confirm dysregulated genes identified in the present study. Conversely, the differentially expressed miRNAs ( > 2 fold-change) were analyzed with miRmap for target prediction. Then, genes with potential miRNA-mRNA interactions were determined by Venn diagram analysis. These miRNA-mRNA interactions were verified by a second miRNA prediction database, TargetScan. Finally, a literature search was performed and a hypothesis was generated. IPF, idiopathic pulmonary fibrosis; IPA, Ingenuity ® Pathway Analysis; DAVID, Database for Annotation, Visualization, and Integrated Discovery; GEO, Gene Expression Omnibus.
    Figure Legend Snippet: Schematic illustration of the study design. Following culture of the IPF fibroblasts and healthy lung fibroblasts, the total RNA were extracted and deep sequenced using a next-generation sequencing platform. The differentially expressed genes ( > 2 fold-change) were identified, and analyzed with IPA and DAVID for pathway analysis and functional interpretation. The GEO IPF databases were analyzed to confirm dysregulated genes identified in the present study. Conversely, the differentially expressed miRNAs ( > 2 fold-change) were analyzed with miRmap for target prediction. Then, genes with potential miRNA-mRNA interactions were determined by Venn diagram analysis. These miRNA-mRNA interactions were verified by a second miRNA prediction database, TargetScan. Finally, a literature search was performed and a hypothesis was generated. IPF, idiopathic pulmonary fibrosis; IPA, Ingenuity ® Pathway Analysis; DAVID, Database for Annotation, Visualization, and Integrated Discovery; GEO, Gene Expression Omnibus.

    Techniques Used: Next-Generation Sequencing, Indirect Immunoperoxidase Assay, Functional Assay, Generated, Expressing

    6) Product Images from "Integration of light and brassinosteroid signaling pathways by a GATA transcription factor in Arabidopsis"

    Article Title: Integration of light and brassinosteroid signaling pathways by a GATA transcription factor in Arabidopsis

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2010.10.023

    Light regulates GATA2 accumulation at the post-translational level (A) Light represses GATA2 and GATA4 transcription levels. Dark-grown Arabidopsis seedlings were treated with white light for indicated time and RNA levels of GATA2 and GATA4 were measured by real-time qRT-PCR. Error bars indicate standard deviation. (B) Light promotes GATA2 protein accumulation. Immunoblot analysis of GATA2 protein in 5-day-old dark-grown GATA2-ox L6 line seedlings treated with white light for the indicated time. (C) qRT-PCR analysis of the levels of RNA expressed from the endogenous GATA2 and GATA4 genes (endo) or total GATA2 RNA level in wild type (WT) and the GATA2-ox transgenic seedlings (L3 and L6). UBC30 was used as internal control. (D) ChIP-qPCR analysis of GATA2 binding to its own promoter. The upper panel shows a diagram of the promoter (open box), 5′UTR (black line) and the first exon (black box) of the GATA2 gene. Black circles indicate positions of putative GATA motifs. Lines marked a to f show GATA2-binding (solid) and non-binding (dashed) regions analyzed by qPCR. The lower panel shows ChIP-qPCR data. Error bars indicate SD.
    Figure Legend Snippet: Light regulates GATA2 accumulation at the post-translational level (A) Light represses GATA2 and GATA4 transcription levels. Dark-grown Arabidopsis seedlings were treated with white light for indicated time and RNA levels of GATA2 and GATA4 were measured by real-time qRT-PCR. Error bars indicate standard deviation. (B) Light promotes GATA2 protein accumulation. Immunoblot analysis of GATA2 protein in 5-day-old dark-grown GATA2-ox L6 line seedlings treated with white light for the indicated time. (C) qRT-PCR analysis of the levels of RNA expressed from the endogenous GATA2 and GATA4 genes (endo) or total GATA2 RNA level in wild type (WT) and the GATA2-ox transgenic seedlings (L3 and L6). UBC30 was used as internal control. (D) ChIP-qPCR analysis of GATA2 binding to its own promoter. The upper panel shows a diagram of the promoter (open box), 5′UTR (black line) and the first exon (black box) of the GATA2 gene. Black circles indicate positions of putative GATA motifs. Lines marked a to f show GATA2-binding (solid) and non-binding (dashed) regions analyzed by qPCR. The lower panel shows ChIP-qPCR data. Error bars indicate SD.

    Techniques Used: Quantitative RT-PCR, Standard Deviation, Transgenic Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    GATA2 is a positive regulator of photomorphogenesis (A), BR treatment reduces GATA2 RNA level. Arabidopsis seedlings grown in the dark (WD) or light (WL) for five days were treated with mock solution or 100 nM 24-epibrassinolide (BR) for 3 hours and the expression of GATA2 was analyzed by qRT-PCR. (B) qRT-PCR analysis of GATA2 and GATA4 RNA levels in 5-day-old dark-grown wild type (Col-0), det2, bri1-116 , and bri1-116 bzr1-1D . (C) Dark-grown phenotypes of three GATA-ox lines. Lower panels show qRT-PCR of GATA2 ). (D) Phenotypes of light-grown (first on left) or dark-grown seedlings of wild type (Col-0), BR mutants and a representative GATA2-ox transgenic line 6. (E) Phenotypes of antisense (AS) or artificial-microRNA (AM) transgenic Arabidopsis seedlings with reduced levels of GATA2 and GATA4 for quantitation data). Lower panel shows qRT-PCR analysis of GATA2 and GATA4 in these transgenic seedlings. All error bars are standard deviation (SD).
    Figure Legend Snippet: GATA2 is a positive regulator of photomorphogenesis (A), BR treatment reduces GATA2 RNA level. Arabidopsis seedlings grown in the dark (WD) or light (WL) for five days were treated with mock solution or 100 nM 24-epibrassinolide (BR) for 3 hours and the expression of GATA2 was analyzed by qRT-PCR. (B) qRT-PCR analysis of GATA2 and GATA4 RNA levels in 5-day-old dark-grown wild type (Col-0), det2, bri1-116 , and bri1-116 bzr1-1D . (C) Dark-grown phenotypes of three GATA-ox lines. Lower panels show qRT-PCR of GATA2 ). (D) Phenotypes of light-grown (first on left) or dark-grown seedlings of wild type (Col-0), BR mutants and a representative GATA2-ox transgenic line 6. (E) Phenotypes of antisense (AS) or artificial-microRNA (AM) transgenic Arabidopsis seedlings with reduced levels of GATA2 and GATA4 for quantitation data). Lower panel shows qRT-PCR analysis of GATA2 and GATA4 in these transgenic seedlings. All error bars are standard deviation (SD).

    Techniques Used: Expressing, Quantitative RT-PCR, Transgenic Assay, Quantitation Assay, Standard Deviation

    7) Product Images from "Interferon-stimulated gene ISG12b2 is localized to the inner mitochondrial membrane and mediates virus-induced cell death"

    Article Title: Interferon-stimulated gene ISG12b2 is localized to the inner mitochondrial membrane and mediates virus-induced cell death

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2010.160

    Knockdown of ISG12b2 reduces DENV- or poly(I:C)-induced caspse-3 activation. ( a ) Hepa 1-6 cells were co-transfected with ISG12b2-HA expression plasmid and non-targeting control siRNA (Ctrl) or targeting ISG12b2 siRNA (ISG) followed by western blot analysis using anti-ISG12b2. Actin was analysed as a loading control. ( b ) Hepa 1-6 cells transfected with non-targeting control siRNA (Ctrl) or targeting ISG12b2 siRNA (ISG) were infected with DENV at a MOI of 5 at 6 h post-transfection. Cells were then lysed using 1% Triton lysis buffer for protein detection or Trizol reagent for RNA extraction at 72 h after DENV infection. Western blot analysis using antibodies against indicated proteins in whole-cell lysates is shown in left panel. Actin was analysed as a loading control. Knockdown of endogenous ISG12b2 by siRNA was measured by real-time quantitative PCR shown in right panel. ( c ) Hepa 1-6 cells transfected with non-targeting control siRNA (Ctrl) or targeting ISG12b2 siRNA (ISG) were infected with DENV at a MOI of 5 and then subjected to Annexin V-Cy5 staining at 66 h post-infection. Data represent the mean±S.E.M. and reflect one representative of three independent experiments. * P
    Figure Legend Snippet: Knockdown of ISG12b2 reduces DENV- or poly(I:C)-induced caspse-3 activation. ( a ) Hepa 1-6 cells were co-transfected with ISG12b2-HA expression plasmid and non-targeting control siRNA (Ctrl) or targeting ISG12b2 siRNA (ISG) followed by western blot analysis using anti-ISG12b2. Actin was analysed as a loading control. ( b ) Hepa 1-6 cells transfected with non-targeting control siRNA (Ctrl) or targeting ISG12b2 siRNA (ISG) were infected with DENV at a MOI of 5 at 6 h post-transfection. Cells were then lysed using 1% Triton lysis buffer for protein detection or Trizol reagent for RNA extraction at 72 h after DENV infection. Western blot analysis using antibodies against indicated proteins in whole-cell lysates is shown in left panel. Actin was analysed as a loading control. Knockdown of endogenous ISG12b2 by siRNA was measured by real-time quantitative PCR shown in right panel. ( c ) Hepa 1-6 cells transfected with non-targeting control siRNA (Ctrl) or targeting ISG12b2 siRNA (ISG) were infected with DENV at a MOI of 5 and then subjected to Annexin V-Cy5 staining at 66 h post-infection. Data represent the mean±S.E.M. and reflect one representative of three independent experiments. * P

    Techniques Used: Activation Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Infection, Lysis, RNA Extraction, Real-time Polymerase Chain Reaction, Staining

    8) Product Images from "Analyzing mRNA expression identifies Smad3 as a microRNA-140 target regulated only at protein level"

    Article Title: Analyzing mRNA expression identifies Smad3 as a microRNA-140 target regulated only at protein level

    Journal: RNA

    doi: 10.1261/rna.1701210

    Accumulation of miR-140 is transiently suppressed by TGFβ. ( A ) Time course Northern blot analysis of miR-140 following TGFβ treatment of 10T1/2 cells. TGFβ was applied once (5 ng/mL) and total RNA was extracted 1, 3, 6, 16, 24, and 48 h after the treatment. The membrane was stripped and reprobed with a miR-21 probe to show that the effect is specific for miR-140 or with a U6 probe to demonstrate equal loading. Three independent results are shown for miR-140 accumulation. ( B ) Accumulation level of miR-140 in 10T1/2 cells is shown without applying TGFβ treatment to demonstrate that the decrease in miR0140 level is due to TGFβ.
    Figure Legend Snippet: Accumulation of miR-140 is transiently suppressed by TGFβ. ( A ) Time course Northern blot analysis of miR-140 following TGFβ treatment of 10T1/2 cells. TGFβ was applied once (5 ng/mL) and total RNA was extracted 1, 3, 6, 16, 24, and 48 h after the treatment. The membrane was stripped and reprobed with a miR-21 probe to show that the effect is specific for miR-140 or with a U6 probe to demonstrate equal loading. Three independent results are shown for miR-140 accumulation. ( B ) Accumulation level of miR-140 in 10T1/2 cells is shown without applying TGFβ treatment to demonstrate that the decrease in miR0140 level is due to TGFβ.

    Techniques Used: Northern Blot

    9) Product Images from "Hepatitis E Virus (HEV) Strains in Serum Samples Can Replicate Efficiently in Cultured Cells Despite the Coexistence of HEV Antibodies: Characterization of HEV Virions in Blood Circulation ▿"

    Article Title: Hepatitis E Virus (HEV) Strains in Serum Samples Can Replicate Efficiently in Cultured Cells Despite the Coexistence of HEV Antibodies: Characterization of HEV Virions in Blood Circulation ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.02002-09

    (a) Quantitation of HEV RNA in culture supernatants of A549 cells inoculated with a serum sample (S1) with a viral load of 5.0 × 10 5 copies/ml that had been mixed with a pooled serum sample obtained during the early convalescent phase (pool 1, 2, or 3) and positive for IgM-, IgA-, and IgG-class anti-HEV antibodies or with an antibody-negative serum and cultured for 50 days. (b) Quantitation of HEV RNA in culture supernatants of A549 cells inoculated with a serum sample (S1) with a viral load of 5.0 × 10 5 ) at a final concentration of 1 mg/ml and cultured for 50 days. (c) Quantitation of HEV RNA in culture supernatants of A549 cells inoculated with a fecal suspension (JE03-1760F), a serum sample (S4a), or a culture supernatant of JE03-1760F (patient 4) origin with an HEV load of 2.0 × 10 5 copies/ml that had been mixed with a serum sample (03-2150) positive for IgM-, IgA-, and IgG-class anti-HEV antibodies but negative for HEV RNA, another serum sample (08-1340) positive only for IgG anti-HEV, or an antibody-negative serum and cultured for the indicated number of days. The fecal suspension, serum sample (S4a), and culture supernatant contained HEV of the same strain (JE03-1760F), and serum samples (S4a, 03-2150, and 08-1340) were obtained from the JE03-1760F patient (patient 4 in this study) 15, 40, and 2,088 days, respectively, after disease onset.
    Figure Legend Snippet: (a) Quantitation of HEV RNA in culture supernatants of A549 cells inoculated with a serum sample (S1) with a viral load of 5.0 × 10 5 copies/ml that had been mixed with a pooled serum sample obtained during the early convalescent phase (pool 1, 2, or 3) and positive for IgM-, IgA-, and IgG-class anti-HEV antibodies or with an antibody-negative serum and cultured for 50 days. (b) Quantitation of HEV RNA in culture supernatants of A549 cells inoculated with a serum sample (S1) with a viral load of 5.0 × 10 5 ) at a final concentration of 1 mg/ml and cultured for 50 days. (c) Quantitation of HEV RNA in culture supernatants of A549 cells inoculated with a fecal suspension (JE03-1760F), a serum sample (S4a), or a culture supernatant of JE03-1760F (patient 4) origin with an HEV load of 2.0 × 10 5 copies/ml that had been mixed with a serum sample (03-2150) positive for IgM-, IgA-, and IgG-class anti-HEV antibodies but negative for HEV RNA, another serum sample (08-1340) positive only for IgG anti-HEV, or an antibody-negative serum and cultured for the indicated number of days. The fecal suspension, serum sample (S4a), and culture supernatant contained HEV of the same strain (JE03-1760F), and serum samples (S4a, 03-2150, and 08-1340) were obtained from the JE03-1760F patient (patient 4 in this study) 15, 40, and 2,088 days, respectively, after disease onset.

    Techniques Used: Quantitation Assay, Cell Culture, Concentration Assay

    Quantitation of HEV RNA in culture supernatants of A549 cells inoculated with a culture supernatant with a viral load of 1.0 × 10 5 copies/ml that had been treated with or without 0.1% NP-40 and 0.1% pronase E, mixed with a normal serum or pooled serum sample obtained at the early convalescent phase (pool 1) and positive for IgM-, IgA-, and IgG-class anti-HEV ORF2 and ORF3 antibodies, and cultured for 30 days.
    Figure Legend Snippet: Quantitation of HEV RNA in culture supernatants of A549 cells inoculated with a culture supernatant with a viral load of 1.0 × 10 5 copies/ml that had been treated with or without 0.1% NP-40 and 0.1% pronase E, mixed with a normal serum or pooled serum sample obtained at the early convalescent phase (pool 1) and positive for IgM-, IgA-, and IgG-class anti-HEV ORF2 and ORF3 antibodies, and cultured for 30 days.

    Techniques Used: Quantitation Assay, Cell Culture

    10) Product Images from "Inhibiting TNF-mediated signaling: a novel therapeutic paradigm for androgen independent prostate cancer"

    Article Title: Inhibiting TNF-mediated signaling: a novel therapeutic paradigm for androgen independent prostate cancer

    Journal: Apoptosis : an international journal on programmed cell death

    doi: 10.1007/s10495-009-0416-9

    Effect of psoralidin on TNF-α and NF-κB in AIPC cells (A) PC-3 and DU-145 cells (70–80% confluency) were treated with 60 and 45 μM psoralidin, respectively, for varying time intervals (3–24 h), and Western blot analysis was performed using TNF-α and TNF-R1 antibodies. Actin was used as the internal loading control. (B) PC-3 and DU-145 cells (70–80% confluency) were treated with 60 and 45 μM psoralidin, respectively, for 12, 24 or 48 h, and the supernatant was collected after the treatment periods, protein concentration in the supernatant was quantified and equal amounts of protein were subjected to sandwich ELISA for quantitation of secreted TNF-α levels in control and psoralidin-treated AIPC cells. (C) PC-3 and DU-145 cells (70–80% confluency) were treated with 60 and 45 μM psoralidin, respectively, for varying time intervals (3-2 h), and total RNA was isolated using Trizol method, cDNA was synthesized using two step RT-PCR and TNF-α mRNA expression was determined. (D). PC-3 and DU-145 cells (70–80% confluency) were treated with 60 and 45 μM psoralidin, respectively, and cell lysates were subjected to sandwich ELISA for quantitation of total NF-κB protein levels in control and psoralidin-treated AIPC cells. Bars represent mean±SD.
    Figure Legend Snippet: Effect of psoralidin on TNF-α and NF-κB in AIPC cells (A) PC-3 and DU-145 cells (70–80% confluency) were treated with 60 and 45 μM psoralidin, respectively, for varying time intervals (3–24 h), and Western blot analysis was performed using TNF-α and TNF-R1 antibodies. Actin was used as the internal loading control. (B) PC-3 and DU-145 cells (70–80% confluency) were treated with 60 and 45 μM psoralidin, respectively, for 12, 24 or 48 h, and the supernatant was collected after the treatment periods, protein concentration in the supernatant was quantified and equal amounts of protein were subjected to sandwich ELISA for quantitation of secreted TNF-α levels in control and psoralidin-treated AIPC cells. (C) PC-3 and DU-145 cells (70–80% confluency) were treated with 60 and 45 μM psoralidin, respectively, for varying time intervals (3-2 h), and total RNA was isolated using Trizol method, cDNA was synthesized using two step RT-PCR and TNF-α mRNA expression was determined. (D). PC-3 and DU-145 cells (70–80% confluency) were treated with 60 and 45 μM psoralidin, respectively, and cell lysates were subjected to sandwich ELISA for quantitation of total NF-κB protein levels in control and psoralidin-treated AIPC cells. Bars represent mean±SD.

    Techniques Used: Western Blot, Protein Concentration, Sandwich ELISA, Quantitation Assay, Isolation, Synthesized, Reverse Transcription Polymerase Chain Reaction, Expressing

    11) Product Images from "A new strategy to amplify degraded RNA from small tissue samples for microarray studies"

    Article Title: A new strategy to amplify degraded RNA from small tissue samples for microarray studies

    Journal: Nucleic Acids Research

    doi:

    RNA degradation and the reliability of microarray data. ( A ) Incubating total RNA from C2 cells in a basic solution at 80°C resulted in a time-dependent breakdown of the sample. ( B ) Probes made from T7dT- or T3N9-amplified samples of degraded RNA from C2 and 3T3 cells were compared to those made from intact, unamplified templates. Even with badly degraded RNA, the T3N9-amplification method could be used to detect most of the differentially expressed genes found with intact, unamplified templates. ( C ) Using total RNAs hydrolyzed in a basic solution for 15 min as templates, we could still detect 80 and 77% of differentially expressed genes after two and three rounds of amplification starting with T3N9. In contrast, only 56 and 54% of the differentially expressed genes could be detected with T7dT.
    Figure Legend Snippet: RNA degradation and the reliability of microarray data. ( A ) Incubating total RNA from C2 cells in a basic solution at 80°C resulted in a time-dependent breakdown of the sample. ( B ) Probes made from T7dT- or T3N9-amplified samples of degraded RNA from C2 and 3T3 cells were compared to those made from intact, unamplified templates. Even with badly degraded RNA, the T3N9-amplification method could be used to detect most of the differentially expressed genes found with intact, unamplified templates. ( C ) Using total RNAs hydrolyzed in a basic solution for 15 min as templates, we could still detect 80 and 77% of differentially expressed genes after two and three rounds of amplification starting with T3N9. In contrast, only 56 and 54% of the differentially expressed genes could be detected with T7dT.

    Techniques Used: Microarray, Amplification

    12) Product Images from "siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs"

    Article Title: siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks239

    An siRNA targeting the WAF1 poly A site also reduces polyadenylated WAF1 mRNA. Wild-type and Ago2 −/− MEF cells were treated with siRNAs and corresponding full MOE ASO as described in ‘Materials and Methods’ section. 3′ RACE was performed using total RNA purified from the cells the following day. Total RNA was also reverse transcribed using oligo dT primer. Oligo dT primed cDNA was then quantitated by qPCR. ( A ) Ethidium bromide stained gel of Il4R-α RACE products in WT and Ago2 −/− cells. ( B ) qPCR of Il4R-α siRNA/MOE treated cells using primer probe sets upstream or downstream of PA site #1. ( C ) Ethidium bromide stained gel of WAF1 RACE products in WT and Ago2 −/− cells. ( D ) qPCR of WAF1 siRNA/MOE treated cells. Primer/probe sets used are indicated at the bottom of the graph.
    Figure Legend Snippet: An siRNA targeting the WAF1 poly A site also reduces polyadenylated WAF1 mRNA. Wild-type and Ago2 −/− MEF cells were treated with siRNAs and corresponding full MOE ASO as described in ‘Materials and Methods’ section. 3′ RACE was performed using total RNA purified from the cells the following day. Total RNA was also reverse transcribed using oligo dT primer. Oligo dT primed cDNA was then quantitated by qPCR. ( A ) Ethidium bromide stained gel of Il4R-α RACE products in WT and Ago2 −/− cells. ( B ) qPCR of Il4R-α siRNA/MOE treated cells using primer probe sets upstream or downstream of PA site #1. ( C ) Ethidium bromide stained gel of WAF1 RACE products in WT and Ago2 −/− cells. ( D ) qPCR of WAF1 siRNA/MOE treated cells. Primer/probe sets used are indicated at the bottom of the graph.

    Techniques Used: Allele-specific Oligonucleotide, Purification, Real-time Polymerase Chain Reaction, Staining

    Treatment Il4R-α siRNA 383281 results in a small increase in polyadenylation at a downstream polyA site. ( A ) PolyA site and RACE primer localization on the Il4R-α transcript. Nucleotide position of 3′ RACE forward primer (FP), and polyA sites is indicated. Expected length of RACE products is given below the transcript. ( B ) Ago2 −/− MEF cells were treated with Il4R-α siRNA 383281 or the corresponding full MOE ASO at a concentration of 100 nM. 3′ RACE was performed using total RNA purified from the cells the following day. Left Panel, ethidium bromide stained gel. Center panel, Southern blot of RACE gel probed with Prb1. Right panel, Southern blot of RACE gel probed with Prb2.
    Figure Legend Snippet: Treatment Il4R-α siRNA 383281 results in a small increase in polyadenylation at a downstream polyA site. ( A ) PolyA site and RACE primer localization on the Il4R-α transcript. Nucleotide position of 3′ RACE forward primer (FP), and polyA sites is indicated. Expected length of RACE products is given below the transcript. ( B ) Ago2 −/− MEF cells were treated with Il4R-α siRNA 383281 or the corresponding full MOE ASO at a concentration of 100 nM. 3′ RACE was performed using total RNA purified from the cells the following day. Left Panel, ethidium bromide stained gel. Center panel, Southern blot of RACE gel probed with Prb1. Right panel, Southern blot of RACE gel probed with Prb2.

    Techniques Used: Allele-specific Oligonucleotide, Concentration Assay, Purification, Staining, Southern Blot

    13) Product Images from "miR-221/222 Compensates for Skp2-Mediated p27 Degradation and Is a Primary Target of Cell Cycle Regulation by Prostacyclin and cAMP"

    Article Title: miR-221/222 Compensates for Skp2-Mediated p27 Degradation and Is a Primary Target of Cell Cycle Regulation by Prostacyclin and cAMP

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056140

    Effect of cAMP elevating agents on miR-221/222 and p27 expression. ( A–B ) Quiescent early passage VSMCs from wild-type were stimulated with 10% FBS in the absence (control) or presence of 50 µM U0126 (U0), 1 mM 8Br-cAMP, or 100 µM Forskolin (Fsk). In A, total RNA was extracted at 24 h, and miR-221/222 expression levels were determined by RT-qPCR. Results show mean ± SE, n = 3−4. In B, total protein was extracted at 24 h and analyzed by western blotting for p27, dually phosphorylated ERK (pERK), total ERK and GAPDH (loading control). ( C–E ) The experiment in A was repeated with wild-type and p27-null VSMCs in 6-well dishes containing coverslips and EdU. In C, coverslips were fixed at 48 h and stained for EdU; results are plotted relative to the FBS-treated control; n = 3. In D-E, total RNA was extracted at 24 h, and miR-221 or miR-222 expression levels were determined by RT-qPCR. Results show mean ± SD, n = 2. ( F ) miR-221/222 regulation by mitogens, ERK, PGI 2 , and cAMP.
    Figure Legend Snippet: Effect of cAMP elevating agents on miR-221/222 and p27 expression. ( A–B ) Quiescent early passage VSMCs from wild-type were stimulated with 10% FBS in the absence (control) or presence of 50 µM U0126 (U0), 1 mM 8Br-cAMP, or 100 µM Forskolin (Fsk). In A, total RNA was extracted at 24 h, and miR-221/222 expression levels were determined by RT-qPCR. Results show mean ± SE, n = 3−4. In B, total protein was extracted at 24 h and analyzed by western blotting for p27, dually phosphorylated ERK (pERK), total ERK and GAPDH (loading control). ( C–E ) The experiment in A was repeated with wild-type and p27-null VSMCs in 6-well dishes containing coverslips and EdU. In C, coverslips were fixed at 48 h and stained for EdU; results are plotted relative to the FBS-treated control; n = 3. In D-E, total RNA was extracted at 24 h, and miR-221 or miR-222 expression levels were determined by RT-qPCR. Results show mean ± SD, n = 2. ( F ) miR-221/222 regulation by mitogens, ERK, PGI 2 , and cAMP.

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Staining

    Transcript profiling reveals that miR-221/222 is induced after vascular injury in vivo. ( A ) Male SMA-GFP mice (5–6 mo) were subjected to fine-wire femoral artery injury. Injured arteries were isolated, carefully opened, and immediately imaged for GFP fluorescence. A representative image of an uninjured and injured femoral artery is shown for a single mouse. The bracket shows a region of vascular injury. ( B ) Uninjured femoral arteries and GFP-negative regions of injured femoral arteries were collected for transcript profiling. Genes differentially expressed in these tissues were plotted against the Gene Ontology (GO) category, Cellular Process. ( C ) Interaction map showing upstream regulators of p27 that are differentially expressed in injured vs. uninjured femoral arteries as determined by Ingenuity Pathway Analysis (IPA) of the microarray data. Green and red represent induction and repression, respectively. Upstream p27 regulators in the IPA database that were not differentially expressed during in vivo response to injury are uncolored. The boxed region of interest at the bottom of the interaction map is expanded below to highlight the induction of miR-221 (green oval). ( D ) Quiescent early passage mouse VSMCs were stimulated with 10% FBS for 24 h. Total RNA was collected, and the levels of miR-221/222 and Skp2 mRNA were determined by RT-qPCR. Results show mean ± SD, n = 2.
    Figure Legend Snippet: Transcript profiling reveals that miR-221/222 is induced after vascular injury in vivo. ( A ) Male SMA-GFP mice (5–6 mo) were subjected to fine-wire femoral artery injury. Injured arteries were isolated, carefully opened, and immediately imaged for GFP fluorescence. A representative image of an uninjured and injured femoral artery is shown for a single mouse. The bracket shows a region of vascular injury. ( B ) Uninjured femoral arteries and GFP-negative regions of injured femoral arteries were collected for transcript profiling. Genes differentially expressed in these tissues were plotted against the Gene Ontology (GO) category, Cellular Process. ( C ) Interaction map showing upstream regulators of p27 that are differentially expressed in injured vs. uninjured femoral arteries as determined by Ingenuity Pathway Analysis (IPA) of the microarray data. Green and red represent induction and repression, respectively. Upstream p27 regulators in the IPA database that were not differentially expressed during in vivo response to injury are uncolored. The boxed region of interest at the bottom of the interaction map is expanded below to highlight the induction of miR-221 (green oval). ( D ) Quiescent early passage mouse VSMCs were stimulated with 10% FBS for 24 h. Total RNA was collected, and the levels of miR-221/222 and Skp2 mRNA were determined by RT-qPCR. Results show mean ± SD, n = 2.

    Techniques Used: In Vivo, Mouse Assay, Isolation, Fluorescence, Indirect Immunoperoxidase Assay, Microarray, Quantitative RT-PCR

    miR-221/222 is a primary target of PGI 2 . Quiescent early passage VSMCs from wild-type or p27-null mice were stimulated with 10% FBS in the absence (control; C) or presence of 200 nM cicaprost (cica). ( A ) Total RNA was extracted at 24 h, and Skp2 mRNA levels were determined by RT-qPCR. Results show mean ± SD, n = 2. ( B ) Total protein was extracted and analyzed by western blotting for p27, Skp2 and actin (loading control). (C) Total RNA was extracted at 24 h, and miR-221/222 levels were determined by RT-qPCR. Results show mean ± SD, n = 2.
    Figure Legend Snippet: miR-221/222 is a primary target of PGI 2 . Quiescent early passage VSMCs from wild-type or p27-null mice were stimulated with 10% FBS in the absence (control; C) or presence of 200 nM cicaprost (cica). ( A ) Total RNA was extracted at 24 h, and Skp2 mRNA levels were determined by RT-qPCR. Results show mean ± SD, n = 2. ( B ) Total protein was extracted and analyzed by western blotting for p27, Skp2 and actin (loading control). (C) Total RNA was extracted at 24 h, and miR-221/222 levels were determined by RT-qPCR. Results show mean ± SD, n = 2.

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

    14) Product Images from "2-Heptyl-Formononetin Increases Cholesterol and Induces Hepatic Steatosis in Mice"

    Article Title: 2-Heptyl-Formononetin Increases Cholesterol and Induces Hepatic Steatosis in Mice

    Journal: BioMed Research International

    doi: 10.1155/2013/926942

    Adipocyte gene expression in C57BL/6 mice fed chow, cholesterol, or cholesterol supplemented formononetin or 2-heptyl-formonetin (C7F) for five weeks (Experiment 2). Gene expression measured by RT-PCR of Srebf1 (sterol regulatory element-binding protein-1c), Pparg (peroxisome proliferator-activated receptor γ ), Cebpa (CCAAT/enhancer-binding protein α ), Acaca (acyl-CoA carboxylase 1), Fasn (fatty acid synthase), Scd1 (stearoyl-CoA desaturase 1), Atgl (adipose triglyceride lipase), Ucp1 (uncoupling protein 1), and Emr1 (F4/80) in (A) eWAT, (B) iWAT, and (C) iBAT measured by RT-PCR. Data is normalised to 18S ribosomal RNA and presented relative to the expression in chow ( n = 6). Graphs show mean ± SEM. Different letters (a, b) denote significant difference ( P ≤ 0.5) between the groups.
    Figure Legend Snippet: Adipocyte gene expression in C57BL/6 mice fed chow, cholesterol, or cholesterol supplemented formononetin or 2-heptyl-formonetin (C7F) for five weeks (Experiment 2). Gene expression measured by RT-PCR of Srebf1 (sterol regulatory element-binding protein-1c), Pparg (peroxisome proliferator-activated receptor γ ), Cebpa (CCAAT/enhancer-binding protein α ), Acaca (acyl-CoA carboxylase 1), Fasn (fatty acid synthase), Scd1 (stearoyl-CoA desaturase 1), Atgl (adipose triglyceride lipase), Ucp1 (uncoupling protein 1), and Emr1 (F4/80) in (A) eWAT, (B) iWAT, and (C) iBAT measured by RT-PCR. Data is normalised to 18S ribosomal RNA and presented relative to the expression in chow ( n = 6). Graphs show mean ± SEM. Different letters (a, b) denote significant difference ( P ≤ 0.5) between the groups.

    Techniques Used: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Binding Assay

    15) Product Images from "Lysophosphatidic Acid Promotes Cell Migration through STIM1- and Orai1-Mediated Ca2+i Mobilization and NFAT2 Activation"

    Article Title: Lysophosphatidic Acid Promotes Cell Migration through STIM1- and Orai1-Mediated Ca2+i Mobilization and NFAT2 Activation

    Journal: The Journal of Investigative Dermatology

    doi: 10.1038/jid.2012.370

    Lysophosphatidic acid (LPA)-induced Ca 2+ entry requires sufficient levels of STIM1 and functional Orai1. ( a – c ) Relative efficiency of small interfering RNA (siRNA)-mediated STIM1 silencing compared to scrambled (scr) control by quantitative PCR and western blotting. ( e ) Fura-PE3-based Ca 2+ imaging showed impaired LPA-induced Ca 2+ entry in STIM1-knockdown cells. ( e , f ) Efficient transfection of Orai1-encoding plasmids demonstrated by western blotting for myc-tag. ( g ) LPA-induced Ca 2+ entry was found to be blocked in Orai1 R91W -expressing cells. ( h ) Keratinocytes were cotransfected with Orai1, nuclear factor of activated T cell (NFAT)-directed, and Renilla luciferase plasmids. Cells were then treated with LPA and 1.2 mℳ Ca 2+ and assessed for NFAT transcriptional activity. Overexpression of Orai1 R91W significantly impaired NFAT activation. Data represent mean±SEM; n ⩾3 unless otherwise stated, * P
    Figure Legend Snippet: Lysophosphatidic acid (LPA)-induced Ca 2+ entry requires sufficient levels of STIM1 and functional Orai1. ( a – c ) Relative efficiency of small interfering RNA (siRNA)-mediated STIM1 silencing compared to scrambled (scr) control by quantitative PCR and western blotting. ( e ) Fura-PE3-based Ca 2+ imaging showed impaired LPA-induced Ca 2+ entry in STIM1-knockdown cells. ( e , f ) Efficient transfection of Orai1-encoding plasmids demonstrated by western blotting for myc-tag. ( g ) LPA-induced Ca 2+ entry was found to be blocked in Orai1 R91W -expressing cells. ( h ) Keratinocytes were cotransfected with Orai1, nuclear factor of activated T cell (NFAT)-directed, and Renilla luciferase plasmids. Cells were then treated with LPA and 1.2 mℳ Ca 2+ and assessed for NFAT transcriptional activity. Overexpression of Orai1 R91W significantly impaired NFAT activation. Data represent mean±SEM; n ⩾3 unless otherwise stated, * P

    Techniques Used: Functional Assay, Small Interfering RNA, Real-time Polymerase Chain Reaction, Western Blot, Imaging, Transfection, Expressing, Luciferase, Activity Assay, Over Expression, Activation Assay

    Lysophosphatidic acid (LPA)-induced keratinocyte migration requires STIM1, calcineurin activity, and sufficient levels of nuclear factor of activated T cell 2 (NFAT2). ( a – d , h , i ) Keratinocyte cultures were subjected to three-dimensional (3D) chemotactic migration assays, which were performed in medium containing 60 μℳ or 1.2 mℳ Ca 2+ . The average numbers of migrated cells are represented as bar graphs±SEM. Keratinocytes were treated with scrambled (scr) or STIM1-targeted small interfering RNA (siRNA) ( a , b ) or NFAT2-targeted siRNA ( h , i ) for 24 hours prior to 3D chemotactic migration assays. In both Ca 2+ conditions, 10 μℳ LPA significantly increased migration rates. RNA interference (RNAi)-mediated knockdown of STIM1 and NFAT2 resulted in a significant impairment of LPA-induced keratinocyte motility ( a , b , h , i , *** P
    Figure Legend Snippet: Lysophosphatidic acid (LPA)-induced keratinocyte migration requires STIM1, calcineurin activity, and sufficient levels of nuclear factor of activated T cell 2 (NFAT2). ( a – d , h , i ) Keratinocyte cultures were subjected to three-dimensional (3D) chemotactic migration assays, which were performed in medium containing 60 μℳ or 1.2 mℳ Ca 2+ . The average numbers of migrated cells are represented as bar graphs±SEM. Keratinocytes were treated with scrambled (scr) or STIM1-targeted small interfering RNA (siRNA) ( a , b ) or NFAT2-targeted siRNA ( h , i ) for 24 hours prior to 3D chemotactic migration assays. In both Ca 2+ conditions, 10 μℳ LPA significantly increased migration rates. RNA interference (RNAi)-mediated knockdown of STIM1 and NFAT2 resulted in a significant impairment of LPA-induced keratinocyte motility ( a , b , h , i , *** P

    Techniques Used: Migration, Activity Assay, Small Interfering RNA

    16) Product Images from "HP1 modulates the transcription of cell-cycle regulators in Drosophila melanogaster"

    Article Title: HP1 modulates the transcription of cell-cycle regulators in Drosophila melanogaster

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki584

    Depletion of HP1 alters cell-cycle progression. ( A ) Expression of HP1 after treatment with dsRNA in Kc cells. Left panel: changes in HP1 expression after RNAi monitored by RT–PCR analysis of RNA extracted from control (Con) and RNAi-treated cells (at 6 and 8 days). Histone H1 was used as a positive control. Right panel: western blotting with anti-HP1 antibodies (C1A9) of extracts from control (Con) and RNAi-treated cells (at 2, 6 and 8 days). Equal loading of cell extracts (15 μg of protein extract in each lane) was monitored with Coomassie blue (Coo.) staining. ( B ) Ablation of HP1 in Kc cells results in loss of cells in S and G2/M phase. Control Kc cells (left panel) and HP1-depleted Kc cells (right panel) were labeled with BrdU and 7-AAD. The fractions of cells in apoptosis (Apo), G1 phase (G1), S phase (S) and G2/M phase (G2/M) are all indicated. R5 represents over-replicated cells. Approximately 25 000 gated cell events were measured in each experiment. The comparison of cell numbers ( n = 2) at different stages of the cell cycle in controls and cells after HP1 depletion is shown on the bottom panel of the figure. %, percentage of cells.
    Figure Legend Snippet: Depletion of HP1 alters cell-cycle progression. ( A ) Expression of HP1 after treatment with dsRNA in Kc cells. Left panel: changes in HP1 expression after RNAi monitored by RT–PCR analysis of RNA extracted from control (Con) and RNAi-treated cells (at 6 and 8 days). Histone H1 was used as a positive control. Right panel: western blotting with anti-HP1 antibodies (C1A9) of extracts from control (Con) and RNAi-treated cells (at 2, 6 and 8 days). Equal loading of cell extracts (15 μg of protein extract in each lane) was monitored with Coomassie blue (Coo.) staining. ( B ) Ablation of HP1 in Kc cells results in loss of cells in S and G2/M phase. Control Kc cells (left panel) and HP1-depleted Kc cells (right panel) were labeled with BrdU and 7-AAD. The fractions of cells in apoptosis (Apo), G1 phase (G1), S phase (S) and G2/M phase (G2/M) are all indicated. R5 represents over-replicated cells. Approximately 25 000 gated cell events were measured in each experiment. The comparison of cell numbers ( n = 2) at different stages of the cell cycle in controls and cells after HP1 depletion is shown on the bottom panel of the figure. %, percentage of cells.

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

    17) Product Images from "hsa-miR29b, a critical downstream target of non-canonical Wnt signaling, plays an anti-proliferative role in non-small cell lung cancer cells via targeting MDM2 expression"

    Article Title: hsa-miR29b, a critical downstream target of non-canonical Wnt signaling, plays an anti-proliferative role in non-small cell lung cancer cells via targeting MDM2 expression

    Journal: Biology Open

    doi: 10.1242/bio.20134507

    hsa-miR29b regulates MDM2 expression in NSCLC cells. ( A ) In silico identification of complimentary sites for hsa-miR29b on the 3′-UTR of MDM2, PTEN and CDK2. A549 or H157 cells were transfected either with empty vector or phsa-miR29b plasmid. After 24 h, total RNA was extracted, reverse transcribed, and real-time PCR analysis was carried out using hsa-miR29b specific primers ( B ) or MDM2 specific primers (forward: 5′-TTGACCTGTCTATAAGAGAATTATATATTTC-3′, reverse: 5′-GTCTTACGGGTAAATGGTGGCT-3′) ( C ). RNU6B and GAPDH were used as internal controls for normalization. Data represent mean ± SEM of three separate experiments performed in duplicates. ** P
    Figure Legend Snippet: hsa-miR29b regulates MDM2 expression in NSCLC cells. ( A ) In silico identification of complimentary sites for hsa-miR29b on the 3′-UTR of MDM2, PTEN and CDK2. A549 or H157 cells were transfected either with empty vector or phsa-miR29b plasmid. After 24 h, total RNA was extracted, reverse transcribed, and real-time PCR analysis was carried out using hsa-miR29b specific primers ( B ) or MDM2 specific primers (forward: 5′-TTGACCTGTCTATAAGAGAATTATATATTTC-3′, reverse: 5′-GTCTTACGGGTAAATGGTGGCT-3′) ( C ). RNU6B and GAPDH were used as internal controls for normalization. Data represent mean ± SEM of three separate experiments performed in duplicates. ** P

    Techniques Used: Expressing, In Silico, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction

    Wnt7a/Fzd9 signaling regulates hsa-miR29b. ( A ) Multiple alignments of hsa-miR29a, hsa-miR29b and hsa-miR29c. Real-time PCR analyses of the expression of hsa-miR29a, hsa-miR29b and hsa-miR29c in NSCLC cell lines. H661 ( B ) or H157 ( C ) cells were transfected either with empty vector, pLNCX-Wnt3-HA or pLNCX-Wnt7a-HA. After 24 h, total RNA was extracted and reverse transcribed. Real-time PCR analysis was carried out using the cDNAs and hsa-miR29a, hsa-miR29b or hsa-miR29c specific primers. RNU6B was used as the internal control for normalization. Data represent mean ± SEM of three separate experiments performed in duplicates. ** P
    Figure Legend Snippet: Wnt7a/Fzd9 signaling regulates hsa-miR29b. ( A ) Multiple alignments of hsa-miR29a, hsa-miR29b and hsa-miR29c. Real-time PCR analyses of the expression of hsa-miR29a, hsa-miR29b and hsa-miR29c in NSCLC cell lines. H661 ( B ) or H157 ( C ) cells were transfected either with empty vector, pLNCX-Wnt3-HA or pLNCX-Wnt7a-HA. After 24 h, total RNA was extracted and reverse transcribed. Real-time PCR analysis was carried out using the cDNAs and hsa-miR29a, hsa-miR29b or hsa-miR29c specific primers. RNU6B was used as the internal control for normalization. Data represent mean ± SEM of three separate experiments performed in duplicates. ** P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Transfection, Plasmid Preparation

    hsa-miR29b regulates NSCLC cell proliferation. ( A ) Real-time PCR analyses of the expression of hsa-miR29b in NSCLC cell lines and non-transformed cell lines. Total RNA was extracted from a non-transformed cell line (Beas2B) or NSCLC cell lines (A549, H157, H661 and H2122) and hsa-miR29b expression was quantified as described in Materials and Methods . RNU6B was used as the internal control for normalization. Data represent mean ± SEM of three separate experiments performed in duplicates. ## P
    Figure Legend Snippet: hsa-miR29b regulates NSCLC cell proliferation. ( A ) Real-time PCR analyses of the expression of hsa-miR29b in NSCLC cell lines and non-transformed cell lines. Total RNA was extracted from a non-transformed cell line (Beas2B) or NSCLC cell lines (A549, H157, H661 and H2122) and hsa-miR29b expression was quantified as described in Materials and Methods . RNU6B was used as the internal control for normalization. Data represent mean ± SEM of three separate experiments performed in duplicates. ## P

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

    ERK5 and PPARγ stimulate hsa-miR29b expression in NSCLC cells. A549 ( A ) or H157 ( B ) cells were transfected either with empty vector or ERK5 expression plasmids. After 24 h, total RNA was extracted, reverse transcribed, and real-time PCR analysis was carried out using hsa-miR29a, hsa-miR29b or hsa-miR29c specific primers as described in Materials and Methods . RNU6B was used as the internal control for normalization. Data represent mean ± SEM of three separate experiments performed in duplicates. ** P
    Figure Legend Snippet: ERK5 and PPARγ stimulate hsa-miR29b expression in NSCLC cells. A549 ( A ) or H157 ( B ) cells were transfected either with empty vector or ERK5 expression plasmids. After 24 h, total RNA was extracted, reverse transcribed, and real-time PCR analysis was carried out using hsa-miR29a, hsa-miR29b or hsa-miR29c specific primers as described in Materials and Methods . RNU6B was used as the internal control for normalization. Data represent mean ± SEM of three separate experiments performed in duplicates. ** P

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction

    18) Product Images from "Annexin A2 Promotes the Migration and Invasion of Human Hepatocellular Carcinoma Cells In Vitro by Regulating the Shedding of CD147-Harboring Microvesicles from Tumor Cells"

    Article Title: Annexin A2 Promotes the Migration and Invasion of Human Hepatocellular Carcinoma Cells In Vitro by Regulating the Shedding of CD147-Harboring Microvesicles from Tumor Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067268

    Expression of ANXA2 in HCC cells was effectively down-regulated by specific si-ANXA2. Forty-eight hours after the transfection of SMMC-7721 or FHCC-98 cells with si-ANXA2 or snc-RNA, ANXA2 expression levels were examined using RT-PCR (A) and Western blot (B). Top, representative image; bottom, quantitative gray scale analysis of at least three independent experiments. Columns, mean; bars, SD; p
    Figure Legend Snippet: Expression of ANXA2 in HCC cells was effectively down-regulated by specific si-ANXA2. Forty-eight hours after the transfection of SMMC-7721 or FHCC-98 cells with si-ANXA2 or snc-RNA, ANXA2 expression levels were examined using RT-PCR (A) and Western blot (B). Top, representative image; bottom, quantitative gray scale analysis of at least three independent experiments. Columns, mean; bars, SD; p

    Techniques Used: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Effects of ANXA2 on the migration and invasion of HCC cells co-cultured with HPF-1 cells. Twenty-four hours after transfection with si-ANXA2 or snc-RNA, HCC cells were co-cultured with an equal number of HPF-1 cells in the upper chamber, which were not coated with Matrigel in the in vitro migration assay (A) but were coated in the in vitro invasion assay (B). Eight (migration assay) or twenty-four (invasion assay) hours later, the cells migrating/invading through the filter were stained and counted. Left, representative images showing the density of cells on the filter. Right, quantitative analyses of the cells migrating/invading through the filter in three independent experiments. Columns, mean; bars, SD. p
    Figure Legend Snippet: Effects of ANXA2 on the migration and invasion of HCC cells co-cultured with HPF-1 cells. Twenty-four hours after transfection with si-ANXA2 or snc-RNA, HCC cells were co-cultured with an equal number of HPF-1 cells in the upper chamber, which were not coated with Matrigel in the in vitro migration assay (A) but were coated in the in vitro invasion assay (B). Eight (migration assay) or twenty-four (invasion assay) hours later, the cells migrating/invading through the filter were stained and counted. Left, representative images showing the density of cells on the filter. Right, quantitative analyses of the cells migrating/invading through the filter in three independent experiments. Columns, mean; bars, SD. p

    Techniques Used: Migration, Cell Culture, Transfection, In Vitro, Invasion Assay, Staining

    19) Product Images from "A mammalian pseudogene lncRNA at the interface of inflammation and anti-inflammatory therapeutics"

    Article Title: A mammalian pseudogene lncRNA at the interface of inflammation and anti-inflammatory therapeutics

    Journal: eLife

    doi: 10.7554/eLife.00762

    ( A ) Western analysis of RelA protein levels. ( B ) PolyA+ Lethe is found on the chromatin. Immunoblot of wildtype and RelA−/− MEFs. Cellular fractionation was performed, total RNA was purified and polyA+ selection was performed. The fraction polyA+ RNA found in the chromatin, nucleus and cytoplasm is shown. MEFs were treated with 20 ng/ml TNFα for 6 hr. Quantitative Taqman real time RT-PCR of the indicated RNAs is shown (mean ± SD is shown). DOI: http://dx.doi.org/10.7554/eLife.00762.006
    Figure Legend Snippet: ( A ) Western analysis of RelA protein levels. ( B ) PolyA+ Lethe is found on the chromatin. Immunoblot of wildtype and RelA−/− MEFs. Cellular fractionation was performed, total RNA was purified and polyA+ selection was performed. The fraction polyA+ RNA found in the chromatin, nucleus and cytoplasm is shown. MEFs were treated with 20 ng/ml TNFα for 6 hr. Quantitative Taqman real time RT-PCR of the indicated RNAs is shown (mean ± SD is shown). DOI: http://dx.doi.org/10.7554/eLife.00762.006

    Techniques Used: Western Blot, Cell Fractionation, Purification, Selection, Quantitative RT-PCR

    20) Product Images from "Global microRNA profiling of well-differentiated small intestinal neuroendocrine tumors"

    Article Title: Global microRNA profiling of well-differentiated small intestinal neuroendocrine tumors

    Journal: Modern Pathology

    doi: 10.1038/modpathol.2012.216

    Quantitative real-time PCR (QRT-PCR) analysis validated the expression of nine selected microRNAs (miRNAs) from the first test group. Total RNA was isolated from microdissected tumor cells and microdissected normal enterochromaffin cells. Analysis was run using three normal enterochromaffin cell, three primary tumor, three mesentery metastasis and three liver metastasis samples. ( a ) Upregulated miRNA expression in tumor cells compared with normal enterochromaffin cells. ( b ) Downregulated miRNA expression in tumor cells compared with normal enterochromaffin cells. Results were plotted using the 2 −ΔΔCt method with RNU48 expression (set to 1) from each individual sample for normalization. Plotted results are mean±s.d. for triplicate wells. Significance was calculated by one-way analysis of variance (ANOVA) followed by Bonferroni test. * P
    Figure Legend Snippet: Quantitative real-time PCR (QRT-PCR) analysis validated the expression of nine selected microRNAs (miRNAs) from the first test group. Total RNA was isolated from microdissected tumor cells and microdissected normal enterochromaffin cells. Analysis was run using three normal enterochromaffin cell, three primary tumor, three mesentery metastasis and three liver metastasis samples. ( a ) Upregulated miRNA expression in tumor cells compared with normal enterochromaffin cells. ( b ) Downregulated miRNA expression in tumor cells compared with normal enterochromaffin cells. Results were plotted using the 2 −ΔΔCt method with RNU48 expression (set to 1) from each individual sample for normalization. Plotted results are mean±s.d. for triplicate wells. Significance was calculated by one-way analysis of variance (ANOVA) followed by Bonferroni test. * P

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Isolation

    Quantitative real-time PCR (QRT-PCR) analysis validated the expression of nine selected microRNAs (miRNAs) from the second test group. Total RNA was isolated from microdissected tumor cells and microdissected normal enterochromaffin cells. Analysis was run using three normal enterochromaffin cell, three primary tumor, three mesentery metastasis and three liver metastasis samples. ( a ) Upregulated miRNA expression in tumor cells compared with normal enterochromaffin cells. ( b ) Downregulated miRNA expression in tumor cells compared with normal enterochromaffin cells. Results were plotted using the 2 −ΔΔCt method with RNU48 expression (set to 1) from each individual sample for normalization. Plotted results are mean±s.d. for triplicate wells. Significance was calculated by one-way analysis of variance (ANOVA) followed by Bonferroni test. * P
    Figure Legend Snippet: Quantitative real-time PCR (QRT-PCR) analysis validated the expression of nine selected microRNAs (miRNAs) from the second test group. Total RNA was isolated from microdissected tumor cells and microdissected normal enterochromaffin cells. Analysis was run using three normal enterochromaffin cell, three primary tumor, three mesentery metastasis and three liver metastasis samples. ( a ) Upregulated miRNA expression in tumor cells compared with normal enterochromaffin cells. ( b ) Downregulated miRNA expression in tumor cells compared with normal enterochromaffin cells. Results were plotted using the 2 −ΔΔCt method with RNU48 expression (set to 1) from each individual sample for normalization. Plotted results are mean±s.d. for triplicate wells. Significance was calculated by one-way analysis of variance (ANOVA) followed by Bonferroni test. * P

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Isolation

    Quantitative real-time PCR (QRT-PCR) analysis validated the expression of the nine selected microRNA (miRNAs) from the first group of specimens. Total RNA was isolated from frozen specimens of three primary tumors (P), three mesentery metastases (M) and three liver metastases (L) to run QRT-PCR analysis. ( a ) Upregulated miRNA expression in metastatic disease compared with primary tumors. ( b ) Downregulated miRNA expression in metastatic disease compared with primary tumors. Results were plotted using the 2 −ΔΔCt method with RNU48 expression (set to 1) from each individual sample for normalization. Plotted results are mean±s.d. for triplicate wells. Significance was calculated by one-way analysis of variance (ANOVA) followed by Bonferroni test. * P
    Figure Legend Snippet: Quantitative real-time PCR (QRT-PCR) analysis validated the expression of the nine selected microRNA (miRNAs) from the first group of specimens. Total RNA was isolated from frozen specimens of three primary tumors (P), three mesentery metastases (M) and three liver metastases (L) to run QRT-PCR analysis. ( a ) Upregulated miRNA expression in metastatic disease compared with primary tumors. ( b ) Downregulated miRNA expression in metastatic disease compared with primary tumors. Results were plotted using the 2 −ΔΔCt method with RNU48 expression (set to 1) from each individual sample for normalization. Plotted results are mean±s.d. for triplicate wells. Significance was calculated by one-way analysis of variance (ANOVA) followed by Bonferroni test. * P

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Isolation

    21) Product Images from "Expression Analysis of Lrrk1, Lrrk2 and Lrrk2 Splice Variants in Mice"

    Article Title: Expression Analysis of Lrrk1, Lrrk2 and Lrrk2 Splice Variants in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063778

    Qualitative expression analysis of alternative Lrrk2 mRNA transcripts. RNA samples from different brain regions and organs were analysed by RT-PCR using a primer combination that amplifies Lrrk2 transcripts between exon 4 and exon 9 ( A ). Note that there is – beside the band of the endogenous Lrrk2 (ex.4–9) – an additional lower band visible in all samples analysed representing a mRNA where exon 5 is spliced out (ex.5 skipped) ( B ). The same primers were used to analyse RNA samples from primary neuronal cultures (i.e. neurons, microglia and astrocytes) ( C ). Interestingly, the band representing the splice variant without exon 5 (E5 skipped) was dominantly expressed in samples from astrocytes, while the band for the endogenous transcript (E4–E9) was almost completely missing in these cells. ( D ) RNA samples were analysed by RT-PCR using primers that amplify Lrrk2 transcripts between exon 39 (E39) and an alternative exon 42a (ex.42a). Note that the alternative exon 42a is present in all samples analysed (ex.39–42a). ( E ) The same primers were used to analyse RNA samples from primary neuronal cultures (i.e. neurons, microglia and astrocytes). Interestingly, the alternative exon 42a could not be amplified from primary microglia cDNA. ( F ) Relative expression level as measured by quantitative RT-PCR with primers and probes specific for either the endogenous Lrrk2 transcript (full-length Lrrk2 ), or for the alternative Lrrk2 mRNA transcripts with skipped exon 5 (ex.5 skipped). The relative expression is depicted as a ratio between endogenous Lrrk2 transcript and the alternative product on a logarithmic scale. ( G ) Accordingly for the alternative Lrrk2 mRNA transcript ending in the alternative exon 42a (alt.ex.42), the relative expression level have been determined by quantitative RT-PCR with specific primers and probes for the alternative product and normalized to the expression of the endogenous Lrrk2 transcript (full-length Lrrk2 ).
    Figure Legend Snippet: Qualitative expression analysis of alternative Lrrk2 mRNA transcripts. RNA samples from different brain regions and organs were analysed by RT-PCR using a primer combination that amplifies Lrrk2 transcripts between exon 4 and exon 9 ( A ). Note that there is – beside the band of the endogenous Lrrk2 (ex.4–9) – an additional lower band visible in all samples analysed representing a mRNA where exon 5 is spliced out (ex.5 skipped) ( B ). The same primers were used to analyse RNA samples from primary neuronal cultures (i.e. neurons, microglia and astrocytes) ( C ). Interestingly, the band representing the splice variant without exon 5 (E5 skipped) was dominantly expressed in samples from astrocytes, while the band for the endogenous transcript (E4–E9) was almost completely missing in these cells. ( D ) RNA samples were analysed by RT-PCR using primers that amplify Lrrk2 transcripts between exon 39 (E39) and an alternative exon 42a (ex.42a). Note that the alternative exon 42a is present in all samples analysed (ex.39–42a). ( E ) The same primers were used to analyse RNA samples from primary neuronal cultures (i.e. neurons, microglia and astrocytes). Interestingly, the alternative exon 42a could not be amplified from primary microglia cDNA. ( F ) Relative expression level as measured by quantitative RT-PCR with primers and probes specific for either the endogenous Lrrk2 transcript (full-length Lrrk2 ), or for the alternative Lrrk2 mRNA transcripts with skipped exon 5 (ex.5 skipped). The relative expression is depicted as a ratio between endogenous Lrrk2 transcript and the alternative product on a logarithmic scale. ( G ) Accordingly for the alternative Lrrk2 mRNA transcript ending in the alternative exon 42a (alt.ex.42), the relative expression level have been determined by quantitative RT-PCR with specific primers and probes for the alternative product and normalized to the expression of the endogenous Lrrk2 transcript (full-length Lrrk2 ).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Variant Assay, Amplification, Quantitative RT-PCR

    22) Product Images from "Additive Protection by Antioxidant and Apoptosis-Inhibiting Effects on Mosquito Cells with Dengue 2 Virus Infection"

    Article Title: Additive Protection by Antioxidant and Apoptosis-Inhibiting Effects on Mosquito Cells with Dengue 2 Virus Infection

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001613

    Imaging evidence of dengue 2 virus (DENV-2) replication within C6/36 cells. (A) Both double-stranded (ds)RNA (green) and the viral E (envelope) protein (red) frequently appeared in C6/36 cells after infection with DENV-2 at 12 and 24 hpi, respectively. This indicates that RNA replication and protein synthesis of the virus were active at these time points. That shown blue represented nuclei stained with DAPI. A merged image was shown at the right bottom of this figure. (B) A great number of virions (Vi) which formed as a crystalline array appeared in membrane-bound vacuoles (arrow) in C6/36 cells infected by the virus for 24 h. N, nucleus, Scare bar = 1 µm.
    Figure Legend Snippet: Imaging evidence of dengue 2 virus (DENV-2) replication within C6/36 cells. (A) Both double-stranded (ds)RNA (green) and the viral E (envelope) protein (red) frequently appeared in C6/36 cells after infection with DENV-2 at 12 and 24 hpi, respectively. This indicates that RNA replication and protein synthesis of the virus were active at these time points. That shown blue represented nuclei stained with DAPI. A merged image was shown at the right bottom of this figure. (B) A great number of virions (Vi) which formed as a crystalline array appeared in membrane-bound vacuoles (arrow) in C6/36 cells infected by the virus for 24 h. N, nucleus, Scare bar = 1 µm.

    Techniques Used: Imaging, Infection, Staining

    23) Product Images from "Selective Hyper-responsiveness of the Interferon System in Major Depressive Disorders and Depression Induced by Interferon Therapy"

    Article Title: Selective Hyper-responsiveness of the Interferon System in Major Depressive Disorders and Depression Induced by Interferon Therapy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038668

    Enhanced ISG expression and IFN-production in psychiatric patients with a severe depressive episode (SDE). Panel A. After 24 h of in vitro incubation without any further stimuli, total RNA was isolated from peripheral blood mononuclear cells of 11 healthy controls (“co-N”) and 22 patients hospitalized for a SDE (“co-D”). Panel B. Total RNA was isolated directly from unseparated peripheral blood of healthy controls (“co-N”, n = 11), SDE patients (“co-D, n = 22”) and HCV patients without (“HCV-N”, n = 11) or with (“HCV-D”, n = 11) IFN-induced depression. Expression of IFN stimulated genes (ISGs) and IFN-β was analyzed by quantitative RT-PCR. Data (copies per 100,000 copies of ACTB ) are shown as box plots (range, 25% and 75% percentile, mean).
    Figure Legend Snippet: Enhanced ISG expression and IFN-production in psychiatric patients with a severe depressive episode (SDE). Panel A. After 24 h of in vitro incubation without any further stimuli, total RNA was isolated from peripheral blood mononuclear cells of 11 healthy controls (“co-N”) and 22 patients hospitalized for a SDE (“co-D”). Panel B. Total RNA was isolated directly from unseparated peripheral blood of healthy controls (“co-N”, n = 11), SDE patients (“co-D, n = 22”) and HCV patients without (“HCV-N”, n = 11) or with (“HCV-D”, n = 11) IFN-induced depression. Expression of IFN stimulated genes (ISGs) and IFN-β was analyzed by quantitative RT-PCR. Data (copies per 100,000 copies of ACTB ) are shown as box plots (range, 25% and 75% percentile, mean).

    Techniques Used: Expressing, In Vitro, Incubation, Isolation, Quantitative RT-PCR

    Enhanced IFN-mediated induction of selective ISGs in HCV patients with IFN-induced depression ( in vivo ) and psychiatric patients with a severe depressive episode (SDE, in vitro ). Total RNA was isolated from peripheral blood of hepatitis C virus (HCV) infected patients with (n = 11, “HCV-D”) or without (n = 11, “HCV-N”) IFN-induced depression 12 hours before and 12 hours after the first injection of pegylated IFN-α2a. Expression of IFN stimulated genes (ISGs) was analyzed by quantitative RT-PCR (panel A: GCH1 , TOR1B ; panel B: DYNLT1 , DISC1 ; panel C: MX1 , ISG15 ). To validate the data in an independent cohort, PBMC were isolated from 11 healthy controls (“co-N”) and 22 patients hospitalized for a SDE (“co-D”) and stimulated with 100 U/mL pegylated IFN-α2a in vitro for 16 h followed by isolation of total RNA. Data are shown as box plots (range, 25% and 75% percentile, mean).
    Figure Legend Snippet: Enhanced IFN-mediated induction of selective ISGs in HCV patients with IFN-induced depression ( in vivo ) and psychiatric patients with a severe depressive episode (SDE, in vitro ). Total RNA was isolated from peripheral blood of hepatitis C virus (HCV) infected patients with (n = 11, “HCV-D”) or without (n = 11, “HCV-N”) IFN-induced depression 12 hours before and 12 hours after the first injection of pegylated IFN-α2a. Expression of IFN stimulated genes (ISGs) was analyzed by quantitative RT-PCR (panel A: GCH1 , TOR1B ; panel B: DYNLT1 , DISC1 ; panel C: MX1 , ISG15 ). To validate the data in an independent cohort, PBMC were isolated from 11 healthy controls (“co-N”) and 22 patients hospitalized for a SDE (“co-D”) and stimulated with 100 U/mL pegylated IFN-α2a in vitro for 16 h followed by isolation of total RNA. Data are shown as box plots (range, 25% and 75% percentile, mean).

    Techniques Used: In Vivo, In Vitro, Isolation, Infection, Injection, Expressing, Quantitative RT-PCR

    24) Product Images from "The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps"

    Article Title: The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20061301

    Secretion of PTX3 by activated neutrophils. (A and B) FACS analysis of intracellular PTX3 expression in neutrophils stimulated for 2 h with 10 μg/ml S. aureus or 10 μg/ml FITC-labeled E. coli (A) or in DCs stimulated or not for 8 h with 80 ng/ml LPS (B). Representative results from one to five experiments are shown. (C–E) Analysis of PTX3 release upon neutrophil stimulation. (C) 2 × 10 6 cells/ml of neutrophils were activated for 16 h with 1 or 10 μg/ml E. coli , S. aureus , or zymosan; 1 or 10 ng/ml PMA; 0.01 or 1 μM ionomycin; or 20 ng/ml TNFα. (D) Time-dependent release of PTX3 in neutrophils (nonstimulated or stimulated with 10 μg/ml S. aureus or 10 ng/ml PMA) is shown. Supernatants were collected at the indicated time points. (E) Induction of PTX3 release by TLR agonists in neutrophils pretreated or not with 50 ng/ml GM-CSF. Supernatants were collected at 16 h. PTX3 was quantified in the supernatants by ELISA. Results are expressed as ng/ml (mean ± SD; n = 6). MPO (F) and MMP-9 (G) were quantified in the supernatants of neutrophils stimulated for 16 h with the indicated stimuli; results are expressed in ng/ml, showing the mean of two representative experiments. (H) PTX3 mRNA expression analyzed by RT-PCR in neutrophils untreated or stimulated for 8 h with 100 ng/ml LPS, 10 μg/ml E. coli , 10 μg/ml S. aureus , 10 μg/ml zymosan, 20 ng/ml TNFα, 10 ng/ml PMA, or 1 μM iononmycin. RNA integrity and cDNA synthesis were verified by amplifying GAPDH cDNA. *, P
    Figure Legend Snippet: Secretion of PTX3 by activated neutrophils. (A and B) FACS analysis of intracellular PTX3 expression in neutrophils stimulated for 2 h with 10 μg/ml S. aureus or 10 μg/ml FITC-labeled E. coli (A) or in DCs stimulated or not for 8 h with 80 ng/ml LPS (B). Representative results from one to five experiments are shown. (C–E) Analysis of PTX3 release upon neutrophil stimulation. (C) 2 × 10 6 cells/ml of neutrophils were activated for 16 h with 1 or 10 μg/ml E. coli , S. aureus , or zymosan; 1 or 10 ng/ml PMA; 0.01 or 1 μM ionomycin; or 20 ng/ml TNFα. (D) Time-dependent release of PTX3 in neutrophils (nonstimulated or stimulated with 10 μg/ml S. aureus or 10 ng/ml PMA) is shown. Supernatants were collected at the indicated time points. (E) Induction of PTX3 release by TLR agonists in neutrophils pretreated or not with 50 ng/ml GM-CSF. Supernatants were collected at 16 h. PTX3 was quantified in the supernatants by ELISA. Results are expressed as ng/ml (mean ± SD; n = 6). MPO (F) and MMP-9 (G) were quantified in the supernatants of neutrophils stimulated for 16 h with the indicated stimuli; results are expressed in ng/ml, showing the mean of two representative experiments. (H) PTX3 mRNA expression analyzed by RT-PCR in neutrophils untreated or stimulated for 8 h with 100 ng/ml LPS, 10 μg/ml E. coli , 10 μg/ml S. aureus , 10 μg/ml zymosan, 20 ng/ml TNFα, 10 ng/ml PMA, or 1 μM iononmycin. RNA integrity and cDNA synthesis were verified by amplifying GAPDH cDNA. *, P

    Techniques Used: FACS, Expressing, Labeling, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

    PTX3 is constitutively expressed in human neutrophils. (A) FACS analysis of PTX3 expression in permeabilized human neutrophils isolated from peripheral blood. (B) Western blot analysis of PTX3 expression in neutrophils and LPS-stimulated DCs. (C) Analysis of PTX3 expression in freshly isolated neutrophils, eosinophils and basophils by confocal microscopy. Fluorescence (left) and differential interference contrasts (right, Nomarski technique) are shown. Bars, 10 μm. (D) Analysis of PTX3 content in freshly isolated neutrophils, eosinophils, and basophils, as well as release by LPS-stimulated DCs, determined by ELISA (mean ± SD). (E) Analysis of PTX3 mRNA expression in freshly isolated human neutrophils by RT-PCR. Results obtained in 2 out of 10 subjects tested are presented. LPS-stimulated DCs are used as a positive control. RNA integrity and cDNA synthesis were verified by amplifying GAPDH cDNA. (F) Analysis of PTX3 mRNA and protein in neutrophil precursors. Promyelocytes (PM), myelocytes/metamyelocytes (MY), and bone marrow–segmented neutrophils (bm-PMN) were analyzed for PTX3 and MPO mRNA expression (left). Expression of PTX3 was evaluated by Western blotting using the anti-PTX3 mAb 16B5 in the three populations of neutrophil precursors (right), and total protein loading was evaluated by analyzing actin expression.
    Figure Legend Snippet: PTX3 is constitutively expressed in human neutrophils. (A) FACS analysis of PTX3 expression in permeabilized human neutrophils isolated from peripheral blood. (B) Western blot analysis of PTX3 expression in neutrophils and LPS-stimulated DCs. (C) Analysis of PTX3 expression in freshly isolated neutrophils, eosinophils and basophils by confocal microscopy. Fluorescence (left) and differential interference contrasts (right, Nomarski technique) are shown. Bars, 10 μm. (D) Analysis of PTX3 content in freshly isolated neutrophils, eosinophils, and basophils, as well as release by LPS-stimulated DCs, determined by ELISA (mean ± SD). (E) Analysis of PTX3 mRNA expression in freshly isolated human neutrophils by RT-PCR. Results obtained in 2 out of 10 subjects tested are presented. LPS-stimulated DCs are used as a positive control. RNA integrity and cDNA synthesis were verified by amplifying GAPDH cDNA. (F) Analysis of PTX3 mRNA and protein in neutrophil precursors. Promyelocytes (PM), myelocytes/metamyelocytes (MY), and bone marrow–segmented neutrophils (bm-PMN) were analyzed for PTX3 and MPO mRNA expression (left). Expression of PTX3 was evaluated by Western blotting using the anti-PTX3 mAb 16B5 in the three populations of neutrophil precursors (right), and total protein loading was evaluated by analyzing actin expression.

    Techniques Used: FACS, Expressing, Isolation, Western Blot, Confocal Microscopy, Fluorescence, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Positive Control

    25) Product Images from "Bridging the Synaptic Gap: Neuroligins and Neurexin I in Apis mellifera"

    Article Title: Bridging the Synaptic Gap: Neuroligins and Neurexin I in Apis mellifera

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0003542

    Developmental Expression Profiles of the Neuroligins and Neurexin I in Honeybee Brain. Honeybee neuroligin and neurexin I expression was assessed by quantitative real time PCR amplification. The ribosomal gene RPL8 was used as the housekeeping gene. Methodology for data analysis and the presentation of results was taken from Collins et al [104] ; where by expression levels were normalised by subtraction against the threshold cycle of the RPL8 . Collins et al [104] found RPL8 to be the best correlate with RNA concentration across varying developmental life stages and varying tissues of the honeybee. Expression levels were examined from whole larvae (5 day old); and brain tissue from pupae (stage P8 as outlined by Ganeshina et al [101] ) 24 hour adult, 7 day adult and forager honeybees. Standards errors were negligible and less than +/−1.18 for all experimental results. The coloured lines illustrate the developmental expression profile of a single gene through development. Data points in columns illustrate the relative levels of neurexin I and neuroligin expression to one another at a particular stage of development. The developmental stage/gene with lowest expression relative to the control gene (neuroligin 1 at 7 days of age) was given an arbitrary expression level of 1. The data values are shown in Supplementary Data Table 3 .
    Figure Legend Snippet: Developmental Expression Profiles of the Neuroligins and Neurexin I in Honeybee Brain. Honeybee neuroligin and neurexin I expression was assessed by quantitative real time PCR amplification. The ribosomal gene RPL8 was used as the housekeeping gene. Methodology for data analysis and the presentation of results was taken from Collins et al [104] ; where by expression levels were normalised by subtraction against the threshold cycle of the RPL8 . Collins et al [104] found RPL8 to be the best correlate with RNA concentration across varying developmental life stages and varying tissues of the honeybee. Expression levels were examined from whole larvae (5 day old); and brain tissue from pupae (stage P8 as outlined by Ganeshina et al [101] ) 24 hour adult, 7 day adult and forager honeybees. Standards errors were negligible and less than +/−1.18 for all experimental results. The coloured lines illustrate the developmental expression profile of a single gene through development. Data points in columns illustrate the relative levels of neurexin I and neuroligin expression to one another at a particular stage of development. The developmental stage/gene with lowest expression relative to the control gene (neuroligin 1 at 7 days of age) was given an arbitrary expression level of 1. The data values are shown in Supplementary Data Table 3 .

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

    26) Product Images from "A bacteria-specific 2[4Fe-4S] ferredoxin is essential in Pseudomonas aeruginosa"

    Article Title: A bacteria-specific 2[4Fe-4S] ferredoxin is essential in Pseudomonas aeruginosa

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-10-271

    Expression of P. aeruginosa fdx1 . (A) For Northern blots, total RNA was hybridized to a [ 32 P]-dCTP-labelled fdx1 -probe after electrophoretic separation and the autoradiogram shown is representative of several experiments. (B) The fdx1 transcript was detected by RT-PCR as a 136 bp amplicon and compared to the reference 350 bp-rRNA. The ratio fdx1 /rRNA was arbitrarily set at 1 for cells at OD = 1, and compared with induced (i.e. calcium-depleted for T3SS induction) cells, and OD = 4.6 cells. Cumulative data from 3 experiments with standard error. (C) Time course evolution of the rRNA control (upper panel) and the fdx1 transcript (lower panel) after OD = 1-cells had infected J774 macrophages at multiplicity of infection of 10. The time of contact with macrophages is indicated in minutes and the size scale in bp is on the left of the panels.
    Figure Legend Snippet: Expression of P. aeruginosa fdx1 . (A) For Northern blots, total RNA was hybridized to a [ 32 P]-dCTP-labelled fdx1 -probe after electrophoretic separation and the autoradiogram shown is representative of several experiments. (B) The fdx1 transcript was detected by RT-PCR as a 136 bp amplicon and compared to the reference 350 bp-rRNA. The ratio fdx1 /rRNA was arbitrarily set at 1 for cells at OD = 1, and compared with induced (i.e. calcium-depleted for T3SS induction) cells, and OD = 4.6 cells. Cumulative data from 3 experiments with standard error. (C) Time course evolution of the rRNA control (upper panel) and the fdx1 transcript (lower panel) after OD = 1-cells had infected J774 macrophages at multiplicity of infection of 10. The time of contact with macrophages is indicated in minutes and the size scale in bp is on the left of the panels.

    Techniques Used: Expressing, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Infection

    27) Product Images from "Regulation of Splicing Factors by Alternative Splicing and NMD Is Conserved between Kingdoms Yet Evolutionarily Flexible"

    Article Title: Regulation of Splicing Factors by Alternative Splicing and NMD Is Conserved between Kingdoms Yet Evolutionarily Flexible

    Journal: Molecular Biology and Evolution

    doi: 10.1093/molbev/msv002

    ( A ) Alignment of SRSF4, SRSF5, and SRSF6 protein sequences indicating exon positions. Colors denote exons (labeled relative to SRSF5). Positions of human cassette exon inclusion are indicated with gray boxes at splice junctions. Sequences were trimmed after the second RNA recognition motif (RRM) domain before alignment. See alignment of all genes in supplementary figure S1 B , Supplementary Material online. ( B ) The exon/intron structure of SRSF5 is conserved in animals and some intron positions are conserved between animals and fungi. Gray bars show corresponding exons with conserved boundaries. An alternative exon in SRSF5 is conserved in animals. Intron retention is shared between animals and fungi. SRSF5 genes in vertebrates including mouse, rat, chicken, frog, and zebrafish were equivalent to human SRSF5 ( supplementary figs. S1 and S2 and table S1 , Supplementary Material online). ( C ) mRNAs of Neurospora crassa srp2 with retained intron 3 (the equivalent of human SRSF5 intron 5) are stabilized in an NMD-deficient strain.
    Figure Legend Snippet: ( A ) Alignment of SRSF4, SRSF5, and SRSF6 protein sequences indicating exon positions. Colors denote exons (labeled relative to SRSF5). Positions of human cassette exon inclusion are indicated with gray boxes at splice junctions. Sequences were trimmed after the second RNA recognition motif (RRM) domain before alignment. See alignment of all genes in supplementary figure S1 B , Supplementary Material online. ( B ) The exon/intron structure of SRSF5 is conserved in animals and some intron positions are conserved between animals and fungi. Gray bars show corresponding exons with conserved boundaries. An alternative exon in SRSF5 is conserved in animals. Intron retention is shared between animals and fungi. SRSF5 genes in vertebrates including mouse, rat, chicken, frog, and zebrafish were equivalent to human SRSF5 ( supplementary figs. S1 and S2 and table S1 , Supplementary Material online). ( C ) mRNAs of Neurospora crassa srp2 with retained intron 3 (the equivalent of human SRSF5 intron 5) are stabilized in an NMD-deficient strain.

    Techniques Used: Labeling

    28) Product Images from "Silencing of Amyloid Precursor Protein Expression Using a New Engineered Delta Ribozyme"

    Article Title: Silencing of Amyloid Precursor Protein Expression Using a New Engineered Delta Ribozyme

    Journal: International Journal of Alzheimer's Disease

    doi: 10.1155/2012/947147

    Selection of APP-SOFA-HDV ribozyme with the greatest potential cleavage. The left portion illustrates the strategy that was used to identify potential cleavage sites. APP mRNA was preincubated in the presence of a 7 nt long randomized DNA oligonucleotide, and RNA/DNA heteroduplexes were hydrolyzed by RNase H. Accessible regions were then visualized by primer extension using one of the four 5′-end-labeled primers (in box) complementary to sequences retrieved in the first ∼ 900 nucleotides of the APP mRNA. Once the most accessible sites were identified, the appropriate SOFA-HDV ribozymes were synthesized and the cleavage activity was tested in vitro using 5′-end-labeled APP mRNA. A typical autoradiogram of a resulting PAGE is indicated in the right panel. The number of each Rz indicates the cleavage position within the APP mRNA. The Rz-HBV, previously used for HBV RNA cleavage [ 15 ], served as an irrelevant Rz. “-” indicates a reaction without Rz.
    Figure Legend Snippet: Selection of APP-SOFA-HDV ribozyme with the greatest potential cleavage. The left portion illustrates the strategy that was used to identify potential cleavage sites. APP mRNA was preincubated in the presence of a 7 nt long randomized DNA oligonucleotide, and RNA/DNA heteroduplexes were hydrolyzed by RNase H. Accessible regions were then visualized by primer extension using one of the four 5′-end-labeled primers (in box) complementary to sequences retrieved in the first ∼ 900 nucleotides of the APP mRNA. Once the most accessible sites were identified, the appropriate SOFA-HDV ribozymes were synthesized and the cleavage activity was tested in vitro using 5′-end-labeled APP mRNA. A typical autoradiogram of a resulting PAGE is indicated in the right panel. The number of each Rz indicates the cleavage position within the APP mRNA. The Rz-HBV, previously used for HBV RNA cleavage [ 15 ], served as an irrelevant Rz. “-” indicates a reaction without Rz.

    Techniques Used: Selection, Labeling, Synthesized, Activity Assay, In Vitro, Polyacrylamide Gel Electrophoresis

    Expression of APP-SOFA-HDV ribozymes in the HEK cell line. Primer extension analysis of total cellular RNA from cells transfected with four selected APP-SOFA-HDV ribozymes from the ribozyme collection (APP-SOFA-HDV-Rz276, APP-SOFA-HDV-Rz753, APP-SOFA-HDV-Rz756, and APP-SOFA-HDV-Rz885). Transcripts corresponding to HDV-ribozymes were detected with HDV-Rz primers (5′-GGGTCCCTTAGCCATGCGCGAACG-3′). U6 primer (5′-GGCCATGCTAATCTTCTCTG-3′) was also used as a positive control, which yielded signals corresponding to endogenous U6 snRNA. Note that all of the 4 selected SOFA-HDV ribozymes were expressed (lanes 1 to 4). A pRNAT empty vector, was used as a negative control.
    Figure Legend Snippet: Expression of APP-SOFA-HDV ribozymes in the HEK cell line. Primer extension analysis of total cellular RNA from cells transfected with four selected APP-SOFA-HDV ribozymes from the ribozyme collection (APP-SOFA-HDV-Rz276, APP-SOFA-HDV-Rz753, APP-SOFA-HDV-Rz756, and APP-SOFA-HDV-Rz885). Transcripts corresponding to HDV-ribozymes were detected with HDV-Rz primers (5′-GGGTCCCTTAGCCATGCGCGAACG-3′). U6 primer (5′-GGCCATGCTAATCTTCTCTG-3′) was also used as a positive control, which yielded signals corresponding to endogenous U6 snRNA. Note that all of the 4 selected SOFA-HDV ribozymes were expressed (lanes 1 to 4). A pRNAT empty vector, was used as a negative control.

    Techniques Used: Expressing, Transfection, Positive Control, Plasmid Preparation, Negative Control

    29) Product Images from "HCMV Protein LUNA Is Required for Viral Reactivation from Latently Infected Primary CD14+ Cells"

    Article Title: HCMV Protein LUNA Is Required for Viral Reactivation from Latently Infected Primary CD14+ Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052827

    LUNA protein expression is not required for lytic gene expression, but does augment the expression of the UL138 transcript. HF cells infected at an MOI = 1 with either FIX-WT, FIX-ΔLUNA and FIX-Rev were harvested at the indicated time points for either RNA or protein analysis. A) Expression of viral RNAs. Total RNA was collected over a 20 d time course together with mock RNA. From all the collected RNA samples cDNA was synthesized and amplified using UL82, UL123, UL81-82ast, UL138 and β-actin specific primers. Negative images were used to visualize weaker bands. B) qRT-PCR analysis was used to assay gene expression of UL138 at the indicated times post infection. Viral mRNA was normalized to actin. Asterisks indicate significant changes (p value
    Figure Legend Snippet: LUNA protein expression is not required for lytic gene expression, but does augment the expression of the UL138 transcript. HF cells infected at an MOI = 1 with either FIX-WT, FIX-ΔLUNA and FIX-Rev were harvested at the indicated time points for either RNA or protein analysis. A) Expression of viral RNAs. Total RNA was collected over a 20 d time course together with mock RNA. From all the collected RNA samples cDNA was synthesized and amplified using UL82, UL123, UL81-82ast, UL138 and β-actin specific primers. Negative images were used to visualize weaker bands. B) qRT-PCR analysis was used to assay gene expression of UL138 at the indicated times post infection. Viral mRNA was normalized to actin. Asterisks indicate significant changes (p value

    Techniques Used: Expressing, Infection, Synthesized, Amplification, Quantitative RT-PCR, Significance Assay

    FIX-ΔLUNA infected CD14+ cells fail to express lytic transcripts following IL6 induced differentiation. A) Diagram of the timeline of infection. Infected cells were collected at 1–20 dpi, IL6 was added at 10 dpi to induce cellular differentiation, after which two additional time points were collected. B) Expression of viral RNAs in FIX-WT, FIX-ΔLUNA and FIX-Rev infected CD14 + cells (MOI = 1). Total RNA was collected over a 20 d time course together with mock RNA. At 10 dpi CD14 + cells were differentiated with IL-6. Five and ten days post differentiation, RNA samples were collected (these samples were termed as 15 d-IL6 and 20 d-IL6). From all the collected RNA samples cDNA was synthesized and amplified using UL82, UL123, UL81-82ast, UL138 and β-actin specific primers. Negative images were used to visualize weaker bands. C) CD14 + cells were infected at an MOI = 1 with FIX-WT, FIX-ΔLUNA or FIX-Rev. D–E) Protein was isolated from infected CD14 + cells at the indicated time points and subjected to western blot analysis. Blots were probed with either a rabbit monoclonal a-LUNA or a mouse monoclonal a-IE1 antibody along with a-Tubulin as a loading control. qRT-PCR analysis was used to assay gene expression of UL138, vIL10 and US28 at the indicated times post infection. Viral mRNA was normalized to actin. All samples were tested in triplicate. Abbreviations used: d, days post infection., Mo; mock. IL6 was added at 10 dpi.
    Figure Legend Snippet: FIX-ΔLUNA infected CD14+ cells fail to express lytic transcripts following IL6 induced differentiation. A) Diagram of the timeline of infection. Infected cells were collected at 1–20 dpi, IL6 was added at 10 dpi to induce cellular differentiation, after which two additional time points were collected. B) Expression of viral RNAs in FIX-WT, FIX-ΔLUNA and FIX-Rev infected CD14 + cells (MOI = 1). Total RNA was collected over a 20 d time course together with mock RNA. At 10 dpi CD14 + cells were differentiated with IL-6. Five and ten days post differentiation, RNA samples were collected (these samples were termed as 15 d-IL6 and 20 d-IL6). From all the collected RNA samples cDNA was synthesized and amplified using UL82, UL123, UL81-82ast, UL138 and β-actin specific primers. Negative images were used to visualize weaker bands. C) CD14 + cells were infected at an MOI = 1 with FIX-WT, FIX-ΔLUNA or FIX-Rev. D–E) Protein was isolated from infected CD14 + cells at the indicated time points and subjected to western blot analysis. Blots were probed with either a rabbit monoclonal a-LUNA or a mouse monoclonal a-IE1 antibody along with a-Tubulin as a loading control. qRT-PCR analysis was used to assay gene expression of UL138, vIL10 and US28 at the indicated times post infection. Viral mRNA was normalized to actin. All samples were tested in triplicate. Abbreviations used: d, days post infection., Mo; mock. IL6 was added at 10 dpi.

    Techniques Used: Infection, Cell Differentiation, Expressing, Synthesized, Amplification, Isolation, Western Blot, Quantitative RT-PCR

    30) Product Images from "Synthetic RNAs Mimicking Structural Domains in the Foot-and-Mouth Disease Virus Genome Elicit a Broad Innate Immune Response in Porcine Cells Triggered by RIG-I and TLR Activation"

    Article Title: Synthetic RNAs Mimicking Structural Domains in the Foot-and-Mouth Disease Virus Genome Elicit a Broad Innate Immune Response in Porcine Cells Triggered by RIG-I and TLR Activation

    Journal: Viruses

    doi: 10.3390/v7072807

    Analysis of the innate immune response of transfected PBMCs from a single pig (six month-old) over time. ( A ) Swine PBMCs were transfected with 20 μg/mL IRES or 3′NCR transcripts, with or without Lipofectin, and RNA was extracted at 2, 4, 8 or 24 h pt. Amplification of Mx1 and cyclophilin mRNAs by RT-PCR is shown; ( B ) Antiviral activity, TNFα, IL-12 and IL-10 levels in supernatants of swine PBMCs (2 × 10 6 ) transfected with 3′NCR or IRES as in A or stimulated with poly I:C (10 μg/mL), and collected at 8 or 24 h pt. Mock transfections with culture medium and Lipofectin were performed as negative controls. Antiviral activity is expressed as the reciprocal of the highest dilution of supernatants from transfected PBMCs causing a 50% reduction of the cytophatic effect induced by infection with FMDV on IBRS-2 cells. The levels of the different cytokines were measured by ELISA; ( C ) Immunoblot detection of Mx1 in lysates of PBMCs transfected with S, IRES, 3′NCR transcripts (20 μg/mL) or mock-transfected using Lipofectin, or stimulated with poly I:C or ODN (both at 10 μg/mL). Cells were lysed 24 h pt. Tubulin was used for normalization.
    Figure Legend Snippet: Analysis of the innate immune response of transfected PBMCs from a single pig (six month-old) over time. ( A ) Swine PBMCs were transfected with 20 μg/mL IRES or 3′NCR transcripts, with or without Lipofectin, and RNA was extracted at 2, 4, 8 or 24 h pt. Amplification of Mx1 and cyclophilin mRNAs by RT-PCR is shown; ( B ) Antiviral activity, TNFα, IL-12 and IL-10 levels in supernatants of swine PBMCs (2 × 10 6 ) transfected with 3′NCR or IRES as in A or stimulated with poly I:C (10 μg/mL), and collected at 8 or 24 h pt. Mock transfections with culture medium and Lipofectin were performed as negative controls. Antiviral activity is expressed as the reciprocal of the highest dilution of supernatants from transfected PBMCs causing a 50% reduction of the cytophatic effect induced by infection with FMDV on IBRS-2 cells. The levels of the different cytokines were measured by ELISA; ( C ) Immunoblot detection of Mx1 in lysates of PBMCs transfected with S, IRES, 3′NCR transcripts (20 μg/mL) or mock-transfected using Lipofectin, or stimulated with poly I:C or ODN (both at 10 μg/mL). Cells were lysed 24 h pt. Tubulin was used for normalization.

    Techniques Used: Transfection, Amplification, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Infection, Enzyme-linked Immunosorbent Assay

    Mx1 induction in porcine cells transfected with the 3′NCR RNA. SK6 cells (1 × 10 6 ) were transfected with 20 μg/mL 3′NCR transcripts. Cells were lysed at 0, 6, 24 or 30 h after transfection. ( A ) RT-PCR detection of IFN-β, Mx1 and GAPDH mRNAs in RNA extracted from SK6 lysates. Negative controls (water) were included in the RT-PCR assays; ( B ) Mx1 detection by immunoblot in transfected SK6 cells. Tubulin was used for normalization.
    Figure Legend Snippet: Mx1 induction in porcine cells transfected with the 3′NCR RNA. SK6 cells (1 × 10 6 ) were transfected with 20 μg/mL 3′NCR transcripts. Cells were lysed at 0, 6, 24 or 30 h after transfection. ( A ) RT-PCR detection of IFN-β, Mx1 and GAPDH mRNAs in RNA extracted from SK6 lysates. Negative controls (water) were included in the RT-PCR assays; ( B ) Mx1 detection by immunoblot in transfected SK6 cells. Tubulin was used for normalization.

    Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction

    31) Product Images from "Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210"

    Article Title: Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-015-0974-3

    BNIP3 and CTSE transcriptomic and western blot analyses with RNAs and protein extracts from L. m. -infected and HBSS-starved BMDM as well as determination of the infection rates of L. m. -infected BMDM after BNIP3 and CTSE downregulation by RNA interference. Methods: ( a , b ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 h or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium or starved for 1 h in HBSS. The Proteins were harvested and subjected to western blot analysis with specific antibodies against BNIP3 and CTSE. Western blots from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Total RNA was harvested from L. m. -infected BMDM and uninfected control BMDM. Affymetrix® chips were hybridized with RNA samples from 2 independent experiments and analyzed densitometrically. c Additionally, L. m. -infected BMDM were transfected with specific siRNAs 20 h p.i. to downregulate the expression of BNIP3 and CTSE. L. m. -infected control BMDM were transfected with negative control siRNA. Infection rates were determined 48 h p.i. in 2 independent experiments. Results: ( a ) BNIP3 and CTSE were significantly overexpressed in L. m. -infected BMDM 24 h p.i. compared to uninfected control BMDM. b Densitometric analyses of western blot experiments confirmed this overexpression 24 h p.i. and showed that CTSE was also overexpressed in L. m. -infected BMDM 1 h p.i.. At the mRNA level, Bnip3 was overexpressed in L. m. -infected BMDM 1 and 24 h p.i. and Ctse was downregulated in L. m. -infected BMDM 24 h p.i.. c A significant increase in the infection rates was detected in L. m. -infected BMDM after downregulation of protein expression of BNIP3 or CTSE compared to L. m. -infected BMDM transfected with negative control siRNA. L. m. -inf. = L. m. -infected, neg. control = negative control, n.s. = not significant, RFU = relative fluorescence units, * p ≤ 0.05, ** p ≤ 0.01,*** p ≤ 0.001
    Figure Legend Snippet: BNIP3 and CTSE transcriptomic and western blot analyses with RNAs and protein extracts from L. m. -infected and HBSS-starved BMDM as well as determination of the infection rates of L. m. -infected BMDM after BNIP3 and CTSE downregulation by RNA interference. Methods: ( a , b ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 h or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium or starved for 1 h in HBSS. The Proteins were harvested and subjected to western blot analysis with specific antibodies against BNIP3 and CTSE. Western blots from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Total RNA was harvested from L. m. -infected BMDM and uninfected control BMDM. Affymetrix® chips were hybridized with RNA samples from 2 independent experiments and analyzed densitometrically. c Additionally, L. m. -infected BMDM were transfected with specific siRNAs 20 h p.i. to downregulate the expression of BNIP3 and CTSE. L. m. -infected control BMDM were transfected with negative control siRNA. Infection rates were determined 48 h p.i. in 2 independent experiments. Results: ( a ) BNIP3 and CTSE were significantly overexpressed in L. m. -infected BMDM 24 h p.i. compared to uninfected control BMDM. b Densitometric analyses of western blot experiments confirmed this overexpression 24 h p.i. and showed that CTSE was also overexpressed in L. m. -infected BMDM 1 h p.i.. At the mRNA level, Bnip3 was overexpressed in L. m. -infected BMDM 1 and 24 h p.i. and Ctse was downregulated in L. m. -infected BMDM 24 h p.i.. c A significant increase in the infection rates was detected in L. m. -infected BMDM after downregulation of protein expression of BNIP3 or CTSE compared to L. m. -infected BMDM transfected with negative control siRNA. L. m. -inf. = L. m. -infected, neg. control = negative control, n.s. = not significant, RFU = relative fluorescence units, * p ≤ 0.05, ** p ≤ 0.01,*** p ≤ 0.001

    Techniques Used: Western Blot, Infection, Mouse Assay, Incubation, Transfection, Expressing, Negative Control, Over Expression, Fluorescence

    MTOR and RPS6 transcriptomic and western blot analyses with RNAs and protein extracts from L. m. -infected and HBSS-starved BMDM, and determination of infection rates of L. m. -infected BMDM after MTOR downregulation by RNA interference. Methods: ( a , b ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium or starved for 1 h in HBSS. Proteins were harvested and subjected to western blot analyses with specific antibodies against MTOR, p-MTOR, RPS6, and p-RPS6. Western blots with proteins from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Total RNA was harvested from L. m. -infected BMDM and uninfected control BMDM. The Affymetrix® chips were hybridized with RNA samples from 2 independent experiments were analyzed densitometrically. c Additionally, BMDM were transfected with specific siRNA 4 h prior to infection to downregulate the expression of MTOR, and the cells were finally infected with L. m. promastigotes. L. m. -infected controls were transfected with negative control siRNA. The infection rates were determined 48 h p.i. in 2 independent experiments. Results: ( a ) A significant hyperphosphorylation was observed for MTOR and RPS6 in samples from L. m. -infected BMDM 1 h p.i. compared to uninfected control BMDM. b Results of densitometric analyses of western blot experiments and Affymetrix® chip analyses showed that MTOR and RPS6 expressions were not regulated at the mRNA or the protein level. However, MTOR and RPS6 were significantly hyperphosphorylated in L. m. -infected BMDM 1 h p.i.. c A significant decrease in the infection rate was detected in L. m. -infected BMDM after downregulation of the protein expression of MTOR compared to L. m. -infected BMDM transfected with negative control siRNA. L. m. -inf. = L. m. -infected, neg. control = negative control, n.s. = not significant, RFU = relative fluorescence units, * p ≤ 0.05, ** p ≤ 0.01
    Figure Legend Snippet: MTOR and RPS6 transcriptomic and western blot analyses with RNAs and protein extracts from L. m. -infected and HBSS-starved BMDM, and determination of infection rates of L. m. -infected BMDM after MTOR downregulation by RNA interference. Methods: ( a , b ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium or starved for 1 h in HBSS. Proteins were harvested and subjected to western blot analyses with specific antibodies against MTOR, p-MTOR, RPS6, and p-RPS6. Western blots with proteins from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Total RNA was harvested from L. m. -infected BMDM and uninfected control BMDM. The Affymetrix® chips were hybridized with RNA samples from 2 independent experiments were analyzed densitometrically. c Additionally, BMDM were transfected with specific siRNA 4 h prior to infection to downregulate the expression of MTOR, and the cells were finally infected with L. m. promastigotes. L. m. -infected controls were transfected with negative control siRNA. The infection rates were determined 48 h p.i. in 2 independent experiments. Results: ( a ) A significant hyperphosphorylation was observed for MTOR and RPS6 in samples from L. m. -infected BMDM 1 h p.i. compared to uninfected control BMDM. b Results of densitometric analyses of western blot experiments and Affymetrix® chip analyses showed that MTOR and RPS6 expressions were not regulated at the mRNA or the protein level. However, MTOR and RPS6 were significantly hyperphosphorylated in L. m. -infected BMDM 1 h p.i.. c A significant decrease in the infection rate was detected in L. m. -infected BMDM after downregulation of the protein expression of MTOR compared to L. m. -infected BMDM transfected with negative control siRNA. L. m. -inf. = L. m. -infected, neg. control = negative control, n.s. = not significant, RFU = relative fluorescence units, * p ≤ 0.05, ** p ≤ 0.01

    Techniques Used: Western Blot, Infection, Mouse Assay, Incubation, Transfection, Expressing, Negative Control, Chromatin Immunoprecipitation, Fluorescence

    32) Product Images from "Inhibition of HIV-1 by curcumin A, a novel curcumin analog"

    Article Title: Inhibition of HIV-1 by curcumin A, a novel curcumin analog

    Journal: Drug Design, Development and Therapy

    doi: 10.2147/DDDT.S86558

    Effect of curcumin and curcumin A on HIV-1 messenger (m)RNA expression, HIV-1 transcription and HIV-1 reverse transcription. Notes: ( A ) Effect of curcumin A on HIV-1 mRNA expr ession. CEM-T cells infected with HIV-1 Luc were treated with DMSO, 1 μM curcumin or 1 μM curcumin A for 48 hours as indicated. RNA was extracted, reverse transcribed, and analyzed with primers for HIV-1 gag and env genes by real-time PCR on Roche 4800 (Hoffman-La Roche Ltd., Basel, Switzerland) using 18S RNA as a reference. ( B , C ). Effect of curcumin and curcumin A on Tat-induced and basal HIV-1 transcription. In panel ( B ) 293T cells were transiently transfected with a vector contacting HIV LTR followed by the luciferase reporter (HIV LTR) and Tat expression vector or pNL4-3.Luc. In panel ( C ) 293T cells were transfected with HIV-1 LTR expression vector or with HIV LTR with the inactivated SP1 sites (NF-κB). For normalization, the cells were also co-transfected with GFP expressing vector. At 24 hours post-transfection the cells were treated with 1 μM curcumin or curcumin A for 24 hours. Then the cells were lyzed and luciferase activity was measured. GFP fluorescence was measured in parallel and used for normalization. ( D ) Effect of curcumin and curcumin A on HIV-1 reverse transcription. CEM-T cells were infected with HIV-1 Luc and then treated with DMSO, AZT, 1 μM curcumin or curcumin A as indicated for 6 hours. DNA was extracted and analyzed by real-time PCR on Roche 4800 using primers for early and late LTR and β-globin gene as a reference. Abbreviations: DMSO, dimethyl sulfoxide; mRNA, messenger RNA; PCR, polymerase chain reaction; AZT, azidothymidine.
    Figure Legend Snippet: Effect of curcumin and curcumin A on HIV-1 messenger (m)RNA expression, HIV-1 transcription and HIV-1 reverse transcription. Notes: ( A ) Effect of curcumin A on HIV-1 mRNA expr ession. CEM-T cells infected with HIV-1 Luc were treated with DMSO, 1 μM curcumin or 1 μM curcumin A for 48 hours as indicated. RNA was extracted, reverse transcribed, and analyzed with primers for HIV-1 gag and env genes by real-time PCR on Roche 4800 (Hoffman-La Roche Ltd., Basel, Switzerland) using 18S RNA as a reference. ( B , C ). Effect of curcumin and curcumin A on Tat-induced and basal HIV-1 transcription. In panel ( B ) 293T cells were transiently transfected with a vector contacting HIV LTR followed by the luciferase reporter (HIV LTR) and Tat expression vector or pNL4-3.Luc. In panel ( C ) 293T cells were transfected with HIV-1 LTR expression vector or with HIV LTR with the inactivated SP1 sites (NF-κB). For normalization, the cells were also co-transfected with GFP expressing vector. At 24 hours post-transfection the cells were treated with 1 μM curcumin or curcumin A for 24 hours. Then the cells were lyzed and luciferase activity was measured. GFP fluorescence was measured in parallel and used for normalization. ( D ) Effect of curcumin and curcumin A on HIV-1 reverse transcription. CEM-T cells were infected with HIV-1 Luc and then treated with DMSO, AZT, 1 μM curcumin or curcumin A as indicated for 6 hours. DNA was extracted and analyzed by real-time PCR on Roche 4800 using primers for early and late LTR and β-globin gene as a reference. Abbreviations: DMSO, dimethyl sulfoxide; mRNA, messenger RNA; PCR, polymerase chain reaction; AZT, azidothymidine.

    Techniques Used: Expressing, Infection, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Fluorescence, Polymerase Chain Reaction

    33) Product Images from "Translational Upregulation of an Individual p21Cip1 Transcript Variant by GCN2 Regulates Cell Proliferation and Survival under Nutrient Stress"

    Article Title: Translational Upregulation of an Individual p21Cip1 Transcript Variant by GCN2 Regulates Cell Proliferation and Survival under Nutrient Stress

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005212

    GCN2 enhances p21 translation independently of p53. A) qPCR for p21 was performed on RNA isolated from wildtype, p53 -/- , p63 -/- , and p73 -/- MEFs deprived of leucine for the indicated times. p21 transcript levels were normalized to 18S rRNA. Left: Results are depicted as fold change over control for each cell line. Right: Results are depicted as absolute levels of normalized transcript. Data represent the average of three independent experiments ± S.E.M. B) Western blot analysis of p21 induction in p53 +/+ and p53 -/- HCT116s. β-actin was used as a loading control. Values below blot represent fold change in total pixel intensity over control of p21 normalized to the loading control for each lane. C) qPCR for p21 was performed on RNA isolated from GCN2 +/+ , GCN2 -/- , and eIF2α S51A MEFs deprived of leucine for the indicated times. p21 transcript levels were normalized to 18S rRNA and are depicted as fold change over control. Data are the average of three independent experiments ± S.E.M. D) Metabolic labeling of p21 under leucine deprivation. Top: Autoradiograph of 35 S-labeled p21 immunoprecipitated from GCN2 +/+ and GCN2 -/- MEFs grown with or without leucine for 16 hours. Bottom: Western blot with cold amino acids to determine immunoprecipitation efficiency and verify induction of p21 in total cell lysates. E) Measurement of p21 protein half-life under replete and leucine starved conditions. GCN2 +/+ MEFs were initially grown in leucine-free media to induce p21 protein levels at time 0. Cells were then switched to complete or leucine-free media containing 50 μg/mL cycloheximide. Top: Western blot analysis of p21 protein levels during a time course of cycloheximide treatment. Total eIF2α was used as a loading control. Values below blot represent fold change in total pixel intensity over control of p21 normalized to the loading control for each lane. Bottom: Normalized p21 protein values were fit to exponential decay curves to calculate protein half-life.
    Figure Legend Snippet: GCN2 enhances p21 translation independently of p53. A) qPCR for p21 was performed on RNA isolated from wildtype, p53 -/- , p63 -/- , and p73 -/- MEFs deprived of leucine for the indicated times. p21 transcript levels were normalized to 18S rRNA. Left: Results are depicted as fold change over control for each cell line. Right: Results are depicted as absolute levels of normalized transcript. Data represent the average of three independent experiments ± S.E.M. B) Western blot analysis of p21 induction in p53 +/+ and p53 -/- HCT116s. β-actin was used as a loading control. Values below blot represent fold change in total pixel intensity over control of p21 normalized to the loading control for each lane. C) qPCR for p21 was performed on RNA isolated from GCN2 +/+ , GCN2 -/- , and eIF2α S51A MEFs deprived of leucine for the indicated times. p21 transcript levels were normalized to 18S rRNA and are depicted as fold change over control. Data are the average of three independent experiments ± S.E.M. D) Metabolic labeling of p21 under leucine deprivation. Top: Autoradiograph of 35 S-labeled p21 immunoprecipitated from GCN2 +/+ and GCN2 -/- MEFs grown with or without leucine for 16 hours. Bottom: Western blot with cold amino acids to determine immunoprecipitation efficiency and verify induction of p21 in total cell lysates. E) Measurement of p21 protein half-life under replete and leucine starved conditions. GCN2 +/+ MEFs were initially grown in leucine-free media to induce p21 protein levels at time 0. Cells were then switched to complete or leucine-free media containing 50 μg/mL cycloheximide. Top: Western blot analysis of p21 protein levels during a time course of cycloheximide treatment. Total eIF2α was used as a loading control. Values below blot represent fold change in total pixel intensity over control of p21 normalized to the loading control for each lane. Bottom: Normalized p21 protein values were fit to exponential decay curves to calculate protein half-life.

    Techniques Used: Real-time Polymerase Chain Reaction, Isolation, Western Blot, Labeling, Autoradiography, Immunoprecipitation

    34) Product Images from "TFAP2C-mediated upregulation of TGFBR1 promotes lung tumorigenesis and epithelial–mesenchymal transition"

    Article Title: TFAP2C-mediated upregulation of TGFBR1 promotes lung tumorigenesis and epithelial–mesenchymal transition

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/emm.2016.125

    TGFBR1 (transforming growth factor-β receptor type 1) was upregulated by TFAP2C (transcription factor-activating enhancer-binding protein 2C) in non-small-cell lung cancer (NSCLC) cells. ( a ) The effects of TFAP2C knockdown by treatment of TFAP2C small interfering RNA (siRNA) (#1 and #2) on the expression of TGFBR1 mRNA and protein in NCI-H292 and NCI-H838 cells were analyzed by real-time quantitative RT-PCR (qRT-PCR) and western blotting, respectively. Based on these results, TFAP2C siRNA no 1 was used for the subsequent experiments. ( b ) The effects of TFAP2C upregulation on the expression of TGFBR1 mRNA and protein in MRC5 and WI-26 VA4 cells were analyzed by real-time qRT-PCR and western blotting, respectively. * P
    Figure Legend Snippet: TGFBR1 (transforming growth factor-β receptor type 1) was upregulated by TFAP2C (transcription factor-activating enhancer-binding protein 2C) in non-small-cell lung cancer (NSCLC) cells. ( a ) The effects of TFAP2C knockdown by treatment of TFAP2C small interfering RNA (siRNA) (#1 and #2) on the expression of TGFBR1 mRNA and protein in NCI-H292 and NCI-H838 cells were analyzed by real-time quantitative RT-PCR (qRT-PCR) and western blotting, respectively. Based on these results, TFAP2C siRNA no 1 was used for the subsequent experiments. ( b ) The effects of TFAP2C upregulation on the expression of TGFBR1 mRNA and protein in MRC5 and WI-26 VA4 cells were analyzed by real-time qRT-PCR and western blotting, respectively. * P

    Techniques Used: Binding Assay, Small Interfering RNA, Expressing, Quantitative RT-PCR, Western Blot

    35) Product Images from "Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis"

    Article Title: Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7833

    Features of intronic location of ultraconserved RNA (uc). 8+ in CASZ1 A. Schematic representation of the transcript including uc.8+ with respect to CASZ1 . J82 RNA was retrotranscribed by using the SMARTer Rapid Amplification of cDNA Ends (RACE) cDNA Amplification kit (Clontech). Primers used for the 5′ RACE were as follows: Universal Primer Mix (UPM) that recognized the SMARTer oligonucleotide added at the 5′ end, gene specific primers 1 (GSP1) that recognized the sense transcript, and GSP2 primers that recognized the antisense transcript. The arrows represent the direction of amplification from the gene-specific primers that successfully amplified the unknown regions of the TUC8 gene. B. 5′- and 3′-RACE polymerase chain reaction (PCR) performed to amplify the uc.8+ cDNA. C. Sequence of the complete uc.8+ transcript (2435 bases) as determined using RACE. The yellow sequence was reported by Bejerano et al , 2004 [v1].
    Figure Legend Snippet: Features of intronic location of ultraconserved RNA (uc). 8+ in CASZ1 A. Schematic representation of the transcript including uc.8+ with respect to CASZ1 . J82 RNA was retrotranscribed by using the SMARTer Rapid Amplification of cDNA Ends (RACE) cDNA Amplification kit (Clontech). Primers used for the 5′ RACE were as follows: Universal Primer Mix (UPM) that recognized the SMARTer oligonucleotide added at the 5′ end, gene specific primers 1 (GSP1) that recognized the sense transcript, and GSP2 primers that recognized the antisense transcript. The arrows represent the direction of amplification from the gene-specific primers that successfully amplified the unknown regions of the TUC8 gene. B. 5′- and 3′-RACE polymerase chain reaction (PCR) performed to amplify the uc.8+ cDNA. C. Sequence of the complete uc.8+ transcript (2435 bases) as determined using RACE. The yellow sequence was reported by Bejerano et al , 2004 [v1].

    Techniques Used: Rapid Amplification of cDNA Ends, Amplification, Polymerase Chain Reaction, Sequencing

    Cellular localization of ultraconserved RNA (uc). 8+ A. Images acquired using inverted fluorescence microscope (magnification, 20×) of J82 control cells (Mock) after transfection with PNA-TO scramble-R8 (PNA-scramble) or with TO-PNA1-R8, the PNA complementary to uc.8+ (PNA-uc.8+). Images were recorded with excitation wavelength (lex)=450–490 nm (DAPI) or lex=510–540 nm (PNA-TO); the superimposition of the images recorded is also reported (Merge). All images were taken with the same confocal microscopy settings. Scale bar, 100 mm. Nuclei of J82 cells were stained with DAPI (blue). B. Inverse correlation between the expression of microRNA (miR)-596 and MMP9 in BlCa samples from 20 patients measured using qRT-PCR. C. Relative expression of MMP9 in siRNA-3 anti-uc.8+ –transfected J82 cells. Endogenous uc.8+ levels in the control cells are shown in grey. Data are expressed as the means ± standard deviation of triplicate values. P values were obtained using the Student t test for independent samples. **P
    Figure Legend Snippet: Cellular localization of ultraconserved RNA (uc). 8+ A. Images acquired using inverted fluorescence microscope (magnification, 20×) of J82 control cells (Mock) after transfection with PNA-TO scramble-R8 (PNA-scramble) or with TO-PNA1-R8, the PNA complementary to uc.8+ (PNA-uc.8+). Images were recorded with excitation wavelength (lex)=450–490 nm (DAPI) or lex=510–540 nm (PNA-TO); the superimposition of the images recorded is also reported (Merge). All images were taken with the same confocal microscopy settings. Scale bar, 100 mm. Nuclei of J82 cells were stained with DAPI (blue). B. Inverse correlation between the expression of microRNA (miR)-596 and MMP9 in BlCa samples from 20 patients measured using qRT-PCR. C. Relative expression of MMP9 in siRNA-3 anti-uc.8+ –transfected J82 cells. Endogenous uc.8+ levels in the control cells are shown in grey. Data are expressed as the means ± standard deviation of triplicate values. P values were obtained using the Student t test for independent samples. **P

    Techniques Used: Fluorescence, Microscopy, Transfection, Confocal Microscopy, Staining, Expressing, Quantitative RT-PCR, Standard Deviation

    Ultraconserved RNA (uc). 8+ and microRNA (miR)-596 interaction and target regulation in bladder cancer (BlCa) cells A. Representative positive correlation between the expression of uc.8+ and that of miR-596 in BlCa samples from 20 patients (Table 1 , dataset 4) measured using qRT-PCR. Correlation was computed using the Spearman correlation coefficient. B. Expression of miR-596 in J82 cell extracts after retrieval of endogenous uc.8+ with a peptide nucleic acid (PNA)/uc.8+ probe. Data are expressed as the means ± standard deviation (SD) of triplicate values. P values were obtained using the Student t test for independent samples. ***P
    Figure Legend Snippet: Ultraconserved RNA (uc). 8+ and microRNA (miR)-596 interaction and target regulation in bladder cancer (BlCa) cells A. Representative positive correlation between the expression of uc.8+ and that of miR-596 in BlCa samples from 20 patients (Table 1 , dataset 4) measured using qRT-PCR. Correlation was computed using the Spearman correlation coefficient. B. Expression of miR-596 in J82 cell extracts after retrieval of endogenous uc.8+ with a peptide nucleic acid (PNA)/uc.8+ probe. Data are expressed as the means ± standard deviation (SD) of triplicate values. P values were obtained using the Student t test for independent samples. ***P

    Techniques Used: Expressing, Quantitative RT-PCR, Standard Deviation

    Independent regulation of ultraconserved RNA (uc). 8+ and CASZ1 in bladder cancer (BlCa) tissues A. Schematic representation of the intronic localization of uc.8+ within CASZ1 . CASZ1 exons are indicated by black boxes. The locations of the uc.8+ forward (F) and reverse (R) primers used for qRT-PCR and the probe used for in situ hybridization are shown. Of the siRNAs targeting CASZ1 , siRNA-1 is located at the 5′ untranslated region (UTR), siRNA-2 is located in exon 6, and siRNA-3 is located at the 3′ UTR. B. RNA levels of CASZ1 and uc.8+ were determined by qRT-PCR in BlCa (n=19, black dots) and control normal bladder epithelium (NBE) samples (n=11, empty circles). Results are presented as means ± standard deviation (SD). Spearman correlation coefficient and P values are indicated. C. CASZ1 expression after silencing of uc.8+. The expression of CASZ1 was not affected in J82 cells transfected with three different siRNAs anti-uc.8+ or siRNA control. D. J82 cells were transfected with siRNA anti- CASZ1 or siRNA control. The CASZ1 level was determined by qRT-PCR. uc.8+ expression was not affected by any of the siRNAs anti- CASZ1 used. Data are expressed as the mean ± SD of triplicate values.
    Figure Legend Snippet: Independent regulation of ultraconserved RNA (uc). 8+ and CASZ1 in bladder cancer (BlCa) tissues A. Schematic representation of the intronic localization of uc.8+ within CASZ1 . CASZ1 exons are indicated by black boxes. The locations of the uc.8+ forward (F) and reverse (R) primers used for qRT-PCR and the probe used for in situ hybridization are shown. Of the siRNAs targeting CASZ1 , siRNA-1 is located at the 5′ untranslated region (UTR), siRNA-2 is located in exon 6, and siRNA-3 is located at the 3′ UTR. B. RNA levels of CASZ1 and uc.8+ were determined by qRT-PCR in BlCa (n=19, black dots) and control normal bladder epithelium (NBE) samples (n=11, empty circles). Results are presented as means ± standard deviation (SD). Spearman correlation coefficient and P values are indicated. C. CASZ1 expression after silencing of uc.8+. The expression of CASZ1 was not affected in J82 cells transfected with three different siRNAs anti-uc.8+ or siRNA control. D. J82 cells were transfected with siRNA anti- CASZ1 or siRNA control. The CASZ1 level was determined by qRT-PCR. uc.8+ expression was not affected by any of the siRNAs anti- CASZ1 used. Data are expressed as the mean ± SD of triplicate values.

    Techniques Used: Quantitative RT-PCR, In Situ Hybridization, Standard Deviation, Expressing, Transfection

    Effect of ultraconserved RNA (uc). 8+ silencing on bladder cancer (BlCa) cell proliferation, migration, and invasion A. J82 cells were transfected with siRNA anti-uc.8+ or siRNA control and were seeded in 96-well plates. Cell proliferation was determined at the indicated time points. The number of cells per well was measured by the absorbance at 595 nm. The results show data from at least three independent experiments. Cell growth after transfection with siRNA-3 anti-uc.8+ was not significantly different from that of cells transfected with siRNA-2 anti-uc.8+. P values were obtained using the Student t test for independent samples. *P
    Figure Legend Snippet: Effect of ultraconserved RNA (uc). 8+ silencing on bladder cancer (BlCa) cell proliferation, migration, and invasion A. J82 cells were transfected with siRNA anti-uc.8+ or siRNA control and were seeded in 96-well plates. Cell proliferation was determined at the indicated time points. The number of cells per well was measured by the absorbance at 595 nm. The results show data from at least three independent experiments. Cell growth after transfection with siRNA-3 anti-uc.8+ was not significantly different from that of cells transfected with siRNA-2 anti-uc.8+. P values were obtained using the Student t test for independent samples. *P

    Techniques Used: Migration, Transfection

    36) Product Images from "Alliin Attenuated RANKL-Induced Osteoclastogenesis by Scavenging Reactive Oxygen Species through Inhibiting Nox1"

    Article Title: Alliin Attenuated RANKL-Induced Osteoclastogenesis by Scavenging Reactive Oxygen Species through Inhibiting Nox1

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17091516

    Alliin inhibited RANKL-induced osteoclast fusion and differentiation in a dose-dependent manner. ( A ) RAW264.7 cells were pretreated with RANKL (50 ng/mL) and M-CSF (50 ng/mL) for about 72 h along with a range of alliin concentrations (0, 1, 10 µg/mL), following focal and adhesion staining, and finally photographed. The nuclei were stained for double immunofluorescence microscopy by DAPI ( blue ) and Vinculin monoclonal antibody ( red ). Each experiment was performed thrice. Scale bar was at 200 µm; ( B ) the quantitative test for the TRAP (+) cells having multiple nuclei in each well of 96-well plate; and ( C ) the total RNA extracted from RAW264.7 cells during RANKL-induced osteoclastogenesis treated with RANKL (50 ng/mL) and M-CSF (50 ng/mL) for 72 h with varying doses of alliin (0, 1, 10, 100 µg/mL). Relative mRNA expression levels of NFATc1, c-Fos, MMP-9, CD9, DC-STAMP, OC-STAMP, TRAP, and RANK against GAPDH are shown. Data in the figures represent the averages ± SD. * ( p -value
    Figure Legend Snippet: Alliin inhibited RANKL-induced osteoclast fusion and differentiation in a dose-dependent manner. ( A ) RAW264.7 cells were pretreated with RANKL (50 ng/mL) and M-CSF (50 ng/mL) for about 72 h along with a range of alliin concentrations (0, 1, 10 µg/mL), following focal and adhesion staining, and finally photographed. The nuclei were stained for double immunofluorescence microscopy by DAPI ( blue ) and Vinculin monoclonal antibody ( red ). Each experiment was performed thrice. Scale bar was at 200 µm; ( B ) the quantitative test for the TRAP (+) cells having multiple nuclei in each well of 96-well plate; and ( C ) the total RNA extracted from RAW264.7 cells during RANKL-induced osteoclastogenesis treated with RANKL (50 ng/mL) and M-CSF (50 ng/mL) for 72 h with varying doses of alliin (0, 1, 10, 100 µg/mL). Relative mRNA expression levels of NFATc1, c-Fos, MMP-9, CD9, DC-STAMP, OC-STAMP, TRAP, and RANK against GAPDH are shown. Data in the figures represent the averages ± SD. * ( p -value

    Techniques Used: Staining, Immunofluorescence, Microscopy, Expressing

    37) Product Images from "The Arabidopsis a zinc finger domain protein ARS1 is essential for seed germination and ROS homeostasis in response to ABA and oxidative stress"

    Article Title: The Arabidopsis a zinc finger domain protein ARS1 is essential for seed germination and ROS homeostasis in response to ABA and oxidative stress

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2015.00963

    Characterization of ARS1. (A) Phylogenetic tree of ARS1 homologs, generated using the BAR Expressolog Tree program. (B) ARS1 encodes an unknown protein with two NLS domains, an NES domain, and a C2H2 type zinc-finger domain. (C) Intracellular localization of the ARS1 protein in the nucleus. Protoplasts prepared from Arabidopsis seedlings were co-transformed with ARS1::sGFP and NLS::RFP . The transformed protoplasts were examined by fluorescence microscopy 12 h after transformation. Green and red images are GFP and RFP signals, respectively. Bars indicate 20 μm. (D) RT-PCR analysis of ARS1 expression in Arabidopsis tissues. TUBULIN2 serves as a control for RNA integrity.
    Figure Legend Snippet: Characterization of ARS1. (A) Phylogenetic tree of ARS1 homologs, generated using the BAR Expressolog Tree program. (B) ARS1 encodes an unknown protein with two NLS domains, an NES domain, and a C2H2 type zinc-finger domain. (C) Intracellular localization of the ARS1 protein in the nucleus. Protoplasts prepared from Arabidopsis seedlings were co-transformed with ARS1::sGFP and NLS::RFP . The transformed protoplasts were examined by fluorescence microscopy 12 h after transformation. Green and red images are GFP and RFP signals, respectively. Bars indicate 20 μm. (D) RT-PCR analysis of ARS1 expression in Arabidopsis tissues. TUBULIN2 serves as a control for RNA integrity.

    Techniques Used: Generated, Transformation Assay, Fluorescence, Microscopy, Reverse Transcription Polymerase Chain Reaction, Expressing

    Expression of ROS-scavenging genes in response to ABA treatment. Total RNA was isolated from 10-d-old seedlings of WT and ars1 mutants with or without ABA (100 μM) treatment for 3 h. Relative transcript levels of CCS (A) , CSD3 (B) , APX1 (C) , and APX2 (D) in Col-0 and ars1 mutants determined by qRT-PCR. Transcript levels were normalized to those of TUBULIN2 . Bars represent mean ± SD of three biological replicates with three technical replicates each. Asterisks represent significant differences from the Col-0 ( ∗ ; 0.01
    Figure Legend Snippet: Expression of ROS-scavenging genes in response to ABA treatment. Total RNA was isolated from 10-d-old seedlings of WT and ars1 mutants with or without ABA (100 μM) treatment for 3 h. Relative transcript levels of CCS (A) , CSD3 (B) , APX1 (C) , and APX2 (D) in Col-0 and ars1 mutants determined by qRT-PCR. Transcript levels were normalized to those of TUBULIN2 . Bars represent mean ± SD of three biological replicates with three technical replicates each. Asterisks represent significant differences from the Col-0 ( ∗ ; 0.01

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR

    The absence of ARS1 leads to ABA-hypersensitive seed germination. (A) T-DNA insertions in the ars1 mutant alleles. (B) RT-PCR analysis of ARS1 expression in WT, ars1-1 , ars1-1 +Vector, and ars1-1 + ARS1 plants. TUBULIN2 serves as a control for RNA integrity. (C) Comparison of seed germination between the WT, ars1-1 , ars1-1 +Vector, and ars1-1 + ARS1 plants exposed to 0 or 1 μM ABA. The photograph shows Arabidopsis seedlings after 5 days of ABA treatment. (D) Expression of ARS1 in WT Col-0 and ars1 allelic mutants determined by RT-PCR. TUBULIN2 serves as a control for RNA integrity. (E) Comparison of seed germination between the WT Col-0 and ars1 mutants exposed to 0 or 1 μM ABA. The photograph shows Arabidopsis seedlings after 5 days of ABA treatment. (F) Quantification of green cotyledons in WT Col-0 and ars1 mutants grown on various concentrations of ABA for 5 days. The data represent the means ± SE of three independent experiments, with 50 seeds per experiment.
    Figure Legend Snippet: The absence of ARS1 leads to ABA-hypersensitive seed germination. (A) T-DNA insertions in the ars1 mutant alleles. (B) RT-PCR analysis of ARS1 expression in WT, ars1-1 , ars1-1 +Vector, and ars1-1 + ARS1 plants. TUBULIN2 serves as a control for RNA integrity. (C) Comparison of seed germination between the WT, ars1-1 , ars1-1 +Vector, and ars1-1 + ARS1 plants exposed to 0 or 1 μM ABA. The photograph shows Arabidopsis seedlings after 5 days of ABA treatment. (D) Expression of ARS1 in WT Col-0 and ars1 allelic mutants determined by RT-PCR. TUBULIN2 serves as a control for RNA integrity. (E) Comparison of seed germination between the WT Col-0 and ars1 mutants exposed to 0 or 1 μM ABA. The photograph shows Arabidopsis seedlings after 5 days of ABA treatment. (F) Quantification of green cotyledons in WT Col-0 and ars1 mutants grown on various concentrations of ABA for 5 days. The data represent the means ± SE of three independent experiments, with 50 seeds per experiment.

    Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Expressing, Plasmid Preparation

    38) Product Images from "Neutrophilic Lung Inflammation Suppressed by Picroside II Is Associated with TGF-β Signaling"

    Article Title: Neutrophilic Lung Inflammation Suppressed by Picroside II Is Associated with TGF-β Signaling

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2015/897272

    PIC II treatment decreases the expression of proinflammatory cytokine gene expression in the lungs of ALI mice. (a) Total RNA extracted from the lung tissues of ALI mice ( n = 5) was analyzed by semiquantitative RT-PCR for the expression of representative proinflammatory cytokine genes. Two representatives in each group are shown. (b) Each band was quantitated by ImageJ, and relative expressions of those cytokine genes were calculated over a house-keeping gene, GAPDH. Data represent the mean ± SEM of each group in three measurements. ∗ P and ∗∗ P were less than 0.05 and 0.001, respectively, compared with the only LPS treated.
    Figure Legend Snippet: PIC II treatment decreases the expression of proinflammatory cytokine gene expression in the lungs of ALI mice. (a) Total RNA extracted from the lung tissues of ALI mice ( n = 5) was analyzed by semiquantitative RT-PCR for the expression of representative proinflammatory cytokine genes. Two representatives in each group are shown. (b) Each band was quantitated by ImageJ, and relative expressions of those cytokine genes were calculated over a house-keeping gene, GAPDH. Data represent the mean ± SEM of each group in three measurements. ∗ P and ∗∗ P were less than 0.05 and 0.001, respectively, compared with the only LPS treated.

    Techniques Used: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    39) Product Images from "Arabidopsis thaliana XRN2 is required for primary cleavage in the pre-ribosomal RNA"

    Article Title: Arabidopsis thaliana XRN2 is required for primary cleavage in the pre-ribosomal RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq172

    Polyadenylated mature, precursor and intermediate rRNAs accumulate in the absence of AtXRN2. ( A and B ) Northern analysis of mature rRNAs in xrn2 and xrn3 mutants. Total RNA was extracted from wild-type, xrn2-3 , xrn3-8 and xrn2-1 xrn3-3 seedlings and separated on 1.1% agarose ( A ) or 6% polyacrylamide ( B ) gels and hybridized with probes specific for 5′ regions of 25S ( p8 ) and 18S rRNAs ( p10 ) as well as 5.8S ( p7 ). Hybridizations for eIF-4A mRNA and 7SL RNA ( p13 ) were used as loading controls. ( C ) Northern analysis of total and poly(A) + RNA extracted from wild-type and xrn2-3 inflorescences, using probes p9 (25S rRNA) and p5 (27S pre-rRNA). rRNA, pre-RNA and intermediates detected in (A–C) are indicated on the right. ( D ) RT-PCR on total RNA from wild-type, xrn2-3 , xrn3-8 and xrn2-1 xrn3-3 lines. Reverse transcription was performed using oligo(dT) 20 and PCR using primers p28 and p23 for detection of the 5′ETS fragment, p10 and p26 for 35S, p29 and p30 for 27SA, and p35 and p36 for both 27SA/27SB pre-rRNAs. RT-PCR for eIF-4A was used as a control. The structure of the pre-rRNA with location of probes (solid lines) and PCR primers (arrows) used is shown below. ( E ) CRT-PCR on poly(A) + RNA extracted from xrn2-3 inflorescences. Top diagram represents the structure of 5′ETS with primers used for PCR. Indicated are: transcription initiation site TIS (+1); conserved cluster A 123 B; cleavage sites P (+1275) and P1 (+1423/+1454); 5′-end of 18S rRNA (+1836). RNA fragments identified as sequenced clones are represented below as horizontal lines, their 5′ -and 3′-ends are shown in italics and the number of adenine residues is indicated at the 3′-end of each molecule.
    Figure Legend Snippet: Polyadenylated mature, precursor and intermediate rRNAs accumulate in the absence of AtXRN2. ( A and B ) Northern analysis of mature rRNAs in xrn2 and xrn3 mutants. Total RNA was extracted from wild-type, xrn2-3 , xrn3-8 and xrn2-1 xrn3-3 seedlings and separated on 1.1% agarose ( A ) or 6% polyacrylamide ( B ) gels and hybridized with probes specific for 5′ regions of 25S ( p8 ) and 18S rRNAs ( p10 ) as well as 5.8S ( p7 ). Hybridizations for eIF-4A mRNA and 7SL RNA ( p13 ) were used as loading controls. ( C ) Northern analysis of total and poly(A) + RNA extracted from wild-type and xrn2-3 inflorescences, using probes p9 (25S rRNA) and p5 (27S pre-rRNA). rRNA, pre-RNA and intermediates detected in (A–C) are indicated on the right. ( D ) RT-PCR on total RNA from wild-type, xrn2-3 , xrn3-8 and xrn2-1 xrn3-3 lines. Reverse transcription was performed using oligo(dT) 20 and PCR using primers p28 and p23 for detection of the 5′ETS fragment, p10 and p26 for 35S, p29 and p30 for 27SA, and p35 and p36 for both 27SA/27SB pre-rRNAs. RT-PCR for eIF-4A was used as a control. The structure of the pre-rRNA with location of probes (solid lines) and PCR primers (arrows) used is shown below. ( E ) CRT-PCR on poly(A) + RNA extracted from xrn2-3 inflorescences. Top diagram represents the structure of 5′ETS with primers used for PCR. Indicated are: transcription initiation site TIS (+1); conserved cluster A 123 B; cleavage sites P (+1275) and P1 (+1423/+1454); 5′-end of 18S rRNA (+1836). RNA fragments identified as sequenced clones are represented below as horizontal lines, their 5′ -and 3′-ends are shown in italics and the number of adenine residues is indicated at the 3′-end of each molecule.

    Techniques Used: Northern Blot, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Clone Assay

    AtXRN2 is not required for processing at site P1 but is involved in the 5′ETS trimming that precedes cleavage at site P. ( A ) Mapping cleavages at sites P1 by primer extension in wild-type and xrn2 plants using primer p24 . Description is as for Figure 5 A. ( B ) Mapping the 5′ and 3′ ends of 5′ETS-P1 fragment by CRT-PCR on total RNA from the xrn2-3 mutant. Description is as for Figure 2 E. Identified termini are shown in italics. ( C ) Northern analysis of the 5′ETS-containing pre-rRNAs in wild-type, xrn2-3 , xrn3-8 and xrn2-1 xrn3-3 plants. Total RNA was separated on a 1.1% agarose gel and hybridized with probes distributed along 5’ETS, shown in the panel below. Pre-rRNA and intermediate species are designated as in Figure 4 . eIF-4A mRNA was used as a loading control. ( D ) Mapping 5′ ends of 35S* pre-rRNA in the xrn2-3 mutant by 5′ RACE. cDNA synthesis was performed with primer p1 and PCR with p25 and a primer complementary to an adaptor ligated to cDNA. Identified 5′ ends are shown in italics. ( E ) Excised P-P1 fragment in wild-type and xrn2-3 plants detected by northern analysis. Total RNA was separated on a 6% polyacrylamide gel and hybridized with probe p23 . 7SL RNA was used as a control. Location of primers used for primer extension, northern blots and CRT-PCR are shown in the schematics below panel A.
    Figure Legend Snippet: AtXRN2 is not required for processing at site P1 but is involved in the 5′ETS trimming that precedes cleavage at site P. ( A ) Mapping cleavages at sites P1 by primer extension in wild-type and xrn2 plants using primer p24 . Description is as for Figure 5 A. ( B ) Mapping the 5′ and 3′ ends of 5′ETS-P1 fragment by CRT-PCR on total RNA from the xrn2-3 mutant. Description is as for Figure 2 E. Identified termini are shown in italics. ( C ) Northern analysis of the 5′ETS-containing pre-rRNAs in wild-type, xrn2-3 , xrn3-8 and xrn2-1 xrn3-3 plants. Total RNA was separated on a 1.1% agarose gel and hybridized with probes distributed along 5’ETS, shown in the panel below. Pre-rRNA and intermediate species are designated as in Figure 4 . eIF-4A mRNA was used as a loading control. ( D ) Mapping 5′ ends of 35S* pre-rRNA in the xrn2-3 mutant by 5′ RACE. cDNA synthesis was performed with primer p1 and PCR with p25 and a primer complementary to an adaptor ligated to cDNA. Identified 5′ ends are shown in italics. ( E ) Excised P-P1 fragment in wild-type and xrn2-3 plants detected by northern analysis. Total RNA was separated on a 6% polyacrylamide gel and hybridized with probe p23 . 7SL RNA was used as a control. Location of primers used for primer extension, northern blots and CRT-PCR are shown in the schematics below panel A.

    Techniques Used: Polymerase Chain Reaction, Mutagenesis, Northern Blot, Agarose Gel Electrophoresis

    40) Product Images from "Identification of FAM111A as an SV40 Host Range Restriction and Adenovirus Helper Factor"

    Article Title: Identification of FAM111A as an SV40 Host Range Restriction and Adenovirus Helper Factor

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002949

    Depletion of FAM111A increases viral gene expression and renders CV-1P cells permissive for host range mutant viruses. (A) U-2 OS cells were co-transfected with host range viral DNA (HR684) and control siRNA (black bars), siRNA targeting FAM111A (white bars) or an expression vector for the C-terminus of LT (grey bars). Quantitative RT-PCR was performed 72 hours post-transfection to determine the expression levels of LT, VP1 and FAM111A (latter not shown) mRNA relative to actin. Error bars represent standard deviation from the mean. U-2 OS (B) and CV-1P (C) cell lines stably expressing two different shRNAs against FAM111A or vector control were generated and the amount of FAM111A RNA remaining (% FAM111A RNA) was confirmed by quantitative RT-PCR. Viral DNA encoding HR684 was transfected into the indicated cell lines and whole cell lysates were harvested at 48 and 96 hours post transfection. (D). CV-1P cells stably expressing two different shRNAs against FAM111A or vector control were transfected with viral DNA and assayed for lytic infection by plaque assay. Plaques were counted 8 days after transfection. Results shown are the average of three independent experiments with standard deviation from the mean denoted by +/−. (E) Control or FAM111A shRNA depleted CV-1P cells were infected at a multiplicity of infection of three with either wild-type SV40 virus or the host range mutant dl1066 virus. Cells were freeze thawed at the indicated time points to extract virus and the viral titer was determined in BSC40 cells. Results shown are the average of three independent experiments with standard deviation from the mean indicated.
    Figure Legend Snippet: Depletion of FAM111A increases viral gene expression and renders CV-1P cells permissive for host range mutant viruses. (A) U-2 OS cells were co-transfected with host range viral DNA (HR684) and control siRNA (black bars), siRNA targeting FAM111A (white bars) or an expression vector for the C-terminus of LT (grey bars). Quantitative RT-PCR was performed 72 hours post-transfection to determine the expression levels of LT, VP1 and FAM111A (latter not shown) mRNA relative to actin. Error bars represent standard deviation from the mean. U-2 OS (B) and CV-1P (C) cell lines stably expressing two different shRNAs against FAM111A or vector control were generated and the amount of FAM111A RNA remaining (% FAM111A RNA) was confirmed by quantitative RT-PCR. Viral DNA encoding HR684 was transfected into the indicated cell lines and whole cell lysates were harvested at 48 and 96 hours post transfection. (D). CV-1P cells stably expressing two different shRNAs against FAM111A or vector control were transfected with viral DNA and assayed for lytic infection by plaque assay. Plaques were counted 8 days after transfection. Results shown are the average of three independent experiments with standard deviation from the mean denoted by +/−. (E) Control or FAM111A shRNA depleted CV-1P cells were infected at a multiplicity of infection of three with either wild-type SV40 virus or the host range mutant dl1066 virus. Cells were freeze thawed at the indicated time points to extract virus and the viral titer was determined in BSC40 cells. Results shown are the average of three independent experiments with standard deviation from the mean indicated.

    Techniques Used: Expressing, Mutagenesis, Transfection, Plasmid Preparation, Quantitative RT-PCR, Standard Deviation, Stable Transfection, Generated, Infection, Plaque Assay, shRNA

    41) Product Images from "LIN-28 co-transcriptionally binds primary let-7 to regulate miRNA maturation in C. elegans"

    Article Title: LIN-28 co-transcriptionally binds primary let-7 to regulate miRNA maturation in C. elegans

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.1986

    Developmentally regulated processing of let-7 pri-miRNA transcripts. ( a ) Depiction of expected Drosha cleavage products: 5′ flanking, let-7 hairpin precursor, and 3′ flanking. The number of sequenced RACE clones that mapped to the precise 3′ and 5′ Drosha cleavage products at each time point from two independent experiments is shown. The sequences of all Drosha cleavage products are shown in Supplementary Figure 3 . ( b ) RT-PCR was performed on two independent 5′ RACE samples from N2 (left panel) or N2 and lin-28(n719) worms (right panel). ( c ) Total RNA was isolated from synchronized eri-1(mg366) RNAi hypersensitive worms at the indicated time points after vector control (-) or pup-2 (+) RNAi treatment, and analyzed by agarose and PAGE northern blotting. Representative blots from three independent experiments are shown.
    Figure Legend Snippet: Developmentally regulated processing of let-7 pri-miRNA transcripts. ( a ) Depiction of expected Drosha cleavage products: 5′ flanking, let-7 hairpin precursor, and 3′ flanking. The number of sequenced RACE clones that mapped to the precise 3′ and 5′ Drosha cleavage products at each time point from two independent experiments is shown. The sequences of all Drosha cleavage products are shown in Supplementary Figure 3 . ( b ) RT-PCR was performed on two independent 5′ RACE samples from N2 (left panel) or N2 and lin-28(n719) worms (right panel). ( c ) Total RNA was isolated from synchronized eri-1(mg366) RNAi hypersensitive worms at the indicated time points after vector control (-) or pup-2 (+) RNAi treatment, and analyzed by agarose and PAGE northern blotting. Representative blots from three independent experiments are shown.

    Techniques Used: Clone Assay, Reverse Transcription Polymerase Chain Reaction, Isolation, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Northern Blot

    42) Product Images from "miR-143 Regulation of Prostaglandin-Endoperoxidase Synthase 2 in the Amnion: Implications for Human Parturition at Term"

    Article Title: miR-143 Regulation of Prostaglandin-Endoperoxidase Synthase 2 in the Amnion: Implications for Human Parturition at Term

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0024131

    miR-143/miR-145 cluster expression in PA and RA. A, qRT-PCR analysis of miR-143 expression in PA and RA obtained from women at term not in labor (TNL; n = 10) and in labor (TIL; n = 10) to confirm microarray results. TNL cases are composed of 5 cases subjected to microarray analysis and 5 additional cases, while TIL cases are composed of 4 cases used in the microarray analysis and 6 additional cases because of RNA availability. miR-143 expression is significantly higher in the PA than in the RA in both groups, and its expression in the RA is significantly higher in TIL cases than in TNL cases. *, p
    Figure Legend Snippet: miR-143/miR-145 cluster expression in PA and RA. A, qRT-PCR analysis of miR-143 expression in PA and RA obtained from women at term not in labor (TNL; n = 10) and in labor (TIL; n = 10) to confirm microarray results. TNL cases are composed of 5 cases subjected to microarray analysis and 5 additional cases, while TIL cases are composed of 4 cases used in the microarray analysis and 6 additional cases because of RNA availability. miR-143 expression is significantly higher in the PA than in the RA in both groups, and its expression in the RA is significantly higher in TIL cases than in TNL cases. *, p

    Techniques Used: Expressing, Quantitative RT-PCR, Microarray

    43) Product Images from "Age-Dependent Decline in Mouse Lung Regeneration with Loss of Lung Fibroblast Clonogenicity and Increased Myofibroblastic Differentiation"

    Article Title: Age-Dependent Decline in Mouse Lung Regeneration with Loss of Lung Fibroblast Clonogenicity and Increased Myofibroblastic Differentiation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0023232

    Study design comparing transcriptomic patterns after PNX in 9 vs 3 month mice. A : Surgeries. Left lung lobe pneumonectomy (PNX) was performed on 18 young (12 week old) and 18 middle-aged (9 month old) female mice. The left lobe of every animal was preserved in RNAlater at the time of surgery. The mice were sacrificed and the remaining right lung lobes were removed at 1, 3 or 7 days post-surgery (n = 6/group). RNA was prepared from the R and L lung lobes of every animal. For each animal, RNA from the left lung lobe was used as the normalizing RNA for microarray analysis. B : Microarray experimental design. At each time point, RNA from two animals was pooled to create one pre-PNX (left lung) sample and one post-PNX (right lung) sample. Therefore, at each time point, 3 pooled samples were created for each group (young pre-PNX; young post-PNX; aged pre-PNX and aged post-PNX). C. Microarray data analysis. We generated two groups of data from the microarray analysis. First, to uncover transcripts that are differentially regulated between young and aged animals, we directly compared the aged pre-PNX samples to the young pre-PNX samples (n = 9 pooled samples/group). Second, to compare the effect of aging on lung regeneration post-PNX, we performed a double comparison by first comparing young post-PNX and aged post-PNX to their own control (pre-PNX) for each pooled sample. (n = 6 data sets/time point), and then using a two-class SAM method to compare these aged vs young data sets to identify preferentially expressed genes in aged vs young post-PNX animals.
    Figure Legend Snippet: Study design comparing transcriptomic patterns after PNX in 9 vs 3 month mice. A : Surgeries. Left lung lobe pneumonectomy (PNX) was performed on 18 young (12 week old) and 18 middle-aged (9 month old) female mice. The left lobe of every animal was preserved in RNAlater at the time of surgery. The mice were sacrificed and the remaining right lung lobes were removed at 1, 3 or 7 days post-surgery (n = 6/group). RNA was prepared from the R and L lung lobes of every animal. For each animal, RNA from the left lung lobe was used as the normalizing RNA for microarray analysis. B : Microarray experimental design. At each time point, RNA from two animals was pooled to create one pre-PNX (left lung) sample and one post-PNX (right lung) sample. Therefore, at each time point, 3 pooled samples were created for each group (young pre-PNX; young post-PNX; aged pre-PNX and aged post-PNX). C. Microarray data analysis. We generated two groups of data from the microarray analysis. First, to uncover transcripts that are differentially regulated between young and aged animals, we directly compared the aged pre-PNX samples to the young pre-PNX samples (n = 9 pooled samples/group). Second, to compare the effect of aging on lung regeneration post-PNX, we performed a double comparison by first comparing young post-PNX and aged post-PNX to their own control (pre-PNX) for each pooled sample. (n = 6 data sets/time point), and then using a two-class SAM method to compare these aged vs young data sets to identify preferentially expressed genes in aged vs young post-PNX animals.

    Techniques Used: Mouse Assay, Microarray, Generated

    44) Product Images from "Calreticulin Promotes Proliferation and Migration But Inhibits Apoptosis in Schwann Cells"

    Article Title: Calreticulin Promotes Proliferation and Migration But Inhibits Apoptosis in Schwann Cells

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.900956

    The difference in calreticulin (CRT) expression in transfected Schwann cells (SCs). SCs were transfected with pcDNA3.1 (pcNC), pcDNA3.1-CRT (pc-CRT), non-silencing small interfering RNA (siNC), or specific small interfering RNA against CRT (siCRT). After transfection, cells were harvested for quantitative real-time (qRT)-PCR ( A ) and Western blot analysis ( B ). Data presented are the mean of at least 3 independent experiments. Error bars indicate SD. ** P
    Figure Legend Snippet: The difference in calreticulin (CRT) expression in transfected Schwann cells (SCs). SCs were transfected with pcDNA3.1 (pcNC), pcDNA3.1-CRT (pc-CRT), non-silencing small interfering RNA (siNC), or specific small interfering RNA against CRT (siCRT). After transfection, cells were harvested for quantitative real-time (qRT)-PCR ( A ) and Western blot analysis ( B ). Data presented are the mean of at least 3 independent experiments. Error bars indicate SD. ** P

    Techniques Used: Expressing, Transfection, Small Interfering RNA, Quantitative RT-PCR, Western Blot

    45) Product Images from "A small set of conserved genes, including sp5 and Hox, are activated by Wnt signaling in the posterior of planarians and acoels"

    Article Title: A small set of conserved genes, including sp5 and Hox, are activated by Wnt signaling in the posterior of planarians and acoels

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008401

    A posterior program of gene expression is acutely down-regulated after β-catenin-1 inhibition in planarians. (A) Experimental scheme for RNA sequencing. Schmidtea mediterranea adults were fed once with control or β-catenin-1 dsRNA. Tails were collected in trizol 1, 2, 4, and 6 days after feeding for RNA sequencing. Red box indicates tissue collected. (B) A cluster of 52 genes is down-regulated early after β-catenin-1 RNAi. Heatmap displaying gene expression counts as z-scores for indicated timepoints post-RNAi feeding. *indicates annotation by best BLAST hit. Gene list provided in S2 Table . (C) Significant down-regulation of gene expression begins at day 1 post- β-catenin-1 RNAi. Mean z-score of gene expression counts for 52 genes in 1(B) at indicated time points post-RNAi feeding. Data is plotted as mean ± S.D.**p
    Figure Legend Snippet: A posterior program of gene expression is acutely down-regulated after β-catenin-1 inhibition in planarians. (A) Experimental scheme for RNA sequencing. Schmidtea mediterranea adults were fed once with control or β-catenin-1 dsRNA. Tails were collected in trizol 1, 2, 4, and 6 days after feeding for RNA sequencing. Red box indicates tissue collected. (B) A cluster of 52 genes is down-regulated early after β-catenin-1 RNAi. Heatmap displaying gene expression counts as z-scores for indicated timepoints post-RNAi feeding. *indicates annotation by best BLAST hit. Gene list provided in S2 Table . (C) Significant down-regulation of gene expression begins at day 1 post- β-catenin-1 RNAi. Mean z-score of gene expression counts for 52 genes in 1(B) at indicated time points post-RNAi feeding. Data is plotted as mean ± S.D.**p

    Techniques Used: Expressing, Inhibition, RNA Sequencing Assay

    A posterior program of gene expression is down-regulated after β-catenin-1 inhibition in acoels. (A) Experimental scheme for RNA sequencing. Hofstenia miamia were injected once a day for three consecutive days with control or β-catenin-1 dsRNA. Tails were collected in trizol at 3, 7, and 14 days after the first injection for RNA sequencing. Red box indicates tissue collected. (B) A cluster of 66 genes is down-regulated early after β-catenin-1 RNAi. Heatmap displaying gene expression counts as z-scores for indicated timepoints post first RNAi injection. *indicates annotation by best BLAST hit. Gene list provided in S5 Table . (C) Left: Validation of β-catenin-1 RNAi-dependent down-regulation of gene expression. FISH for indicated transcripts at day 9 post-RNAi initiation in homeostasis. Images are presented in grayscale with color inverted. Right: Genes down-regulated after β-catenin-1 RNAi are expressed in the posterior. FISH for indicated transcripts in two week old hatchlings. Transcript ID provided in parentheses. Images are presented in grayscale with color inverted. Scale bars, 200μm.
    Figure Legend Snippet: A posterior program of gene expression is down-regulated after β-catenin-1 inhibition in acoels. (A) Experimental scheme for RNA sequencing. Hofstenia miamia were injected once a day for three consecutive days with control or β-catenin-1 dsRNA. Tails were collected in trizol at 3, 7, and 14 days after the first injection for RNA sequencing. Red box indicates tissue collected. (B) A cluster of 66 genes is down-regulated early after β-catenin-1 RNAi. Heatmap displaying gene expression counts as z-scores for indicated timepoints post first RNAi injection. *indicates annotation by best BLAST hit. Gene list provided in S5 Table . (C) Left: Validation of β-catenin-1 RNAi-dependent down-regulation of gene expression. FISH for indicated transcripts at day 9 post-RNAi initiation in homeostasis. Images are presented in grayscale with color inverted. Right: Genes down-regulated after β-catenin-1 RNAi are expressed in the posterior. FISH for indicated transcripts in two week old hatchlings. Transcript ID provided in parentheses. Images are presented in grayscale with color inverted. Scale bars, 200μm.

    Techniques Used: Expressing, Inhibition, RNA Sequencing Assay, Injection, Fluorescence In Situ Hybridization

    An anterior program of gene expression is acutely up-regulated after β-catenin-1 inhibition in planarians. (A) Experimental scheme for RNA sequencing. Schmidtea mediterranea adults were fed once with control or β-catenin-1 dsRNA. Tails were collected in trizol 1, 2, 4, and 6 days after feeding for RNA sequencing. Red box indicates tissue collected. (B) 56 genes are up-regulated early after β-catenin-1 RNAi in planarian tails. Heatmap displaying gene expression counts as z-scores for indicated timepoints post-RNAi feeding. *indicates annotation by best BLAST hit. Gene list provided in S2 Table . (C) Up-regulation of gene expression begins by day 2 post- β-catenin-1 RNAi. Mean z-score of gene expression counts is plotted for 56 genes up-regulated in 2(B) at indicated time points post-RNAi feeding. Data is plotted as mean ± S.D. *p
    Figure Legend Snippet: An anterior program of gene expression is acutely up-regulated after β-catenin-1 inhibition in planarians. (A) Experimental scheme for RNA sequencing. Schmidtea mediterranea adults were fed once with control or β-catenin-1 dsRNA. Tails were collected in trizol 1, 2, 4, and 6 days after feeding for RNA sequencing. Red box indicates tissue collected. (B) 56 genes are up-regulated early after β-catenin-1 RNAi in planarian tails. Heatmap displaying gene expression counts as z-scores for indicated timepoints post-RNAi feeding. *indicates annotation by best BLAST hit. Gene list provided in S2 Table . (C) Up-regulation of gene expression begins by day 2 post- β-catenin-1 RNAi. Mean z-score of gene expression counts is plotted for 56 genes up-regulated in 2(B) at indicated time points post-RNAi feeding. Data is plotted as mean ± S.D. *p

    Techniques Used: Expressing, Inhibition, RNA Sequencing Assay

    An anterior program of gene expression is up-regulated after β-catenin-1 inhibition in acoels. (A) Experimental scheme for RNA sequencing. Hofstenia miamia were injected once a day for three consecutive days with control or β-catenin-1 dsRNA. Tails were collected in trizol at 3, 7, and 14 days after the first injection for RNA sequencing. Red box indicates tissue collected. (B) 92 genes are up-regulated early after β-catenin-1 RNAi. Heatmap displaying gene expression counts as a z-score for indicated time-points post-RNAi initiation. *indicates annotation by best BLAST hit. Gene list provided in S5 Table . (C) Top: Validation of β-catenin-1 RNAi dependent up-regulation of gene expression. FISH for indicated transcripts at day 9 post-RNAi initiation in homeostasis. Images are presented in grayscale with color inverted. Bottom: Genes up-regulated after β-catenin-1 RNAi are expressed in the anterior. FISH for indicated transcripts in two week old hatchlings. Transcript ID provided in parentheses. Images are presented in grayscale with color inverted. All scale bars, 200μm.
    Figure Legend Snippet: An anterior program of gene expression is up-regulated after β-catenin-1 inhibition in acoels. (A) Experimental scheme for RNA sequencing. Hofstenia miamia were injected once a day for three consecutive days with control or β-catenin-1 dsRNA. Tails were collected in trizol at 3, 7, and 14 days after the first injection for RNA sequencing. Red box indicates tissue collected. (B) 92 genes are up-regulated early after β-catenin-1 RNAi. Heatmap displaying gene expression counts as a z-score for indicated time-points post-RNAi initiation. *indicates annotation by best BLAST hit. Gene list provided in S5 Table . (C) Top: Validation of β-catenin-1 RNAi dependent up-regulation of gene expression. FISH for indicated transcripts at day 9 post-RNAi initiation in homeostasis. Images are presented in grayscale with color inverted. Bottom: Genes up-regulated after β-catenin-1 RNAi are expressed in the anterior. FISH for indicated transcripts in two week old hatchlings. Transcript ID provided in parentheses. Images are presented in grayscale with color inverted. All scale bars, 200μm.

    Techniques Used: Expressing, Inhibition, RNA Sequencing Assay, Injection, Fluorescence In Situ Hybridization

    46) Product Images from "Influenza A Virus PA Antagonizes Interferon-β by Interacting with Interferon Regulatory Factor 3"

    Article Title: Influenza A Virus PA Antagonizes Interferon-β by Interacting with Interferon Regulatory Factor 3

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01051

    The binding activity of pdm/09 polymerase acid protein (PA) to interferon regulatory factor 3 (IRF3) is dependent on Asp108. (A) 293T cells in 12-well plates were transfected with 0.5 µg of Flag-tagged pdm/09 PA, pdm/09 PA-D108A mutant or empty vector, together with 0.3 µg of interferon-β (IFN-β)-luc and 0.02 µg of RL-TK. After 24 h, cells were infected with Sendai virus (SEV) or were left uninfected for 8 h and were then lysed for the luciferase assay. (B) A549 cells in 6-well plates were transfected with 2 µg of Flag-tagged pdm/09 PA, pdm/09 PA-D108A mutant or an empty vector. After 24 h, cells were infected with SEV or were left uninfected for 8 h. The cells were harvested, and total RNA was extracted for detection of IFN-β, CXCL-10, ISG-15, and ISG-56 expression levels by real-time q-PCR. (C) 293T cells in 6-well plates were transfected with 2 µg of Flag-tagged pdm/09 PA, pdm/09 PA-D108A mutant or an empty vector, and HA-tagged IRF3. After 24 h, cells were lysed and precipitated with anti-Flag antibody. The cell lysates and immunoprecipitates (IPs) were analyzed by Western blotting using anti-Flag and anti-HA antibodies. The bars represent the SEs of the means, based on three experiments. * p
    Figure Legend Snippet: The binding activity of pdm/09 polymerase acid protein (PA) to interferon regulatory factor 3 (IRF3) is dependent on Asp108. (A) 293T cells in 12-well plates were transfected with 0.5 µg of Flag-tagged pdm/09 PA, pdm/09 PA-D108A mutant or empty vector, together with 0.3 µg of interferon-β (IFN-β)-luc and 0.02 µg of RL-TK. After 24 h, cells were infected with Sendai virus (SEV) or were left uninfected for 8 h and were then lysed for the luciferase assay. (B) A549 cells in 6-well plates were transfected with 2 µg of Flag-tagged pdm/09 PA, pdm/09 PA-D108A mutant or an empty vector. After 24 h, cells were infected with SEV or were left uninfected for 8 h. The cells were harvested, and total RNA was extracted for detection of IFN-β, CXCL-10, ISG-15, and ISG-56 expression levels by real-time q-PCR. (C) 293T cells in 6-well plates were transfected with 2 µg of Flag-tagged pdm/09 PA, pdm/09 PA-D108A mutant or an empty vector, and HA-tagged IRF3. After 24 h, cells were lysed and precipitated with anti-Flag antibody. The cell lysates and immunoprecipitates (IPs) were analyzed by Western blotting using anti-Flag and anti-HA antibodies. The bars represent the SEs of the means, based on three experiments. * p

    Techniques Used: Binding Assay, Activity Assay, Transfection, Mutagenesis, Plasmid Preparation, Infection, Luciferase, Expressing, Polymerase Chain Reaction, Western Blot

    Interferon-β (IFN-β) induction was suppressed by the N-terminus of pdm/09 polymerase acid protein (PA). (A) 293T cells in 12-well plates were transfected with 0.5 µg of Flag-tagged pdm/09 PA, pdm/09 N-terminal PA fragment (PAN), pdm/09 C-terminal PA fragment (PAC), or an empty vector, together with 0.3 µg of IFN-β-luc and 0.02 µg of RL-TK. After 24 h, cells were infected with SEV or were left uninfected for 8 h and then they were lysed for the luciferase assay. (B) A549 cells in 6-well plates were transfected with 1 µg of Flag-tagged pdm/09 PA, pdm/09 PAN, pdm/09 PAC, or empty vector. After 24 h, cells were infected with SEV for 8 h and subsequently harvested, and total RNA was then extracted for detection of IFN-β and ISG-56 expression levels by real-time q-PCR. (C) 293T cells in 12-well plates were transfected with HA-tagged PAN and PAC in increasing quantities and co-transfected with 0.3 µg of IFN-β-luc and 0.02 µg of RL-TK. After 24 h, cells were infected with SEV for 8 h and then lysed for use in the luciferase assay. (D) 293T cells in 6-well plates were transfected with 2 µg of HA-tagged pdm/09 PA, pdm/09 PAN, pdm/09 PAC, or an empty vector, and Flag-tagged IRF3. After 24 h, cells were lysed and precipitated with anti-Flag antibody. The cell lysates and IPs were analyzed by Western blotting using anti-Flag and anti-HA antibodies. (E) 293T cells were seeded onto coverslips and placed into 12-well plates and were transfected with Flag-tagged pdm/09 PA, pdm/09 PAN, pdm/09 PAC, or empty vector. After 24 h, cells were infected with SEV or were left uninfected for 8 h and were fixed for IFA, with endogenous IRF3 (red) and nuclei (blue) shown with anti-IRF3 antibody and DAPI via confocal microscopy. The bars represent the SEs of the means, based on three experiments. * p
    Figure Legend Snippet: Interferon-β (IFN-β) induction was suppressed by the N-terminus of pdm/09 polymerase acid protein (PA). (A) 293T cells in 12-well plates were transfected with 0.5 µg of Flag-tagged pdm/09 PA, pdm/09 N-terminal PA fragment (PAN), pdm/09 C-terminal PA fragment (PAC), or an empty vector, together with 0.3 µg of IFN-β-luc and 0.02 µg of RL-TK. After 24 h, cells were infected with SEV or were left uninfected for 8 h and then they were lysed for the luciferase assay. (B) A549 cells in 6-well plates were transfected with 1 µg of Flag-tagged pdm/09 PA, pdm/09 PAN, pdm/09 PAC, or empty vector. After 24 h, cells were infected with SEV for 8 h and subsequently harvested, and total RNA was then extracted for detection of IFN-β and ISG-56 expression levels by real-time q-PCR. (C) 293T cells in 12-well plates were transfected with HA-tagged PAN and PAC in increasing quantities and co-transfected with 0.3 µg of IFN-β-luc and 0.02 µg of RL-TK. After 24 h, cells were infected with SEV for 8 h and then lysed for use in the luciferase assay. (D) 293T cells in 6-well plates were transfected with 2 µg of HA-tagged pdm/09 PA, pdm/09 PAN, pdm/09 PAC, or an empty vector, and Flag-tagged IRF3. After 24 h, cells were lysed and precipitated with anti-Flag antibody. The cell lysates and IPs were analyzed by Western blotting using anti-Flag and anti-HA antibodies. (E) 293T cells were seeded onto coverslips and placed into 12-well plates and were transfected with Flag-tagged pdm/09 PA, pdm/09 PAN, pdm/09 PAC, or empty vector. After 24 h, cells were infected with SEV or were left uninfected for 8 h and were fixed for IFA, with endogenous IRF3 (red) and nuclei (blue) shown with anti-IRF3 antibody and DAPI via confocal microscopy. The bars represent the SEs of the means, based on three experiments. * p

    Techniques Used: Transfection, Plasmid Preparation, Infection, Luciferase, Expressing, Polymerase Chain Reaction, Western Blot, Immunofluorescence, Confocal Microscopy

    47) Product Images from "miRNA-148a regulates the expression of the estrogen receptor through DNMT1-mediated DNA methylation in breast cancer cells"

    Article Title: miRNA-148a regulates the expression of the estrogen receptor through DNMT1-mediated DNA methylation in breast cancer cells

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.6803

    DNMT1 is a target of miRNA-148a. (A) TargetScan was used to predict miRNA-148a target genes. The DNMT1 gene 3′UTR included 7 sequential pairing bases with the 5′ of miRNR-148a, indicating that DNMT1 may be a potential target of miRNA-148a. (B) In order to confirm that DNMT1 is a target, an miRNA-148a mimic or inhibitor was transfected into MCF7 cells. At 48 h after transfection, the detection of DNMT protein levels by western blot analysis indicated that the expression of DNMT1 decreased upon miRNA-148a mimic transfection and increased upon miRNA-148a inhibitor transfection. DNMT1, DNA methyltransferase 1; miRNA, micro RNA; 3′UTR, 3′ untranslated region.
    Figure Legend Snippet: DNMT1 is a target of miRNA-148a. (A) TargetScan was used to predict miRNA-148a target genes. The DNMT1 gene 3′UTR included 7 sequential pairing bases with the 5′ of miRNR-148a, indicating that DNMT1 may be a potential target of miRNA-148a. (B) In order to confirm that DNMT1 is a target, an miRNA-148a mimic or inhibitor was transfected into MCF7 cells. At 48 h after transfection, the detection of DNMT protein levels by western blot analysis indicated that the expression of DNMT1 decreased upon miRNA-148a mimic transfection and increased upon miRNA-148a inhibitor transfection. DNMT1, DNA methyltransferase 1; miRNA, micro RNA; 3′UTR, 3′ untranslated region.

    Techniques Used: Transfection, Western Blot, Expressing

    48) Product Images from "An endoplasmic reticulum stress-regulated lncRNA hosting a microRNA megacluster induces early features of diabetic nephropathy"

    Article Title: An endoplasmic reticulum stress-regulated lncRNA hosting a microRNA megacluster induces early features of diabetic nephropathy

    Journal: Nature Communications

    doi: 10.1038/ncomms12864

    The miR-379 miRNA megacluster is increased in the glomeruli of diabetic mice. Small RNA (smRNA) sequencing was performed as previously described 22 . ( a ) Scatter plot of miRNAs in kidney glomeruli from control (CTR, vehicle injected) and diabetic mice (STZ injected; 4 weeks post diabetes). The expression of each detectable miRNA in the form of log scaled reads was plotted with x axis for CTR and y axis for STZ. Each dot represents one miRNA. miRNAs in the miR-379 cluster were presented in red. Among these, the miRNAs upregulated by fold change ≥2 are highlighted with bigger size dots and labelled with the corresponding miRNA names. miR-882 was plotted in blue as a negative control (outside the miR-379 cluster). ( b ) Heatmap of the miR-379 cluster miRNAs in CTR and STZ samples. The expression of each detectable miRNA within the miR-379 cluster in the two samples (CTR and STZ) were ordered by log2 fold change from low to high, mean-centered and shown in the heatmap. Blue represents lower than average expression in the two samples and yellow presents higher than average expression level. The expression of the detected miRNAs in this cluster was higher in STZ than CTR. ( c ) Genome structure of the mouse miR-379 megacluster region. This cluster is located within the largest miRNA cluster currently identified in the genome. It maps within the DLK-DIO3 genomic region (mouse chr 12, human chr14), which is home to several miRNAs and lncRNAs. TSS, transcription start site. miR-882 is located far-upstream of the miR-379 cluster and not covered by lnc-MGC. ( d ) Gene Set Enrichment Analysis (GSEA). All the miRNAs detected by smRNA-seq with at least 5 scaled reads in at least one sample are ranked by log2 fold change between STZ and CTR samples to generate ranked list and all the detectable miRNAs in the cluster are considered as a gene set. Pre-ranked gene set analysis (GSEA) applied on the gene set using the ranked list of all the miRNAs revealed that miRNAs in the miR-379 cluster were significantly enriched within the miRNAs upregulated in the STZ diabetic mice, with normalized enrichment score of 1.56 ( P =0.004).
    Figure Legend Snippet: The miR-379 miRNA megacluster is increased in the glomeruli of diabetic mice. Small RNA (smRNA) sequencing was performed as previously described 22 . ( a ) Scatter plot of miRNAs in kidney glomeruli from control (CTR, vehicle injected) and diabetic mice (STZ injected; 4 weeks post diabetes). The expression of each detectable miRNA in the form of log scaled reads was plotted with x axis for CTR and y axis for STZ. Each dot represents one miRNA. miRNAs in the miR-379 cluster were presented in red. Among these, the miRNAs upregulated by fold change ≥2 are highlighted with bigger size dots and labelled with the corresponding miRNA names. miR-882 was plotted in blue as a negative control (outside the miR-379 cluster). ( b ) Heatmap of the miR-379 cluster miRNAs in CTR and STZ samples. The expression of each detectable miRNA within the miR-379 cluster in the two samples (CTR and STZ) were ordered by log2 fold change from low to high, mean-centered and shown in the heatmap. Blue represents lower than average expression in the two samples and yellow presents higher than average expression level. The expression of the detected miRNAs in this cluster was higher in STZ than CTR. ( c ) Genome structure of the mouse miR-379 megacluster region. This cluster is located within the largest miRNA cluster currently identified in the genome. It maps within the DLK-DIO3 genomic region (mouse chr 12, human chr14), which is home to several miRNAs and lncRNAs. TSS, transcription start site. miR-882 is located far-upstream of the miR-379 cluster and not covered by lnc-MGC. ( d ) Gene Set Enrichment Analysis (GSEA). All the miRNAs detected by smRNA-seq with at least 5 scaled reads in at least one sample are ranked by log2 fold change between STZ and CTR samples to generate ranked list and all the detectable miRNAs in the cluster are considered as a gene set. Pre-ranked gene set analysis (GSEA) applied on the gene set using the ranked list of all the miRNAs revealed that miRNAs in the miR-379 cluster were significantly enriched within the miRNAs upregulated in the STZ diabetic mice, with normalized enrichment score of 1.56 ( P =0.004).

    Techniques Used: Mouse Assay, Sequencing, Injection, Expressing, Negative Control

    49) Product Images from "Eicosapentaenoic acid prevents arterial calcification in klotho mutant mice"

    Article Title: Eicosapentaenoic acid prevents arterial calcification in klotho mutant mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0181009

    GPR120 and NADPH oxidase (NOX) in arterial smooth muscle cells. A, Expression of the GPR120 gene. Lane 1, maker; lane 2, wild-type (WT) mouse; lane 3, klotho mutant ( kl/kl) mouse and lane 4, C57/BL6 mouse (positive control). B, GPR120 downregulation by siRNA. Lane 1, si-GPR120 RNA and lane 2, s-scramble RNA. C, Effects of GPR120 gene knockdown on NOX4 gene expression and NOX activity in arterial SMCs of kl/kl mice (n = 6, each).
    Figure Legend Snippet: GPR120 and NADPH oxidase (NOX) in arterial smooth muscle cells. A, Expression of the GPR120 gene. Lane 1, maker; lane 2, wild-type (WT) mouse; lane 3, klotho mutant ( kl/kl) mouse and lane 4, C57/BL6 mouse (positive control). B, GPR120 downregulation by siRNA. Lane 1, si-GPR120 RNA and lane 2, s-scramble RNA. C, Effects of GPR120 gene knockdown on NOX4 gene expression and NOX activity in arterial SMCs of kl/kl mice (n = 6, each).

    Techniques Used: Expressing, Mutagenesis, Positive Control, Activity Assay, Mouse Assay

    50) Product Images from "Secreted and Tissue miRNAs as Diagnosis Biomarkers of Malignant Pleural Mesothelioma"

    Article Title: Secreted and Tissue miRNAs as Diagnosis Biomarkers of Malignant Pleural Mesothelioma

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19020595

    Biogenesis of miRNAs in the cell. miRNA precursors (pre-miRNAs) are transcribed in the nucleus and processed by the Drosha complex to generate pre-miRNAs. Pre-miRNAs are exported to the cytoplasm via exportin-5 and excised by DICER into a mature form of double-stranded RNA ~22 nt long. Double-stranded RNA is loaded onto AGO2 that is the catalytic component of the miRISC complex. One strand is removed from the duplex (the passenger strand) and the remaining RNA strand (the guide strand) binds to complementary sequences typically located in the 3’ untranslated region (UTR) of target mRNAs to repress translation or trigger mRNA cleavage.
    Figure Legend Snippet: Biogenesis of miRNAs in the cell. miRNA precursors (pre-miRNAs) are transcribed in the nucleus and processed by the Drosha complex to generate pre-miRNAs. Pre-miRNAs are exported to the cytoplasm via exportin-5 and excised by DICER into a mature form of double-stranded RNA ~22 nt long. Double-stranded RNA is loaded onto AGO2 that is the catalytic component of the miRISC complex. One strand is removed from the duplex (the passenger strand) and the remaining RNA strand (the guide strand) binds to complementary sequences typically located in the 3’ untranslated region (UTR) of target mRNAs to repress translation or trigger mRNA cleavage.

    Techniques Used:

    Mechanisms of secretion of miRNAs. ( A ) Secretion of the miRNAs associated with Argonaute2 protein (AGO2); ( B ) Secretion of the miRNAs by exosomes; ( C ) Secretion of the miRNAs associated with high-density lipoprotein (HDL); ( D ) Secretion of the miRNAs associated with the RNA binding protein nucleophosmin (NPM1). ( A ) miRNAs associated with AGO2, a main component of the RISC, can be stably exported into plasma samples. ( B ) miRNAs are sorted into multivesicular bodies (MVBs) derived from early endosomes. This mechanism requires ceramide production on the cytosolic side by neutral sphingomyelinase 2 (nSMase2), ESCRT machinery, and sumoylated hnRNPA2B1 protein, which specifically binds mature miRNAs and controls their loading into MVBs. MVBs are enriched with GW182 and AGO2, which are known to regulate the function of miRNAs. MVBs fuse with the plasma membrane and release exosomes into the extracellular medium. ( C ) miRNAs bound to HDL also can be stably exported into plasma samples via a mechanism repressed by nSMase2. ( D ) NPM1 binds miRNAs from the culture supernatants of tumor cell lines and fibroblasts while protecting them from RNase activity.
    Figure Legend Snippet: Mechanisms of secretion of miRNAs. ( A ) Secretion of the miRNAs associated with Argonaute2 protein (AGO2); ( B ) Secretion of the miRNAs by exosomes; ( C ) Secretion of the miRNAs associated with high-density lipoprotein (HDL); ( D ) Secretion of the miRNAs associated with the RNA binding protein nucleophosmin (NPM1). ( A ) miRNAs associated with AGO2, a main component of the RISC, can be stably exported into plasma samples. ( B ) miRNAs are sorted into multivesicular bodies (MVBs) derived from early endosomes. This mechanism requires ceramide production on the cytosolic side by neutral sphingomyelinase 2 (nSMase2), ESCRT machinery, and sumoylated hnRNPA2B1 protein, which specifically binds mature miRNAs and controls their loading into MVBs. MVBs are enriched with GW182 and AGO2, which are known to regulate the function of miRNAs. MVBs fuse with the plasma membrane and release exosomes into the extracellular medium. ( C ) miRNAs bound to HDL also can be stably exported into plasma samples via a mechanism repressed by nSMase2. ( D ) NPM1 binds miRNAs from the culture supernatants of tumor cell lines and fibroblasts while protecting them from RNase activity.

    Techniques Used: RNA Binding Assay, Stable Transfection, Derivative Assay, Activity Assay

    51) Product Images from "Identification of genes directly responding to DLK1 signaling in Callipyge sheep"

    Article Title: Identification of genes directly responding to DLK1 signaling in Callipyge sheep

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-4682-1

    Transcript abundance of genes from the DLK1-DIO3 locus. a DLK1 ; b RTL1 ; c MEG8 and d MEG3. Least square means and standard errors for log transcript abundance are shown for each muscle and genotype, callipyge (+/C) and normal (+/+). The hypertrophied muscles are LD, SM, ST, and TB and the non-hypertrophied muscles are SS, IS and HT. The increased expression of paternally allele-specific genes DLK1 and RTL1 , and the maternal allele-specific non-coding RNA MEG8 and MEG3 in callipyge hypertrophied muscles are shown. Significant differences are indicated by (*; P
    Figure Legend Snippet: Transcript abundance of genes from the DLK1-DIO3 locus. a DLK1 ; b RTL1 ; c MEG8 and d MEG3. Least square means and standard errors for log transcript abundance are shown for each muscle and genotype, callipyge (+/C) and normal (+/+). The hypertrophied muscles are LD, SM, ST, and TB and the non-hypertrophied muscles are SS, IS and HT. The increased expression of paternally allele-specific genes DLK1 and RTL1 , and the maternal allele-specific non-coding RNA MEG8 and MEG3 in callipyge hypertrophied muscles are shown. Significant differences are indicated by (*; P

    Techniques Used: Expressing

    52) Product Images from "Inhibition of endo-lysosomal function exacerbates vascular calcification"

    Article Title: Inhibition of endo-lysosomal function exacerbates vascular calcification

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17540-6

    LAMP2 is increased in calcifying rodent aortas. Rat aortic calcification was induced by subcutaneous injection with 1.2 × 10 5 IU/kg/day vitamin D 3 or vehicle for 3 days. After total 14 days, aortas were dissected and total RNA was isolated. qPCR results revealed that LAMP2 was increased concomitant with osteogenic transformation showing increased RUNX2 and BMP2 , and decreased TAGLN . Six rats for each group were analyzed. Values are presented as mean ± SEM. * P
    Figure Legend Snippet: LAMP2 is increased in calcifying rodent aortas. Rat aortic calcification was induced by subcutaneous injection with 1.2 × 10 5 IU/kg/day vitamin D 3 or vehicle for 3 days. After total 14 days, aortas were dissected and total RNA was isolated. qPCR results revealed that LAMP2 was increased concomitant with osteogenic transformation showing increased RUNX2 and BMP2 , and decreased TAGLN . Six rats for each group were analyzed. Values are presented as mean ± SEM. * P

    Techniques Used: Injection, Isolation, Real-time Polymerase Chain Reaction, Transformation Assay

    53) Product Images from "Transcriptome analysis of mRNA and microRNAs in intramuscular fat tissues of castrated and intact male Chinese Qinchuan cattle"

    Article Title: Transcriptome analysis of mRNA and microRNAs in intramuscular fat tissues of castrated and intact male Chinese Qinchuan cattle

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0185961

    Differential miRNAs expression between steers and bulls. A: Length distribution of the clean reads. Most of the reads had a length of 21–22 nt. B: Number and percentage of identified miRNAs. A total of 5,530 unique miRNAs were identified. C: The Global miRNA expression profile of the IMF tissue from steers and bulls was analyzed by RNA-seq analysis. The heat map shows the differentially expressed (DE) miRNA patterns (|log2 (FC)| > 1and FDR
    Figure Legend Snippet: Differential miRNAs expression between steers and bulls. A: Length distribution of the clean reads. Most of the reads had a length of 21–22 nt. B: Number and percentage of identified miRNAs. A total of 5,530 unique miRNAs were identified. C: The Global miRNA expression profile of the IMF tissue from steers and bulls was analyzed by RNA-seq analysis. The heat map shows the differentially expressed (DE) miRNA patterns (|log2 (FC)| > 1and FDR

    Techniques Used: Expressing, RNA Sequencing Assay

    54) Product Images from "Overexpression of SphK2 contributes to ATRA resistance in colon cancer through rapid degradation of cytoplasmic RXRα by K48/K63-linked polyubiquitination"

    Article Title: Overexpression of SphK2 contributes to ATRA resistance in colon cancer through rapid degradation of cytoplasmic RXRα by K48/K63-linked polyubiquitination

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17174

    Sphk2 enhances the ATRA-induced degradation of RARβ and RXRα in HCT-116 Sphk2 cells (A) Western blots of cells exposed to vehicle or ATRA (5 μM) for 24 h to analyze the expression of RARβ and RXRα. (B) Western blots of xenografts of HCT-116 Sphk2 or HCT-116 removed from nude mice treated with ATRA for three weeks. Reverse transcriptase and qPCR assays measured RXRα (C) and RARβ mRNA. (D) HCT-116 Sphk2 or HCT-116 cells were exposed to ATRA (5 μM) for 24 h and then total RNA was extracted for reverse transcription and qPCR assays to determine the level of RXRα and RARβ mRNAs. The bars indicate means ± S.D (n = 3). *, p
    Figure Legend Snippet: Sphk2 enhances the ATRA-induced degradation of RARβ and RXRα in HCT-116 Sphk2 cells (A) Western blots of cells exposed to vehicle or ATRA (5 μM) for 24 h to analyze the expression of RARβ and RXRα. (B) Western blots of xenografts of HCT-116 Sphk2 or HCT-116 removed from nude mice treated with ATRA for three weeks. Reverse transcriptase and qPCR assays measured RXRα (C) and RARβ mRNA. (D) HCT-116 Sphk2 or HCT-116 cells were exposed to ATRA (5 μM) for 24 h and then total RNA was extracted for reverse transcription and qPCR assays to determine the level of RXRα and RARβ mRNAs. The bars indicate means ± S.D (n = 3). *, p

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

    55) Product Images from "Zbtb38 is a novel target for spinal cord injury"

    Article Title: Zbtb38 is a novel target for spinal cord injury

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17487

    Restoration of Zbtb38 expression attenuates SCI-induced apoptosis in vivo RNA was collected from the samples used in Figure 4 for QRT-PCR analysis. * p
    Figure Legend Snippet: Restoration of Zbtb38 expression attenuates SCI-induced apoptosis in vivo RNA was collected from the samples used in Figure 4 for QRT-PCR analysis. * p

    Techniques Used: Expressing, In Vivo, Quantitative RT-PCR

    Decreased Zbtb38 expression levels are associated with increased ER stress-associated apoptosis in SCI mice ( A ) Kunming mice were divided into two groups underwent sham injuries (Sham) and SCI, respectively ( n = 10). The spinal cord samples were harvested from these mice on the day 4 after injury and cell lysates were collected for Western blot analysis. 1, 2, 3 indicate three different mice in each group. ( B, C ) RNA from these samples was collected for QRT-PCR analysis to determine the expression levels of ER stress markers (B) and pro-apoptotic genes (C). * p
    Figure Legend Snippet: Decreased Zbtb38 expression levels are associated with increased ER stress-associated apoptosis in SCI mice ( A ) Kunming mice were divided into two groups underwent sham injuries (Sham) and SCI, respectively ( n = 10). The spinal cord samples were harvested from these mice on the day 4 after injury and cell lysates were collected for Western blot analysis. 1, 2, 3 indicate three different mice in each group. ( B, C ) RNA from these samples was collected for QRT-PCR analysis to determine the expression levels of ER stress markers (B) and pro-apoptotic genes (C). * p

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

    ATF4 regulates Zbtb38 expression in SCI animal model through direct binding ( A ) The cartoon showed the Zbtb38 promoter region upstream of the start codon contains a putative ATF4 and ATF6 binding sites. ( B, C ) The cell lysates from spinal cord samples were pulled down by ATF4 (B) or ATF6 (C) antibody and qPCR primers were used to amplify indicated different regions within the Zbtb38 promoter. The level of enrichment of the indicated DNA sequence was determined relative to the total amount of input DNA (% input). ( D ) SH-SY5Y cells were transfected with scramble siRNA (siCONT) or siRNA against ATF4 (siATF4-1, siATF4-2 and siATF4-3) for 72 hours and RNA was collected from these cells for QRT-PCR analysis. ** p
    Figure Legend Snippet: ATF4 regulates Zbtb38 expression in SCI animal model through direct binding ( A ) The cartoon showed the Zbtb38 promoter region upstream of the start codon contains a putative ATF4 and ATF6 binding sites. ( B, C ) The cell lysates from spinal cord samples were pulled down by ATF4 (B) or ATF6 (C) antibody and qPCR primers were used to amplify indicated different regions within the Zbtb38 promoter. The level of enrichment of the indicated DNA sequence was determined relative to the total amount of input DNA (% input). ( D ) SH-SY5Y cells were transfected with scramble siRNA (siCONT) or siRNA against ATF4 (siATF4-1, siATF4-2 and siATF4-3) for 72 hours and RNA was collected from these cells for QRT-PCR analysis. ** p

    Techniques Used: Expressing, Animal Model, Binding Assay, Real-time Polymerase Chain Reaction, Sequencing, Transfection, Quantitative RT-PCR

    ER stress triggers Zbtb38-mediated apoptosis in SH-SY5Y cells ( A ) SH-SY5Y cells were treated with 1 μM TG for 24 hours and RNA was collected for QRT-PCR analysis. ( B ) Apoptosis of SH-SY5Y cells in the presence or absence of TG was determined by The Cell Death Detection Elisa Kit. ( C ) SH-SY5Y cells were transfected with scramble shRNA (shNC-1 and shNC-2) or shRNA against Zbtb38 (shZbtb38-1 and shZbtb38-2) for 72 hours and cell lysates were collected for Western blot analysis. ( D ) RNA from Zbtb38 knockdown SH-SY5Y cells (shZbtb38) and control cells (shNC) was collected for QRT-PCR analysis to determine the expression levels of pro-apoptotic genes. * p
    Figure Legend Snippet: ER stress triggers Zbtb38-mediated apoptosis in SH-SY5Y cells ( A ) SH-SY5Y cells were treated with 1 μM TG for 24 hours and RNA was collected for QRT-PCR analysis. ( B ) Apoptosis of SH-SY5Y cells in the presence or absence of TG was determined by The Cell Death Detection Elisa Kit. ( C ) SH-SY5Y cells were transfected with scramble shRNA (shNC-1 and shNC-2) or shRNA against Zbtb38 (shZbtb38-1 and shZbtb38-2) for 72 hours and cell lysates were collected for Western blot analysis. ( D ) RNA from Zbtb38 knockdown SH-SY5Y cells (shZbtb38) and control cells (shNC) was collected for QRT-PCR analysis to determine the expression levels of pro-apoptotic genes. * p

    Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection, shRNA, Western Blot, Expressing

    56) Product Images from "HVC1 ameliorates hyperlipidemia and inflammation in LDLR−/− mice"

    Article Title: HVC1 ameliorates hyperlipidemia and inflammation in LDLR−/− mice

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/s12906-017-1734-z

    HVC1 regulation of cholesterol metabolism and lipid synthesis in LDLR −/− mice. Total RNA was subjected to real-time PCR as described in the Methods section. Cholesterol and lipid metabolism-related genes mRNA levels were analyzed by real-time PCR analysis. ( a ) SREBP-2, ( b ) HMGCR, ( c ) LPL, ( d ) apoB, and ( e ) LXR. Protein levels of ( f ) SREBP-2, ( g ) HMGCR, (H) p-AMPK, and AMPK in liver tissues were analyzed by western blot. Proteins were determined by western blot assay using specific antibody. β-actin was used as a loading control. Values represent mean ± S.D. of three independent experiments (significant as compared to HCD, * p
    Figure Legend Snippet: HVC1 regulation of cholesterol metabolism and lipid synthesis in LDLR −/− mice. Total RNA was subjected to real-time PCR as described in the Methods section. Cholesterol and lipid metabolism-related genes mRNA levels were analyzed by real-time PCR analysis. ( a ) SREBP-2, ( b ) HMGCR, ( c ) LPL, ( d ) apoB, and ( e ) LXR. Protein levels of ( f ) SREBP-2, ( g ) HMGCR, (H) p-AMPK, and AMPK in liver tissues were analyzed by western blot. Proteins were determined by western blot assay using specific antibody. β-actin was used as a loading control. Values represent mean ± S.D. of three independent experiments (significant as compared to HCD, * p

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

    Inhibitory effects of HVC1 on mRNA expression of inflammatory cytokines in HCD induced LDLR −/− mice. Inflammatory cytokine genes mRNA levels were analyzed by real-time PCR analysis. ( a ) TNF-α, ( b ) IL-1β, ( c ) IL-6.Total RNA was subjected to real-time PCR as described in the Methods section. Values represent mean ± S.D. of three independent experiments (significant as compared to HCD, * p
    Figure Legend Snippet: Inhibitory effects of HVC1 on mRNA expression of inflammatory cytokines in HCD induced LDLR −/− mice. Inflammatory cytokine genes mRNA levels were analyzed by real-time PCR analysis. ( a ) TNF-α, ( b ) IL-1β, ( c ) IL-6.Total RNA was subjected to real-time PCR as described in the Methods section. Values represent mean ± S.D. of three independent experiments (significant as compared to HCD, * p

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

    57) Product Images from "Different murine-derived feeder cells alter the definitive endoderm differentiation of human induced pluripotent stem cells"

    Article Title: Different murine-derived feeder cells alter the definitive endoderm differentiation of human induced pluripotent stem cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201239

    Changes in gene-expression of hXIST exons and of the undifferentiated stem cell marker genes of SNL- and MEFP1-201B7 cells. (A–J) RT-qPCR analysis was used to evaluate the mRNA expression of hXIST exons or undifferentiated stem cell marker genes in SNL- and MEFP1-201B7 cells. Total RNA of SNL- and MEFP1-201B7 cells was used for cDNA synthesis. The relative mRNA expression levels of hXIST exons 1–3 ( ex1-3 ) (A), exon 4 ( ex4 ) (B), and exons 5–6 ( ex5-6 ) (C) (n = 12 each) or the undifferentiated stem cell markers KLF4 (D), KLF5 (E), OCT3/4 (F), SOX2 (G), NANOG (H), UTF1 (I), GRB7 (J), NODAL (K), LEFTY1 (L), and LEFTY2 (M) (n = 6 each) were determined by RT-qPCR, normalized to that of human GAPDH , and expressed in relation to the levels in SNL-201B7 cells (set as 1). Data represent the mean ± SEM and are representative of two or three experiments. n.d.: not detected. *p
    Figure Legend Snippet: Changes in gene-expression of hXIST exons and of the undifferentiated stem cell marker genes of SNL- and MEFP1-201B7 cells. (A–J) RT-qPCR analysis was used to evaluate the mRNA expression of hXIST exons or undifferentiated stem cell marker genes in SNL- and MEFP1-201B7 cells. Total RNA of SNL- and MEFP1-201B7 cells was used for cDNA synthesis. The relative mRNA expression levels of hXIST exons 1–3 ( ex1-3 ) (A), exon 4 ( ex4 ) (B), and exons 5–6 ( ex5-6 ) (C) (n = 12 each) or the undifferentiated stem cell markers KLF4 (D), KLF5 (E), OCT3/4 (F), SOX2 (G), NANOG (H), UTF1 (I), GRB7 (J), NODAL (K), LEFTY1 (L), and LEFTY2 (M) (n = 6 each) were determined by RT-qPCR, normalized to that of human GAPDH , and expressed in relation to the levels in SNL-201B7 cells (set as 1). Data represent the mean ± SEM and are representative of two or three experiments. n.d.: not detected. *p

    Techniques Used: Expressing, Marker, Quantitative RT-PCR

    58) Product Images from "Different murine-derived feeder cells alter the definitive endoderm differentiation of human induced pluripotent stem cells"

    Article Title: Different murine-derived feeder cells alter the definitive endoderm differentiation of human induced pluripotent stem cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201239

    Changes in gene-expression of hXIST exons and of the undifferentiated stem cell marker genes of SNL- and MEFP1-201B7 cells. (A–J) RT-qPCR analysis was used to evaluate the mRNA expression of hXIST exons or undifferentiated stem cell marker genes in SNL- and MEFP1-201B7 cells. Total RNA of SNL- and MEFP1-201B7 cells was used for cDNA synthesis. The relative mRNA expression levels of hXIST exons 1–3 ( ex1-3 ) (A), exon 4 ( ex4 ) (B), and exons 5–6 ( ex5-6 ) (C) (n = 12 each) or the undifferentiated stem cell markers KLF4 (D), KLF5 (E), OCT3/4 (F), SOX2 (G), NANOG (H), UTF1 (I), GRB7 (J), NODAL (K), LEFTY1 (L), and LEFTY2 (M) (n = 6 each) were determined by RT-qPCR, normalized to that of human GAPDH , and expressed in relation to the levels in SNL-201B7 cells (set as 1). Data represent the mean ± SEM and are representative of two or three experiments. n.d.: not detected. *p
    Figure Legend Snippet: Changes in gene-expression of hXIST exons and of the undifferentiated stem cell marker genes of SNL- and MEFP1-201B7 cells. (A–J) RT-qPCR analysis was used to evaluate the mRNA expression of hXIST exons or undifferentiated stem cell marker genes in SNL- and MEFP1-201B7 cells. Total RNA of SNL- and MEFP1-201B7 cells was used for cDNA synthesis. The relative mRNA expression levels of hXIST exons 1–3 ( ex1-3 ) (A), exon 4 ( ex4 ) (B), and exons 5–6 ( ex5-6 ) (C) (n = 12 each) or the undifferentiated stem cell markers KLF4 (D), KLF5 (E), OCT3/4 (F), SOX2 (G), NANOG (H), UTF1 (I), GRB7 (J), NODAL (K), LEFTY1 (L), and LEFTY2 (M) (n = 6 each) were determined by RT-qPCR, normalized to that of human GAPDH , and expressed in relation to the levels in SNL-201B7 cells (set as 1). Data represent the mean ± SEM and are representative of two or three experiments. n.d.: not detected. *p

    Techniques Used: Expressing, Marker, Quantitative RT-PCR

    59) Product Images from "Human embryonic stem cells extracellular vesicles and their effects on immortalized human retinal Müller cells"

    Article Title: Human embryonic stem cells extracellular vesicles and their effects on immortalized human retinal Müller cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0194004

    Size, markers and total RNA profiles of MVs and EXOs. (A) TEM images of H9 hESC-derived MVs and EXOs after negative staining with uranyl acetate. Scale bars: MVs, 500 nm and EXOs, 200 nm. (B) Bar graphs show vesicles’ size distribution (diameter) in the TEM images of MVs and EXOs from 3 independent hESEV fractionations. (C) Diameters of the two main MV and EXO populations from 3 independent hESEV fractionations recorded by light scattering. Numbers above bars are the mean ± SEM. (D) Representative Western blot of CD9, CD63, CD81 and CD90 in H9 hESCs and in the MVs and EXOs derived from them. (E) Electrophoresis of total RNA from hESEVs, MVs and EXOs.
    Figure Legend Snippet: Size, markers and total RNA profiles of MVs and EXOs. (A) TEM images of H9 hESC-derived MVs and EXOs after negative staining with uranyl acetate. Scale bars: MVs, 500 nm and EXOs, 200 nm. (B) Bar graphs show vesicles’ size distribution (diameter) in the TEM images of MVs and EXOs from 3 independent hESEV fractionations. (C) Diameters of the two main MV and EXO populations from 3 independent hESEV fractionations recorded by light scattering. Numbers above bars are the mean ± SEM. (D) Representative Western blot of CD9, CD63, CD81 and CD90 in H9 hESCs and in the MVs and EXOs derived from them. (E) Electrophoresis of total RNA from hESEVs, MVs and EXOs.

    Techniques Used: Transmission Electron Microscopy, Derivative Assay, Negative Staining, Western Blot, Electrophoresis

    60) Product Images from "Ribothrypsis, a novel process of canonical mRNA decay, mediates ribosome-phased mRNA endonucleolysis"

    Article Title: Ribothrypsis, a novel process of canonical mRNA decay, mediates ribosome-phased mRNA endonucleolysis

    Journal: Nature structural & molecular biology

    doi: 10.1038/s41594-018-0042-8

    Ribothrypsis associates with RNA G-quadruplexes (rG4) and conserved RNA areas ( a ) Centered coverage of predicted rG4s, located in indicated positions relative to reading frame (ORF) or cumulative (all), upstream and downstream of Akron5 ends that map in CDS, versus random and shuffled controls. ( b, c ) Centered coverage of predicted rG4s upstream and downstream of PARE-Seq 5′ ends that map in CDS, versus random and shuffled controls, in indicated knockdowns (KD). ( d, e ) Centered coverage of rG4-Seq from Balasubramanian lab 53 ( d ) or Bartel lab 54 ( e ), upstream and downstream of Akron5 5′ ends that map in CDS with corresponding random control. ( f ) Evolutionary conservation for 100 vertebrates (PhastCons) upstream and downstream of Akron5 ends (blue) and Akron5 ends with rG4s (green) in CDS. A random control that maintains the nucleotide (nt) and ORF context of Akron5 ends (dashed pink) and a completely random control (dashed grey) are shown.
    Figure Legend Snippet: Ribothrypsis associates with RNA G-quadruplexes (rG4) and conserved RNA areas ( a ) Centered coverage of predicted rG4s, located in indicated positions relative to reading frame (ORF) or cumulative (all), upstream and downstream of Akron5 ends that map in CDS, versus random and shuffled controls. ( b, c ) Centered coverage of predicted rG4s upstream and downstream of PARE-Seq 5′ ends that map in CDS, versus random and shuffled controls, in indicated knockdowns (KD). ( d, e ) Centered coverage of rG4-Seq from Balasubramanian lab 53 ( d ) or Bartel lab 54 ( e ), upstream and downstream of Akron5 5′ ends that map in CDS with corresponding random control. ( f ) Evolutionary conservation for 100 vertebrates (PhastCons) upstream and downstream of Akron5 ends (blue) and Akron5 ends with rG4s (green) in CDS. A random control that maintains the nucleotide (nt) and ORF context of Akron5 ends (dashed pink) and a completely random control (dashed grey) are shown.

    Techniques Used:

    Ribothrypsis is not mediated by XRN1, the exosome or the SMG6 endonuclease and its end products are captured in small RNA sequencing libraries ( a , b ) 5′ end density upstream and downstream of stop codon (last nucleotide of stop codon set as position 0) and DFT upstream and downstream of position −17 (corresponding to the terminating ribosome 5′ end, marked with a green dotted line) for Akron5 ( a ), and PARE-Seq after XRN1 knockdown (KD) ( b ). ( c – h ) Density and DFT around the RPFs’ 5′ end (position 0) of 5′ or 3′ ends from indicated libraries in CDS; sRNA = small RNA libraries.
    Figure Legend Snippet: Ribothrypsis is not mediated by XRN1, the exosome or the SMG6 endonuclease and its end products are captured in small RNA sequencing libraries ( a , b ) 5′ end density upstream and downstream of stop codon (last nucleotide of stop codon set as position 0) and DFT upstream and downstream of position −17 (corresponding to the terminating ribosome 5′ end, marked with a green dotted line) for Akron5 ( a ), and PARE-Seq after XRN1 knockdown (KD) ( b ). ( c – h ) Density and DFT around the RPFs’ 5′ end (position 0) of 5′ or 3′ ends from indicated libraries in CDS; sRNA = small RNA libraries.

    Techniques Used: RNA Sequencing Assay

    61) Product Images from "The nonstop decay and the RNA silencing systems operate cooperatively in plants"

    Article Title: The nonstop decay and the RNA silencing systems operate cooperatively in plants

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky279

    Nonstop decay operates in plants. ( A ) In N. benthamiana , the 5′ and the 3′ cleavage fragments of PDS viral siRNA-programmed RISC are eliminated by SKI2- and XRN4-dependent pathways, respectively. VIGS-agroinfiltration assays were conducted to study the degradation of vsiRISC cleavage products. Leaves of PDS-silenced (PDS) negative control, and PDS + XRN4 or PDS + SKI2 silenced test plants (P-XRN4 and P-SKI2, respectively) were agroinfiltrated with P14 internal control and PPG sensor construct (P14 + PPG). Schematic, non-proportional representation of the PPG vsiRISC sensor transcript. Two samples isolated from different plants were run in parallel to illustrate experimental variations. RNA gel blots were hybridized with P14 and PHA (upper panel) or P14 and GFP (bottom panel) probes (probe names are in italics). The stabilized 5′ and 3′ cleavage products of PPG (5′ and 3′ Cl.) are indicated. ( B ) NSD reporter mRNAs accumulate to low levels in plants. The GFP-nst and PHA-nst NSD reporter mRNAs (G-nst and P-nst) and the corresponding stop codon containing control mRNAs (G-st and P-st) were agroinfiltrated with P14 internal control into the leaves of wild-type N. benthamiana plants. ( C ) NSD is a translation-dependent system in plants. G-nst NSD reporter was co-expressed with P14 internal control and a cycloheximide sensitive positive control (G-600) in N. benthamiana leaves. The samples were treated with cycloheximide translation inhibitor or with buffer. G-600 mRNA is targeted by NMD, another translation-dependent quality control system therefore sensitive to cycloheximide (also see below at Supplementary Figure S14 ). Note that both the G-nst and the G-600 mRNAs are overexpressed in the cycloheximide treated samples.
    Figure Legend Snippet: Nonstop decay operates in plants. ( A ) In N. benthamiana , the 5′ and the 3′ cleavage fragments of PDS viral siRNA-programmed RISC are eliminated by SKI2- and XRN4-dependent pathways, respectively. VIGS-agroinfiltration assays were conducted to study the degradation of vsiRISC cleavage products. Leaves of PDS-silenced (PDS) negative control, and PDS + XRN4 or PDS + SKI2 silenced test plants (P-XRN4 and P-SKI2, respectively) were agroinfiltrated with P14 internal control and PPG sensor construct (P14 + PPG). Schematic, non-proportional representation of the PPG vsiRISC sensor transcript. Two samples isolated from different plants were run in parallel to illustrate experimental variations. RNA gel blots were hybridized with P14 and PHA (upper panel) or P14 and GFP (bottom panel) probes (probe names are in italics). The stabilized 5′ and 3′ cleavage products of PPG (5′ and 3′ Cl.) are indicated. ( B ) NSD reporter mRNAs accumulate to low levels in plants. The GFP-nst and PHA-nst NSD reporter mRNAs (G-nst and P-nst) and the corresponding stop codon containing control mRNAs (G-st and P-st) were agroinfiltrated with P14 internal control into the leaves of wild-type N. benthamiana plants. ( C ) NSD is a translation-dependent system in plants. G-nst NSD reporter was co-expressed with P14 internal control and a cycloheximide sensitive positive control (G-600) in N. benthamiana leaves. The samples were treated with cycloheximide translation inhibitor or with buffer. G-600 mRNA is targeted by NMD, another translation-dependent quality control system therefore sensitive to cycloheximide (also see below at Supplementary Figure S14 ). Note that both the G-nst and the G-600 mRNAs are overexpressed in the cycloheximide treated samples.

    Techniques Used: Negative Control, Construct, Isolation, Positive Control

    NSD is involved in the degradation of 5′ cleavage fragments of several endogenous miRNA targets. ( A ) The schematic, non-proportional representation of the N. benthamiana SCL6-IV miRNA171 target transcript. The primer pairs that were used to measure the accumulation of the 5′ (5′F and 5′R) and 3′ (3′F and 3′R) cleavage fragments and the probes that were used for the northern assay are shown. ( B and C ) The 5′ cleavage fragments of SCL6-IV miR171 target mRNAs are overaccumulated in NSD deficient leaves. qRT-PCR assays were carried out from PDS-, P-Pelota- and P-HBS1-silenced leaves, and from Pelota1-complemented P-Pelota- and P-HBS1-silenced leaves. ( D ) qRT-PCR experiments show that the 3′ cleavage fragments of SCL6-IV mRNAs are overaccumulated in P-XRN4-silenced leaves. ( E ) RNA gel blot assay was carried out to study the accumulation of the 5′ and the 3′ cleavage products (5′ Cl. and 3′ Cl.) of SCL6-IV mRNA in PDS-, P-XRN4-, P-Pelota- and P-HBS1-silenced leaves. Note that VIGS did not alter the expression of the miRNA171. ( F – H ) Comparative RNA-seq assay conducted from PDS- and P-Pelota-silenced plants show that Pelota plays a role in the degradation of a subset of endogenous miRNA target transcripts. ( F ) The coverage of the SCL6-IV transcript. Yellow columns show the number of reads at a given position from P-Pelota, while blue line indicates number of reads from PDS-silenced control. The degradome peak (red column) and the miRNA cleavage site (red arrow) are marked. ( G and H ) The 5′/3′coverage ratio (see main text) of miRISC targets from PDS- and P-Pelota silenced plants were compared. Transcript whose 5′/3′ coverage ratio was at least 1.5-fold higher in the P-Pelota–silenced plants were defined as Pelota-dependent.
    Figure Legend Snippet: NSD is involved in the degradation of 5′ cleavage fragments of several endogenous miRNA targets. ( A ) The schematic, non-proportional representation of the N. benthamiana SCL6-IV miRNA171 target transcript. The primer pairs that were used to measure the accumulation of the 5′ (5′F and 5′R) and 3′ (3′F and 3′R) cleavage fragments and the probes that were used for the northern assay are shown. ( B and C ) The 5′ cleavage fragments of SCL6-IV miR171 target mRNAs are overaccumulated in NSD deficient leaves. qRT-PCR assays were carried out from PDS-, P-Pelota- and P-HBS1-silenced leaves, and from Pelota1-complemented P-Pelota- and P-HBS1-silenced leaves. ( D ) qRT-PCR experiments show that the 3′ cleavage fragments of SCL6-IV mRNAs are overaccumulated in P-XRN4-silenced leaves. ( E ) RNA gel blot assay was carried out to study the accumulation of the 5′ and the 3′ cleavage products (5′ Cl. and 3′ Cl.) of SCL6-IV mRNA in PDS-, P-XRN4-, P-Pelota- and P-HBS1-silenced leaves. Note that VIGS did not alter the expression of the miRNA171. ( F – H ) Comparative RNA-seq assay conducted from PDS- and P-Pelota-silenced plants show that Pelota plays a role in the degradation of a subset of endogenous miRNA target transcripts. ( F ) The coverage of the SCL6-IV transcript. Yellow columns show the number of reads at a given position from P-Pelota, while blue line indicates number of reads from PDS-silenced control. The degradome peak (red column) and the miRNA cleavage site (red arrow) are marked. ( G and H ) The 5′/3′coverage ratio (see main text) of miRISC targets from PDS- and P-Pelota silenced plants were compared. Transcript whose 5′/3′ coverage ratio was at least 1.5-fold higher in the P-Pelota–silenced plants were defined as Pelota-dependent.

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

    SKI2 is required for the elimination of NSD target mRNAs. ( A and B ) NSD plays a role in the decay of 5′ cleavage fragments of miRISC in Arabidopsis . Comparative RNA-seq experiment was conducted from Pelota1 and Hbs1 T-DNA mutants ( pelota1, hbs1 ) and from wild-type Arabidopsis plants. ( A ) Venn diagram shows the Pelota-, Hbs1- and the NSD-dependent Arabidopsis transcripts. The 5′/3′ coverage ratio of 73 miRNA target transcripts was analyzed. Pelota- or HBS1-dependent mRNAs are defined when the 5′ fragment overaccumulate > 1.5-fold in the mutant relative to the wild type. If the 5′ fragment overaccumulate in both mutants, the mRNA was categorized as NSD-dependent. ( B ) The coverage of the SCL6-IV transcript. Yellow and green columns show the number of reads at a given position from pelota1 and hbs1 Arabidopsis mutants, while blue columns indicate the number of reads from wild type control. The degradome peak (red column) and the miRNA cleavage site (red arrow) are marked. ( C ) NSD reporter mRNAs accumulate to high levels in SKI2-deficient leaves. GFP-nst (G-nst) nonstop reporter construct was co-agroinfiltrated with P14 into the leaves of PDS-silenced (negative control), P-Pelota-silenced (positive control) and P-SKI2-silenced test plants. As controls, the same leaves were also co-agroinfiltrated with P14 + G-st control and with P14+PPG vsiRISC sensor constructs. ( D ) SKI2 is involved in the decay of 5′ cleavage products of miRISC. Leaves of P-Pelota- and P-SKI2-silenced plants were agroinfiltrated in the presence or in the absence of amir-GFP with P14+PHA-GFP (P-G) fusion constructs, or with P14+P-st-G construct, in which a stop codon separates the PHA and the GFP reporter genes. The blot was hybridized with GFP 5′ fragment probe, stripped, and then reprobed for P14.
    Figure Legend Snippet: SKI2 is required for the elimination of NSD target mRNAs. ( A and B ) NSD plays a role in the decay of 5′ cleavage fragments of miRISC in Arabidopsis . Comparative RNA-seq experiment was conducted from Pelota1 and Hbs1 T-DNA mutants ( pelota1, hbs1 ) and from wild-type Arabidopsis plants. ( A ) Venn diagram shows the Pelota-, Hbs1- and the NSD-dependent Arabidopsis transcripts. The 5′/3′ coverage ratio of 73 miRNA target transcripts was analyzed. Pelota- or HBS1-dependent mRNAs are defined when the 5′ fragment overaccumulate > 1.5-fold in the mutant relative to the wild type. If the 5′ fragment overaccumulate in both mutants, the mRNA was categorized as NSD-dependent. ( B ) The coverage of the SCL6-IV transcript. Yellow and green columns show the number of reads at a given position from pelota1 and hbs1 Arabidopsis mutants, while blue columns indicate the number of reads from wild type control. The degradome peak (red column) and the miRNA cleavage site (red arrow) are marked. ( C ) NSD reporter mRNAs accumulate to high levels in SKI2-deficient leaves. GFP-nst (G-nst) nonstop reporter construct was co-agroinfiltrated with P14 into the leaves of PDS-silenced (negative control), P-Pelota-silenced (positive control) and P-SKI2-silenced test plants. As controls, the same leaves were also co-agroinfiltrated with P14 + G-st control and with P14+PPG vsiRISC sensor constructs. ( D ) SKI2 is involved in the decay of 5′ cleavage products of miRISC. Leaves of P-Pelota- and P-SKI2-silenced plants were agroinfiltrated in the presence or in the absence of amir-GFP with P14+PHA-GFP (P-G) fusion constructs, or with P14+P-st-G construct, in which a stop codon separates the PHA and the GFP reporter genes. The blot was hybridized with GFP 5′ fragment probe, stripped, and then reprobed for P14.

    Techniques Used: RNA Sequencing Assay, Mutagenesis, Construct, Negative Control, Positive Control

    NSD eliminates the 5′ cleavage fragments of miRISC when the cleavage occurs in the coding region. ( A ) The role of NSD components and XRN4 in the degradation of miRISC-generated cleavage fragments. PDS-, P-XRN4-, P-Pelota- and P-HBS1-silenced plants were co-agroinfiltrated with P14 internal control and GFP-171.1 miRNA sensor or P14 and GFP-171.1 constructs were co-expressed with either amiR-GFP or pri-miR171c constructs. amiR-GFP targets the sensor mRNA within the coding region, while pri-miR171c targets the sensor within the 3′UTR. Note that the weak miR171 signals in the samples without the agroinfiltrated pri-miR171c originates from the endogenous locus. ( B ) The effect of PeloDN on the accumulation of 5′ cleavage fragments of amiR-GFP-programmed RISC. PHA-GFP fusion (P-G) construct and PHA-st-GFP (P-st-G), a similar sensor construct in which a stop codon is present between the PHA and GFP reporter genes, were co-agroinfiltrated with amiR-GFP, or with amir-GFP and a dominant-negative version of Arabidopsis Pelota1 (PeloDN) (p14 was used as internal control). 5′ cleavage product accumulates only when PeloDN is present and the amir-GFP–directed cleveage occurs within the coding region. ( C ) NSD does not affect the tasiRNA silencing pathway. N. benthamiana leaves were co-agroinfiltrated with TAS1b mRNA and miR173 expressing constructs, or with TAS1b, miR173 and PeloDN expressing constructs. RNA gel blot assays were conducted to monitor the abundance of miR173, TAS1b and the tasiR255 RNAs. TAS1b is cleaved by miR173-programmed RISC, and then tasiR255 is generated by RDR6 and DCL4 from the 3′ cleavage product. The presence of PeloDN does not stabilize the miR173-generated TAS1b cleavage product. As P14 modifies the tasiRNA pathway, P14 was not co-expressed.
    Figure Legend Snippet: NSD eliminates the 5′ cleavage fragments of miRISC when the cleavage occurs in the coding region. ( A ) The role of NSD components and XRN4 in the degradation of miRISC-generated cleavage fragments. PDS-, P-XRN4-, P-Pelota- and P-HBS1-silenced plants were co-agroinfiltrated with P14 internal control and GFP-171.1 miRNA sensor or P14 and GFP-171.1 constructs were co-expressed with either amiR-GFP or pri-miR171c constructs. amiR-GFP targets the sensor mRNA within the coding region, while pri-miR171c targets the sensor within the 3′UTR. Note that the weak miR171 signals in the samples without the agroinfiltrated pri-miR171c originates from the endogenous locus. ( B ) The effect of PeloDN on the accumulation of 5′ cleavage fragments of amiR-GFP-programmed RISC. PHA-GFP fusion (P-G) construct and PHA-st-GFP (P-st-G), a similar sensor construct in which a stop codon is present between the PHA and GFP reporter genes, were co-agroinfiltrated with amiR-GFP, or with amir-GFP and a dominant-negative version of Arabidopsis Pelota1 (PeloDN) (p14 was used as internal control). 5′ cleavage product accumulates only when PeloDN is present and the amir-GFP–directed cleveage occurs within the coding region. ( C ) NSD does not affect the tasiRNA silencing pathway. N. benthamiana leaves were co-agroinfiltrated with TAS1b mRNA and miR173 expressing constructs, or with TAS1b, miR173 and PeloDN expressing constructs. RNA gel blot assays were conducted to monitor the abundance of miR173, TAS1b and the tasiR255 RNAs. TAS1b is cleaved by miR173-programmed RISC, and then tasiR255 is generated by RDR6 and DCL4 from the 3′ cleavage product. The presence of PeloDN does not stabilize the miR173-generated TAS1b cleavage product. As P14 modifies the tasiRNA pathway, P14 was not co-expressed.

    Techniques Used: Generated, Construct, Dominant Negative Mutation, Expressing, Western Blot

    Pelota and Hbs1 are required for plant NSD. ( A and B ) NSD reporter mRNAs accumulate to high levels in deadenylated form in Pelota-silenced plants. ( A ) GFP-nst and PHA-nst NSD reporter (G-nst and P-nst) and the corresponding control (P-st and G-st) constructs were agroinfiltrated with the P14 internal control into the leaves of PDS-silenced control (PDS) or PDS+Pelota silenced (P-Pelota) test plants. Note that in the P-Pelota plants, the NSD reporter mRNAs migrate faster than the stop-codon containing control transcripts. ( B ) NSD reporter mRNA is present in deadenylated form in P-Pelota-silenced leaves. P14 was co-expressed with G-st control or with G-nst NSD reporter mRNAs in P-Pelota-silenced leaves. RNAs were isolated and incubated with oligo(dT) cellulose, and then the (dT)-bound and the unbound (supernatant) fractions were subjected to RNA gel blot assay. Note that the G-nst NSD reporter transcript accumulates mainly in the supernatant, while the P14 and the G-st control mRNAs predominantly accumulate in the (dT)-bound fraction. ( C ) NSD reporter mRNAs accumulate to high levels in the P-HBS1-silenced plants. G-nst reporter and the G-st control constructs were agroinfiltrated with P14 into PDS– and PDS + HBS1 silenced (P-HBS1) plants. Note that the NSD reporter mRNAs migrate faster relative to the controls in P-HBS1 plants. ( D ) Pelota overexpression complements both the Pelota- and the Hbs1-deficient lines. G-nst reporter and P14 control constructs were expressed in the absence (–) or presence of the Arabidopsis AtPelota1 (Pelota1) in the leaves of P-Pelota- and P-HBS1-silenced plants. Reduced G-nst level indicates that Pelota1 complemented the NSD deficiency of the silenced plants. ( E and F ) Pelota and Hbs1 are required for the elimination of the 5′ but not the 3′ cleavage products of vsiRISC. PPG vsiRNA sensor and P14 (-) or PPG, P14 and Pelota1 were co-agroinfiltrated into PDS- and P-Pelota-silenced ( E ) and into P-HBS1-silenced ( F ) plants. Note that the 5′ cleavage fragment of vsiRISC accumulates to high levels in both P-Pelota and P-HBS1 plants and that Pelota overexpression complements the absence of endogenous Pelota and Hbs1 in the silenced plants.
    Figure Legend Snippet: Pelota and Hbs1 are required for plant NSD. ( A and B ) NSD reporter mRNAs accumulate to high levels in deadenylated form in Pelota-silenced plants. ( A ) GFP-nst and PHA-nst NSD reporter (G-nst and P-nst) and the corresponding control (P-st and G-st) constructs were agroinfiltrated with the P14 internal control into the leaves of PDS-silenced control (PDS) or PDS+Pelota silenced (P-Pelota) test plants. Note that in the P-Pelota plants, the NSD reporter mRNAs migrate faster than the stop-codon containing control transcripts. ( B ) NSD reporter mRNA is present in deadenylated form in P-Pelota-silenced leaves. P14 was co-expressed with G-st control or with G-nst NSD reporter mRNAs in P-Pelota-silenced leaves. RNAs were isolated and incubated with oligo(dT) cellulose, and then the (dT)-bound and the unbound (supernatant) fractions were subjected to RNA gel blot assay. Note that the G-nst NSD reporter transcript accumulates mainly in the supernatant, while the P14 and the G-st control mRNAs predominantly accumulate in the (dT)-bound fraction. ( C ) NSD reporter mRNAs accumulate to high levels in the P-HBS1-silenced plants. G-nst reporter and the G-st control constructs were agroinfiltrated with P14 into PDS– and PDS + HBS1 silenced (P-HBS1) plants. Note that the NSD reporter mRNAs migrate faster relative to the controls in P-HBS1 plants. ( D ) Pelota overexpression complements both the Pelota- and the Hbs1-deficient lines. G-nst reporter and P14 control constructs were expressed in the absence (–) or presence of the Arabidopsis AtPelota1 (Pelota1) in the leaves of P-Pelota- and P-HBS1-silenced plants. Reduced G-nst level indicates that Pelota1 complemented the NSD deficiency of the silenced plants. ( E and F ) Pelota and Hbs1 are required for the elimination of the 5′ but not the 3′ cleavage products of vsiRISC. PPG vsiRNA sensor and P14 (-) or PPG, P14 and Pelota1 were co-agroinfiltrated into PDS- and P-Pelota-silenced ( E ) and into P-HBS1-silenced ( F ) plants. Note that the 5′ cleavage fragment of vsiRISC accumulates to high levels in both P-Pelota and P-HBS1 plants and that Pelota overexpression complements the absence of endogenous Pelota and Hbs1 in the silenced plants.

    Techniques Used: Construct, Isolation, Incubation, Western Blot, Over Expression

    Model of plant NSD. ( A ) In NSD-deficient cells, the 5′ cleavage fragments of miRISC are associated with ribosomes. Extracts were isolated with cycloheximide (CHX) or EDTA containing buffer (upper and bottom panel) from P-Pelota-silenced (P-Pelota) leaves, in which GFP, amiR-GFP and P14 were co-expressed. These extracts were subjected to sucrose gradient sedimentation, and then the fractions were analyzed by RNA gel blot assays. The blots were hybridized with GFP 5′ fragment probe. Note that the 5′ cleavage fragment (5′ Cl.) accumulates in the polysomal fractions in the CHX containing samples, while polysomal migration of 5′ fragment is disrupted by the addition of EDTA. Mon indicates monosomal fraction. ( B ) The role of NSD in the decay of nonstop and ( C ) RISC cleavage generated stop codon-less transcripts. It is not known whether SKI-exosome mediated decay occurs when the mRNA is still associated with ribosomes or SKI-exosome degrades mRNAs, from which the ribosomes have already removed. ( D ) NSD is not required for the 5′ fragment decay when si/miRISC cuts outside of the coding region or if the target transcript is not translated. For details, see the main text.
    Figure Legend Snippet: Model of plant NSD. ( A ) In NSD-deficient cells, the 5′ cleavage fragments of miRISC are associated with ribosomes. Extracts were isolated with cycloheximide (CHX) or EDTA containing buffer (upper and bottom panel) from P-Pelota-silenced (P-Pelota) leaves, in which GFP, amiR-GFP and P14 were co-expressed. These extracts were subjected to sucrose gradient sedimentation, and then the fractions were analyzed by RNA gel blot assays. The blots were hybridized with GFP 5′ fragment probe. Note that the 5′ cleavage fragment (5′ Cl.) accumulates in the polysomal fractions in the CHX containing samples, while polysomal migration of 5′ fragment is disrupted by the addition of EDTA. Mon indicates monosomal fraction. ( B ) The role of NSD in the decay of nonstop and ( C ) RISC cleavage generated stop codon-less transcripts. It is not known whether SKI-exosome mediated decay occurs when the mRNA is still associated with ribosomes or SKI-exosome degrades mRNAs, from which the ribosomes have already removed. ( D ) NSD is not required for the 5′ fragment decay when si/miRISC cuts outside of the coding region or if the target transcript is not translated. For details, see the main text.

    Techniques Used: Isolation, Sedimentation, Western Blot, Migration, Generated

    62) Product Images from "Up-regulation of microRNA-203 in influenza A virus infection inhibits viral replication by targeting DR1"

    Article Title: Up-regulation of microRNA-203 in influenza A virus infection inhibits viral replication by targeting DR1

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25073-9

    IAV-mediated demethylation of the miR-203 promoter region up-regulates expression of miR-203. ( A ) Bisulfite sequencing PCR of the miR-203 promoter region was performed in A549 cells mock-infected or infected with H5N1 (MOI = 2) viruses for 24 h. The sequenced region covered −600 to −10 bp within the miR-203 promoter region. Three lines denote three replicates in per group. Open circles, unmethylated; solid circles, methylated. ( B ) A549 cells were treated for 48 h with 5-aza-2′-deoxycytidine, a methylation inhibitor, at a final concentration of 0.5, 1, or 2 μM, and total RNA was purified to analyze abundance of miR-203 by quantitative real-time PCR (qPCR). ( C ) Vero cells were treated for 48 h with 5-aza-2′-deoxycytidine (final concentration, 1 μM) and total RNA purified. The abundance of miR-203 was measured by qPCR. Data are expressed as the mean + SD of three independent experiments. * p
    Figure Legend Snippet: IAV-mediated demethylation of the miR-203 promoter region up-regulates expression of miR-203. ( A ) Bisulfite sequencing PCR of the miR-203 promoter region was performed in A549 cells mock-infected or infected with H5N1 (MOI = 2) viruses for 24 h. The sequenced region covered −600 to −10 bp within the miR-203 promoter region. Three lines denote three replicates in per group. Open circles, unmethylated; solid circles, methylated. ( B ) A549 cells were treated for 48 h with 5-aza-2′-deoxycytidine, a methylation inhibitor, at a final concentration of 0.5, 1, or 2 μM, and total RNA was purified to analyze abundance of miR-203 by quantitative real-time PCR (qPCR). ( C ) Vero cells were treated for 48 h with 5-aza-2′-deoxycytidine (final concentration, 1 μM) and total RNA purified. The abundance of miR-203 was measured by qPCR. Data are expressed as the mean + SD of three independent experiments. * p

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

    63) Product Images from "Hypoxia-Inducible Factor-1α Mediates Hypoxia-Induced Delayed Neuronal Death That Involves p53"

    Article Title: Hypoxia-Inducible Factor-1α Mediates Hypoxia-Induced Delayed Neuronal Death That Involves p53

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.19-16-06818.1999

    HIF-1α activity in human neuroblastoma lines and cultured mouse primary cortical neurons. A , Normoxic cultured lines and primary neurons express HIF-1α mRNA. Total RNA was harvested from the cell lines Hep3B ( lane 1 ), SHEP-1 ( lane 2 ), SY5Y ( lane 3 ), and mouse cortical neurons ( lane 4 ). Samples (20 μg) were probed by using human and mouse HIF-1α cDNA fragments as described in Materials and Methods. B, HIF-1-responsive reporter plasmids are activated in hypoxic human neuroblastoma lines. SHEP-1 ( SH ), SY5Y ( SY ), and Hep3B ( HB ) cell lines were transfected with the HRE-containing reporter plasmid 18–123F-pXP2 and exposed to normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions. Then 36 hr later the lysates were tested for hypoxia-dependent reporter activation by luciferase assay. Results from quadruplicate samples are presented in absolute light units × 1000 (mean ± SD).
    Figure Legend Snippet: HIF-1α activity in human neuroblastoma lines and cultured mouse primary cortical neurons. A , Normoxic cultured lines and primary neurons express HIF-1α mRNA. Total RNA was harvested from the cell lines Hep3B ( lane 1 ), SHEP-1 ( lane 2 ), SY5Y ( lane 3 ), and mouse cortical neurons ( lane 4 ). Samples (20 μg) were probed by using human and mouse HIF-1α cDNA fragments as described in Materials and Methods. B, HIF-1-responsive reporter plasmids are activated in hypoxic human neuroblastoma lines. SHEP-1 ( SH ), SY5Y ( SY ), and Hep3B ( HB ) cell lines were transfected with the HRE-containing reporter plasmid 18–123F-pXP2 and exposed to normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions. Then 36 hr later the lysates were tested for hypoxia-dependent reporter activation by luciferase assay. Results from quadruplicate samples are presented in absolute light units × 1000 (mean ± SD).

    Techniques Used: Activity Assay, Cell Culture, Transfection, Plasmid Preparation, Activation Assay, Luciferase

    64) Product Images from "Hepatitis Delta Virus histone mimicry drives the recruitment of chromatin remodelers for viral RNA replication"

    Article Title: Hepatitis Delta Virus histone mimicry drives the recruitment of chromatin remodelers for viral RNA replication

    Journal: Nature Communications

    doi: 10.1038/s41467-020-14299-9

    The chromatin remodeling complexes BRF-1 and BRF-5 interact with the HDV RNP. a Interaction of wt S-HDAg with endogenous SNF2L, SNF2H, and BAZ2B. Nuclear extracts from Huh7 cells stably expressing wt S-HDAg or Huh7 cells were subjected to immunoprecipitation (IP) with α-HDAg (IP-HDAg), α-SNF2L (IP-SNF2L), α-SNF2H (IP-SNF2H), and αBAZ2B (IP-BAZ2B). Nonspecific antibody (IP-IgG) was used as a negative immunoprecipitation control. Input and elutions were analyzed by immunoblotting (IB) using the indicated antibodies. Input and HDAg lanes represent 10% and 20% volume compared with the other lanes, respectively. b Amplification of SNF2L variants from hepatocyte-derived cell lines. Left: PCR products demonstrating that both SNF2L and SNF2L(ex13) isoforms were observed in each cell line. Right: schematic representation of PCR products in the presence or absence of exon 13. Arrows represent primer positions. c , d HDV RNA immunoprecipitation assays (RIP). Glutaraldehyde-crosslinked nuclei from Huh7 cells transfected with the replication competent pSVLD3 plasmid ( c ) and PHHs infected with HDV (m.o.i. = 10) ( d ) were immunoprecipitated with antibodies directed against HDAg, RNA Pol II, SNF2L, SNF2H, and BAZ2B. HDV RNA was detected by qRT-PCR using HDV-specific primers. Histone H3 served as a negative RIP control. Error bars indicate SEMs from at least three independent experiments. e , f Genomic (g) or antigenomic (ag) HDV RIP assay. Glutaraldehyde-crosslinked nuclei from PHHs infected with HDV (m.o.i. = 10) were immunoprecipitated with antibodies directed against HDAg, RNA Pol II, SNF2L, SNF2H, BAZ2B, and the negative control H3. Genomic HDV ( e ) or ag HDV ( f ) RNA were detected by the biotinylated magnetic beads-based qRT-PCR assay using specific biotinylated ag HDV or g HDV primers, respectively. Values represent the mean ± SEM ( n = 4 with the exception of n = 2 for Pol II RIP). Source data are provided as a Source Data file.
    Figure Legend Snippet: The chromatin remodeling complexes BRF-1 and BRF-5 interact with the HDV RNP. a Interaction of wt S-HDAg with endogenous SNF2L, SNF2H, and BAZ2B. Nuclear extracts from Huh7 cells stably expressing wt S-HDAg or Huh7 cells were subjected to immunoprecipitation (IP) with α-HDAg (IP-HDAg), α-SNF2L (IP-SNF2L), α-SNF2H (IP-SNF2H), and αBAZ2B (IP-BAZ2B). Nonspecific antibody (IP-IgG) was used as a negative immunoprecipitation control. Input and elutions were analyzed by immunoblotting (IB) using the indicated antibodies. Input and HDAg lanes represent 10% and 20% volume compared with the other lanes, respectively. b Amplification of SNF2L variants from hepatocyte-derived cell lines. Left: PCR products demonstrating that both SNF2L and SNF2L(ex13) isoforms were observed in each cell line. Right: schematic representation of PCR products in the presence or absence of exon 13. Arrows represent primer positions. c , d HDV RNA immunoprecipitation assays (RIP). Glutaraldehyde-crosslinked nuclei from Huh7 cells transfected with the replication competent pSVLD3 plasmid ( c ) and PHHs infected with HDV (m.o.i. = 10) ( d ) were immunoprecipitated with antibodies directed against HDAg, RNA Pol II, SNF2L, SNF2H, and BAZ2B. HDV RNA was detected by qRT-PCR using HDV-specific primers. Histone H3 served as a negative RIP control. Error bars indicate SEMs from at least three independent experiments. e , f Genomic (g) or antigenomic (ag) HDV RIP assay. Glutaraldehyde-crosslinked nuclei from PHHs infected with HDV (m.o.i. = 10) were immunoprecipitated with antibodies directed against HDAg, RNA Pol II, SNF2L, SNF2H, BAZ2B, and the negative control H3. Genomic HDV ( e ) or ag HDV ( f ) RNA were detected by the biotinylated magnetic beads-based qRT-PCR assay using specific biotinylated ag HDV or g HDV primers, respectively. Values represent the mean ± SEM ( n = 4 with the exception of n = 2 for Pol II RIP). Source data are provided as a Source Data file.

    Techniques Used: Stable Transfection, Expressing, Immunoprecipitation, Amplification, Derivative Assay, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Negative Control, Magnetic Beads

    65) Product Images from "Transcriptional activation of USP16 gene expression by NFκB signaling"

    Article Title: Transcriptional activation of USP16 gene expression by NFκB signaling

    Journal: Molecular Brain

    doi: 10.1186/s13041-019-0535-3

    Enhancement of USP16 transcription in response to p65, LPS and TNFα. ( a ) HEK293 Cells were transfected with either empty vector (pMTF) or the p65 expression plasmids (pMTF-p65) for 24 h, and USP16 mRNA levels were determined by qRT-PCR. ( b ) SH-SY5Y cells were exposed to LPS at 50 ng/ml for 24 h. Total RNA was extracted. The mRNA levels of USP16 were determined by qRT-PCR and normalized against the levels of GAPDH. HEK293 cells ( c ) and SH-SY5Y cells ( d ) were exposed to TNFα at 10 ng/ml for 24 h. The mRNA levels of endogenous USP16 gene were determined by qRT-PCR and normalized against the levels of GAPDH. All data are presented as mean ± SEM. n = 3, * p
    Figure Legend Snippet: Enhancement of USP16 transcription in response to p65, LPS and TNFα. ( a ) HEK293 Cells were transfected with either empty vector (pMTF) or the p65 expression plasmids (pMTF-p65) for 24 h, and USP16 mRNA levels were determined by qRT-PCR. ( b ) SH-SY5Y cells were exposed to LPS at 50 ng/ml for 24 h. Total RNA was extracted. The mRNA levels of USP16 were determined by qRT-PCR and normalized against the levels of GAPDH. HEK293 cells ( c ) and SH-SY5Y cells ( d ) were exposed to TNFα at 10 ng/ml for 24 h. The mRNA levels of endogenous USP16 gene were determined by qRT-PCR and normalized against the levels of GAPDH. All data are presented as mean ± SEM. n = 3, * p

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR

    66) Product Images from "TLR7 Modulated T Cell Response in the Mesenteric Lymph Node of Schistosoma japonicum-Infected C57BL/6 Mice"

    Article Title: TLR7 Modulated T Cell Response in the Mesenteric Lymph Node of Schistosoma japonicum-Infected C57BL/6 Mice

    Journal: Journal of Immunology Research

    doi: 10.1155/2019/2691808

    Expression of TLRs in the MLN of S. japonicum- infected mouse. Six weeks after S. japonicum infection, the mice were sacrificed. Single mononuclear cell suspensions from normal and infected mice were prepared as described in Materials and Methods. Total RNA was collected and purified, and cDNA was synthesized. (a, b) The relative mRNA expression of TLRs and β -actin genes was detected. Data was from three independent experiments with 5 mice per group and shown as the mean ± SEM. ∗ P
    Figure Legend Snippet: Expression of TLRs in the MLN of S. japonicum- infected mouse. Six weeks after S. japonicum infection, the mice were sacrificed. Single mononuclear cell suspensions from normal and infected mice were prepared as described in Materials and Methods. Total RNA was collected and purified, and cDNA was synthesized. (a, b) The relative mRNA expression of TLRs and β -actin genes was detected. Data was from three independent experiments with 5 mice per group and shown as the mean ± SEM. ∗ P

    Techniques Used: Expressing, Infection, Mouse Assay, Purification, Synthesized

    67) Product Images from "T cell mediated suppression of neurotropic coronavirus replication in neural precursor cells"

    Article Title: T cell mediated suppression of neurotropic coronavirus replication in neural precursor cells

    Journal: Virology

    doi: 10.1016/j.virol.2013.11.025

    Cultured NPCs express CEACAM1a ( A ) PCR amplification revealed that cultured C57BL/6 NPCs express transcripts specific for the JHMV receptor CEACAM1a. A 250 bp amplicon, specific for CEACAM1a, was amplified from cDNA generated from total RNA extracted from cultured NPCs; controls included water and splenocytes isolated from non-infected mice. ( B ) Representative dot plots showing CEACAM1a expression on the cell surface of C57BL/6 splenocytes (control) and cultured NPCs.
    Figure Legend Snippet: Cultured NPCs express CEACAM1a ( A ) PCR amplification revealed that cultured C57BL/6 NPCs express transcripts specific for the JHMV receptor CEACAM1a. A 250 bp amplicon, specific for CEACAM1a, was amplified from cDNA generated from total RNA extracted from cultured NPCs; controls included water and splenocytes isolated from non-infected mice. ( B ) Representative dot plots showing CEACAM1a expression on the cell surface of C57BL/6 splenocytes (control) and cultured NPCs.

    Techniques Used: Cell Culture, Polymerase Chain Reaction, Amplification, Generated, Isolation, Infection, Mouse Assay, Expressing

    68) Product Images from "Polyphenoloxidase Silencing Affects Latex Coagulation in Taraxacum Species 1 Species 1 [W]"

    Article Title: Polyphenoloxidase Silencing Affects Latex Coagulation in Taraxacum Species 1 Species 1 [W]

    Journal: Plant Physiology

    doi: 10.1104/pp.109.138743

    Analysis of PPO-1 expression in planta. A, Detection of ToPPO-1 and TkPPO-1 mRNA by RT-PCR, demonstrating laticifer-specific gene expression. Total RNA was isolated from leaves (lanes 1 and 3) and latex (lanes 2 and 4) of T. officinale ( T.o. ) and T. kok-saghyz
    Figure Legend Snippet: Analysis of PPO-1 expression in planta. A, Detection of ToPPO-1 and TkPPO-1 mRNA by RT-PCR, demonstrating laticifer-specific gene expression. Total RNA was isolated from leaves (lanes 1 and 3) and latex (lanes 2 and 4) of T. officinale ( T.o. ) and T. kok-saghyz

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

    69) Product Images from "Severe liver degeneration and lack of NF-?B activation in NEMO/IKKγ-deficient mice"

    Article Title: Severe liver degeneration and lack of NF-?B activation in NEMO/IKKγ-deficient mice

    Journal: Genes & Development

    doi:

    Impaired expression of NF-κB target genes in NEMO/IKKγ -deficient MEFs. ( A ) TNFα-induced IκBα mRNA synthesis. Wild-type and NEMO/IKKγ -deficient MEFs were stimulated with TNFα (10 ng/ml) for the indicated times and 20 μg of RNA was analyzed by Northern blot analysis using a 32 P-labeled random priming probe corresponding to the full-length IκBα cDNA. β-actin , loading control. ( B ) IL-1 induced IL-6 production. Wild-type and NEMO/IKKγ -deficient MEFs (3 × 10 4 ) were left untreated or stimulated with IL-1 (10 ng/ml) for 16 hr. Levels of IL-6 (pg/ml) in culture supernatants were determined by ELISA. Values shown are the mean and standard deviation of triplicate samples.
    Figure Legend Snippet: Impaired expression of NF-κB target genes in NEMO/IKKγ -deficient MEFs. ( A ) TNFα-induced IκBα mRNA synthesis. Wild-type and NEMO/IKKγ -deficient MEFs were stimulated with TNFα (10 ng/ml) for the indicated times and 20 μg of RNA was analyzed by Northern blot analysis using a 32 P-labeled random priming probe corresponding to the full-length IκBα cDNA. β-actin , loading control. ( B ) IL-1 induced IL-6 production. Wild-type and NEMO/IKKγ -deficient MEFs (3 × 10 4 ) were left untreated or stimulated with IL-1 (10 ng/ml) for 16 hr. Levels of IL-6 (pg/ml) in culture supernatants were determined by ELISA. Values shown are the mean and standard deviation of triplicate samples.

    Techniques Used: Expressing, Northern Blot, Labeling, Enzyme-linked Immunosorbent Assay, Standard Deviation

    70) Product Images from "Transient expression of Ngn3 in Xenopus endoderm promotes early and ectopic development of pancreatic beta and delta cells"

    Article Title: Transient expression of Ngn3 in Xenopus endoderm promotes early and ectopic development of pancreatic beta and delta cells

    Journal: Genesis (New York, N.y. : 2000)

    doi: 10.1002/dvg.20828

    Summary of results and schematic diagram of microarray experiment (A) Summary of ectopic expression of endocrine markers. Red boxes indicate time of Dex treatment. Activation of Ngn3-GR for 1 or 4 hours beginning at stage 12 resulted in increased insulin and somatostatin expression, whereas continuous activation beginning at stage 12 or a 4 hour activation beginning at stage 15 resulted in increased expression of insulin , somatostatin and glucagon . (B,C) Diagram of microarray experiment. Ngn3-GR mRNA was injected in the two dorsal vegetal blastomeres at the eight cell stage. Embryos were grown until stage 12 activated with dexamethasone for four hours and confirmed targeting to the anterior endoderm at stage 15 with GFP fluorescence. All dorsal structures were removed and RNA was extracted immediately after. Four replicates of ten embryos were used to hybridize to the Affymetrix Xenopus laevis GeneChip 2.0.
    Figure Legend Snippet: Summary of results and schematic diagram of microarray experiment (A) Summary of ectopic expression of endocrine markers. Red boxes indicate time of Dex treatment. Activation of Ngn3-GR for 1 or 4 hours beginning at stage 12 resulted in increased insulin and somatostatin expression, whereas continuous activation beginning at stage 12 or a 4 hour activation beginning at stage 15 resulted in increased expression of insulin , somatostatin and glucagon . (B,C) Diagram of microarray experiment. Ngn3-GR mRNA was injected in the two dorsal vegetal blastomeres at the eight cell stage. Embryos were grown until stage 12 activated with dexamethasone for four hours and confirmed targeting to the anterior endoderm at stage 15 with GFP fluorescence. All dorsal structures were removed and RNA was extracted immediately after. Four replicates of ten embryos were used to hybridize to the Affymetrix Xenopus laevis GeneChip 2.0.

    Techniques Used: Microarray, Expressing, Activation Assay, Injection, Fluorescence

    71) Product Images from "DRB2, DRB3 and DRB5 function in a non-canonical microRNA pathway in Arabidopsis thaliana"

    Article Title: DRB2, DRB3 and DRB5 function in a non-canonical microRNA pathway in Arabidopsis thaliana

    Journal: Plant Signaling & Behavior

    doi: 10.4161/psb.21518

    Figure 2. Artificial miRNA-directed RNA silencing in drb mutants. (A) northern blot and RT-PCR analyses revealed that miR159 accumulation is reduced in the SAM region and rosette leaf petioles of four week old drb1 , drb2 and drb235 plants due
    Figure Legend Snippet: Figure 2. Artificial miRNA-directed RNA silencing in drb mutants. (A) northern blot and RT-PCR analyses revealed that miR159 accumulation is reduced in the SAM region and rosette leaf petioles of four week old drb1 , drb2 and drb235 plants due

    Techniques Used: Northern Blot, Reverse Transcription Polymerase Chain Reaction

    72) Product Images from "Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis"

    Article Title: Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis

    Journal: Genes & Development

    doi: 10.1101/gad.348405

    Differential role of the two RNAPIVs on siRNA accumulation. ( A ) Small RNA blot assays for miR-159 and various endogenous siRNAs in various nrpd1 mutants. Blots in A were stripped and reprobed multiple times as indicated on the right . ( Right panel) Small
    Figure Legend Snippet: Differential role of the two RNAPIVs on siRNA accumulation. ( A ) Small RNA blot assays for miR-159 and various endogenous siRNAs in various nrpd1 mutants. Blots in A were stripped and reprobed multiple times as indicated on the right . ( Right panel) Small

    Techniques Used: Northern blot

    73) Product Images from "Toxoplasma gondii Induces B7-2 Expression through Activation of JNK Signal Transduction ▿"

    Article Title: Toxoplasma gondii Induces B7-2 Expression through Activation of JNK Signal Transduction ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.05562-11

    Genome-wide transcriptional profiling of T. gondii -infected macrophages. BMdM were mock treated or infected with T. gondii (RH strain), and RNA was harvested at 6 hpi. cDNA was synthesized and labeled for hybridization on Affymetrix mouse 430 2.0 microarrays.
    Figure Legend Snippet: Genome-wide transcriptional profiling of T. gondii -infected macrophages. BMdM were mock treated or infected with T. gondii (RH strain), and RNA was harvested at 6 hpi. cDNA was synthesized and labeled for hybridization on Affymetrix mouse 430 2.0 microarrays.

    Techniques Used: Genome Wide, Infection, Synthesized, Labeling, Hybridization

    74) Product Images from "Persisting Murine Cytomegalovirus Can Reactivate and Has Unique Transcriptional Activity in Ocular Tissue"

    Article Title: Persisting Murine Cytomegalovirus Can Reactivate and Has Unique Transcriptional Activity in Ocular Tissue

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.18.9165-9175.2002

    Persistence of MCMV in ocular tissue 12 months p.i. DNA and RNA were extracted from the eyes of MCMV-infected (+CMV) mice ( n = 6) or mock-infected (−CMV) mice ( n = 3) 12 months p.i. The DNA was analyzed by PCR for ie-1 DNA (A), and the RNA was reverse transcribed and analyzed by RT-PCR for ie-1 RNA (B). Both PCR and RT-PCR analyses included direct incorporation of [α- 32 P]dATP into the PCR products and visualization by autoradiography. Serially diluted plasmid pRE1 of known copy number was used as a positive control for DNA analysis, and serially diluted in vitro-transcribed RNA of known copy number was reverse transcribed and used as a positive control for RNA analysis. Water was used as a PCR negative control, and APRT DNA or RNA was used as internal sample controls.
    Figure Legend Snippet: Persistence of MCMV in ocular tissue 12 months p.i. DNA and RNA were extracted from the eyes of MCMV-infected (+CMV) mice ( n = 6) or mock-infected (−CMV) mice ( n = 3) 12 months p.i. The DNA was analyzed by PCR for ie-1 DNA (A), and the RNA was reverse transcribed and analyzed by RT-PCR for ie-1 RNA (B). Both PCR and RT-PCR analyses included direct incorporation of [α- 32 P]dATP into the PCR products and visualization by autoradiography. Serially diluted plasmid pRE1 of known copy number was used as a positive control for DNA analysis, and serially diluted in vitro-transcribed RNA of known copy number was reverse transcribed and used as a positive control for RNA analysis. Water was used as a PCR negative control, and APRT DNA or RNA was used as internal sample controls.

    Techniques Used: Infection, Mouse Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Autoradiography, Plasmid Preparation, Positive Control, In Vitro, Negative Control

    75) Product Images from "Suppression of progenitor differentiation requires the long noncoding RNA ANCR"

    Article Title: Suppression of progenitor differentiation requires the long noncoding RNA ANCR

    Journal: Genes & Development

    doi: 10.1101/gad.182121.111

    Transcriptome characterization identifies ANCR as a lncRNA suppressed during terminal differentiation. ( A ) High-throughput paired-end RNA-Seq during three time points (days 0, 3, and 6) of human keratinocyte terminal differentiation was performed. Transcriptome
    Figure Legend Snippet: Transcriptome characterization identifies ANCR as a lncRNA suppressed during terminal differentiation. ( A ) High-throughput paired-end RNA-Seq during three time points (days 0, 3, and 6) of human keratinocyte terminal differentiation was performed. Transcriptome

    Techniques Used: High Throughput Screening Assay, RNA Sequencing Assay

    Related Articles

    Clone Assay:

    Article Title: GmWRKY16 Enhances Drought and Salt Tolerance Through an ABA-Mediated Pathway in Arabidopsis thaliana
    Article Snippet: The seedlings of HC2 under the treatment of 100 mM NaCl were used to extract total RNA by TRIzol reagent (Invitrogen). .. The purified PCR product was then inserted into the multiple cloning sites of the pLB vector (Tiangen Rapid DNA Ligation Kit, Beijing, China).

    Article Title: Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells
    Article Snippet: To analyze the primary structure of tRNA products in EtBr-treated cells, total RNA was isolated, tRNAs deacylated and circularized by T4 RNA ligase (MBI Fermentas) as described , reverse-transcribed with oligonucleotides cser1 or cleu1 and PCR-amplified using oligonucleotides cser1 and cser2 for analysis of tRNASer(UCN) or cleu1 and cleu2 for analysis of tRNALeu(UUR) , essentially as described previously ( ). .. PCR products were cloned (TOPO TA cloning kit, Invitrogen) and inserts Sanger-sequenced using M13 forward primer and standard dye-terminator technology as described previously ( ).

    Amplification:

    Article Title: GmWRKY16 Enhances Drought and Salt Tolerance Through an ABA-Mediated Pathway in Arabidopsis thaliana
    Article Snippet: The full-length sequence of GmWRKY16 was amplified by RT-PCR using specific primers (Supplementary Table ) and Super-Fidelity DNA polymerase (Phanta Max, Vazyme Biotech Co., Ltd.; Nanjing, China). .. The seedlings of HC2 under the treatment of 100 mM NaCl were used to extract total RNA by TRIzol reagent (Invitrogen).

    Article Title: Hepatitis E Virus (HEV) Strains in Serum Samples Can Replicate Efficiently in Cultured Cells Despite the Coexistence of HEV Antibodies: Characterization of HEV Virions in Blood Circulation ▿
    Article Snippet: For detection of HEV RNA, total RNA was extracted from 10 to 100 μl of serum sample or culture supernatant by use of TRIzol-LS reagent (Invitrogen, Carlsbad, CA) and subjected to a nested reverse transcription-PCR (RT-PCR) that amplifies a 457-nucleotide (nt) sequence in ORF2, as described previously ( ). .. The amplification product was sequenced directly on both strands, using a BigDye Terminator v3.1 cycle sequencing kit on an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA).

    Article Title: A new strategy to amplify degraded RNA from small tissue samples for microarray studies
    Article Snippet: Paragraph title: RNA sample preparation and amplification ... Total RNA was extracted from mouse C2 and NIH 3T3 cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

    Article Title: Interferon-stimulated gene ISG12b2 is localized to the inner mitochondrial membrane and mediates virus-induced cell death
    Article Snippet: Total RNA was extracted from mouse livers or Hepa 1-6 cells by Trizol reagent (Invitrogen), and 3 μ g total RNA was subjected to reverse transcription using an oligo(dT) primer and SuperScript III reverse transcriptase (Invitrogen). .. For real-time quantitative PCR amplification of cDNA, the 2–ΔΔCT method was used to quantify the relative changes in gene expression.

    Article Title: Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells
    Article Snippet: To analyze the primary structure of tRNA products in EtBr-treated cells, total RNA was isolated, tRNAs deacylated and circularized by T4 RNA ligase (MBI Fermentas) as described , reverse-transcribed with oligonucleotides cser1 or cleu1 and PCR-amplified using oligonucleotides cser1 and cser2 for analysis of tRNASer(UCN) or cleu1 and cleu2 for analysis of tRNALeu(UUR) , essentially as described previously ( ). .. The high-molecular weight tRNALeu(UUR) products induced by doxycycline treatment were gel-extracted, circularized and amplified similarly.

    DNA Ligation:

    Article Title: GmWRKY16 Enhances Drought and Salt Tolerance Through an ABA-Mediated Pathway in Arabidopsis thaliana
    Article Snippet: The seedlings of HC2 under the treatment of 100 mM NaCl were used to extract total RNA by TRIzol reagent (Invitrogen). .. The purified PCR product was then inserted into the multiple cloning sites of the pLB vector (Tiangen Rapid DNA Ligation Kit, Beijing, China).

    Synthesized:

    Article Title: Inhibiting TNF-mediated signaling: a novel therapeutic paradigm for androgen independent prostate cancer
    Article Snippet: Total RNA was isolated from control and psoralidin (15 min to 2 h)-treated PC-3 and DU-14 cells using Trizol reagent from Invitrogen Corporation (Carlsbad, CA). .. The RNA (1 μg) was subjected to reverse transcription using the Two-Step RT-PCR kit from USB (Cleveland, OH) and cDNA was synthesized.

    Article Title: Integration of light and brassinosteroid signaling pathways by a GATA transcription factor in Arabidopsis
    Article Snippet: Total RNA was extracted from Arabidopsis seedlings using the Trizol RNA extraction kit (Invitrogen, USA). .. The first-strand cDNA was synthesized by using M-MLV reverse transcriptase (Promega, USA) and used as RT-PCR templates.

    TA Cloning:

    Article Title: Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells
    Article Snippet: To analyze the primary structure of tRNA products in EtBr-treated cells, total RNA was isolated, tRNAs deacylated and circularized by T4 RNA ligase (MBI Fermentas) as described , reverse-transcribed with oligonucleotides cser1 or cleu1 and PCR-amplified using oligonucleotides cser1 and cser2 for analysis of tRNASer(UCN) or cleu1 and cleu2 for analysis of tRNALeu(UUR) , essentially as described previously ( ). .. PCR products were cloned (TOPO TA cloning kit, Invitrogen) and inserts Sanger-sequenced using M13 forward primer and standard dye-terminator technology as described previously ( ).

    Construct:

    Article Title: Bioinformatic analysis of next-generation sequencing data to identify dysregulated genes in fibroblasts of idiopathic pulmonary fibrosis
    Article Snippet: In brief, total RNA from the DHLF-IPF and NHLF cells were extracted with TRIzol® reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at Welgene Biotech Co., Ltd. (Taipei, Taiwan). .. For small RNA sequencing, the harvested cDNA constructs containing 18-40-nucleotide RNA fragments (140-155 nucleotides in length with all adapters) were selected.

    Article Title: Hepatitis E Virus (HEV) Strains in Serum Samples Can Replicate Efficiently in Cultured Cells Despite the Coexistence of HEV Antibodies: Characterization of HEV Virions in Blood Circulation ▿
    Article Snippet: For detection of HEV RNA, total RNA was extracted from 10 to 100 μl of serum sample or culture supernatant by use of TRIzol-LS reagent (Invitrogen, Carlsbad, CA) and subjected to a nested reverse transcription-PCR (RT-PCR) that amplifies a 457-nucleotide (nt) sequence in ORF2, as described previously ( ). .. A phylogenetic tree was constructed by the neighbor-joining method , based on the 412-nt ORF2 sequence.

    Real-time Polymerase Chain Reaction:

    Article Title: Involvement of Up-Regulation of DR5 Expression and Down-Regulation of c-FLIP in Niclosamide-Mediated TRAIL Sensitization in Human Renal Carcinoma Caki Cells
    Article Snippet: .. RT-PCR and Quantitative PCR (qPCR) To isolate total RNA, we used TriZol reagent (Life Technologies, Gaithersburg, MD, USA), and using Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase, we made (Gibco-BRL, Gaithersburg, MD, USA) [ ]. ..

    Article Title: Hemi-methylated CpG sites connect Dnmt1-knockdown-induced and Tet1-induced DNA demethylation during somatic cell reprogramming
    Article Snippet: .. Quantitative RT-PCR (qPCR) For the qPCR, total RNA was extracted from the cells using TRIzol (Thermo Fisher), and 5 μg of RNA were used to synthesize cDNA with ReverTra Ace® (Toyobo) and oligo-dT (Takara) according to the manufacturers’ instructions. .. The transcript levels of the genes were determined using SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara) and a CFX-96 Real-Time system (Bio-Rad).

    Article Title: Integration of light and brassinosteroid signaling pathways by a GATA transcription factor in Arabidopsis
    Article Snippet: Total RNA was extracted from Arabidopsis seedlings using the Trizol RNA extraction kit (Invitrogen, USA). .. Quantitative real-time PCR analyses were carried out on Mx3000P (Stratagene, USA) by using the SYBR® Green reagent (TOYOBO, JAPAN) according to the manufacturer’s instructions.

    Article Title: Interferon-stimulated gene ISG12b2 is localized to the inner mitochondrial membrane and mediates virus-induced cell death
    Article Snippet: Paragraph title: Real-time quantitative PCR analysis ... Total RNA was extracted from mouse livers or Hepa 1-6 cells by Trizol reagent (Invitrogen), and 3 μ g total RNA was subjected to reverse transcription using an oligo(dT) primer and SuperScript III reverse transcriptase (Invitrogen).

    Luciferase:

    Article Title: Analyzing mRNA expression identifies Smad3 as a microRNA-140 target regulated only at protein level
    Article Snippet: Total RNA was extracted from 10T1/2 cells using TRIzol reagent, following the recommendations of the supplier (Invitrogen). .. The Smad3 probe was the same 2935-bp fragment used in the luciferase assays.

    Expressing:

    Article Title: Bioinformatic analysis of next-generation sequencing data to identify dysregulated genes in fibroblasts of idiopathic pulmonary fibrosis
    Article Snippet: NGS The expression profiles of miRNAs and mRNAs were examined using NGS. .. In brief, total RNA from the DHLF-IPF and NHLF cells were extracted with TRIzol® reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at Welgene Biotech Co., Ltd. (Taipei, Taiwan).

    Article Title: Interferon-stimulated gene ISG12b2 is localized to the inner mitochondrial membrane and mediates virus-induced cell death
    Article Snippet: Total RNA was extracted from mouse livers or Hepa 1-6 cells by Trizol reagent (Invitrogen), and 3 μ g total RNA was subjected to reverse transcription using an oligo(dT) primer and SuperScript III reverse transcriptase (Invitrogen). .. For real-time quantitative PCR amplification of cDNA, the 2–ΔΔCT method was used to quantify the relative changes in gene expression.

    Western Blot:

    Article Title: Analyzing mRNA expression identifies Smad3 as a microRNA-140 target regulated only at protein level
    Article Snippet: Paragraph title: Western and Northern blot ... Total RNA was extracted from 10T1/2 cells using TRIzol reagent, following the recommendations of the supplier (Invitrogen).

    Allele-specific Oligonucleotide:

    Article Title: siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs
    Article Snippet: RLM-RACE mapping of polyadenylation sites Ago2−/− MEF cells were treated with Il4R-α siRNA 383281 or the corresponding full MOE ASO at a concentration of 100 nM. .. 3′ RACE was performed using 1 µg total RNA purified from the cells the following day using a First Choice RLM-RACE kit according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA).

    Hybridization:

    Article Title: Analyzing mRNA expression identifies Smad3 as a microRNA-140 target regulated only at protein level
    Article Snippet: Total RNA was extracted from 10T1/2 cells using TRIzol reagent, following the recommendations of the supplier (Invitrogen). .. To detect Smad3 mRNA, 20 μg of total RNA from each sample were separated on 1% denaturing agarose gel, blotted to membrane and hybridized to radioactively labeled probes overnight at 37°C in ULTRAhyb hybridization buffer (Ambion).

    Countercurrent Chromatography:

    Article Title: Inhibiting TNF-mediated signaling: a novel therapeutic paradigm for androgen independent prostate cancer
    Article Snippet: Total RNA was isolated from control and psoralidin (15 min to 2 h)-treated PC-3 and DU-14 cells using Trizol reagent from Invitrogen Corporation (Carlsbad, CA). .. TNF cDNA was synthesized using the forward primer (5′-TGA ACC AGC CTT TAG TGC CTA CCA -3′) and the reverse primer (5′-ACC ATG GTA CCC AGA CAT GCT CAA -3′) using DNA Engine (MJ Research) thermo cycler.

    Transfection:

    Article Title: Analyzing mRNA expression identifies Smad3 as a microRNA-140 target regulated only at protein level
    Article Snippet: Whole cell lysates were extracted 48 h after the second transfection and Western blotting was performed using standard procedures. .. Total RNA was extracted from 10T1/2 cells using TRIzol reagent, following the recommendations of the supplier (Invitrogen).

    Northern Blot:

    Article Title: Analyzing mRNA expression identifies Smad3 as a microRNA-140 target regulated only at protein level
    Article Snippet: Paragraph title: Western and Northern blot ... Total RNA was extracted from 10T1/2 cells using TRIzol reagent, following the recommendations of the supplier (Invitrogen).

    Generated:

    Article Title: GmWRKY16 Enhances Drought and Salt Tolerance Through an ABA-Mediated Pathway in Arabidopsis thaliana
    Article Snippet: The seedlings of HC2 under the treatment of 100 mM NaCl were used to extract total RNA by TRIzol reagent (Invitrogen). .. The methods of generated cDNA, RT-PCR reaction and agarose gel electrophoresis were described in detail previously ( ).

    Polymerase Chain Reaction:

    Article Title: Inhibiting TNF-mediated signaling: a novel therapeutic paradigm for androgen independent prostate cancer
    Article Snippet: Total RNA was isolated from control and psoralidin (15 min to 2 h)-treated PC-3 and DU-14 cells using Trizol reagent from Invitrogen Corporation (Carlsbad, CA). .. PCR primers were designed (PrimerQuest) and purchased from IDT (Coralville, IA), and GAPDH was used as the internal control for PCR.

    Article Title: GmWRKY16 Enhances Drought and Salt Tolerance Through an ABA-Mediated Pathway in Arabidopsis thaliana
    Article Snippet: The seedlings of HC2 under the treatment of 100 mM NaCl were used to extract total RNA by TRIzol reagent (Invitrogen). .. The purified PCR product was then inserted into the multiple cloning sites of the pLB vector (Tiangen Rapid DNA Ligation Kit, Beijing, China).

    Article Title: siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs
    Article Snippet: 3′ RACE was performed using 1 µg total RNA purified from the cells the following day using a First Choice RLM-RACE kit according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA). .. The sequence of the Il4R-α forward primer for the 3′RACE PCR reaction was GGAAGCTGGGCCTAGAAACT.

    Article Title: Interferon-stimulated gene ISG12b2 is localized to the inner mitochondrial membrane and mediates virus-induced cell death
    Article Snippet: Total RNA was extracted from mouse livers or Hepa 1-6 cells by Trizol reagent (Invitrogen), and 3 μ g total RNA was subjected to reverse transcription using an oligo(dT) primer and SuperScript III reverse transcriptase (Invitrogen). .. Amplification of ISG12b2 and GAPDH cDNAs was performed using a TaqMan Gene Expression assay kit containing a mixture of unlabeled PCR primers and TaqMan FAM-labeled MGB probe specific for each gene (Applied Biosystems, Foster City, CA, USA).

    Article Title: Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells
    Article Snippet: .. To analyze the primary structure of tRNA products in EtBr-treated cells, total RNA was isolated, tRNAs deacylated and circularized by T4 RNA ligase (MBI Fermentas) as described , reverse-transcribed with oligonucleotides cser1 or cleu1 and PCR-amplified using oligonucleotides cser1 and cser2 for analysis of tRNASer(UCN) or cleu1 and cleu2 for analysis of tRNALeu(UUR) , essentially as described previously ( ). .. The high-molecular weight tRNALeu(UUR) products induced by doxycycline treatment were gel-extracted, circularized and amplified similarly.

    Imaging:

    Article Title: Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells
    Article Snippet: Signals were visualized by sensitive X-ray film or, where indicated in legends, by phosphorimaging (Typhoon™ imaging system, GE Healthcare). .. To analyze the primary structure of tRNA products in EtBr-treated cells, total RNA was isolated, tRNAs deacylated and circularized by T4 RNA ligase (MBI Fermentas) as described , reverse-transcribed with oligonucleotides cser1 or cleu1 and PCR-amplified using oligonucleotides cser1 and cser2 for analysis of tRNASer(UCN) or cleu1 and cleu2 for analysis of tRNALeu(UUR) , essentially as described previously ( ).

    DNA Labeling:

    Article Title: Analyzing mRNA expression identifies Smad3 as a microRNA-140 target regulated only at protein level
    Article Snippet: Total RNA was extracted from 10T1/2 cells using TRIzol reagent, following the recommendations of the supplier (Invitrogen). .. Probes were labeled with [α-32P]dCTP using Ready-to-Go DNA labeling beads (Amersham Pharmacia Biotech), following the instructions of the supplier.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Involvement of Up-Regulation of DR5 Expression and Down-Regulation of c-FLIP in Niclosamide-Mediated TRAIL Sensitization in Human Renal Carcinoma Caki Cells
    Article Snippet: .. RT-PCR and Quantitative PCR (qPCR) To isolate total RNA, we used TriZol reagent (Life Technologies, Gaithersburg, MD, USA), and using Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase, we made (Gibco-BRL, Gaithersburg, MD, USA) [ ]. ..

    Article Title: Inhibiting TNF-mediated signaling: a novel therapeutic paradigm for androgen independent prostate cancer
    Article Snippet: Paragraph title: RNA extraction and RT-PCR analysis ... Total RNA was isolated from control and psoralidin (15 min to 2 h)-treated PC-3 and DU-14 cells using Trizol reagent from Invitrogen Corporation (Carlsbad, CA).

    Article Title: GmWRKY16 Enhances Drought and Salt Tolerance Through an ABA-Mediated Pathway in Arabidopsis thaliana
    Article Snippet: The full-length sequence of GmWRKY16 was amplified by RT-PCR using specific primers (Supplementary Table ) and Super-Fidelity DNA polymerase (Phanta Max, Vazyme Biotech Co., Ltd.; Nanjing, China). .. The seedlings of HC2 under the treatment of 100 mM NaCl were used to extract total RNA by TRIzol reagent (Invitrogen).

    Article Title: Hepatitis E Virus (HEV) Strains in Serum Samples Can Replicate Efficiently in Cultured Cells Despite the Coexistence of HEV Antibodies: Characterization of HEV Virions in Blood Circulation ▿
    Article Snippet: .. For detection of HEV RNA, total RNA was extracted from 10 to 100 μl of serum sample or culture supernatant by use of TRIzol-LS reagent (Invitrogen, Carlsbad, CA) and subjected to a nested reverse transcription-PCR (RT-PCR) that amplifies a 457-nucleotide (nt) sequence in ORF2, as described previously ( ). .. The amplification product was sequenced directly on both strands, using a BigDye Terminator v3.1 cycle sequencing kit on an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA).

    Article Title: Integration of light and brassinosteroid signaling pathways by a GATA transcription factor in Arabidopsis
    Article Snippet: Total RNA was extracted from Arabidopsis seedlings using the Trizol RNA extraction kit (Invitrogen, USA). .. The first-strand cDNA was synthesized by using M-MLV reverse transcriptase (Promega, USA) and used as RT-PCR templates.

    Cellular Antioxidant Activity Assay:

    Article Title: Inhibiting TNF-mediated signaling: a novel therapeutic paradigm for androgen independent prostate cancer
    Article Snippet: Total RNA was isolated from control and psoralidin (15 min to 2 h)-treated PC-3 and DU-14 cells using Trizol reagent from Invitrogen Corporation (Carlsbad, CA). .. TNF cDNA was synthesized using the forward primer (5′-TGA ACC AGC CTT TAG TGC CTA CCA -3′) and the reverse primer (5′-ACC ATG GTA CCC AGA CAT GCT CAA -3′) using DNA Engine (MJ Research) thermo cycler.

    RNA Sequencing Assay:

    Article Title: Bioinformatic analysis of next-generation sequencing data to identify dysregulated genes in fibroblasts of idiopathic pulmonary fibrosis
    Article Snippet: In brief, total RNA from the DHLF-IPF and NHLF cells were extracted with TRIzol® reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at Welgene Biotech Co., Ltd. (Taipei, Taiwan). .. For small RNA sequencing, the harvested cDNA constructs containing 18-40-nucleotide RNA fragments (140-155 nucleotides in length with all adapters) were selected.

    Isolation:

    Article Title: Inhibiting TNF-mediated signaling: a novel therapeutic paradigm for androgen independent prostate cancer
    Article Snippet: .. Total RNA was isolated from control and psoralidin (15 min to 2 h)-treated PC-3 and DU-14 cells using Trizol reagent from Invitrogen Corporation (Carlsbad, CA). .. The RNA (1 μg) was subjected to reverse transcription using the Two-Step RT-PCR kit from USB (Cleveland, OH) and cDNA was synthesized.

    Article Title: GmWRKY16 Enhances Drought and Salt Tolerance Through an ABA-Mediated Pathway in Arabidopsis thaliana
    Article Snippet: Paragraph title: GmWRKY16 Gene Isolation and Sequence Analysis ... The seedlings of HC2 under the treatment of 100 mM NaCl were used to extract total RNA by TRIzol reagent (Invitrogen).

    Article Title: Integration of light and brassinosteroid signaling pathways by a GATA transcription factor in Arabidopsis
    Article Snippet: Paragraph title: Total RNA isolation and quantitative RT-PCR analysis ... Total RNA was extracted from Arabidopsis seedlings using the Trizol RNA extraction kit (Invitrogen, USA).

    Article Title: Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells
    Article Snippet: .. To analyze the primary structure of tRNA products in EtBr-treated cells, total RNA was isolated, tRNAs deacylated and circularized by T4 RNA ligase (MBI Fermentas) as described , reverse-transcribed with oligonucleotides cser1 or cleu1 and PCR-amplified using oligonucleotides cser1 and cser2 for analysis of tRNASer(UCN) or cleu1 and cleu2 for analysis of tRNALeu(UUR) , essentially as described previously ( ). .. The high-molecular weight tRNALeu(UUR) products induced by doxycycline treatment were gel-extracted, circularized and amplified similarly.

    Labeling:

    Article Title: Analyzing mRNA expression identifies Smad3 as a microRNA-140 target regulated only at protein level
    Article Snippet: Total RNA was extracted from 10T1/2 cells using TRIzol reagent, following the recommendations of the supplier (Invitrogen). .. To detect Smad3 mRNA, 20 μg of total RNA from each sample were separated on 1% denaturing agarose gel, blotted to membrane and hybridized to radioactively labeled probes overnight at 37°C in ULTRAhyb hybridization buffer (Ambion).

    Purification:

    Article Title: Bioinformatic analysis of next-generation sequencing data to identify dysregulated genes in fibroblasts of idiopathic pulmonary fibrosis
    Article Snippet: In brief, total RNA from the DHLF-IPF and NHLF cells were extracted with TRIzol® reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at Welgene Biotech Co., Ltd. (Taipei, Taiwan). .. Purified RNA was quantified at an optical density of 260 nm using an ND-1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.) and the RNA quality was examined using a Bioanalyzer 2100 (Agilent Technologies, Inc., Santa Clara, CA, USA) with RNA 6000 LabChip® kit (Agilent Technologies, Inc.).

    Article Title: GmWRKY16 Enhances Drought and Salt Tolerance Through an ABA-Mediated Pathway in Arabidopsis thaliana
    Article Snippet: The seedlings of HC2 under the treatment of 100 mM NaCl were used to extract total RNA by TRIzol reagent (Invitrogen). .. The purified PCR product was then inserted into the multiple cloning sites of the pLB vector (Tiangen Rapid DNA Ligation Kit, Beijing, China).

    Article Title: siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs
    Article Snippet: .. 3′ RACE was performed using 1 µg total RNA purified from the cells the following day using a First Choice RLM-RACE kit according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA). .. The sequence of the Il4R-α forward primer for the 3′RACE PCR reaction was GGAAGCTGGGCCTAGAAACT.

    Sequencing:

    Article Title: Bioinformatic analysis of next-generation sequencing data to identify dysregulated genes in fibroblasts of idiopathic pulmonary fibrosis
    Article Snippet: In brief, total RNA from the DHLF-IPF and NHLF cells were extracted with TRIzol® reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at Welgene Biotech Co., Ltd. (Taipei, Taiwan). .. Next, the samples were prepared for library construction and sequencing using an Illumina preparation kit (Illumina, Inc., San Diego, CA, USA).

    Article Title: GmWRKY16 Enhances Drought and Salt Tolerance Through an ABA-Mediated Pathway in Arabidopsis thaliana
    Article Snippet: Paragraph title: GmWRKY16 Gene Isolation and Sequence Analysis ... The seedlings of HC2 under the treatment of 100 mM NaCl were used to extract total RNA by TRIzol reagent (Invitrogen).

    Article Title: siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs
    Article Snippet: 3′ RACE was performed using 1 µg total RNA purified from the cells the following day using a First Choice RLM-RACE kit according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA). .. The sequence of the Il4R-α forward primer for the 3′RACE PCR reaction was GGAAGCTGGGCCTAGAAACT.

    Article Title: Hepatitis E Virus (HEV) Strains in Serum Samples Can Replicate Efficiently in Cultured Cells Despite the Coexistence of HEV Antibodies: Characterization of HEV Virions in Blood Circulation ▿
    Article Snippet: .. For detection of HEV RNA, total RNA was extracted from 10 to 100 μl of serum sample or culture supernatant by use of TRIzol-LS reagent (Invitrogen, Carlsbad, CA) and subjected to a nested reverse transcription-PCR (RT-PCR) that amplifies a 457-nucleotide (nt) sequence in ORF2, as described previously ( ). .. The amplification product was sequenced directly on both strands, using a BigDye Terminator v3.1 cycle sequencing kit on an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA).

    Quantitative RT-PCR:

    Article Title: Hemi-methylated CpG sites connect Dnmt1-knockdown-induced and Tet1-induced DNA demethylation during somatic cell reprogramming
    Article Snippet: .. Quantitative RT-PCR (qPCR) For the qPCR, total RNA was extracted from the cells using TRIzol (Thermo Fisher), and 5 μg of RNA were used to synthesize cDNA with ReverTra Ace® (Toyobo) and oligo-dT (Takara) according to the manufacturers’ instructions. .. The transcript levels of the genes were determined using SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara) and a CFX-96 Real-Time system (Bio-Rad).

    Article Title: Integration of light and brassinosteroid signaling pathways by a GATA transcription factor in Arabidopsis
    Article Snippet: Paragraph title: Total RNA isolation and quantitative RT-PCR analysis ... Total RNA was extracted from Arabidopsis seedlings using the Trizol RNA extraction kit (Invitrogen, USA).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells
    Article Snippet: For aminoacylation analyses, total RNA was dissolved on ice in 0.1 M sodium acetate, pH 5.2 and fractionated on acidic 6.5% PAGE-7 M urea gels essentially as described previously ( , ). .. To analyze the primary structure of tRNA products in EtBr-treated cells, total RNA was isolated, tRNAs deacylated and circularized by T4 RNA ligase (MBI Fermentas) as described , reverse-transcribed with oligonucleotides cser1 or cleu1 and PCR-amplified using oligonucleotides cser1 and cser2 for analysis of tRNASer(UCN) or cleu1 and cleu2 for analysis of tRNALeu(UUR) , essentially as described previously ( ).

    IA:

    Article Title: Inhibiting TNF-mediated signaling: a novel therapeutic paradigm for androgen independent prostate cancer
    Article Snippet: Total RNA was isolated from control and psoralidin (15 min to 2 h)-treated PC-3 and DU-14 cells using Trizol reagent from Invitrogen Corporation (Carlsbad, CA). .. PCR primers were designed (PrimerQuest) and purchased from IDT (Coralville, IA), and GAPDH was used as the internal control for PCR.

    Agarose Gel Electrophoresis:

    Article Title: GmWRKY16 Enhances Drought and Salt Tolerance Through an ABA-Mediated Pathway in Arabidopsis thaliana
    Article Snippet: The seedlings of HC2 under the treatment of 100 mM NaCl were used to extract total RNA by TRIzol reagent (Invitrogen). .. The methods of generated cDNA, RT-PCR reaction and agarose gel electrophoresis were described in detail previously ( ).

    Article Title: siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs
    Article Snippet: 3′ RACE was performed using 1 µg total RNA purified from the cells the following day using a First Choice RLM-RACE kit according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA). .. Following 35 cycles of PCR, 10 µl was run on a 2% agarose gel in 1 × TBE buffer and visualized by ethidium bromide staining.

    Article Title: Analyzing mRNA expression identifies Smad3 as a microRNA-140 target regulated only at protein level
    Article Snippet: Total RNA was extracted from 10T1/2 cells using TRIzol reagent, following the recommendations of the supplier (Invitrogen). .. To detect Smad3 mRNA, 20 μg of total RNA from each sample were separated on 1% denaturing agarose gel, blotted to membrane and hybridized to radioactively labeled probes overnight at 37°C in ULTRAhyb hybridization buffer (Ambion).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Inhibiting TNF-mediated signaling: a novel therapeutic paradigm for androgen independent prostate cancer
    Article Snippet: Total RNA was isolated from control and psoralidin (15 min to 2 h)-treated PC-3 and DU-14 cells using Trizol reagent from Invitrogen Corporation (Carlsbad, CA). .. TNF cDNA was synthesized using the forward primer (5′-TGA ACC AGC CTT TAG TGC CTA CCA -3′) and the reverse primer (5′-ACC ATG GTA CCC AGA CAT GCT CAA -3′) using DNA Engine (MJ Research) thermo cycler.

    Plasmid Preparation:

    Article Title: GmWRKY16 Enhances Drought and Salt Tolerance Through an ABA-Mediated Pathway in Arabidopsis thaliana
    Article Snippet: The seedlings of HC2 under the treatment of 100 mM NaCl were used to extract total RNA by TRIzol reagent (Invitrogen). .. The purified PCR product was then inserted into the multiple cloning sites of the pLB vector (Tiangen Rapid DNA Ligation Kit, Beijing, China).

    SYBR Green Assay:

    Article Title: Integration of light and brassinosteroid signaling pathways by a GATA transcription factor in Arabidopsis
    Article Snippet: Total RNA was extracted from Arabidopsis seedlings using the Trizol RNA extraction kit (Invitrogen, USA). .. Quantitative real-time PCR analyses were carried out on Mx3000P (Stratagene, USA) by using the SYBR® Green reagent (TOYOBO, JAPAN) according to the manufacturer’s instructions.

    RNA Extraction:

    Article Title: Inhibiting TNF-mediated signaling: a novel therapeutic paradigm for androgen independent prostate cancer
    Article Snippet: Paragraph title: RNA extraction and RT-PCR analysis ... Total RNA was isolated from control and psoralidin (15 min to 2 h)-treated PC-3 and DU-14 cells using Trizol reagent from Invitrogen Corporation (Carlsbad, CA).

    Article Title: Integration of light and brassinosteroid signaling pathways by a GATA transcription factor in Arabidopsis
    Article Snippet: .. Total RNA was extracted from Arabidopsis seedlings using the Trizol RNA extraction kit (Invitrogen, USA). .. The first-strand cDNA was synthesized by using M-MLV reverse transcriptase (Promega, USA) and used as RT-PCR templates.

    Sample Prep:

    Article Title: A new strategy to amplify degraded RNA from small tissue samples for microarray studies
    Article Snippet: Paragraph title: RNA sample preparation and amplification ... Total RNA was extracted from mouse C2 and NIH 3T3 cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

    Next-Generation Sequencing:

    Article Title: Bioinformatic analysis of next-generation sequencing data to identify dysregulated genes in fibroblasts of idiopathic pulmonary fibrosis
    Article Snippet: Paragraph title: NGS ... In brief, total RNA from the DHLF-IPF and NHLF cells were extracted with TRIzol® reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at Welgene Biotech Co., Ltd. (Taipei, Taiwan).

    Spectrophotometry:

    Article Title: Bioinformatic analysis of next-generation sequencing data to identify dysregulated genes in fibroblasts of idiopathic pulmonary fibrosis
    Article Snippet: In brief, total RNA from the DHLF-IPF and NHLF cells were extracted with TRIzol® reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at Welgene Biotech Co., Ltd. (Taipei, Taiwan). .. Purified RNA was quantified at an optical density of 260 nm using an ND-1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.) and the RNA quality was examined using a Bioanalyzer 2100 (Agilent Technologies, Inc., Santa Clara, CA, USA) with RNA 6000 LabChip® kit (Agilent Technologies, Inc.).

    Concentration Assay:

    Article Title: siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs
    Article Snippet: RLM-RACE mapping of polyadenylation sites Ago2−/− MEF cells were treated with Il4R-α siRNA 383281 or the corresponding full MOE ASO at a concentration of 100 nM. .. 3′ RACE was performed using 1 µg total RNA purified from the cells the following day using a First Choice RLM-RACE kit according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA).

    Staining:

    Article Title: siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs
    Article Snippet: 3′ RACE was performed using 1 µg total RNA purified from the cells the following day using a First Choice RLM-RACE kit according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA). .. Following 35 cycles of PCR, 10 µl was run on a 2% agarose gel in 1 × TBE buffer and visualized by ethidium bromide staining.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher quantitative rt pcr analysis total rna
    Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger <t>RNA</t> (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time <t>(qRT)‐PCR.</t> ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P
    Quantitative Rt Pcr Analysis Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative rt pcr analysis total rna/product/Thermo Fisher
    Average 99 stars, based on 105 article reviews
    Price from $9.99 to $1999.99
    quantitative rt pcr analysis total rna - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher total rna
    Demethylation of hemi-methylated CpG sites up-regulates pluripotent genes. a – c Approximately 20,000 protein-coding genes were divided into different groups based on their methylation levels and CpG densities near TSS (−1.5 ~ + 2.0 kb). The number of genes in the different groups were summarized in a . Genes with 42–83 CpG sites near their TSS (−1.5 ~ + 2.0 kb) were highlighted as in Zone I in a . Of the genes in Zone I, the genes with methylation levels between 40 and 60% were highlighted in Zone II. The changes in methylation of approximately 14,500 genes detected in the current RRBS were summarized in b , while the changes in the expression of approximately 11,000 genes detected in the current <t>RNA-seq</t> were summarized in c . Groups with less than 50 genes detected in RRBS or RNA-seq were not listed in b and c . d – f Demethylation of genes highlighted in Zone I and Zone II in a were compared to the average demethylation of all genes in d , e . The expression changes of 698 genes in Zone II were compared to the expression changes of all genes ( f ). g – i GO analysis results of the 698 genes in Zone II ( g ). The 22 genes that undergo significant up-regulation were selected and their demethylation was listed in h . Of these 22 genes, the expression changes of 6 pluripotent genes were determined by <t>qPCR</t> ( i )
    Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 277 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/total rna/product/Thermo Fisher
    Average 99 stars, based on 277 article reviews
    Price from $9.99 to $1999.99
    total rna - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher quantitative real time polymerase chain reaction qrt pcr total rna
    Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by <t>qRT-PCR;</t> b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P
    Quantitative Real Time Polymerase Chain Reaction Qrt Pcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 364 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative real time polymerase chain reaction qrt pcr total rna/product/Thermo Fisher
    Average 99 stars, based on 364 article reviews
    Price from $9.99 to $1999.99
    quantitative real time polymerase chain reaction qrt pcr total rna - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    Image Search Results


    Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger RNA (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time (qRT)‐PCR. ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P

    Journal: Thoracic Cancer

    Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB

    doi: 10.1111/1759-7714.12895

    Figure Lengend Snippet: Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger RNA (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time (qRT)‐PCR. ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P

    Article Snippet: Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific).

    Techniques: Expressing, Migration, In Vitro, Quantitative RT-PCR, Western Blot

    Downregulation of SUMO1 suppresses the proliferation, cell cycle progression, colony formation, migration, and invasion of Calu‐1 cells in vitro. ( a ) Quantitative real time‐PCR analysis revealed that the messenger RNA (mRNA) expression levels of SUMO1 in short hairpin RNA (shRNA)‐SUMO1‐1 Calu‐1 cells were significantly downregulated. ( b ) Similar results were obtained through Western blot analysis. ( c ) Downregulation of SUMO1 expression significantly inhibited the ( c ) proliferation, ( e , f ) colony‐formation ability, ( g , h ) migration, and ( i , j ) invasion of Calu‐1 cells. ( d ) Downregulation of SUMO1 reduced the number of Calu‐1 cells in the S phase of the cell cycle.

    Journal: Thoracic Cancer

    Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB

    doi: 10.1111/1759-7714.12895

    Figure Lengend Snippet: Downregulation of SUMO1 suppresses the proliferation, cell cycle progression, colony formation, migration, and invasion of Calu‐1 cells in vitro. ( a ) Quantitative real time‐PCR analysis revealed that the messenger RNA (mRNA) expression levels of SUMO1 in short hairpin RNA (shRNA)‐SUMO1‐1 Calu‐1 cells were significantly downregulated. ( b ) Similar results were obtained through Western blot analysis. ( c ) Downregulation of SUMO1 expression significantly inhibited the ( c ) proliferation, ( e , f ) colony‐formation ability, ( g , h ) migration, and ( i , j ) invasion of Calu‐1 cells. ( d ) Downregulation of SUMO1 reduced the number of Calu‐1 cells in the S phase of the cell cycle.

    Article Snippet: Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific).

    Techniques: Migration, In Vitro, Real-time Polymerase Chain Reaction, Expressing, shRNA, Western Blot

    Demethylation of hemi-methylated CpG sites up-regulates pluripotent genes. a – c Approximately 20,000 protein-coding genes were divided into different groups based on their methylation levels and CpG densities near TSS (−1.5 ~ + 2.0 kb). The number of genes in the different groups were summarized in a . Genes with 42–83 CpG sites near their TSS (−1.5 ~ + 2.0 kb) were highlighted as in Zone I in a . Of the genes in Zone I, the genes with methylation levels between 40 and 60% were highlighted in Zone II. The changes in methylation of approximately 14,500 genes detected in the current RRBS were summarized in b , while the changes in the expression of approximately 11,000 genes detected in the current RNA-seq were summarized in c . Groups with less than 50 genes detected in RRBS or RNA-seq were not listed in b and c . d – f Demethylation of genes highlighted in Zone I and Zone II in a were compared to the average demethylation of all genes in d , e . The expression changes of 698 genes in Zone II were compared to the expression changes of all genes ( f ). g – i GO analysis results of the 698 genes in Zone II ( g ). The 22 genes that undergo significant up-regulation were selected and their demethylation was listed in h . Of these 22 genes, the expression changes of 6 pluripotent genes were determined by qPCR ( i )

    Journal: Cell Discovery

    Article Title: Hemi-methylated CpG sites connect Dnmt1-knockdown-induced and Tet1-induced DNA demethylation during somatic cell reprogramming

    doi: 10.1038/s41421-018-0074-6

    Figure Lengend Snippet: Demethylation of hemi-methylated CpG sites up-regulates pluripotent genes. a – c Approximately 20,000 protein-coding genes were divided into different groups based on their methylation levels and CpG densities near TSS (−1.5 ~ + 2.0 kb). The number of genes in the different groups were summarized in a . Genes with 42–83 CpG sites near their TSS (−1.5 ~ + 2.0 kb) were highlighted as in Zone I in a . Of the genes in Zone I, the genes with methylation levels between 40 and 60% were highlighted in Zone II. The changes in methylation of approximately 14,500 genes detected in the current RRBS were summarized in b , while the changes in the expression of approximately 11,000 genes detected in the current RNA-seq were summarized in c . Groups with less than 50 genes detected in RRBS or RNA-seq were not listed in b and c . d – f Demethylation of genes highlighted in Zone I and Zone II in a were compared to the average demethylation of all genes in d , e . The expression changes of 698 genes in Zone II were compared to the expression changes of all genes ( f ). g – i GO analysis results of the 698 genes in Zone II ( g ). The 22 genes that undergo significant up-regulation were selected and their demethylation was listed in h . Of these 22 genes, the expression changes of 6 pluripotent genes were determined by qPCR ( i )

    Article Snippet: Quantitative RT-PCR (qPCR) For the qPCR, total RNA was extracted from the cells using TRIzol (Thermo Fisher), and 5 μg of RNA were used to synthesize cDNA with ReverTra Ace® (Toyobo) and oligo-dT (Takara) according to the manufacturers’ instructions.

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

    Two types of DNA demethylation differentially influence gene expression. The results obtained from the WGBS, ATAC-seq, and RNA-seq in assays of MEFs were analyzed together in a – g . The results obtained from WGBS (MEFs) and RRBS/RNA-seq (control, Tet1, and sh-Dnmt1 groups in MEFs) were analyzed in h , i . a First, genes were divided into 102 groups based their methylation levels (0%, 0–1%, …, 99–100%, and 100%). Then, the genes in each group were equally separated into two sub-groups, i.e., high enrichment and low enrichment, based on their AMDs. The genes from all high/low enrichment sub-groups were combined and sorted based on their methylation levels, and were presented along with their chromatin accessibility. The chromatin accessibilities generated from ATAC-seq were listed near TSS (−2.0 ~ + 2.0 kb). Gene methylation was plotted on the left. b First, the genes were divided into 20 groups (0–5%, …, and 95–100%). Then, the averages of gene methylation and chromatin accessibility were plotted. c – g Six sets of genes with particular methylation levels and enrichment of hemi-methylation were selected ( c ). The average methylation levels ( d ), enrichment of hemi-methylation ( e ), chromatin accessibilities ( f ), and gene expression ( g ) in MEFs were listed. The number of genes in each group was provided. h – i Of the 300 selected genes, the expression of 197 genes was detected in the current RNA-seq. The expression changes induced by Tet1 and sh-Dnmt1 were plotted in h . In total, 20 genes were randomly selected from these 197 genes, and the chromatin accessibilities of the genes in the Tet1 and sh-Dnmt1 groups were determined by ATAC-qPCR, compared, and plotted in i

    Journal: Cell Discovery

    Article Title: Hemi-methylated CpG sites connect Dnmt1-knockdown-induced and Tet1-induced DNA demethylation during somatic cell reprogramming

    doi: 10.1038/s41421-018-0074-6

    Figure Lengend Snippet: Two types of DNA demethylation differentially influence gene expression. The results obtained from the WGBS, ATAC-seq, and RNA-seq in assays of MEFs were analyzed together in a – g . The results obtained from WGBS (MEFs) and RRBS/RNA-seq (control, Tet1, and sh-Dnmt1 groups in MEFs) were analyzed in h , i . a First, genes were divided into 102 groups based their methylation levels (0%, 0–1%, …, 99–100%, and 100%). Then, the genes in each group were equally separated into two sub-groups, i.e., high enrichment and low enrichment, based on their AMDs. The genes from all high/low enrichment sub-groups were combined and sorted based on their methylation levels, and were presented along with their chromatin accessibility. The chromatin accessibilities generated from ATAC-seq were listed near TSS (−2.0 ~ + 2.0 kb). Gene methylation was plotted on the left. b First, the genes were divided into 20 groups (0–5%, …, and 95–100%). Then, the averages of gene methylation and chromatin accessibility were plotted. c – g Six sets of genes with particular methylation levels and enrichment of hemi-methylation were selected ( c ). The average methylation levels ( d ), enrichment of hemi-methylation ( e ), chromatin accessibilities ( f ), and gene expression ( g ) in MEFs were listed. The number of genes in each group was provided. h – i Of the 300 selected genes, the expression of 197 genes was detected in the current RNA-seq. The expression changes induced by Tet1 and sh-Dnmt1 were plotted in h . In total, 20 genes were randomly selected from these 197 genes, and the chromatin accessibilities of the genes in the Tet1 and sh-Dnmt1 groups were determined by ATAC-qPCR, compared, and plotted in i

    Article Snippet: Quantitative RT-PCR (qPCR) For the qPCR, total RNA was extracted from the cells using TRIzol (Thermo Fisher), and 5 μg of RNA were used to synthesize cDNA with ReverTra Ace® (Toyobo) and oligo-dT (Takara) according to the manufacturers’ instructions.

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

    Schematic illustration of the study design. Following culture of the IPF fibroblasts and healthy lung fibroblasts, the total RNA were extracted and deep sequenced using a next-generation sequencing platform. The differentially expressed genes ( > 2 fold-change) were identified, and analyzed with IPA and DAVID for pathway analysis and functional interpretation. The GEO IPF databases were analyzed to confirm dysregulated genes identified in the present study. Conversely, the differentially expressed miRNAs ( > 2 fold-change) were analyzed with miRmap for target prediction. Then, genes with potential miRNA-mRNA interactions were determined by Venn diagram analysis. These miRNA-mRNA interactions were verified by a second miRNA prediction database, TargetScan. Finally, a literature search was performed and a hypothesis was generated. IPF, idiopathic pulmonary fibrosis; IPA, Ingenuity ® Pathway Analysis; DAVID, Database for Annotation, Visualization, and Integrated Discovery; GEO, Gene Expression Omnibus.

    Journal: International Journal of Molecular Medicine

    Article Title: Bioinformatic analysis of next-generation sequencing data to identify dysregulated genes in fibroblasts of idiopathic pulmonary fibrosis

    doi: 10.3892/ijmm.2019.4086

    Figure Lengend Snippet: Schematic illustration of the study design. Following culture of the IPF fibroblasts and healthy lung fibroblasts, the total RNA were extracted and deep sequenced using a next-generation sequencing platform. The differentially expressed genes ( > 2 fold-change) were identified, and analyzed with IPA and DAVID for pathway analysis and functional interpretation. The GEO IPF databases were analyzed to confirm dysregulated genes identified in the present study. Conversely, the differentially expressed miRNAs ( > 2 fold-change) were analyzed with miRmap for target prediction. Then, genes with potential miRNA-mRNA interactions were determined by Venn diagram analysis. These miRNA-mRNA interactions were verified by a second miRNA prediction database, TargetScan. Finally, a literature search was performed and a hypothesis was generated. IPF, idiopathic pulmonary fibrosis; IPA, Ingenuity ® Pathway Analysis; DAVID, Database for Annotation, Visualization, and Integrated Discovery; GEO, Gene Expression Omnibus.

    Article Snippet: In brief, total RNA from the DHLF-IPF and NHLF cells were extracted with TRIzol® reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at Welgene Biotech Co., Ltd. (Taipei, Taiwan).

    Techniques: Next-Generation Sequencing, Indirect Immunoperoxidase Assay, Functional Assay, Generated, Expressing

    Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by qRT-PCR; b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P

    Journal: Cancer Cell International

    Article Title: miR-96 exerts carcinogenic effect by activating AKT/GSK-3β/β-catenin signaling pathway through targeting inhibition of FOXO1 in hepatocellular carcinoma

    doi: 10.1186/s12935-019-0756-7

    Figure Lengend Snippet: Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by qRT-PCR; b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P

    Article Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) Total RNA in tumor tissues and cells was obtained by Trizol kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the the instruction manual.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    FOXO1 was directly suppressed by miR-96. a Prediction of binding sites for FOXO1 and miR-96 by Target Scan; b Luciferase reporter assay; c FOXO1 mRNA expression by qRT-PCR; d FOXO1 protein expression by Western blot. ## P

    Journal: Cancer Cell International

    Article Title: miR-96 exerts carcinogenic effect by activating AKT/GSK-3β/β-catenin signaling pathway through targeting inhibition of FOXO1 in hepatocellular carcinoma

    doi: 10.1186/s12935-019-0756-7

    Figure Lengend Snippet: FOXO1 was directly suppressed by miR-96. a Prediction of binding sites for FOXO1 and miR-96 by Target Scan; b Luciferase reporter assay; c FOXO1 mRNA expression by qRT-PCR; d FOXO1 protein expression by Western blot. ## P

    Article Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) Total RNA in tumor tissues and cells was obtained by Trizol kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the the instruction manual.

    Techniques: Binding Assay, Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR, Western Blot