qrt pcr assays  (Thermo Fisher)


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    Structured Review

    Thermo Fisher qrt pcr assays
    Relative levels of gag RNA expression in FBL-3 tumor cells compared to liver and thymus cells from gag-Tg mice. Total RNA was extracted from FBL-3 cells, thymus or liver biopsies from gag-Tg mice; samples were isolated from either two independent FBL-3 cell cultures (#1, #2) or two individual mice (#1, #2). Following cDNA synthesis, <t>qRT-PCR</t> was used to compare the relative levels of gag gene expression between samples; qRT-PCR reactions were run in triplicate for each sample and combined; error bars: SEM. -actin transcript amplification was detected in all cDNA samples and was used to normalize gene expression levels.
    Qrt Pcr Assays, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 437 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    qrt pcr assays - by Bioz Stars, 2020-10
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    Images

    1) Product Images from "DNA fusion gene vaccination mobilizes effective anti-leukemic cytotoxic T lymphocytes from a tolerized repertoire"

    Article Title: DNA fusion gene vaccination mobilizes effective anti-leukemic cytotoxic T lymphocytes from a tolerized repertoire

    Journal: European journal of immunology

    doi: 10.1002/eji.200838213

    Relative levels of gag RNA expression in FBL-3 tumor cells compared to liver and thymus cells from gag-Tg mice. Total RNA was extracted from FBL-3 cells, thymus or liver biopsies from gag-Tg mice; samples were isolated from either two independent FBL-3 cell cultures (#1, #2) or two individual mice (#1, #2). Following cDNA synthesis, qRT-PCR was used to compare the relative levels of gag gene expression between samples; qRT-PCR reactions were run in triplicate for each sample and combined; error bars: SEM. -actin transcript amplification was detected in all cDNA samples and was used to normalize gene expression levels.
    Figure Legend Snippet: Relative levels of gag RNA expression in FBL-3 tumor cells compared to liver and thymus cells from gag-Tg mice. Total RNA was extracted from FBL-3 cells, thymus or liver biopsies from gag-Tg mice; samples were isolated from either two independent FBL-3 cell cultures (#1, #2) or two individual mice (#1, #2). Following cDNA synthesis, qRT-PCR was used to compare the relative levels of gag gene expression between samples; qRT-PCR reactions were run in triplicate for each sample and combined; error bars: SEM. -actin transcript amplification was detected in all cDNA samples and was used to normalize gene expression levels.

    Techniques Used: RNA Expression, Mouse Assay, Isolation, Quantitative RT-PCR, Expressing, Amplification

    2) Product Images from "Diurnal and Circadian Rhythms in the Tomato Transcriptome and Their Modulation by Cryptochrome Photoreceptors"

    Article Title: Diurnal and Circadian Rhythms in the Tomato Transcriptome and Their Modulation by Cryptochrome Photoreceptors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002798

    Effect of CRY1a loss-of function and CRY2 over-expression on circadian expression of tomato cryptochrome (A) and phytochrome (B) genes in LL. Wt, cry1a - and CRY2-OX tomato plants were entrained under LD cycles and then transferred to LL. The abundance of the mRNAs were measured by QRT-PCR. Results are presented as a proportion of the highest value after normalization with β-actin. Open and hatched bars along the horizontal axis represent light and subjective night periods, respectively. Time points are measured in hours from dawn (zeitgeber Time [ZT]). An additional panel depicts CRY2 transcript values in wt and cry1a - genotypes to avoid the masking effect of CRY2-OX values. Data shown are the average of two biological replicates, with error bars representing SEM. Circles (O) indicate time points of CRY2-OX and cry1a- genotypes, significantly different from the corresponding ones in wt genotype (Student's t test, P ≤ 0.05). For each genotype, X indicate time points significantly different from the highest transcription value (Student's t test, P ≤ 0.05).
    Figure Legend Snippet: Effect of CRY1a loss-of function and CRY2 over-expression on circadian expression of tomato cryptochrome (A) and phytochrome (B) genes in LL. Wt, cry1a - and CRY2-OX tomato plants were entrained under LD cycles and then transferred to LL. The abundance of the mRNAs were measured by QRT-PCR. Results are presented as a proportion of the highest value after normalization with β-actin. Open and hatched bars along the horizontal axis represent light and subjective night periods, respectively. Time points are measured in hours from dawn (zeitgeber Time [ZT]). An additional panel depicts CRY2 transcript values in wt and cry1a - genotypes to avoid the masking effect of CRY2-OX values. Data shown are the average of two biological replicates, with error bars representing SEM. Circles (O) indicate time points of CRY2-OX and cry1a- genotypes, significantly different from the corresponding ones in wt genotype (Student's t test, P ≤ 0.05). For each genotype, X indicate time points significantly different from the highest transcription value (Student's t test, P ≤ 0.05).

    Techniques Used: Over Expression, Expressing, Quantitative RT-PCR

    Diurnal oscillations of Cryptochrome and Phytochrome transcripts analyzed by QRT-PCR in tomato plants grown in LD conditions. Results are presented as a proportion of the highest value after normalization with β-actin. Open and closed bars along the horizontal axis represent light and dark periods, respectively. Time points are measured in hours from dawn (zeitgeber Time [ZT]). Data shown are the average of two biological replicates, with error bars representing SEM. Time points of CRY1a , CRY2 , PHYA , PHYB1 , PHYB2 , PHYE and PHYF transcripts significantly different from the corresponding ones of the CRY1b gene (Student's t test, P ≤ 0.05) are marked with an O. Time points significantly different from the highest transcription value (Student's t test, P ≤ 0.05) are marked with an X.
    Figure Legend Snippet: Diurnal oscillations of Cryptochrome and Phytochrome transcripts analyzed by QRT-PCR in tomato plants grown in LD conditions. Results are presented as a proportion of the highest value after normalization with β-actin. Open and closed bars along the horizontal axis represent light and dark periods, respectively. Time points are measured in hours from dawn (zeitgeber Time [ZT]). Data shown are the average of two biological replicates, with error bars representing SEM. Time points of CRY1a , CRY2 , PHYA , PHYB1 , PHYB2 , PHYE and PHYF transcripts significantly different from the corresponding ones of the CRY1b gene (Student's t test, P ≤ 0.05) are marked with an O. Time points significantly different from the highest transcription value (Student's t test, P ≤ 0.05) are marked with an X.

    Techniques Used: Quantitative RT-PCR

    Pearson's correlations between gene expression levels of photoreceptors determined by QRT-PCR and the TOM2 microarray.
    Figure Legend Snippet: Pearson's correlations between gene expression levels of photoreceptors determined by QRT-PCR and the TOM2 microarray.

    Techniques Used: Expressing, Quantitative RT-PCR, Microarray

    Effect of CRY1a loss and CRY2 over-expression on light induced transcription of tomato GI and LHC4 genes. Wt, cry1a - and CRY2-OX tomato plants were grown under LD (A) and LL (B) conditions. The abundance of the mRNAs of GI and LHC4 genes were measured by QRT-PCR. Results are presented as a proportion of the highest value after normalization with β-actin. Open, closed and hatched bars along the horizontal axis represent light, dark and subjective night periods, respectively. Time points are measured in hours from dawn (zeitgeber Time [ZT]). Data shown are the average of two biological replicates, with error bars representing SEM. Circles (O) indicate time points of CRY2-OX and cry1a- genotypes, significantly different from the corresponding ones in wt (Student's t test, P ≤ 0.05). For each genotype, X indicate time points significantly different from the highest transcription value (Student's t test, P ≤ 0.05).
    Figure Legend Snippet: Effect of CRY1a loss and CRY2 over-expression on light induced transcription of tomato GI and LHC4 genes. Wt, cry1a - and CRY2-OX tomato plants were grown under LD (A) and LL (B) conditions. The abundance of the mRNAs of GI and LHC4 genes were measured by QRT-PCR. Results are presented as a proportion of the highest value after normalization with β-actin. Open, closed and hatched bars along the horizontal axis represent light, dark and subjective night periods, respectively. Time points are measured in hours from dawn (zeitgeber Time [ZT]). Data shown are the average of two biological replicates, with error bars representing SEM. Circles (O) indicate time points of CRY2-OX and cry1a- genotypes, significantly different from the corresponding ones in wt (Student's t test, P ≤ 0.05). For each genotype, X indicate time points significantly different from the highest transcription value (Student's t test, P ≤ 0.05).

    Techniques Used: Over Expression, Quantitative RT-PCR

    Effect of CRY1a loss-of-function and CRY2 over-expression on diurnal expression of tomato cryptochrome (A) and phytochrome (B) genes. Wt, cry1a - and CRY2-OX tomato plants were grown under LD conditions. The abundance of the mRNAs were measured by QRT-PCR. Results are presented as a proportion of the highest value after normalization with β-actin. Open and closed bars along the horizontal axis represent light and dark periods, respectively. Time points are measured in hours from dawn (zeitgeber Time [ZT]). An additional panel depicts CRY2 transcript values in wt and cry1a - genotypes to avoid the masking effect of CRY2-OX values. Data shown are the average of two biological replicates, with error bars representing SEM. Circles (O) indicate time points of CRY2-OX and cry1a- genotypes, significantly different from the corresponding ones in wt genotype (Student's t test, P ≤ 0.05). For each genotype X indicate time points significantly different from the highest transcription value (Student's t test, P ≤ 0.05).
    Figure Legend Snippet: Effect of CRY1a loss-of-function and CRY2 over-expression on diurnal expression of tomato cryptochrome (A) and phytochrome (B) genes. Wt, cry1a - and CRY2-OX tomato plants were grown under LD conditions. The abundance of the mRNAs were measured by QRT-PCR. Results are presented as a proportion of the highest value after normalization with β-actin. Open and closed bars along the horizontal axis represent light and dark periods, respectively. Time points are measured in hours from dawn (zeitgeber Time [ZT]). An additional panel depicts CRY2 transcript values in wt and cry1a - genotypes to avoid the masking effect of CRY2-OX values. Data shown are the average of two biological replicates, with error bars representing SEM. Circles (O) indicate time points of CRY2-OX and cry1a- genotypes, significantly different from the corresponding ones in wt genotype (Student's t test, P ≤ 0.05). For each genotype X indicate time points significantly different from the highest transcription value (Student's t test, P ≤ 0.05).

    Techniques Used: Over Expression, Expressing, Quantitative RT-PCR

    3) Product Images from "Proteomics and Transcriptomics of BJAB Cells Expressing the Epstein-Barr Virus Noncoding RNAs EBER1 and EBER2"

    Article Title: Proteomics and Transcriptomics of BJAB Cells Expressing the Epstein-Barr Virus Noncoding RNAs EBER1 and EBER2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0124638

    WB and qRT-PCR validation assays. (A) Representative precursor ion SILAC pair MS spectra and WB assay for ADAD2 in the BJAB-EBER1/2 vs BJAB-CTL comparison. (B) WB assays showing the measured fold-changes for La and L22 in the BJAB-EBER1/2 vs BJAB-CTL comparison, which in our SILAC data do not change. (C) Representative precursor ion SILAC pair MS spectra for EIF2B4, plus the corresponding WB and qRT-PCR assays in the BJAB-EBER1/2 vs BJAB-CTL comparison. (D) Representative precursor ion SILAC pair MS spectra and WB assay for the FCRLA/1 protein group in the BJAB-EBER1/2 vs BJAB-CTL comparison. Total mRNA level fold-changes based on qRT-PCR assays for the family of FCRLs expressed in BJAB cells in the two indicated comparisons. (E) Representative precursor ion SILAC pair MS spectra for PIK3AP1, plus the corresponding WB and qRT-PCR assays in the BJAB-EBER1/2 vs BJAB-CTL comparison. (F) WB assays for total AKT and pAKT in the three indicated comparisons. In all panels, the WB densitometry and qRT-PCR bar plots are based on three independent biological replicates. Error bars indicate the standard deviation. In the qRT-PCR measurements, the values obtained were normalized to ACTB as a control. (G) WB assays using antibodies for ADAD2 and EIF2B4 in total cell lysates from 293T cells transiently transfected (24 hours) with a control plasmid (empty FRT) or one encoding the EBERs (FRT-EBER1/2). Also shown are the WB assays for PIK3AP1, ADAD2 and EIF2B4 in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison. Error bars reflect the standard deviation of three biological replicates.
    Figure Legend Snippet: WB and qRT-PCR validation assays. (A) Representative precursor ion SILAC pair MS spectra and WB assay for ADAD2 in the BJAB-EBER1/2 vs BJAB-CTL comparison. (B) WB assays showing the measured fold-changes for La and L22 in the BJAB-EBER1/2 vs BJAB-CTL comparison, which in our SILAC data do not change. (C) Representative precursor ion SILAC pair MS spectra for EIF2B4, plus the corresponding WB and qRT-PCR assays in the BJAB-EBER1/2 vs BJAB-CTL comparison. (D) Representative precursor ion SILAC pair MS spectra and WB assay for the FCRLA/1 protein group in the BJAB-EBER1/2 vs BJAB-CTL comparison. Total mRNA level fold-changes based on qRT-PCR assays for the family of FCRLs expressed in BJAB cells in the two indicated comparisons. (E) Representative precursor ion SILAC pair MS spectra for PIK3AP1, plus the corresponding WB and qRT-PCR assays in the BJAB-EBER1/2 vs BJAB-CTL comparison. (F) WB assays for total AKT and pAKT in the three indicated comparisons. In all panels, the WB densitometry and qRT-PCR bar plots are based on three independent biological replicates. Error bars indicate the standard deviation. In the qRT-PCR measurements, the values obtained were normalized to ACTB as a control. (G) WB assays using antibodies for ADAD2 and EIF2B4 in total cell lysates from 293T cells transiently transfected (24 hours) with a control plasmid (empty FRT) or one encoding the EBERs (FRT-EBER1/2). Also shown are the WB assays for PIK3AP1, ADAD2 and EIF2B4 in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison. Error bars reflect the standard deviation of three biological replicates.

    Techniques Used: Western Blot, Quantitative RT-PCR, Mass Spectrometry, CTL Assay, Standard Deviation, Transfection, Plasmid Preparation

    GO analysis and tumorigenic signature. Using the DAVID web-based portal ( http://david.abcc.ncifcrf.gov/ ), we performed a GO analysis of the two datasets (low and high EBER1/2 expression) based on the main PANTHER classification terms, Molecular Function (MF) and Biological Property (BP). (A) GO analysis of the BJAB-EBER1/2 vs BJAB-CTL comparison (low EBER1/2 expression levels). Right and left plots indicate the enrichment in MF and BP terms, respectively. (B) As in the previous panel, GO analysis of the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison (high EBER1/2 levels). (C) Figure adapted from Hanahan and Weinberg [ 37 ] to highlight the oncogenic signature enriched in BJAB-EBNA1-EBER1/2 cells. (D) qRT-PCR validation assays for il10 , vegfa and zeb1 mRNAs from three biological replicates per comparison. The left plot shows an overlay of the BJAB-EBER1/2 vs BJAB-CTL (blue) and BJAB-EBNA1-EBER1-/2 vs BJAB-EBNA1 (red) fold-changes. The right plot shows the BJAB-B1 vs BJAB-parental fold-changes. All values were normalized to ACTB as a control. (E) WB assays with an antibody specific for ZEB1 in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison. Densitometry values and error bars reflect three independent biological replicates.
    Figure Legend Snippet: GO analysis and tumorigenic signature. Using the DAVID web-based portal ( http://david.abcc.ncifcrf.gov/ ), we performed a GO analysis of the two datasets (low and high EBER1/2 expression) based on the main PANTHER classification terms, Molecular Function (MF) and Biological Property (BP). (A) GO analysis of the BJAB-EBER1/2 vs BJAB-CTL comparison (low EBER1/2 expression levels). Right and left plots indicate the enrichment in MF and BP terms, respectively. (B) As in the previous panel, GO analysis of the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison (high EBER1/2 levels). (C) Figure adapted from Hanahan and Weinberg [ 37 ] to highlight the oncogenic signature enriched in BJAB-EBNA1-EBER1/2 cells. (D) qRT-PCR validation assays for il10 , vegfa and zeb1 mRNAs from three biological replicates per comparison. The left plot shows an overlay of the BJAB-EBER1/2 vs BJAB-CTL (blue) and BJAB-EBNA1-EBER1-/2 vs BJAB-EBNA1 (red) fold-changes. The right plot shows the BJAB-B1 vs BJAB-parental fold-changes. All values were normalized to ACTB as a control. (E) WB assays with an antibody specific for ZEB1 in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison. Densitometry values and error bars reflect three independent biological replicates.

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

    Validation by qRT-PCR of vegfa alternative isoform abundances in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison. (A) The cartoon shows the mRNA-seq paired-end reads aligned by TopHat to the vegfa gene and indicates the alternative isoforms identified by Cufflinks in the bioinformatics analysis. The isoforms with a significant fold-change in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison are colored blue. (B) Isoform abundances calculated by Cufflinks for each alternative isoform identified. The isoforms calculated to have changed significantly by Cufflinks are colored blue. (C) Validation by qRT-PCR of the indicated isoforms. The qRT-PCR values were normalized to 18S rRNA.
    Figure Legend Snippet: Validation by qRT-PCR of vegfa alternative isoform abundances in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison. (A) The cartoon shows the mRNA-seq paired-end reads aligned by TopHat to the vegfa gene and indicates the alternative isoforms identified by Cufflinks in the bioinformatics analysis. The isoforms with a significant fold-change in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison are colored blue. (B) Isoform abundances calculated by Cufflinks for each alternative isoform identified. The isoforms calculated to have changed significantly by Cufflinks are colored blue. (C) Validation by qRT-PCR of the indicated isoforms. The qRT-PCR values were normalized to 18S rRNA.

    Techniques Used: Quantitative RT-PCR

    Expression levels of EBER1 and EBER2 in BJAB cells. (A) In BJAB-EBER1/2 cells (FRT), EBER1 and EBER2 are expressed to about one tenth of their levels found in EBV-infected BJAB cells (BJAB-B1) set to 1. The densitometry values are the average of three biological replicates. (B) BJAB cells stably transfected with the ppCEP4 vector containing four (4X), six (6X) or eight (8X) copies of the EcoRI-J fragment expressed similar levels of EBER1 and EBER2, compared to BJAB-B1 cells. The densitometry values are the average of three biological replicates. (C) qRT-PCR assays specific for ebna1 mRNA in BJAB-EBNA1 and BJAB-EBNA1-EBER1/2, compared to BJAB-B1 cells. (D) FISH image shows the consistent expression of EBER1 in the nucleus of BJAB-EBNA1-EBER1/2 cells. In (A-C) the values are normalized to actin (ACTB) as a control and the error bars reflect the standard deviation from three biological replicates. (E ) Proliferation assays of BJAB cell lines. Cells were counted in triplicate every 24 hours.
    Figure Legend Snippet: Expression levels of EBER1 and EBER2 in BJAB cells. (A) In BJAB-EBER1/2 cells (FRT), EBER1 and EBER2 are expressed to about one tenth of their levels found in EBV-infected BJAB cells (BJAB-B1) set to 1. The densitometry values are the average of three biological replicates. (B) BJAB cells stably transfected with the ppCEP4 vector containing four (4X), six (6X) or eight (8X) copies of the EcoRI-J fragment expressed similar levels of EBER1 and EBER2, compared to BJAB-B1 cells. The densitometry values are the average of three biological replicates. (C) qRT-PCR assays specific for ebna1 mRNA in BJAB-EBNA1 and BJAB-EBNA1-EBER1/2, compared to BJAB-B1 cells. (D) FISH image shows the consistent expression of EBER1 in the nucleus of BJAB-EBNA1-EBER1/2 cells. In (A-C) the values are normalized to actin (ACTB) as a control and the error bars reflect the standard deviation from three biological replicates. (E ) Proliferation assays of BJAB cell lines. Cells were counted in triplicate every 24 hours.

    Techniques Used: Expressing, Infection, Stable Transfection, Transfection, Plasmid Preparation, Quantitative RT-PCR, Fluorescence In Situ Hybridization, Standard Deviation

    Validation by qRT-PCR of zeb1 alternative isoform abundances in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison. (A) The cartoon shows the mRNA-seq paired-end reads aligned by TopHat to the zeb1 gene and indicates the alternative isoforms identified by Cufflinks in the bioinformatics analysis. The isoforms with a significant fold-change in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison are colored blue. (B) Isoform abundances calculated by Cufflinks for each alternative isoform identified. The isoforms calculated to have changed significantly by Cufflinks are colored blue. (C) Validation by qRT-PCR of the indicated isoforms. The qRT-PCR values were normalized to 18S rRNA.
    Figure Legend Snippet: Validation by qRT-PCR of zeb1 alternative isoform abundances in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison. (A) The cartoon shows the mRNA-seq paired-end reads aligned by TopHat to the zeb1 gene and indicates the alternative isoforms identified by Cufflinks in the bioinformatics analysis. The isoforms with a significant fold-change in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison are colored blue. (B) Isoform abundances calculated by Cufflinks for each alternative isoform identified. The isoforms calculated to have changed significantly by Cufflinks are colored blue. (C) Validation by qRT-PCR of the indicated isoforms. The qRT-PCR values were normalized to 18S rRNA.

    Techniques Used: Quantitative RT-PCR

    4) Product Images from "Glipizide suppresses prostate cancer progression in the TRAMP model by inhibiting angiogenesis"

    Article Title: Glipizide suppresses prostate cancer progression in the TRAMP model by inhibiting angiogenesis

    Journal: Scientific Reports

    doi: 10.1038/srep27819

    Glipizide inhibits angiogenesis through down-regulation of ANGPT1 expression. ( A ) ANGPT1 expression is down-regulated in HUVECs treated with glipizide by qRT-PCR array analysis. ( B , C ) To further determine the qRT-PCR array results, qRT-PCR ( B ) and Western blotting ( C ) were performed. ( D , E ) Immunofluorescent staining shows that the levels of ANGPT1 expression are high in the blood vessels of tumor tissues of TRAMP mice treated with DMSO. However, ANGPT1 expression is significantly decreased in the blood vessels of tumor tissues of TRAMP mice treated with glipizide. ( F ) ANGPT1 silencing and glipizide significantly inhibit HUVEC tube formation. In addition, glipizide does not affect the ability of HUVECs to form tubular structures after ANGPT1 is silenced. Scale bars, 20 μm in E and 50 μm in F. * p
    Figure Legend Snippet: Glipizide inhibits angiogenesis through down-regulation of ANGPT1 expression. ( A ) ANGPT1 expression is down-regulated in HUVECs treated with glipizide by qRT-PCR array analysis. ( B , C ) To further determine the qRT-PCR array results, qRT-PCR ( B ) and Western blotting ( C ) were performed. ( D , E ) Immunofluorescent staining shows that the levels of ANGPT1 expression are high in the blood vessels of tumor tissues of TRAMP mice treated with DMSO. However, ANGPT1 expression is significantly decreased in the blood vessels of tumor tissues of TRAMP mice treated with glipizide. ( F ) ANGPT1 silencing and glipizide significantly inhibit HUVEC tube formation. In addition, glipizide does not affect the ability of HUVECs to form tubular structures after ANGPT1 is silenced. Scale bars, 20 μm in E and 50 μm in F. * p

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

    5) Product Images from "Identification of HDA15-PIF1 as a key repression module directing the transcriptional network of seed germination in the dark"

    Article Title: Identification of HDA15-PIF1 as a key repression module directing the transcriptional network of seed germination in the dark

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx283

    HDA15 directly represses GA3OX1 and GA3OX2 expression by histone deacetylation. ( A ) qRT-PCR analysis of the expression levels of GA3OX1 and GA3OX2 in Col-0 and hda15 seeds under FR and FR/R conditions. PP2A was used as an internal control. Values are shown as means ± SD ( t -test, * P
    Figure Legend Snippet: HDA15 directly represses GA3OX1 and GA3OX2 expression by histone deacetylation. ( A ) qRT-PCR analysis of the expression levels of GA3OX1 and GA3OX2 in Col-0 and hda15 seeds under FR and FR/R conditions. PP2A was used as an internal control. Values are shown as means ± SD ( t -test, * P

    Techniques Used: Expressing, Quantitative RT-PCR

    HDA15 and PIF1 decrease the histone H3 acetylation levels of the target genes. ( A ) qRT-PCR analysis of the expression levels of PINs, EXPs and XTHs under FR and FR/R conditions. PP2A was used as an internal control. Values are shown as means ± SD ( t -test, * P
    Figure Legend Snippet: HDA15 and PIF1 decrease the histone H3 acetylation levels of the target genes. ( A ) qRT-PCR analysis of the expression levels of PINs, EXPs and XTHs under FR and FR/R conditions. PP2A was used as an internal control. Values are shown as means ± SD ( t -test, * P

    Techniques Used: Quantitative RT-PCR, Expressing

    HDA15-PIF1 co-represses the genes involved in plant hormones and cellular processes in imbibed seeds. ( A ) HDA15-PIF1 co-repressed genes related to plant hormones and cellular processes. ( B ) qRT-PCR analysis of the expression levels of PINs, XTHs and EXPs in Col-0, hda15, pif1 and hda15 pif1 seeds in FR conditions. PP2A was used as an internal control. Values are shown as means ± SD ( t -test, * P
    Figure Legend Snippet: HDA15-PIF1 co-represses the genes involved in plant hormones and cellular processes in imbibed seeds. ( A ) HDA15-PIF1 co-repressed genes related to plant hormones and cellular processes. ( B ) qRT-PCR analysis of the expression levels of PINs, XTHs and EXPs in Col-0, hda15, pif1 and hda15 pif1 seeds in FR conditions. PP2A was used as an internal control. Values are shown as means ± SD ( t -test, * P

    Techniques Used: Quantitative RT-PCR, Expressing

    6) Product Images from "Circular RNA hsa_circ_0000467 Promotes the Development of Gastric Cancer by Competitively Binding to MicroRNA miR-326-3p"

    Article Title: Circular RNA hsa_circ_0000467 Promotes the Development of Gastric Cancer by Competitively Binding to MicroRNA miR-326-3p

    Journal: BioMed Research International

    doi: 10.1155/2020/4030826

    Hsa_circ_0000467 has binding sites with miR-326-3p. (a) Differential miRNA expression in gastric cancer cells in which hsa_circ_0000467 was decreased was detected by qRT-PCR. (b) Schematic model showing the putative binding sites for miR-326-3p and hsa_circ_0000467. (c) Construction of dual-luciferase reporter plasmids containing the WT or Mut hsa_circ_0000467 sequence. (d) The luciferase activities of the blank controls or miR-326-3p mimics and the WT or Mut hsa_circ_0000467 cotransfected BGC-823 cells were determined by luciferase reporter assays. ns: not significant, ∗ P
    Figure Legend Snippet: Hsa_circ_0000467 has binding sites with miR-326-3p. (a) Differential miRNA expression in gastric cancer cells in which hsa_circ_0000467 was decreased was detected by qRT-PCR. (b) Schematic model showing the putative binding sites for miR-326-3p and hsa_circ_0000467. (c) Construction of dual-luciferase reporter plasmids containing the WT or Mut hsa_circ_0000467 sequence. (d) The luciferase activities of the blank controls or miR-326-3p mimics and the WT or Mut hsa_circ_0000467 cotransfected BGC-823 cells were determined by luciferase reporter assays. ns: not significant, ∗ P

    Techniques Used: Binding Assay, Expressing, Quantitative RT-PCR, Luciferase, Sequencing

    Changes in the proliferation and invasion of BGC-823 and SGC-7901 cells caused by low has_circ_0000647 expression could be reversed by knocking down miR-326-3p. (a) Expression of hsa_circ_0000467 and miR-326-3p in different groups of BGC-823 and SGC-7901 cells was detected by qRT-PCR. (b) The proliferation activities of different groups of these two cell lines were measured by CCK8 assays. (c) The invasion ability of different groups of these two cell lines was detected by Transwell assays. The data are presented as the mean ± SD. ns: not significant, ∗ P
    Figure Legend Snippet: Changes in the proliferation and invasion of BGC-823 and SGC-7901 cells caused by low has_circ_0000647 expression could be reversed by knocking down miR-326-3p. (a) Expression of hsa_circ_0000467 and miR-326-3p in different groups of BGC-823 and SGC-7901 cells was detected by qRT-PCR. (b) The proliferation activities of different groups of these two cell lines were measured by CCK8 assays. (c) The invasion ability of different groups of these two cell lines was detected by Transwell assays. The data are presented as the mean ± SD. ns: not significant, ∗ P

    Techniques Used: Expressing, Quantitative RT-PCR

    Expression of hsa_circ_0000467 in GC tissues and cell lines. (a) The relative hsa_circ_0000467 expression in tissues was detected by qRT-PCR. (b) Hsa_circ_0000467 expression in cell lines was detected by qRT-PCR. The results are shown as the fold change (2 −ΔΔCT ) and the mean ± SD from three times experiments. ∗ P
    Figure Legend Snippet: Expression of hsa_circ_0000467 in GC tissues and cell lines. (a) The relative hsa_circ_0000467 expression in tissues was detected by qRT-PCR. (b) Hsa_circ_0000467 expression in cell lines was detected by qRT-PCR. The results are shown as the fold change (2 −ΔΔCT ) and the mean ± SD from three times experiments. ∗ P

    Techniques Used: Expressing, Quantitative RT-PCR

    Effect of hsa_circ_0000467 silencing on GC proliferation, invasion ability, and cell cycle. (a) The interference efficacy of siRNA targeting hsa_circ_0000467 was measured by qRT-PCR. (b) The proliferation activity of BGC-823 and SGC-7901 cells transfected with si-NC or si-hsa_circ_0000467 was detected by CCK8 assays. (c) The invasion ability of BGC-823 and SGC-7901 cells transfected with si-NC or si-hsa_circ_0000467 was detected by Transwell assays. (d) The cell cycle of BGC-823 and SGC-7901 cells transfected with si-NC or si-hsa_circ_0000467 was detected by flow cytometry. The results are represented as the means ± SD of at least three times. ns: not significant, ∗ P
    Figure Legend Snippet: Effect of hsa_circ_0000467 silencing on GC proliferation, invasion ability, and cell cycle. (a) The interference efficacy of siRNA targeting hsa_circ_0000467 was measured by qRT-PCR. (b) The proliferation activity of BGC-823 and SGC-7901 cells transfected with si-NC or si-hsa_circ_0000467 was detected by CCK8 assays. (c) The invasion ability of BGC-823 and SGC-7901 cells transfected with si-NC or si-hsa_circ_0000467 was detected by Transwell assays. (d) The cell cycle of BGC-823 and SGC-7901 cells transfected with si-NC or si-hsa_circ_0000467 was detected by flow cytometry. The results are represented as the means ± SD of at least three times. ns: not significant, ∗ P

    Techniques Used: Quantitative RT-PCR, Activity Assay, Transfection, Flow Cytometry

    7) Product Images from "Curcumin Inhibits Rift Valley Fever Virus Replication in Human Cells *"

    Article Title: Curcumin Inhibits Rift Valley Fever Virus Replication in Human Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.356535

    MP-12 infection caused phosphorylation of p65 by the classical pathway. A , HSAECs were infected with MP-12 or UV-inactivated MP-12 virus and analyzed for phosphorylation of p65 and IκBα by Western blot analysis. Mock-infected cells were maintained alongside as negative controls. Total p65 and β-actin Western blots were carried out as controls. B , extent of phosphorylation of p65 was quantified by averaging signal intensities observed in three different experiments after infection by MP-12 or UV-MP-12 virus. Phosphorylation is represented as -fold increase over that of uninfected cells. h.p.i ., (hours post-infection). C , phosphorylation of p65 and IκBα after infection by wild type MP-12 virus, NSs mutant (rMP-12-NSdel), and NSm mutant (arMP-12-del21/384) were analyzed by Western blot. Total p65 and β-actin levels were analyzed as controls. D , intracellular RNA levels of MP-12 virus or the NSs and NSm mutant viruses were determined by qRT-PCR using total RNA extracted from infected cells at 6 h post-infection.
    Figure Legend Snippet: MP-12 infection caused phosphorylation of p65 by the classical pathway. A , HSAECs were infected with MP-12 or UV-inactivated MP-12 virus and analyzed for phosphorylation of p65 and IκBα by Western blot analysis. Mock-infected cells were maintained alongside as negative controls. Total p65 and β-actin Western blots were carried out as controls. B , extent of phosphorylation of p65 was quantified by averaging signal intensities observed in three different experiments after infection by MP-12 or UV-MP-12 virus. Phosphorylation is represented as -fold increase over that of uninfected cells. h.p.i ., (hours post-infection). C , phosphorylation of p65 and IκBα after infection by wild type MP-12 virus, NSs mutant (rMP-12-NSdel), and NSm mutant (arMP-12-del21/384) were analyzed by Western blot. Total p65 and β-actin levels were analyzed as controls. D , intracellular RNA levels of MP-12 virus or the NSs and NSm mutant viruses were determined by qRT-PCR using total RNA extracted from infected cells at 6 h post-infection.

    Techniques Used: Infection, Western Blot, Mutagenesis, Quantitative RT-PCR

    Down-regulation of extracellular genomic RNA by curcumin. A , HSAECs were pre- and post-treated with curcumin, and supernatants were analyzed for viral genomic RNA copies by qRT-PCR in comparison with untreated and DMSO-treated, MP-12-infected cells. Viral genomic copy numbers in the context of inhibition by synthetic curcumin or dimethoxycurcumin were determined. Supernatants were obtained from infected, inhibitor-treated cells at 24 h post-infection and analyzed by qRT-PCR. B , HSAECs were either pretreated (2 h) or post-treated (3 h) with curcumin, and the viral genomic copy number in the supernatant was determined by qRT-PCR.
    Figure Legend Snippet: Down-regulation of extracellular genomic RNA by curcumin. A , HSAECs were pre- and post-treated with curcumin, and supernatants were analyzed for viral genomic RNA copies by qRT-PCR in comparison with untreated and DMSO-treated, MP-12-infected cells. Viral genomic copy numbers in the context of inhibition by synthetic curcumin or dimethoxycurcumin were determined. Supernatants were obtained from infected, inhibitor-treated cells at 24 h post-infection and analyzed by qRT-PCR. B , HSAECs were either pretreated (2 h) or post-treated (3 h) with curcumin, and the viral genomic copy number in the supernatant was determined by qRT-PCR.

    Techniques Used: Quantitative RT-PCR, Infection, Inhibition

    8) Product Images from "In vitro infectivity and differential gene expression of Leishmania infantum metacyclic promastigotes: negative selection with peanut agglutinin in culture versus isolation from the stomodeal valve of Phlebotomus perniciosus"

    Article Title: In vitro infectivity and differential gene expression of Leishmania infantum metacyclic promastigotes: negative selection with peanut agglutinin in culture versus isolation from the stomodeal valve of Phlebotomus perniciosus

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-2672-8

    Up-regulation of genes encoding amastins, GPI biosynthetic proteins and the IF2 in Pro-Pper. The results of the qRT-PCR and microarray hybridization analyses (Table 2 ) are compared
    Figure Legend Snippet: Up-regulation of genes encoding amastins, GPI biosynthetic proteins and the IF2 in Pro-Pper. The results of the qRT-PCR and microarray hybridization analyses (Table 2 ) are compared

    Techniques Used: Quantitative RT-PCR, Microarray, Hybridization

    9) Product Images from "Tetraspanin 1 promotes epithelial-to-mesenchymal transition and metastasis of cholangiocarcinoma via PI3K/AKT signaling"

    Article Title: Tetraspanin 1 promotes epithelial-to-mesenchymal transition and metastasis of cholangiocarcinoma via PI3K/AKT signaling

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0969-y

    Tetraspanin 1 (TSPAN1) is frequently upregulated in human cholangiocarcinoma (CCA). ( a ) TSPAN1 mRNA level was analyzed in 60 CCA and paracancerous, 40 normal liver, and 10 normal bile duct tissue specimens using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). ( b and c ) Western blot and immunohistochemical (IHC) analyses of TSPAN1 protein expression in CCA tissues and adjacent normal tissues. Scale bars: 100× = 100 μm; 200× = 50 μm. ( d ) TSPAN1 positive staining was associated with poor clinicopathological features, including TNM stage (I–II and III–IV), lymph node status (N0 or N1), and metastasis status (M0 or M1). ( e ) A Kaplan-Meier analysis of overall survival (OS) in patients with different staining of TSPAN1. ( f and g ) Relative TSPAN1 levels in L02, HIBEpiC, and six CCA cells were analyzed using qRT-PCR and western blot. Data are means ± SD of three independent experiments. * p
    Figure Legend Snippet: Tetraspanin 1 (TSPAN1) is frequently upregulated in human cholangiocarcinoma (CCA). ( a ) TSPAN1 mRNA level was analyzed in 60 CCA and paracancerous, 40 normal liver, and 10 normal bile duct tissue specimens using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). ( b and c ) Western blot and immunohistochemical (IHC) analyses of TSPAN1 protein expression in CCA tissues and adjacent normal tissues. Scale bars: 100× = 100 μm; 200× = 50 μm. ( d ) TSPAN1 positive staining was associated with poor clinicopathological features, including TNM stage (I–II and III–IV), lymph node status (N0 or N1), and metastasis status (M0 or M1). ( e ) A Kaplan-Meier analysis of overall survival (OS) in patients with different staining of TSPAN1. ( f and g ) Relative TSPAN1 levels in L02, HIBEpiC, and six CCA cells were analyzed using qRT-PCR and western blot. Data are means ± SD of three independent experiments. * p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Expressing, Staining

    10) Product Images from "The MADS transcription factor CmANR1 positively modulates root system development by directly regulating CmPIN2 in chrysanthemum"

    Article Title: The MADS transcription factor CmANR1 positively modulates root system development by directly regulating CmPIN2 in chrysanthemum

    Journal: Horticulture Research

    doi: 10.1038/s41438-018-0061-y

    Verification of the RNA-Seq results by qRT-PCR. a The relative transcript abundance of some IAA-responsive unigenes that were selected based on their differential expression in the roots of WT vs. CmANR1 -transgenetic plants. b The relative expression level of the unigenes in Ca 2+ signaling-related, ethylene-related, and cell cycle-related groups in the roots of WT and CmANR1 -transgenetic plants. c Comparison of the expression level of unigenes between the RNA-Seq and qRT-PCR. d Scatter diagram of the log ratios (log 2 FC) of the unigenes. The qRT-PCR data were normalized to the internal control CmUBI . Note that in a and b , data are shown as the mean ± SE based on three or more replicate. Statistical significance was determined using Student’s t -test. n.s.: p > 0.01; * p
    Figure Legend Snippet: Verification of the RNA-Seq results by qRT-PCR. a The relative transcript abundance of some IAA-responsive unigenes that were selected based on their differential expression in the roots of WT vs. CmANR1 -transgenetic plants. b The relative expression level of the unigenes in Ca 2+ signaling-related, ethylene-related, and cell cycle-related groups in the roots of WT and CmANR1 -transgenetic plants. c Comparison of the expression level of unigenes between the RNA-Seq and qRT-PCR. d Scatter diagram of the log ratios (log 2 FC) of the unigenes. The qRT-PCR data were normalized to the internal control CmUBI . Note that in a and b , data are shown as the mean ± SE based on three or more replicate. Statistical significance was determined using Student’s t -test. n.s.: p > 0.01; * p

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

    Heatmaps of four groups of candidate unigenes associated with root system development in the DEGs. a Heatmap of auxin-responsive unigenes. b Heatmap of Ca 2+ signaling-related unigenes. c Heatmap of ethylene-related unigenes. d Heatmap of cell cycle-related unigenes. Differences in expression levels are represented by color gradients. Red and orange strips indicate highly to moderately up-regulated unigenes, while dark blue to light blue strips represent highly to moderately down-regulated unigenes. The ID numbers of the four groups of unigenes are provided in Supplementary Appendix S4 . The red unigenes in the four groups were selected for qRT-PCR
    Figure Legend Snippet: Heatmaps of four groups of candidate unigenes associated with root system development in the DEGs. a Heatmap of auxin-responsive unigenes. b Heatmap of Ca 2+ signaling-related unigenes. c Heatmap of ethylene-related unigenes. d Heatmap of cell cycle-related unigenes. Differences in expression levels are represented by color gradients. Red and orange strips indicate highly to moderately up-regulated unigenes, while dark blue to light blue strips represent highly to moderately down-regulated unigenes. The ID numbers of the four groups of unigenes are provided in Supplementary Appendix S4 . The red unigenes in the four groups were selected for qRT-PCR

    Techniques Used: Expressing, Quantitative RT-PCR

    11) Product Images from "Tetraspanin 1 promotes epithelial-to-mesenchymal transition and metastasis of cholangiocarcinoma via PI3K/AKT signaling"

    Article Title: Tetraspanin 1 promotes epithelial-to-mesenchymal transition and metastasis of cholangiocarcinoma via PI3K/AKT signaling

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0969-y

    Tetraspanin 1 (TSPAN1) is frequently upregulated in human cholangiocarcinoma (CCA). ( a ) TSPAN1 mRNA level was analyzed in 60 CCA and paracancerous, 40 normal liver, and 10 normal bile duct tissue specimens using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). ( b and c ) Western blot and immunohistochemical (IHC) analyses of TSPAN1 protein expression in CCA tissues and adjacent normal tissues. Scale bars: 100× = 100 μm; 200× = 50 μm. ( d ) TSPAN1 positive staining was associated with poor clinicopathological features, including TNM stage (I–II and III–IV), lymph node status (N0 or N1), and metastasis status (M0 or M1). ( e ) A Kaplan-Meier analysis of overall survival (OS) in patients with different staining of TSPAN1. ( f and g ) Relative TSPAN1 levels in L02, HIBEpiC, and six CCA cells were analyzed using qRT-PCR and western blot. Data are means ± SD of three independent experiments. * p
    Figure Legend Snippet: Tetraspanin 1 (TSPAN1) is frequently upregulated in human cholangiocarcinoma (CCA). ( a ) TSPAN1 mRNA level was analyzed in 60 CCA and paracancerous, 40 normal liver, and 10 normal bile duct tissue specimens using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). ( b and c ) Western blot and immunohistochemical (IHC) analyses of TSPAN1 protein expression in CCA tissues and adjacent normal tissues. Scale bars: 100× = 100 μm; 200× = 50 μm. ( d ) TSPAN1 positive staining was associated with poor clinicopathological features, including TNM stage (I–II and III–IV), lymph node status (N0 or N1), and metastasis status (M0 or M1). ( e ) A Kaplan-Meier analysis of overall survival (OS) in patients with different staining of TSPAN1. ( f and g ) Relative TSPAN1 levels in L02, HIBEpiC, and six CCA cells were analyzed using qRT-PCR and western blot. Data are means ± SD of three independent experiments. * p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Expressing, Staining

    12) Product Images from "Adipocyte-derived IL-6 and leptin promote breast Cancer metastasis via upregulation of Lysyl Hydroxylase-2 expression"

    Article Title: Adipocyte-derived IL-6 and leptin promote breast Cancer metastasis via upregulation of Lysyl Hydroxylase-2 expression

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-018-0309-z

    PLOD2 knockdown attenuates breast cancer cell migration in vitro. a PLOD2 was knocked down using two independent short hairpin RNAs (SHC and SHD) in MDA-MB-231 (MB-231) cells. qRT-PCR and Western blotting were used to detect PLOD2 expression in scramble (SCR) and PLOD2-knockdown cells (SHC, SHD). Error bars represent means ± SD. ** P
    Figure Legend Snippet: PLOD2 knockdown attenuates breast cancer cell migration in vitro. a PLOD2 was knocked down using two independent short hairpin RNAs (SHC and SHD) in MDA-MB-231 (MB-231) cells. qRT-PCR and Western blotting were used to detect PLOD2 expression in scramble (SCR) and PLOD2-knockdown cells (SHC, SHD). Error bars represent means ± SD. ** P

    Techniques Used: Migration, In Vitro, Multiple Displacement Amplification, Quantitative RT-PCR, Western Blot, Expressing

    Adipocyte-derived IL-6 and leptin regulate PLOD2 expression. a qRT-PCR analysis of the relative expression levels of IGF-BP1, PAI-1, IL-6, MIF, TIMP-1, TIMP-2 and Leptin in 3 T3-L1 preadipocytes, adipocytes and adipocytes cocultured with MDA-MB-231 (MB-231) breast cancer cells. Error bars represent means ± SD. ** P
    Figure Legend Snippet: Adipocyte-derived IL-6 and leptin regulate PLOD2 expression. a qRT-PCR analysis of the relative expression levels of IGF-BP1, PAI-1, IL-6, MIF, TIMP-1, TIMP-2 and Leptin in 3 T3-L1 preadipocytes, adipocytes and adipocytes cocultured with MDA-MB-231 (MB-231) breast cancer cells. Error bars represent means ± SD. ** P

    Techniques Used: Derivative Assay, Expressing, Quantitative RT-PCR, Multiple Displacement Amplification

    13) Product Images from "A Calpain-Like Protein Is Involved in the Execution Phase of Programmed Cell Death of Entamoeba histolytica"

    Article Title: A Calpain-Like Protein Is Involved in the Execution Phase of Programmed Cell Death of Entamoeba histolytica

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00339

    Silencing of the calpain-like gene. (A) qRT-PCR expression of calpain-like gene from trophozoites incubated with 50 μg/ml of the calpain-like siRNA sequence for 16 and 24 h. As control, trophozoites were incubated with a non-related sequence (NRS). Negative control represents trophozoites without siRNA incubation. (B) WB of calpain-like protein. (A) Negative control, untreated trophozoites; (B) Trophozoites after a 9-h incubation with 10 μg/ml of G418; (C,D) Trophozoites pre-incubated for 24 h with NRS (C) or calpain-like (D) siRNAs sequences and incubated 9 h with 10 μg/ml G418. The graph shows the densitometry analysis of calpain-like protein expression levels. *indicate statistically significant difference ( P
    Figure Legend Snippet: Silencing of the calpain-like gene. (A) qRT-PCR expression of calpain-like gene from trophozoites incubated with 50 μg/ml of the calpain-like siRNA sequence for 16 and 24 h. As control, trophozoites were incubated with a non-related sequence (NRS). Negative control represents trophozoites without siRNA incubation. (B) WB of calpain-like protein. (A) Negative control, untreated trophozoites; (B) Trophozoites after a 9-h incubation with 10 μg/ml of G418; (C,D) Trophozoites pre-incubated for 24 h with NRS (C) or calpain-like (D) siRNAs sequences and incubated 9 h with 10 μg/ml G418. The graph shows the densitometry analysis of calpain-like protein expression levels. *indicate statistically significant difference ( P

    Techniques Used: Quantitative RT-PCR, Expressing, Incubation, Sequencing, Negative Control, Western Blot

    14) Product Images from "The long non-coding RNA CRNDE acts as a ceRNA and promotes glioma malignancy by preventing miR-136-5p-mediated downregulation of Bcl-2 and Wnt2"

    Article Title: The long non-coding RNA CRNDE acts as a ceRNA and promotes glioma malignancy by preventing miR-136-5p-mediated downregulation of Bcl-2 and Wnt2

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21513

    CRNDE overexpression and silencing in glioma cells (A) Assessment by qRT-PCR of CRNDE expression in four cell lines (U87, U251, A172, and T98G) compared with normal brain tissue. Data are presented as mean ± SD (n = 3, each group). ** P
    Figure Legend Snippet: CRNDE overexpression and silencing in glioma cells (A) Assessment by qRT-PCR of CRNDE expression in four cell lines (U87, U251, A172, and T98G) compared with normal brain tissue. Data are presented as mean ± SD (n = 3, each group). ** P

    Techniques Used: Over Expression, Quantitative RT-PCR, Expressing

    CRNDE upregulation in human glioma specimens (A) CRNDE levels in 47 clinical glioma specimens and 9 normal brain samples, assessed by qRT-PCR. ** P
    Figure Legend Snippet: CRNDE upregulation in human glioma specimens (A) CRNDE levels in 47 clinical glioma specimens and 9 normal brain samples, assessed by qRT-PCR. ** P

    Techniques Used: Quantitative RT-PCR

    15) Product Images from "miR824/AGAMOUS-LIKE16 Module Integrates Recurring Environmental Heat Stress Changes to Fine-Tune Poststress Development"

    Article Title: miR824/AGAMOUS-LIKE16 Module Integrates Recurring Environmental Heat Stress Changes to Fine-Tune Poststress Development

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2019.01454

    miR824 transcriptional induction requires heat stress elements and heat shock factors. (A) Schematic representation of MIR824 gene promoter: heat stress element, HSE; heat stress element-like motifs, HSE-like; dehydration-responsive element, DRE; TATA-box, FLC-binding site (based on Deng et al., 2011) , arrow shows transcription start site, TSS; nucleotide base numbers are relative to TSS. (B) Northern blot data of GUS expression using the wild-type ( p824 WT ::GUS ), HSE single mutant ( p824 HSE1 ::GUS ), and triple mutant ( p824 HSE123 ::GUS ) variants of miR824 promoter were quantified to ACTIN2 and normalized to nontreated control; nontreated, NT, acclimated, ACC; bars represent standard errors calculated based on six representative independent lines each. (C) miR824-precursor induction and miR824 accumulation are faulty or partial in miR824-mutant Δ824 , triple mutants of HSFA1 family ( aTK , bTK , dTK , and eTK ) and HSFA2 mutant hsfa2 . miR159, ACTIN2 , and ethidium-bromide (EtBr) staining are shown as loading controls. (D) Quantification of pri-miR824 signals of Northern blots; bars represent standard errors based on three biological replicates; Col-0 NT value was set to 1. (E) chromatin immunoprecipitation (ChIP) qRT-PCR using 3xHA-tagged HsfA1a expressing transgenic plants: HsfA1a binds to the HSE-containing region of miR824 promoter; data were normalized to no-tag ChIP control; bars represent standard errors calculated based on two biological and two technical reps, schematic representation of MIR824 locus is shown below: A, B, C, D, E segments show the locations of PCR amplicons; black boxes show the location of HSE elements; green arrowheads show locations of miR824- 5p and miR824- 3p ; p values based on two-tailed Student's t -test.
    Figure Legend Snippet: miR824 transcriptional induction requires heat stress elements and heat shock factors. (A) Schematic representation of MIR824 gene promoter: heat stress element, HSE; heat stress element-like motifs, HSE-like; dehydration-responsive element, DRE; TATA-box, FLC-binding site (based on Deng et al., 2011) , arrow shows transcription start site, TSS; nucleotide base numbers are relative to TSS. (B) Northern blot data of GUS expression using the wild-type ( p824 WT ::GUS ), HSE single mutant ( p824 HSE1 ::GUS ), and triple mutant ( p824 HSE123 ::GUS ) variants of miR824 promoter were quantified to ACTIN2 and normalized to nontreated control; nontreated, NT, acclimated, ACC; bars represent standard errors calculated based on six representative independent lines each. (C) miR824-precursor induction and miR824 accumulation are faulty or partial in miR824-mutant Δ824 , triple mutants of HSFA1 family ( aTK , bTK , dTK , and eTK ) and HSFA2 mutant hsfa2 . miR159, ACTIN2 , and ethidium-bromide (EtBr) staining are shown as loading controls. (D) Quantification of pri-miR824 signals of Northern blots; bars represent standard errors based on three biological replicates; Col-0 NT value was set to 1. (E) chromatin immunoprecipitation (ChIP) qRT-PCR using 3xHA-tagged HsfA1a expressing transgenic plants: HsfA1a binds to the HSE-containing region of miR824 promoter; data were normalized to no-tag ChIP control; bars represent standard errors calculated based on two biological and two technical reps, schematic representation of MIR824 locus is shown below: A, B, C, D, E segments show the locations of PCR amplicons; black boxes show the location of HSE elements; green arrowheads show locations of miR824- 5p and miR824- 3p ; p values based on two-tailed Student's t -test.

    Techniques Used: Binding Assay, Northern Blot, Expressing, Mutagenesis, Staining, Chromatin Immunoprecipitation, Quantitative RT-PCR, Transgenic Assay, Polymerase Chain Reaction, Two Tailed Test

    16) Product Images from "A Single Agent Dual Specificity Targeting of FOLR1 and DR5 as an Effective Strategy for Ovarian Cancer"

    Article Title: A Single Agent Dual Specificity Targeting of FOLR1 and DR5 as an Effective Strategy for Ovarian Cancer

    Journal: Cancer cell

    doi: 10.1016/j.ccell.2018.07.005

    BaCa activity is highly selective towards FOLR1 overexpressing OvCa cancer cells. (A) Cell viability analysis of lexatumumab and lexatumumab containing BaCa antibody ± anti-Fc crosslinking (n=3). (B) Cell viability analysis of AMG-655 and AMG-655 containing BaCa antibody ± anti-Fc crosslinking (n=3). (C) qRT-PCR analysis of FOLR1 and TRAIL-R2 transcripts in OVCAR-4 and Colo-205 cells (n=5). (D) Cell viability assays using BaCa antibody in OVCAR-4 and Colo-205 cells. IC 50 values are shown in right (n=3). (E) Schematic of results described in F. 50% GFP − OVCAR-4 and 50% GFP + Colo-205 cells were co-cultured. After 24 hr, cells were treated with indicated antibodies at constant 0.1 nM. After 36–48 hr, cells were analyzed using fluorescent microscope. (F) Represented images as described in E with indicated antibody treatment (scale bar represent 400 μm). (G) Immunoblot analysis for GFP and tubulin of GFP + Colo-205 cells co-cultured with equal number of GFP − OVCAR-4 or GFP − Colo-205 cells and treated with the indicated concentrations of BaCa antibody (H) The normalized relative intensities of GFP signal were plotted for the increasing BaCa dose in co-cultured conditions (filled black circles: 50% GFP − OVCAR-4 and 50% GFP + Colo-205) against constant 10 nM dose (filled red circle: 50% GFP − Colo-205 and 50% GFP + Colo-205). (I) Co-cultured MC38 (GFP − ) and Colo-205 (GFP + ) cells were treated with 50 nM of indicated antibodies. After 48 hr, lysates were run on gel and blotted for tubulin and GFP. (J) Cell viability of MC38 cells treated with LK26, LK26+anti-Fc, LK26-AMG-655 bispecific and BaCa antibody (n=3). (K) Cell viability of OVCAR-3 cells treated with AMG-655, AMG-655+anti-Fc, LK26-AMG-655 bispecific and BaCa antibody (n=3). (L) Co-cultured MC38 and OVCAR-3 cells were treated with the increasing concentration of indicated antibodies. After 48 hr, cell viability was analyzed using MTT assays. Error bars in A, B, C, D, J, K and L represent SEM. .
    Figure Legend Snippet: BaCa activity is highly selective towards FOLR1 overexpressing OvCa cancer cells. (A) Cell viability analysis of lexatumumab and lexatumumab containing BaCa antibody ± anti-Fc crosslinking (n=3). (B) Cell viability analysis of AMG-655 and AMG-655 containing BaCa antibody ± anti-Fc crosslinking (n=3). (C) qRT-PCR analysis of FOLR1 and TRAIL-R2 transcripts in OVCAR-4 and Colo-205 cells (n=5). (D) Cell viability assays using BaCa antibody in OVCAR-4 and Colo-205 cells. IC 50 values are shown in right (n=3). (E) Schematic of results described in F. 50% GFP − OVCAR-4 and 50% GFP + Colo-205 cells were co-cultured. After 24 hr, cells were treated with indicated antibodies at constant 0.1 nM. After 36–48 hr, cells were analyzed using fluorescent microscope. (F) Represented images as described in E with indicated antibody treatment (scale bar represent 400 μm). (G) Immunoblot analysis for GFP and tubulin of GFP + Colo-205 cells co-cultured with equal number of GFP − OVCAR-4 or GFP − Colo-205 cells and treated with the indicated concentrations of BaCa antibody (H) The normalized relative intensities of GFP signal were plotted for the increasing BaCa dose in co-cultured conditions (filled black circles: 50% GFP − OVCAR-4 and 50% GFP + Colo-205) against constant 10 nM dose (filled red circle: 50% GFP − Colo-205 and 50% GFP + Colo-205). (I) Co-cultured MC38 (GFP − ) and Colo-205 (GFP + ) cells were treated with 50 nM of indicated antibodies. After 48 hr, lysates were run on gel and blotted for tubulin and GFP. (J) Cell viability of MC38 cells treated with LK26, LK26+anti-Fc, LK26-AMG-655 bispecific and BaCa antibody (n=3). (K) Cell viability of OVCAR-3 cells treated with AMG-655, AMG-655+anti-Fc, LK26-AMG-655 bispecific and BaCa antibody (n=3). (L) Co-cultured MC38 and OVCAR-3 cells were treated with the increasing concentration of indicated antibodies. After 48 hr, cell viability was analyzed using MTT assays. Error bars in A, B, C, D, J, K and L represent SEM. .

    Techniques Used: Activity Assay, Quantitative RT-PCR, Cell Culture, Microscopy, Concentration Assay, MTT Assay

    17) Product Images from "Identification and characterization of miRNAs in two closely related C4 and C3 species of Cleome by high-throughput sequencing"

    Article Title: Identification and characterization of miRNAs in two closely related C4 and C3 species of Cleome by high-throughput sequencing

    Journal: Scientific Reports

    doi: 10.1038/srep46552

    Quantitative analysis of the fourteen miRNAs levels by poly-A tail extension qRT-PCR in the leaf of Cleome gynandra and Cleome hassleriana . U6 was used as the internal control. The line represents the fold change of the expression level by sRNA-seq.
    Figure Legend Snippet: Quantitative analysis of the fourteen miRNAs levels by poly-A tail extension qRT-PCR in the leaf of Cleome gynandra and Cleome hassleriana . U6 was used as the internal control. The line represents the fold change of the expression level by sRNA-seq.

    Techniques Used: Quantitative RT-PCR, Expressing

    18) Product Images from "MicroRNA-448 suppresses osteosarcoma cell proliferation and invasion through targeting EPHA7"

    Article Title: MicroRNA-448 suppresses osteosarcoma cell proliferation and invasion through targeting EPHA7

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0175553

    Overexpression of miR-448 suppressed osteosarcoma cell proliferation, colony formation and migration. (A) The expression level of miR-448 in osteosarcoma cell lines (U2OS, MG-63, SAOS-2 and SOSP-9607) and the osteoblast cell line (hFOB) was determined by qRT-PCR. (B) miR-448 expression was significantly upregulated in the MG-63 cells after treatment with miR-448 mimic. (C) Elevated expression of miR-448 suppressed MG-63 cell proliferation. (D) Overexpression of miR-448 also decreased cyclin D1 expression in the MG-63 cells.(E) miR-448 expression was significantly upregulated in the U2OS cells after treatment with miR-448 mimic.(F) Elevated expression of miR-448 suppressed U2OS cell proliferation.(G) Overexpression of miR-448 inhibited MG-63 cell colony formation. The relative cell colony formation is shown. (H) Overexpression of miR-448 inhibited U2OS cell colony formation. The relative cell colony formation is shown. (I) Ectopic expression of miR-448 suppressed MG-63 cell migration. The relative open wound is shown. (J)Ectopic expression of miR-448 suppressed U2OS cell migration. The relative open wound is shown.*p
    Figure Legend Snippet: Overexpression of miR-448 suppressed osteosarcoma cell proliferation, colony formation and migration. (A) The expression level of miR-448 in osteosarcoma cell lines (U2OS, MG-63, SAOS-2 and SOSP-9607) and the osteoblast cell line (hFOB) was determined by qRT-PCR. (B) miR-448 expression was significantly upregulated in the MG-63 cells after treatment with miR-448 mimic. (C) Elevated expression of miR-448 suppressed MG-63 cell proliferation. (D) Overexpression of miR-448 also decreased cyclin D1 expression in the MG-63 cells.(E) miR-448 expression was significantly upregulated in the U2OS cells after treatment with miR-448 mimic.(F) Elevated expression of miR-448 suppressed U2OS cell proliferation.(G) Overexpression of miR-448 inhibited MG-63 cell colony formation. The relative cell colony formation is shown. (H) Overexpression of miR-448 inhibited U2OS cell colony formation. The relative cell colony formation is shown. (I) Ectopic expression of miR-448 suppressed MG-63 cell migration. The relative open wound is shown. (J)Ectopic expression of miR-448 suppressed U2OS cell migration. The relative open wound is shown.*p

    Techniques Used: Over Expression, Migration, Expressing, Quantitative RT-PCR

    Elevated expression of miR-448 suppressedosteosarcoma cell proliferation and invasion by targeting EPHA7. (A) The expression level of EPHA7 in the osteosarcoma cell lines (U2OS, MG-63, SAOS-2 and SOSP-9607)and osteoblast cell line (hFOB) was measured by qRT-PCR. (B) The EPHA7 mRNA expression was significantly upregulated in the MG-63 cells after treatment with EPHA7 vector. (C) The protein expression of EPHA7 was determined by Western blot. (D) CCK8 assay results demonstrated that EPHA7 overexpression restored miR-448 overexpression MG-63 cell proliferation. (E) Overexpression of EPHA7 promoted cyclin D1 expression in the miR-448 overexpressing MG-63 cells. (F) Overexpression of EPHA7 promoted miR-448 overexpressing MG-63 cell migration. (G) The relative migrative wound was shown. *p
    Figure Legend Snippet: Elevated expression of miR-448 suppressedosteosarcoma cell proliferation and invasion by targeting EPHA7. (A) The expression level of EPHA7 in the osteosarcoma cell lines (U2OS, MG-63, SAOS-2 and SOSP-9607)and osteoblast cell line (hFOB) was measured by qRT-PCR. (B) The EPHA7 mRNA expression was significantly upregulated in the MG-63 cells after treatment with EPHA7 vector. (C) The protein expression of EPHA7 was determined by Western blot. (D) CCK8 assay results demonstrated that EPHA7 overexpression restored miR-448 overexpression MG-63 cell proliferation. (E) Overexpression of EPHA7 promoted cyclin D1 expression in the miR-448 overexpressing MG-63 cells. (F) Overexpression of EPHA7 promoted miR-448 overexpressing MG-63 cell migration. (G) The relative migrative wound was shown. *p

    Techniques Used: Expressing, Quantitative RT-PCR, Plasmid Preparation, Western Blot, CCK-8 Assay, Over Expression, Migration

    EPHA7 expression was upregulated in osteosarcoma tissues. (A) The expression level of EPHA7 in osteosarcoma tissues and their related normal tissues was measured by qRT-PCR. (B) The expression level of EPHA7 was higher in the osteosarcoma tissues compared to that in the related normal tissues. (C) The expression level of EPHA7 was inversely correlated with that of miR-448 in osteosarcoma tissues.
    Figure Legend Snippet: EPHA7 expression was upregulated in osteosarcoma tissues. (A) The expression level of EPHA7 in osteosarcoma tissues and their related normal tissues was measured by qRT-PCR. (B) The expression level of EPHA7 was higher in the osteosarcoma tissues compared to that in the related normal tissues. (C) The expression level of EPHA7 was inversely correlated with that of miR-448 in osteosarcoma tissues.

    Techniques Used: Expressing, Quantitative RT-PCR

    miR-448 expression level was downregulated in osteosarcoma tissues. (A) The expression level of miR-448 in the osteosarcoma tissues and their related normal tissues was determined by qRT-PCR. (B) The expression level of miR-448 was lower in osteosarcoma tissues compared to that in the related normal tissues.
    Figure Legend Snippet: miR-448 expression level was downregulated in osteosarcoma tissues. (A) The expression level of miR-448 in the osteosarcoma tissues and their related normal tissues was determined by qRT-PCR. (B) The expression level of miR-448 was lower in osteosarcoma tissues compared to that in the related normal tissues.

    Techniques Used: Expressing, Quantitative RT-PCR

    19) Product Images from "Regulation of Serum Amyloid A3 (SAA3) in Mouse Colonic Epithelium and Adipose Tissue by the Intestinal Microbiota"

    Article Title: Regulation of Serum Amyloid A3 (SAA3) in Mouse Colonic Epithelium and Adipose Tissue by the Intestinal Microbiota

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005842

    TNF-α mRNA is increased in the mouse colon in the presence of microbiota. qRT-PCR analysis of SAA3 mRNA expression levels in colonic tissue of germ-free (GF) and conventionally raised (CONV-R) mice (n = 10 mice per group). Data are means±SEM. ** P
    Figure Legend Snippet: TNF-α mRNA is increased in the mouse colon in the presence of microbiota. qRT-PCR analysis of SAA3 mRNA expression levels in colonic tissue of germ-free (GF) and conventionally raised (CONV-R) mice (n = 10 mice per group). Data are means±SEM. ** P

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

    SAA3 mRNA expression is increased in mouse colon in presence of microbiota. (A) qRT-PCR analysis of SAA3 mRNA expression levels along the length of the gut in germ-free (GF) and conventionally raised (CONV-R) mice using pooled cDNA samples (n = 5 mice per group). Duo , duodenum; Jej , jejunum; Ile , ileum; Col , proximal colon. Data are means±SD. (B) Colonic SAA3 mRNA expression assayed in individual mice (n = 8−10 mice per group). Data are means±SEM. * P
    Figure Legend Snippet: SAA3 mRNA expression is increased in mouse colon in presence of microbiota. (A) qRT-PCR analysis of SAA3 mRNA expression levels along the length of the gut in germ-free (GF) and conventionally raised (CONV-R) mice using pooled cDNA samples (n = 5 mice per group). Duo , duodenum; Jej , jejunum; Ile , ileum; Col , proximal colon. Data are means±SD. (B) Colonic SAA3 mRNA expression assayed in individual mice (n = 8−10 mice per group). Data are means±SEM. * P

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

    SAA3 expression in the mouse colon may be stimulated by TLR/MyD88/NF-κB signaling. (A) Representative immunostaining for TLR4 ( red ) and bis -benzimide-positive nuclei ( blue ) in CONV-R mice. (B) Primary antibody omitted. Scale bars = 100 µm. (C) qRT-PCR analysis of colonic SAA3 mRNA expression levels in microbiota-associated wild-type (WT) and Myd88−/− mice (n = 10−13 mice per group). *** P
    Figure Legend Snippet: SAA3 expression in the mouse colon may be stimulated by TLR/MyD88/NF-κB signaling. (A) Representative immunostaining for TLR4 ( red ) and bis -benzimide-positive nuclei ( blue ) in CONV-R mice. (B) Primary antibody omitted. Scale bars = 100 µm. (C) qRT-PCR analysis of colonic SAA3 mRNA expression levels in microbiota-associated wild-type (WT) and Myd88−/− mice (n = 10−13 mice per group). *** P

    Techniques Used: Expressing, Immunostaining, Mouse Assay, Quantitative RT-PCR

    SAA3 but not SAA1/2 mRNA expression is increased in mouse adipose tissue in the presence of microbiota. (A) qRT-PCR analysis of SAA3 mRNA expression levels in epididymal adipose tissue of germ-free (GF) and conventionally raised (CONV-R) mice (n = 10 mice per group). (B) qRT-PCR analysis of SAA1/2 mRNA expression levels in epididymal adipose tissue of germ-free (GF) and conventionally raised (CONV-R) mice (n = 10 mice per group). Data are means±SEM. *** P
    Figure Legend Snippet: SAA3 but not SAA1/2 mRNA expression is increased in mouse adipose tissue in the presence of microbiota. (A) qRT-PCR analysis of SAA3 mRNA expression levels in epididymal adipose tissue of germ-free (GF) and conventionally raised (CONV-R) mice (n = 10 mice per group). (B) qRT-PCR analysis of SAA1/2 mRNA expression levels in epididymal adipose tissue of germ-free (GF) and conventionally raised (CONV-R) mice (n = 10 mice per group). Data are means±SEM. *** P

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

    LPS induces SAA3 and TNF-α mRNA expression in CMT-93 colonic epithelial cells and RAW 264.7 macrophages. (A) qRT-PCR analysis of SAA3 mRNA expression levels in CMT-93 cells and RAW 264.7 macrophages after 1 h LPS at the indicated concentrations. (B) qRT-PCR analysis of TNF-α mRNA expression levels in CMT-93 cells and RAW 264.7 macrophages after 1 h LPS at the indicated concentrations. (C) SAA3 mRNA expression levels in CMT-93 cells and RAW 264.7 macrophages after 1 h treatment with recombinant TNF-α (10 ng/ml). n = 6 biological replicates per treatment. * P
    Figure Legend Snippet: LPS induces SAA3 and TNF-α mRNA expression in CMT-93 colonic epithelial cells and RAW 264.7 macrophages. (A) qRT-PCR analysis of SAA3 mRNA expression levels in CMT-93 cells and RAW 264.7 macrophages after 1 h LPS at the indicated concentrations. (B) qRT-PCR analysis of TNF-α mRNA expression levels in CMT-93 cells and RAW 264.7 macrophages after 1 h LPS at the indicated concentrations. (C) SAA3 mRNA expression levels in CMT-93 cells and RAW 264.7 macrophages after 1 h treatment with recombinant TNF-α (10 ng/ml). n = 6 biological replicates per treatment. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Recombinant

    20) Product Images from "Transcriptional Maintenance of Pancreatic Acinar Identity, Differentiation, and Homeostasis by PTF1A"

    Article Title: Transcriptional Maintenance of Pancreatic Acinar Identity, Differentiation, and Homeostasis by PTF1A

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00358-16

    Loss of acinar cell differentiation: manufacture of secretory proteins. (A and B) Fold changes in the expression of genes for Ptf1a-cKO at 6 and 14 days relative to control TAM-treated, Ptf1a + / CreER pancreases. Asterisks indicate genes with bound PTF1A in pancreatic chromatin. (C) qRT-PCR measurements of total amylase or trypsinogen mRNAs ( n = 3; *, P
    Figure Legend Snippet: Loss of acinar cell differentiation: manufacture of secretory proteins. (A and B) Fold changes in the expression of genes for Ptf1a-cKO at 6 and 14 days relative to control TAM-treated, Ptf1a + / CreER pancreases. Asterisks indicate genes with bound PTF1A in pancreatic chromatin. (C) qRT-PCR measurements of total amylase or trypsinogen mRNAs ( n = 3; *, P

    Techniques Used: Cell Differentiation, Expressing, Quantitative RT-PCR

    21) Product Images from "Mechanisms underlying the resistance to diet-induced obesity in germ-free mice"

    Article Title: Mechanisms underlying the resistance to diet-induced obesity in germ-free mice

    Journal:

    doi: 10.1073/pnas.0605374104

    GF Fiaf −/− mice are not protected against diet-induced obesity and have lower expression of Pgc-1α and genes involved in fatty acid oxidation in their gastrocnemius muscles. ( A ) qRT-PCR assays of Fiaf expression in the distal small
    Figure Legend Snippet: GF Fiaf −/− mice are not protected against diet-induced obesity and have lower expression of Pgc-1α and genes involved in fatty acid oxidation in their gastrocnemius muscles. ( A ) qRT-PCR assays of Fiaf expression in the distal small

    Techniques Used: Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Quantitative RT-PCR

    22) Product Images from "Epigenetic silencing of miR-200b is associated with cisplatin resistance in bladder cancer"

    Article Title: Epigenetic silencing of miR-200b is associated with cisplatin resistance in bladder cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25326

    Epigenetic silencing of miR-200b in CDDP-resistant BCa cells ( A ) Schematic representation of the miR-200b, -200a and -429 genomic loci. Location of the CpG island and regions analyzed using the indicated methods are also shown. ( B ) Results of bisulfite pyrosequencing in the indicated BCa cells. ( C ) Results of bisulfite sequencing in the indicated cells. The region analyzed with bisulfite pyrosequencing is indicated by an arrow. ( D ) qRT-PCR analysis of miR-200b in CDDP-resistant BCa cells treated with or without 5-aza-dC (Aza). ( E ) MSP analysis in T24RC cells treated with or without 5-aza-dC. Bands in the “M” lanes are PCR products obtained with methylation-specific primers; those in the “U” lanes are products obtained with unmethylated-specific primers. In vitro methylated DNA and DNMT1/DNMT3B double knockout HCT116 cells serve as methylated and unmethylated controls. ( F ) ChIP-PCR analysis in the indicated cells. Levels of H3K9ac and H3K27me3 are shown. ( G ) Proliferation of T24RC cells treated with or without 5-aza-dC (Aza) and/or CDDP. Numbers of cells with the indicated treatments are shown relative to the numbers on day 0 (5 × 10 3 cells). Shown in B, D, F and G are means of 3 replications; error bars represent SDs. NS, not significant; * P
    Figure Legend Snippet: Epigenetic silencing of miR-200b in CDDP-resistant BCa cells ( A ) Schematic representation of the miR-200b, -200a and -429 genomic loci. Location of the CpG island and regions analyzed using the indicated methods are also shown. ( B ) Results of bisulfite pyrosequencing in the indicated BCa cells. ( C ) Results of bisulfite sequencing in the indicated cells. The region analyzed with bisulfite pyrosequencing is indicated by an arrow. ( D ) qRT-PCR analysis of miR-200b in CDDP-resistant BCa cells treated with or without 5-aza-dC (Aza). ( E ) MSP analysis in T24RC cells treated with or without 5-aza-dC. Bands in the “M” lanes are PCR products obtained with methylation-specific primers; those in the “U” lanes are products obtained with unmethylated-specific primers. In vitro methylated DNA and DNMT1/DNMT3B double knockout HCT116 cells serve as methylated and unmethylated controls. ( F ) ChIP-PCR analysis in the indicated cells. Levels of H3K9ac and H3K27me3 are shown. ( G ) Proliferation of T24RC cells treated with or without 5-aza-dC (Aza) and/or CDDP. Numbers of cells with the indicated treatments are shown relative to the numbers on day 0 (5 × 10 3 cells). Shown in B, D, F and G are means of 3 replications; error bars represent SDs. NS, not significant; * P

    Techniques Used: Methylation Sequencing, Quantitative RT-PCR, Polymerase Chain Reaction, Methylation, In Vitro, Double Knockout, Chromatin Immunoprecipitation

    Downregulation of miR-200b in CDDP-resistant BCa cells ( A ) Anti-tumor effects of CDDP in T24 and T24RC cells. Numbers of cells treated with the indicated concentrations of CDDP are shown relative to those without treatment. ( B ) qRT-PCR analysis of miR-200b in T24 and T24RC cells. ( C ) Effects of miR-200b on CDDP sensitivity in T24RC cells. Numbers of cells transfected with a miR-200b mimic or negative control (NC) and then treated with the indicated concentrations of CDDP are shown relative to cells without treatment. ( D ) Effects of miR-200b inhibition on CDDP sensitivity in T24 cells. Cells were transfected with a miR-200b inhibitor or negative control (NC) and then treated with the indicated concentrations of CDDP. ( E ) Anti-tumor effects of the indicated concentrations of CDDP on EJ138 and EJ138RC cells. ( F ) qRT-PCR analysis of miR-200b in EJ138 and EJ138RC cells. Shown are means of 6 (A, C–E) or 3 (B, F) replications; error bars represent SDs. * P
    Figure Legend Snippet: Downregulation of miR-200b in CDDP-resistant BCa cells ( A ) Anti-tumor effects of CDDP in T24 and T24RC cells. Numbers of cells treated with the indicated concentrations of CDDP are shown relative to those without treatment. ( B ) qRT-PCR analysis of miR-200b in T24 and T24RC cells. ( C ) Effects of miR-200b on CDDP sensitivity in T24RC cells. Numbers of cells transfected with a miR-200b mimic or negative control (NC) and then treated with the indicated concentrations of CDDP are shown relative to cells without treatment. ( D ) Effects of miR-200b inhibition on CDDP sensitivity in T24 cells. Cells were transfected with a miR-200b inhibitor or negative control (NC) and then treated with the indicated concentrations of CDDP. ( E ) Anti-tumor effects of the indicated concentrations of CDDP on EJ138 and EJ138RC cells. ( F ) qRT-PCR analysis of miR-200b in EJ138 and EJ138RC cells. Shown are means of 6 (A, C–E) or 3 (B, F) replications; error bars represent SDs. * P

    Techniques Used: Quantitative RT-PCR, Transfection, Negative Control, Inhibition

    Effects of miR-200b on gene expression profiles in CDDP-resistant BCa cells ( A ) Heat map showing expression of 733 probe sets (595 genes) identified through microarray analysis of T24RC cells transfected with a miR-200b mimic or negative control (NC) and then treated with or without CDDP. ( B ) Results of gene ontology (GO, upper) and pathway (lower) analyses of the 595 selected genes. ( C ) Heat map showing expression of miR-200b target genes predicted by TargetScan. ( D ) qRT-PCR analysis of the indicated genes in T24RC cells with or without miR-200b and/or CDDP. Shown are means of 3 replications; error bars represent SDs.
    Figure Legend Snippet: Effects of miR-200b on gene expression profiles in CDDP-resistant BCa cells ( A ) Heat map showing expression of 733 probe sets (595 genes) identified through microarray analysis of T24RC cells transfected with a miR-200b mimic or negative control (NC) and then treated with or without CDDP. ( B ) Results of gene ontology (GO, upper) and pathway (lower) analyses of the 595 selected genes. ( C ) Heat map showing expression of miR-200b target genes predicted by TargetScan. ( D ) qRT-PCR analysis of the indicated genes in T24RC cells with or without miR-200b and/or CDDP. Shown are means of 3 replications; error bars represent SDs.

    Techniques Used: Expressing, Microarray, Transfection, Negative Control, Quantitative RT-PCR

    Effects of miR-200b and CDDP on gene expression profiles in CDDP-resistant BCa cells ( A ) Heat map showing expression of 551 probe sets (509 genes) identified through microarray analysis of T24RC cells transfected with a miR-200b mimic or negative control (NC) and then treated with or without CDDP. ( B ) Results of gene ontology (GO, upper) and pathway analyses (lower) of the selected 509 genes. ( C ) qRT-PCR analysis of the indicated genes in T24RC cells with or without miR-200b and/or CDDP. Shown are means of 3 replications; error bars represent SDs.
    Figure Legend Snippet: Effects of miR-200b and CDDP on gene expression profiles in CDDP-resistant BCa cells ( A ) Heat map showing expression of 551 probe sets (509 genes) identified through microarray analysis of T24RC cells transfected with a miR-200b mimic or negative control (NC) and then treated with or without CDDP. ( B ) Results of gene ontology (GO, upper) and pathway analyses (lower) of the selected 509 genes. ( C ) qRT-PCR analysis of the indicated genes in T24RC cells with or without miR-200b and/or CDDP. Shown are means of 3 replications; error bars represent SDs.

    Techniques Used: Expressing, Microarray, Transfection, Negative Control, Quantitative RT-PCR

    23) Product Images from "The Three Flagellar Loci of Brucella ovis PA Are Dispensable for Virulence in Cellular Models and Mice"

    Article Title: The Three Flagellar Loci of Brucella ovis PA Are Dispensable for Virulence in Cellular Models and Mice

    Journal: Frontiers in Veterinary Science

    doi: 10.3389/fvets.2020.00441

    Genomic organization (A) and transcription (B,C) of the three main flagellar loci detected in the B. ovis genome. Flagellar genes targeted in transcription analysis with B. ovis PA strains (B,C) are represented with a black pattern (A) . Mutagenesis procedure deleted 99% of each locus (A) in flagellar mutants listed in Table 2 . End-point RT-PCR (B) was performed with cDNA obtained by retrotranscription of RNA extracted at t16 from B. ovis PA or the Δ flg1 Δ flg2 Δ flg3 triple mutant (RT+ reactions). Reactions of cDNA synthesis lacking RT were used as controls of DNA absence (RT- reactions). Amplification with fliC primers in the Δ flg1 Δ flg2 Δ flg3 triple mutant (B) was expected, since both primers hybridize adjacent but externally to the deleted fragment (the deletion removes 80% of fliC ). qRT-PCR (C) with fliF was performed with RNA extracted at t16 and t49 from B. ovis PA parental strain. Gene expression levels (C) were determined, with the StepOne TM software v2.3, by the 2 −ΔΔCt method with the 16S gene as internal reference and t16 results as control condition. Four biological replicates, with three technical replicates each, were analyzed and the results are expressed as means ± SD.
    Figure Legend Snippet: Genomic organization (A) and transcription (B,C) of the three main flagellar loci detected in the B. ovis genome. Flagellar genes targeted in transcription analysis with B. ovis PA strains (B,C) are represented with a black pattern (A) . Mutagenesis procedure deleted 99% of each locus (A) in flagellar mutants listed in Table 2 . End-point RT-PCR (B) was performed with cDNA obtained by retrotranscription of RNA extracted at t16 from B. ovis PA or the Δ flg1 Δ flg2 Δ flg3 triple mutant (RT+ reactions). Reactions of cDNA synthesis lacking RT were used as controls of DNA absence (RT- reactions). Amplification with fliC primers in the Δ flg1 Δ flg2 Δ flg3 triple mutant (B) was expected, since both primers hybridize adjacent but externally to the deleted fragment (the deletion removes 80% of fliC ). qRT-PCR (C) with fliF was performed with RNA extracted at t16 and t49 from B. ovis PA parental strain. Gene expression levels (C) were determined, with the StepOne TM software v2.3, by the 2 −ΔΔCt method with the 16S gene as internal reference and t16 results as control condition. Four biological replicates, with three technical replicates each, were analyzed and the results are expressed as means ± SD.

    Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, Expressing, Software

    24) Product Images from "MYB97, MYB101 and MYB120 Function as Male Factors That Control Pollen Tube-Synergid Interaction in Arabidopsis thaliana Fertilization"

    Article Title: MYB97, MYB101 and MYB120 Function as Male Factors That Control Pollen Tube-Synergid Interaction in Arabidopsis thaliana Fertilization

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003933

    Molecular characterization of myb97 , myb101 and myb120 mutants. ( A ) Schematic diagrams of the MYB gene structures and T-DNA insertion sites in the mutants. The gray and white boxes indicate the translated and untranslated regions, respectively. ( B ) The expression levels of MYB97 , MYB101 and MYB120 genes in wild type (WT) and the triple homozygous mutants, as revealed by qRT-PCR.
    Figure Legend Snippet: Molecular characterization of myb97 , myb101 and myb120 mutants. ( A ) Schematic diagrams of the MYB gene structures and T-DNA insertion sites in the mutants. The gray and white boxes indicate the translated and untranslated regions, respectively. ( B ) The expression levels of MYB97 , MYB101 and MYB120 genes in wild type (WT) and the triple homozygous mutants, as revealed by qRT-PCR.

    Techniques Used: Expressing, Quantitative RT-PCR

    MYB101 binds to the MYBGAHV ( TAACAAA ) cis -element in the DG1 , DG2 and DG3 promoters in vitro . ( A ) to ( B ) The expression of DG1 , DG2 , and DG3 were significantly reduced in the myb97 myb101 myb120 triple mutant, as revealed by RT-PCR ( A ) and qRT-PCR ( B ). ( C ) The sequences of the oligonucleotides used in the EMSA experiments. DG1WT, DG2WT and DG3WT are the wild-type versions of the MYBGAHV ( TAACAAA ) cis -elements (underlined) in the DG1 , DG2 and DG3 promoters, respectively. The TAACAAA motifs were mutated as indicated by lowercase letters in the mDG1WT, mDG2WT and mDG3WT sequences. ( D ) MYB101 is able to bind to the MYBGAHV cis -element ( TAACAAA ) in the DG1 promoter. Lanes 1 and 2 show reactions to which the MYB101 protein or the biotin-labeled DG1WT oligonucleotide was added, respectively. As shown in lane 3, a shift (black triangle) was observed when the MYB101 protein was added to the reaction containing the biotin-labeled DG1WT oligonucleotide. Lanes 4 to 7 show reactions in which unlabeled oligonucleotides were added to the binding reactions to compete with biotin-labeled DG1WT oligonucleotide. The competition becomes increasingly apparent with the unlabeled DG1WT oligonucleotide added at 50×, 250× and 500× molar excess in lanes 4, 5 and 6, respectively. Lane 7 shows that the unlabeled mDG1WT oligonucleotide competed only weakly, even at 500× molar excess. These results were reconfirmed in independent EMSA experiments. ( E ) MYB101 is able to bind to the MYBGAHV cis -element in the DG2 promoter. The binding reaction containing the MYB101 protein and the biotin-labeled DG2WT oligonucleotide causes a clear shift. The unlabeled DG2WT oligonucleotide competed fully at 500× molar excess. No competition was observed when the unlabeled mDG2WT oligonucleotide was used at 500× molar excess. These results were reconfirmed in independent EMSA experiments. ( F ) MYB101 is able to bind to the MYBGAHV cis -element in the DG3 promoter. The biotin-labeled DG3WT oligonucleotide was mixed with the MYB101 protein, and a shift was observed in the binding reaction. The unlabeled DG3WT oligonucleotide competed fully at 500× molar excess. Only weak competition was observed when unlabeled mDG3WT oligonucleotide was used at 500× molar excess. These results were reconfirmed in independent EMSA experiments.
    Figure Legend Snippet: MYB101 binds to the MYBGAHV ( TAACAAA ) cis -element in the DG1 , DG2 and DG3 promoters in vitro . ( A ) to ( B ) The expression of DG1 , DG2 , and DG3 were significantly reduced in the myb97 myb101 myb120 triple mutant, as revealed by RT-PCR ( A ) and qRT-PCR ( B ). ( C ) The sequences of the oligonucleotides used in the EMSA experiments. DG1WT, DG2WT and DG3WT are the wild-type versions of the MYBGAHV ( TAACAAA ) cis -elements (underlined) in the DG1 , DG2 and DG3 promoters, respectively. The TAACAAA motifs were mutated as indicated by lowercase letters in the mDG1WT, mDG2WT and mDG3WT sequences. ( D ) MYB101 is able to bind to the MYBGAHV cis -element ( TAACAAA ) in the DG1 promoter. Lanes 1 and 2 show reactions to which the MYB101 protein or the biotin-labeled DG1WT oligonucleotide was added, respectively. As shown in lane 3, a shift (black triangle) was observed when the MYB101 protein was added to the reaction containing the biotin-labeled DG1WT oligonucleotide. Lanes 4 to 7 show reactions in which unlabeled oligonucleotides were added to the binding reactions to compete with biotin-labeled DG1WT oligonucleotide. The competition becomes increasingly apparent with the unlabeled DG1WT oligonucleotide added at 50×, 250× and 500× molar excess in lanes 4, 5 and 6, respectively. Lane 7 shows that the unlabeled mDG1WT oligonucleotide competed only weakly, even at 500× molar excess. These results were reconfirmed in independent EMSA experiments. ( E ) MYB101 is able to bind to the MYBGAHV cis -element in the DG2 promoter. The binding reaction containing the MYB101 protein and the biotin-labeled DG2WT oligonucleotide causes a clear shift. The unlabeled DG2WT oligonucleotide competed fully at 500× molar excess. No competition was observed when the unlabeled mDG2WT oligonucleotide was used at 500× molar excess. These results were reconfirmed in independent EMSA experiments. ( F ) MYB101 is able to bind to the MYBGAHV cis -element in the DG3 promoter. The biotin-labeled DG3WT oligonucleotide was mixed with the MYB101 protein, and a shift was observed in the binding reaction. The unlabeled DG3WT oligonucleotide competed fully at 500× molar excess. Only weak competition was observed when unlabeled mDG3WT oligonucleotide was used at 500× molar excess. These results were reconfirmed in independent EMSA experiments.

    Techniques Used: In Vitro, Expressing, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Labeling, Binding Assay

    Identification of the MYB genes. ( A ) Phylogenetic analysis of the MYB proteins. ( B ) Expression patterns of the MYB genes, as revealed by RT-PCR. ( C ) Expression of the MYB genes in mature pollen grains, as revealed by qRT-PCR. gDNA, genomic DNA; Rt, roots; St, stems; Lf, leaves; If, inflorescences; Mp, mature pollen grains; Sq, siliques and Sl, seedlings.
    Figure Legend Snippet: Identification of the MYB genes. ( A ) Phylogenetic analysis of the MYB proteins. ( B ) Expression patterns of the MYB genes, as revealed by RT-PCR. ( C ) Expression of the MYB genes in mature pollen grains, as revealed by qRT-PCR. gDNA, genomic DNA; Rt, roots; St, stems; Lf, leaves; If, inflorescences; Mp, mature pollen grains; Sq, siliques and Sl, seedlings.

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

    25) Product Images from "A five-microRNA panel in plasma was identified as potential biomarker for early detection of gastric cancer"

    Article Title: A five-microRNA panel in plasma was identified as potential biomarker for early detection of gastric cancer

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2014.119

    Overview of the study design. Abbreviations: GCA= gastric cardia adenocarcinoma; GNCA= gastric non-cardia adenocarcinoma; TLDA= TaqMan low density array; qRT–PCR= quantitative reverse-transcriptase PCR assay; ROC= receiver operating characteristic curve.
    Figure Legend Snippet: Overview of the study design. Abbreviations: GCA= gastric cardia adenocarcinoma; GNCA= gastric non-cardia adenocarcinoma; TLDA= TaqMan low density array; qRT–PCR= quantitative reverse-transcriptase PCR assay; ROC= receiver operating characteristic curve.

    Techniques Used: TLDA Assay, Quantitative RT-PCR, Polymerase Chain Reaction

    26) Product Images from "VmPacC Is Required for Acidification and Virulence in Valsa mali"

    Article Title: VmPacC Is Required for Acidification and Virulence in Valsa mali

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01981

    Expression levels of pectinase genes in wild-type, deletion mutant, and complemented mutant strains determined by qRT-PCR. RNA samples were isolated from lesion borders of apple tree bark at 3 days post-inoculation. Wild-type expression levels were arbitrarily set to 1. The mean was calculated using data from three independent biological replicates. The asterisk represents a statistically significant difference compared to wild-type.
    Figure Legend Snippet: Expression levels of pectinase genes in wild-type, deletion mutant, and complemented mutant strains determined by qRT-PCR. RNA samples were isolated from lesion borders of apple tree bark at 3 days post-inoculation. Wild-type expression levels were arbitrarily set to 1. The mean was calculated using data from three independent biological replicates. The asterisk represents a statistically significant difference compared to wild-type.

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

    Negative regulation of pectinase production by VmPacC dominant activated mutants C27-2 and C27-3. (A) Phenotypes of twigs inoculated with wild-type, VmPacC deletion mutant, and activated mutants C27-2 and C27-3; length of lesions was measured at 9 days post-inoculation. (B) Mycelial growth on SM supplemented with pectin for 6 days. Expression levels of pectinase genes in wild-type, deletion mutant, and activated mutant strains cultured in SM medium supplemented with pectin at pH 4 (C) and pH 7 (D) determined using qRT-PCR. (E) Pectinase activity of different strains cultured in MS supplemented pectin for 12 h. (F) Assays for protein concentration of wild-type, deletion mutant, and activated mutant strains in 1% pectin inducing medium after culture for 12 h. Bars marked by asterisks in each group differ significantly from wild-type (LSD, P
    Figure Legend Snippet: Negative regulation of pectinase production by VmPacC dominant activated mutants C27-2 and C27-3. (A) Phenotypes of twigs inoculated with wild-type, VmPacC deletion mutant, and activated mutants C27-2 and C27-3; length of lesions was measured at 9 days post-inoculation. (B) Mycelial growth on SM supplemented with pectin for 6 days. Expression levels of pectinase genes in wild-type, deletion mutant, and activated mutant strains cultured in SM medium supplemented with pectin at pH 4 (C) and pH 7 (D) determined using qRT-PCR. (E) Pectinase activity of different strains cultured in MS supplemented pectin for 12 h. (F) Assays for protein concentration of wild-type, deletion mutant, and activated mutant strains in 1% pectin inducing medium after culture for 12 h. Bars marked by asterisks in each group differ significantly from wild-type (LSD, P

    Techniques Used: Mutagenesis, Expressing, Cell Culture, Quantitative RT-PCR, Activity Assay, Mass Spectrometry, Protein Concentration

    27) Product Images from "Organoid modelling identifies that DACH1 functions as a tumour promoter in colorectal cancer by modulating BMP signalling"

    Article Title: Organoid modelling identifies that DACH1 functions as a tumour promoter in colorectal cancer by modulating BMP signalling

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2020.102800

    DACH1 overexpression enhances the growth of organoids derived from normal colon crypts and colorectal adenomas. DACH1 overexpression efficiency was verified by qRT-PCR (experiments were performed in triplicate) (a) and western blot (b). c. Overexpression of DACH1 enhanced the sphere formation of organoids derived from normal colon crypts (colonoid) compared with the control; scale bars=200 μm d. Overexpression of DACH1 significantly enhanced colonoid proliferation compared with the vector group. e. Overexpression of DACH1 significantly enhanced the colonoid formation rate compared with the control. DACH1 overexpression was verified by qRT-PCR (experiments were performed in triplicate) and western blot (f and g). h. Adenoma organoids with DACH1 overexpression expanded to almost twice the size of the control organoids; scale bars=200 μm. i. Overexpression of DACH1 significantly enhanced the proliferation of organoids derived from colorectal adenoma compared with the control. j. The adenoma organoid formation efficiency was improved by DACH1 overexpression. k-l. The organoid areas of DACH1-overexpressing organoids were within the lower quartile more than the control; scale bars=200 μm. m-n. The average adenoma organoid areas in DACH1-overexpressing organoids were significantly larger than those in the control; scale bars=100 μm. For 4a, 4d, 4e, 4f, 4i, 4j and 4n, * P
    Figure Legend Snippet: DACH1 overexpression enhances the growth of organoids derived from normal colon crypts and colorectal adenomas. DACH1 overexpression efficiency was verified by qRT-PCR (experiments were performed in triplicate) (a) and western blot (b). c. Overexpression of DACH1 enhanced the sphere formation of organoids derived from normal colon crypts (colonoid) compared with the control; scale bars=200 μm d. Overexpression of DACH1 significantly enhanced colonoid proliferation compared with the vector group. e. Overexpression of DACH1 significantly enhanced the colonoid formation rate compared with the control. DACH1 overexpression was verified by qRT-PCR (experiments were performed in triplicate) and western blot (f and g). h. Adenoma organoids with DACH1 overexpression expanded to almost twice the size of the control organoids; scale bars=200 μm. i. Overexpression of DACH1 significantly enhanced the proliferation of organoids derived from colorectal adenoma compared with the control. j. The adenoma organoid formation efficiency was improved by DACH1 overexpression. k-l. The organoid areas of DACH1-overexpressing organoids were within the lower quartile more than the control; scale bars=200 μm. m-n. The average adenoma organoid areas in DACH1-overexpressing organoids were significantly larger than those in the control; scale bars=100 μm. For 4a, 4d, 4e, 4f, 4i, 4j and 4n, * P

    Techniques Used: Over Expression, Derivative Assay, Quantitative RT-PCR, Western Blot, Plasmid Preparation

    DACH1 marks crypt base cells in intestines and predicts poor outcomes in colorectal cancer patients. DACH1 expression in the mouse small intestine (a) and large intestine (b); scale bars=20 μm. In the mouse small intestine, DACH1 is expressed in crypt base cells interspersed between Paneth cells. Lysozymes (a marker of Paneth cells) were stained red, and DACH1 was stained green, which merged into cyan with DAPI (blue); scale bar=20 μm (c). DACH1 is also expressed in the human large intestine; scale bar=20 μm (d). DACH1 mRNA is overexpressed in colorectal cancer tissues compared to adjacent normal tissues, as detected by qRT-PCR (e), and IHC revealed that the expression of DACH1 increased in all stages of CRC when compared with the normal tissue (f) (* P
    Figure Legend Snippet: DACH1 marks crypt base cells in intestines and predicts poor outcomes in colorectal cancer patients. DACH1 expression in the mouse small intestine (a) and large intestine (b); scale bars=20 μm. In the mouse small intestine, DACH1 is expressed in crypt base cells interspersed between Paneth cells. Lysozymes (a marker of Paneth cells) were stained red, and DACH1 was stained green, which merged into cyan with DAPI (blue); scale bar=20 μm (c). DACH1 is also expressed in the human large intestine; scale bar=20 μm (d). DACH1 mRNA is overexpressed in colorectal cancer tissues compared to adjacent normal tissues, as detected by qRT-PCR (e), and IHC revealed that the expression of DACH1 increased in all stages of CRC when compared with the normal tissue (f) (* P

    Techniques Used: Expressing, Marker, Staining, Quantitative RT-PCR, Immunohistochemistry

    28) Product Images from "Runx2 Regulates G Protein-coupled Signaling Pathways to Control Growth of Osteoblast Progenitors *"

    Article Title: Runx2 Regulates G Protein-coupled Signaling Pathways to Control Growth of Osteoblast Progenitors *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M802453200

    Expression of G protein-coupled receptor signaling components at distinct biological stages of osteogenic differentiation. A, expression of Runx2 -dependent genes related to G protein-coupled receptor signaling and other markers was examined by qRT-PCR in mesenchymal progenitor cells obtained from the calvarial regions of Runx2 wild type and Runx2 null ( KO ) mice at embryonic day 17.5 and maintained in subconfluent culture for three passages. Several housekeeping genes were used as internal controls to validate differences in mRNA levels ( Gapdh, Mcox, rRNA, and Hprt ). mRNA levels of all different genes were plotted as fold change relative to each other (Gpr64 expression in Runx2 KO cells was arbitrary set as 1). Error bars represent standard error between four different mRNA preparations, each derived from multiple mouse embryos. Statistical significance of the data was determined by Student's t test, and values with p
    Figure Legend Snippet: Expression of G protein-coupled receptor signaling components at distinct biological stages of osteogenic differentiation. A, expression of Runx2 -dependent genes related to G protein-coupled receptor signaling and other markers was examined by qRT-PCR in mesenchymal progenitor cells obtained from the calvarial regions of Runx2 wild type and Runx2 null ( KO ) mice at embryonic day 17.5 and maintained in subconfluent culture for three passages. Several housekeeping genes were used as internal controls to validate differences in mRNA levels ( Gapdh, Mcox, rRNA, and Hprt ). mRNA levels of all different genes were plotted as fold change relative to each other (Gpr64 expression in Runx2 KO cells was arbitrary set as 1). Error bars represent standard error between four different mRNA preparations, each derived from multiple mouse embryos. Statistical significance of the data was determined by Student's t test, and values with p

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

    Runx2 reconstitution in osteoprogenitors from Runx2 null mouse calvaria. Cells were infected with adenoviral vectors expressing an IRES-driven GFP marker and either Runx2 wild type ( WT ) or different mutants defective for interactions with distinct regulatory cofactors. Virus expressing GFP alone was used as a control ( GFP-EV ). A, schematic representation of specific Runx2 mutations used in the context of functional protein domains of Runx2. Numbering is for the P1 isoform of Runx2 that starts with the amino acids MASNS and ends with the conserved residues VWRPY. Also indicated are a poly(Q/A) stretch, the runt homology domain ( RHD ) that controls DNA binding, C-terminal activation, and repression domains, as well as a nuclear matrix targeting signal. Micrographs were taken of transfected cells expressing an IRES-driven GFP at day 2 after infection to establish comparable infection efficiencies of each adenoviral vector (data not shown). B, Western blot analysis of exogenous Runx2 protein levels and different mutants shows comparable expression of Runx2 at day 1 after infection. The figure is a representative collage of comparable exposures of four distinct experiments with different sets of adenoviral vectors. Spliced lanes are indicated with dashed lines . C, expression of Runx2 mRNA levels in Runx2 null cells and induction of bone marker genes were monitored by qRT-PCR analysis. The mRNA levels for exogenous Runx2 were plotted relative to the average endogenous mRNA level in an equivalent amount of RNA from MC3T3 cells that was examined in parallel as external standard. RNA quantity was normalized relative to GAPDH as internal control. The mRNA levels of osteocalcin and osteopontin in control cells (no virus) are minimal, and values of 1 and below are close to background levels.
    Figure Legend Snippet: Runx2 reconstitution in osteoprogenitors from Runx2 null mouse calvaria. Cells were infected with adenoviral vectors expressing an IRES-driven GFP marker and either Runx2 wild type ( WT ) or different mutants defective for interactions with distinct regulatory cofactors. Virus expressing GFP alone was used as a control ( GFP-EV ). A, schematic representation of specific Runx2 mutations used in the context of functional protein domains of Runx2. Numbering is for the P1 isoform of Runx2 that starts with the amino acids MASNS and ends with the conserved residues VWRPY. Also indicated are a poly(Q/A) stretch, the runt homology domain ( RHD ) that controls DNA binding, C-terminal activation, and repression domains, as well as a nuclear matrix targeting signal. Micrographs were taken of transfected cells expressing an IRES-driven GFP at day 2 after infection to establish comparable infection efficiencies of each adenoviral vector (data not shown). B, Western blot analysis of exogenous Runx2 protein levels and different mutants shows comparable expression of Runx2 at day 1 after infection. The figure is a representative collage of comparable exposures of four distinct experiments with different sets of adenoviral vectors. Spliced lanes are indicated with dashed lines . C, expression of Runx2 mRNA levels in Runx2 null cells and induction of bone marker genes were monitored by qRT-PCR analysis. The mRNA levels for exogenous Runx2 were plotted relative to the average endogenous mRNA level in an equivalent amount of RNA from MC3T3 cells that was examined in parallel as external standard. RNA quantity was normalized relative to GAPDH as internal control. The mRNA levels of osteocalcin and osteopontin in control cells (no virus) are minimal, and values of 1 and below are close to background levels.

    Techniques Used: Infection, Expressing, Marker, Functional Assay, Binding Assay, Activation Assay, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR

    29) Product Images from "Evaluation of Two Triplex One-Step qRT-PCR Assays for the Quantification of Human Enteric Viruses in Environmental Samples"

    Article Title: Evaluation of Two Triplex One-Step qRT-PCR Assays for the Quantification of Human Enteric Viruses in Environmental Samples

    Journal: Food and Environmental Virology

    doi: 10.1007/s12560-017-9293-5

    Standard curves of the qRT-PCR assays for the target viruses in singleplex ( black filled circle ) and multiplex ( open circle ) qRT-PCR assays. Norovirus GII (NoV GII), hepatitis A virus (HAV and mengovirus (MgV) assays were run using the annealing temperature (Ta) of 60 °C, whereas NoV GI, sapovirus (SaV) and Hepatitis E virus (HEV) assays were run using Ta of 56 °C. The grey circle represents the results for the norovirus GI standards when a Ta of 60 °C was used. Symbols and error bars represent the mean and standard deviation of the triplicated experiments
    Figure Legend Snippet: Standard curves of the qRT-PCR assays for the target viruses in singleplex ( black filled circle ) and multiplex ( open circle ) qRT-PCR assays. Norovirus GII (NoV GII), hepatitis A virus (HAV and mengovirus (MgV) assays were run using the annealing temperature (Ta) of 60 °C, whereas NoV GI, sapovirus (SaV) and Hepatitis E virus (HEV) assays were run using Ta of 56 °C. The grey circle represents the results for the norovirus GI standards when a Ta of 60 °C was used. Symbols and error bars represent the mean and standard deviation of the triplicated experiments

    Techniques Used: Quantitative RT-PCR, Multiplex Assay, Standard Deviation

    30) Product Images from "Novel Biomarkers of Arterial and Venous Ischemia in Microvascular Flaps"

    Article Title: Novel Biomarkers of Arterial and Venous Ischemia in Microvascular Flaps

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071628

    Temporal expression profile of selected genes under venous congestion. qRT-PCR performed on Prol1, Muc1, Vcsa1, Fcnb, Il1b for flaps undergoing venous congestion compared to control at t = 1, 3, 4 hrs. Data are expressed as means ± SE and analyzed by unpaired t -test. * P
    Figure Legend Snippet: Temporal expression profile of selected genes under venous congestion. qRT-PCR performed on Prol1, Muc1, Vcsa1, Fcnb, Il1b for flaps undergoing venous congestion compared to control at t = 1, 3, 4 hrs. Data are expressed as means ± SE and analyzed by unpaired t -test. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    Validation of microarray data using qRT-PCR analysis. qRT-PCR performed on 5 differentially expressed genes ( Prol1, Muc1, Vcsa1, Fcnb, Il1b ) for flaps undergoing both arterial ischemia and venous congestion for 4 hours compared to control. n = 5 and performed in duplicate. Data are expressed as means ± SE and analyzed by unpaired t -test. * P
    Figure Legend Snippet: Validation of microarray data using qRT-PCR analysis. qRT-PCR performed on 5 differentially expressed genes ( Prol1, Muc1, Vcsa1, Fcnb, Il1b ) for flaps undergoing both arterial ischemia and venous congestion for 4 hours compared to control. n = 5 and performed in duplicate. Data are expressed as means ± SE and analyzed by unpaired t -test. * P

    Techniques Used: Microarray, Quantitative RT-PCR

    31) Product Images from "MicroRNA-486-5p is an erythroid oncomiR of the myeloid leukemias of Down syndrome"

    Article Title: MicroRNA-486-5p is an erythroid oncomiR of the myeloid leukemias of Down syndrome

    Journal: Blood

    doi: 10.1182/blood-2014-06-581892

    miR-486-5p expression in the erythroid lin. (A) qRT-PCR analysis of miR-486-5p expression (i) and Gata1 (or Gata1s) expression in different BM cells populations separated by sorting. LIN − = lineage negative cells; MEP = megakaryocytic-erythroid
    Figure Legend Snippet: miR-486-5p expression in the erythroid lin. (A) qRT-PCR analysis of miR-486-5p expression (i) and Gata1 (or Gata1s) expression in different BM cells populations separated by sorting. LIN − = lineage negative cells; MEP = megakaryocytic-erythroid

    Techniques Used: Expressing, Quantitative RT-PCR

    32) Product Images from "The MADS transcription factor CmANR1 positively modulates root system development by directly regulating CmPIN2 in chrysanthemum"

    Article Title: The MADS transcription factor CmANR1 positively modulates root system development by directly regulating CmPIN2 in chrysanthemum

    Journal: Horticulture Research

    doi: 10.1038/s41438-018-0061-y

    Verification of the RNA-Seq results by qRT-PCR. a The relative transcript abundance of some IAA-responsive unigenes that were selected based on their differential expression in the roots of WT vs. CmANR1 -transgenetic plants. b The relative expression level of the unigenes in Ca 2+ signaling-related, ethylene-related, and cell cycle-related groups in the roots of WT and CmANR1 -transgenetic plants. c Comparison of the expression level of unigenes between the RNA-Seq and qRT-PCR. d Scatter diagram of the log ratios (log 2 FC) of the unigenes. The qRT-PCR data were normalized to the internal control CmUBI . Note that in a and b , data are shown as the mean ± SE based on three or more replicate. Statistical significance was determined using Student’s t -test. n.s.: p > 0.01; * p
    Figure Legend Snippet: Verification of the RNA-Seq results by qRT-PCR. a The relative transcript abundance of some IAA-responsive unigenes that were selected based on their differential expression in the roots of WT vs. CmANR1 -transgenetic plants. b The relative expression level of the unigenes in Ca 2+ signaling-related, ethylene-related, and cell cycle-related groups in the roots of WT and CmANR1 -transgenetic plants. c Comparison of the expression level of unigenes between the RNA-Seq and qRT-PCR. d Scatter diagram of the log ratios (log 2 FC) of the unigenes. The qRT-PCR data were normalized to the internal control CmUBI . Note that in a and b , data are shown as the mean ± SE based on three or more replicate. Statistical significance was determined using Student’s t -test. n.s.: p > 0.01; * p

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

    Heatmaps of four groups of candidate unigenes associated with root system development in the DEGs. a Heatmap of auxin-responsive unigenes. b Heatmap of Ca 2+ signaling-related unigenes. c Heatmap of ethylene-related unigenes. d . The red unigenes in the four groups were selected for qRT-PCR
    Figure Legend Snippet: Heatmaps of four groups of candidate unigenes associated with root system development in the DEGs. a Heatmap of auxin-responsive unigenes. b Heatmap of Ca 2+ signaling-related unigenes. c Heatmap of ethylene-related unigenes. d . The red unigenes in the four groups were selected for qRT-PCR

    Techniques Used: Quantitative RT-PCR

    33) Product Images from "MIKCC-type MADS-box genes in Rosa chinensis: the remarkable expansion of ABCDE model genes and their roles in floral organogenesis"

    Article Title: MIKCC-type MADS-box genes in Rosa chinensis: the remarkable expansion of ABCDE model genes and their roles in floral organogenesis

    Journal: Horticulture Research

    doi: 10.1038/s41438-018-0031-4

    The expression profiles of rose MIKC C genes from AP1/FUL, AP3/PI, AG, and SEP clades (ABCDE model genes) in response to floral organogenesis in R. chinensis cv. Old Blush and R. chinensis cv. Viridiflora. a , b Rose flower development stages of cultivars Old Blush and Viridiflora, respectively. L, leaves; DBO, vegetative meristem stage; Stage 1–4, initiation stages of sepals (stage 1), petals/petal-like (stage 2), stamens/stamen-like (stage 3), and carpels/carpel-like (stage 4) in Old Blush and Viridiflora; Stage 5, hypanthium starts to sink below the perianth and stamens; Se, sepals; Pe, petals; St, stamens; Pi, pistils. c , d Heatmaps of the relative gene expression levels of MIKC C genes in R. chinensis cv. Old Blush and R. chinensis cv. Viridiflora, respectively, as determined by qRT-PCR. The relative gene expression levels were normalized with the RcTUBULIN , RcGAPDH , and RcTCTP . e Fold change of gene expression for the sepal, petal, stamen, and pistil differentiation phases, which were calculated via comparison with the previous development phase
    Figure Legend Snippet: The expression profiles of rose MIKC C genes from AP1/FUL, AP3/PI, AG, and SEP clades (ABCDE model genes) in response to floral organogenesis in R. chinensis cv. Old Blush and R. chinensis cv. Viridiflora. a , b Rose flower development stages of cultivars Old Blush and Viridiflora, respectively. L, leaves; DBO, vegetative meristem stage; Stage 1–4, initiation stages of sepals (stage 1), petals/petal-like (stage 2), stamens/stamen-like (stage 3), and carpels/carpel-like (stage 4) in Old Blush and Viridiflora; Stage 5, hypanthium starts to sink below the perianth and stamens; Se, sepals; Pe, petals; St, stamens; Pi, pistils. c , d Heatmaps of the relative gene expression levels of MIKC C genes in R. chinensis cv. Old Blush and R. chinensis cv. Viridiflora, respectively, as determined by qRT-PCR. The relative gene expression levels were normalized with the RcTUBULIN , RcGAPDH , and RcTCTP . e Fold change of gene expression for the sepal, petal, stamen, and pistil differentiation phases, which were calculated via comparison with the previous development phase

    Techniques Used: Expressing, Quantitative RT-PCR

    34) Product Images from "Cxcr1 mediates recruitment of neutrophils and supports proliferation of tumor-initiating astrocytes in vivo"

    Article Title: Cxcr1 mediates recruitment of neutrophils and supports proliferation of tumor-initiating astrocytes in vivo

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31675-0

    Expression of oncogenic Kras from an astrocyte-specific promoter drives transformation in the larval hindbrain. Live-imaging of mosaic gfap :GFP, gfap :GFP-kras WT , and gfap :GFP-kras G12V expression at 3 days post-fertilization (dpf) in the larval zebrafish hindbrain ( A ). Red dashed outline in schematic of A shows approximate field of view and image orientation for all figures. White dashed lines in representative images denote the hindbrain region of interest in all figures. Morphology of expressing cells was categorized based on characteristics observed throughout the population of injected larvae and larvae were assigned to category of most severe cell morphology identified ( B , n = 19 gfap :GFP, 17 gfap :kras WT , and 38 gfap :kras G12V larvae; Chi-squared analysis performed). gfap :H2B-mCherry was mosaically co-expressed with gfap :kras plasmids to label nuclei of Kras-expressing cells ( C ). qRT-PCR was performed on whole-larvae isolates for EMT genes including vimentin , mmp9 , cdh1 ( e-cadherin ), and cdh2 ( n-cadherin ). Cq were normalized to housekeeping gene rps11 and then normalized to kras WT condition ( D , n = 5 kras WT and 5 kras G12V replicates). Larvae were fixed at 3 dpf or 6 dpf and immunostained for N-cadherin ( E ). N-cadherin co-localization was observed in gfap :kras G12V -expressing masses at 6 dpf (yellow asterisks). Kras-expressing larvae were fixed at 3 dpf and immunostained for proliferation marker phospho-Histone H3 (pH3, F ). pH3/GFP double positive cells were quantified using confocal optical sectioning (single optical slice from yellow dashed box shown in inset) and normalized to the total gfap -expressing area (G, n = 16 larvae/condition). *p
    Figure Legend Snippet: Expression of oncogenic Kras from an astrocyte-specific promoter drives transformation in the larval hindbrain. Live-imaging of mosaic gfap :GFP, gfap :GFP-kras WT , and gfap :GFP-kras G12V expression at 3 days post-fertilization (dpf) in the larval zebrafish hindbrain ( A ). Red dashed outline in schematic of A shows approximate field of view and image orientation for all figures. White dashed lines in representative images denote the hindbrain region of interest in all figures. Morphology of expressing cells was categorized based on characteristics observed throughout the population of injected larvae and larvae were assigned to category of most severe cell morphology identified ( B , n = 19 gfap :GFP, 17 gfap :kras WT , and 38 gfap :kras G12V larvae; Chi-squared analysis performed). gfap :H2B-mCherry was mosaically co-expressed with gfap :kras plasmids to label nuclei of Kras-expressing cells ( C ). qRT-PCR was performed on whole-larvae isolates for EMT genes including vimentin , mmp9 , cdh1 ( e-cadherin ), and cdh2 ( n-cadherin ). Cq were normalized to housekeeping gene rps11 and then normalized to kras WT condition ( D , n = 5 kras WT and 5 kras G12V replicates). Larvae were fixed at 3 dpf or 6 dpf and immunostained for N-cadherin ( E ). N-cadherin co-localization was observed in gfap :kras G12V -expressing masses at 6 dpf (yellow asterisks). Kras-expressing larvae were fixed at 3 dpf and immunostained for proliferation marker phospho-Histone H3 (pH3, F ). pH3/GFP double positive cells were quantified using confocal optical sectioning (single optical slice from yellow dashed box shown in inset) and normalized to the total gfap -expressing area (G, n = 16 larvae/condition). *p

    Techniques Used: Expressing, Transformation Assay, Imaging, Injection, Quantitative RT-PCR, Marker

    35) Product Images from "Translational Regulation of the Mitochondrial Genome Following Redistribution of Mitochondrial MicroRNA (MitomiR) in the Diabetic Heart"

    Article Title: Translational Regulation of the Mitochondrial Genome Following Redistribution of Mitochondrial MicroRNA (MitomiR) in the Diabetic Heart

    Journal: Circulation. Cardiovascular genetics

    doi: 10.1161/CIRCGENETICS.115.001067

    Crosslinked immunoprecipitation (CLIP) in cardiac mitochondrial subpopulations and MitomiR-378 RISC constituent interactions with the mitochondrial genome. (A) Western blots of biotinylated RNA from CLIP-Ago2 and CLIP-FXR1 reactions illustrating crosslinked protein/RNA and the associated gel shift from 80 kDa to 95–110 kDa. (B) Western blot analyses of CLIP-Ago2 and CLIP-FXR1 subjected to RNAase I treatment at 1:50 dilution (high RNAase) illustrating interaction between the two proteins in the absence of RNA. (C) CLIP-Ago2 and CLIP-FXR1 associated enrichment analyses of mitomiR-378 analyzed by qRT-PCR in control and diabetic cardiac mitochondrial subpopulations. Values are presented as means ± SE; n = 2 where each individual sample represents a pool of 5 individual animals. (D) CLIP-Ago2 and CLIP-FXR1 associated enrichment analysis of transcripts for mitochondrial encoded ATP6 mRNA levels as assessed by qRT-PCR analysis in control and diabetic cardiac mitochondrial subpopulations. Values are presented as means ± SE; n = 2 where each individual sample represents a pool of 5 individual animals.
    Figure Legend Snippet: Crosslinked immunoprecipitation (CLIP) in cardiac mitochondrial subpopulations and MitomiR-378 RISC constituent interactions with the mitochondrial genome. (A) Western blots of biotinylated RNA from CLIP-Ago2 and CLIP-FXR1 reactions illustrating crosslinked protein/RNA and the associated gel shift from 80 kDa to 95–110 kDa. (B) Western blot analyses of CLIP-Ago2 and CLIP-FXR1 subjected to RNAase I treatment at 1:50 dilution (high RNAase) illustrating interaction between the two proteins in the absence of RNA. (C) CLIP-Ago2 and CLIP-FXR1 associated enrichment analyses of mitomiR-378 analyzed by qRT-PCR in control and diabetic cardiac mitochondrial subpopulations. Values are presented as means ± SE; n = 2 where each individual sample represents a pool of 5 individual animals. (D) CLIP-Ago2 and CLIP-FXR1 associated enrichment analysis of transcripts for mitochondrial encoded ATP6 mRNA levels as assessed by qRT-PCR analysis in control and diabetic cardiac mitochondrial subpopulations. Values are presented as means ± SE; n = 2 where each individual sample represents a pool of 5 individual animals.

    Techniques Used: Immunoprecipitation, Cross-linking Immunoprecipitation, Western Blot, Electrophoretic Mobility Shift Assay, Quantitative RT-PCR

    Validation of miR-378 mitochondrial targeting in vitro . (A) RT-PCR analyses for miR-378 levels in isolated mitochondria from HL-1 (Control) and miR-378 cells; n = 3. (B) QRT-PCR analyses for ATP6 mRNA in isolated mitochondria from Control and miR-378 cells; n = 4. (C) Representative Western blot analyses of ATP6 and GW182 protein levels in isolated mitochondria from Control and miR-378 cells. COX IV protein expression is utilized as a loading control. (D) Quantitative analysis of ATP6 protein levels in isolated mitochondria from Control and miR-378 cells. Values are expressed per COX IV protein levels; n = 6. (e) ATP synthase activity in control HL-1 cells and in miR-378 cells; n = 4. Values are means ± SE. * P
    Figure Legend Snippet: Validation of miR-378 mitochondrial targeting in vitro . (A) RT-PCR analyses for miR-378 levels in isolated mitochondria from HL-1 (Control) and miR-378 cells; n = 3. (B) QRT-PCR analyses for ATP6 mRNA in isolated mitochondria from Control and miR-378 cells; n = 4. (C) Representative Western blot analyses of ATP6 and GW182 protein levels in isolated mitochondria from Control and miR-378 cells. COX IV protein expression is utilized as a loading control. (D) Quantitative analysis of ATP6 protein levels in isolated mitochondria from Control and miR-378 cells. Values are expressed per COX IV protein levels; n = 6. (e) ATP synthase activity in control HL-1 cells and in miR-378 cells; n = 4. Values are means ± SE. * P

    Techniques Used: In Vitro, Reverse Transcription Polymerase Chain Reaction, Isolation, Quantitative RT-PCR, Western Blot, Expressing, Activity Assay

    Relative normalized mitomiR expression patterns in cardiac mitochondrial subpopulations. (A) Hierarchical clustering heat map for the microarray analysis of mitomiR expression profiles in cardiac mitochondrial subpopulations. CS = control SSM, DS = diabetic SSM, CI = control IFM, DI = diabetic IFM; n = 4. All mitomiRs reported in the heat map are expressed as normalized intensities. (B) QRT-PCR analyses of miR-378 in control and diabetic whole heart tissue. Values are represented as mean ± SE, n = 4. U6 mRNA served as control. (C) QRT-PCR analyses of mitomiR-378 in SSM and IFM diabetic mitochondrial subpopulations as compared with control. Values are represented as mean ± SE, n = 6. U6 mRNA served as control. (D) QRT-PCR analyses for pre-miR-378 in SSM and IFM diabetic mitochondrial subpopulations as compared with control. Values are represented as mean ± SE, n = 6 for SSM and IFM. AMA mRNA served as a reference control; cytoplasm served as a positive control; no template served as a negative control.
    Figure Legend Snippet: Relative normalized mitomiR expression patterns in cardiac mitochondrial subpopulations. (A) Hierarchical clustering heat map for the microarray analysis of mitomiR expression profiles in cardiac mitochondrial subpopulations. CS = control SSM, DS = diabetic SSM, CI = control IFM, DI = diabetic IFM; n = 4. All mitomiRs reported in the heat map are expressed as normalized intensities. (B) QRT-PCR analyses of miR-378 in control and diabetic whole heart tissue. Values are represented as mean ± SE, n = 4. U6 mRNA served as control. (C) QRT-PCR analyses of mitomiR-378 in SSM and IFM diabetic mitochondrial subpopulations as compared with control. Values are represented as mean ± SE, n = 6. U6 mRNA served as control. (D) QRT-PCR analyses for pre-miR-378 in SSM and IFM diabetic mitochondrial subpopulations as compared with control. Values are represented as mean ± SE, n = 6 for SSM and IFM. AMA mRNA served as a reference control; cytoplasm served as a positive control; no template served as a negative control.

    Techniques Used: Expressing, Microarray, Quantitative RT-PCR, Positive Control, Negative Control

    MitomiR-378 and its targeting to the mitochondrial genome. (A) Schematic representation of miR-378 location in the PPARGC1b (PGC-1b) gene. (B) QRT-PCR quantification of PGC1α and PGC1β transcripts in control and diabetic hearts. GAPDH served as control. Values are represented as mean ± SE; n = 4 for each group. * P
    Figure Legend Snippet: MitomiR-378 and its targeting to the mitochondrial genome. (A) Schematic representation of miR-378 location in the PPARGC1b (PGC-1b) gene. (B) QRT-PCR quantification of PGC1α and PGC1β transcripts in control and diabetic hearts. GAPDH served as control. Values are represented as mean ± SE; n = 4 for each group. * P

    Techniques Used: Pyrolysis Gas Chromatography, Quantitative RT-PCR

    36) Product Images from "Cxcr1 mediates recruitment of neutrophils and supports proliferation of tumor-initiating astrocytes in vivo"

    Article Title: Cxcr1 mediates recruitment of neutrophils and supports proliferation of tumor-initiating astrocytes in vivo

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31675-0

    Neutrophils are recruited to the tumor-initiating microenvironment. Live-imaging of neutrophils in the hindbrain of Tg( mpx :mCherry) larvae expressing gfap :kras WT or kras G12V at 3 dpf ( A ). Neutrophils within the field of view were quantified and normalized to the total gfap -expressing area within the same field of view at 3 and 6 dpf ( B , n = 24 kras WT 3dpf, 42 kras G12V 3 dpf, 24 kras WT 6 dpf, and 30 kras G12V 6 dpf larvae). Live time-lapse imaging was performed for 9 hours beginning at 3–3.5 dpf and total neutrophils recruited to the hindbrain during the period of imaging were quantified ( C , n = 9 kras WT and 11 kras G12V larvae). qRT-PCR was performed on whole-larvae mRNA isolates for several neutrophil recruiting chemokines implicated in the tumor microenvironment including cxcl8a , cxcl8b , and il1b ( D , n = 6 replicates for cxcl8a/b , 5 replicates for il1b , normalized as in Fig. 1D ). *p
    Figure Legend Snippet: Neutrophils are recruited to the tumor-initiating microenvironment. Live-imaging of neutrophils in the hindbrain of Tg( mpx :mCherry) larvae expressing gfap :kras WT or kras G12V at 3 dpf ( A ). Neutrophils within the field of view were quantified and normalized to the total gfap -expressing area within the same field of view at 3 and 6 dpf ( B , n = 24 kras WT 3dpf, 42 kras G12V 3 dpf, 24 kras WT 6 dpf, and 30 kras G12V 6 dpf larvae). Live time-lapse imaging was performed for 9 hours beginning at 3–3.5 dpf and total neutrophils recruited to the hindbrain during the period of imaging were quantified ( C , n = 9 kras WT and 11 kras G12V larvae). qRT-PCR was performed on whole-larvae mRNA isolates for several neutrophil recruiting chemokines implicated in the tumor microenvironment including cxcl8a , cxcl8b , and il1b ( D , n = 6 replicates for cxcl8a/b , 5 replicates for il1b , normalized as in Fig. 1D ). *p

    Techniques Used: Imaging, Expressing, Quantitative RT-PCR

    Expression of oncogenic Kras from an astrocyte-specific promoter drives transformation in the larval hindbrain. Live-imaging of mosaic gfap :GFP, gfap :GFP-kras WT , and gfap :GFP-kras G12V expression at 3 days post-fertilization (dpf) in the larval zebrafish hindbrain ( A ). Red dashed outline in schematic of A shows approximate field of view and image orientation for all figures. White dashed lines in representative images denote the hindbrain region of interest in all figures. Morphology of expressing cells was categorized based on characteristics observed throughout the population of injected larvae and larvae were assigned to category of most severe cell morphology identified ( B , n = 19 gfap :GFP, 17 gfap :kras WT , and 38 gfap :kras G12V larvae; Chi-squared analysis performed). gfap :H2B-mCherry was mosaically co-expressed with gfap :kras plasmids to label nuclei of Kras-expressing cells ( C ). qRT-PCR was performed on whole-larvae isolates for EMT genes including vimentin , mmp9 , cdh1 ( e-cadherin ), and cdh2 ( n-cadherin ). Cq were normalized to housekeeping gene rps11 and then normalized to kras WT condition ( D , n = 5 kras WT and 5 kras G12V replicates). Larvae were fixed at 3 dpf or 6 dpf and immunostained for N-cadherin ( E ). N-cadherin co-localization was observed in gfap :kras G12V -expressing masses at 6 dpf (yellow asterisks). Kras-expressing larvae were fixed at 3 dpf and immunostained for proliferation marker phospho-Histone H3 (pH3, F ). pH3/GFP double positive cells were quantified using confocal optical sectioning (single optical slice from yellow dashed box shown in inset) and normalized to the total gfap -expressing area (G, n = 16 larvae/condition). *p
    Figure Legend Snippet: Expression of oncogenic Kras from an astrocyte-specific promoter drives transformation in the larval hindbrain. Live-imaging of mosaic gfap :GFP, gfap :GFP-kras WT , and gfap :GFP-kras G12V expression at 3 days post-fertilization (dpf) in the larval zebrafish hindbrain ( A ). Red dashed outline in schematic of A shows approximate field of view and image orientation for all figures. White dashed lines in representative images denote the hindbrain region of interest in all figures. Morphology of expressing cells was categorized based on characteristics observed throughout the population of injected larvae and larvae were assigned to category of most severe cell morphology identified ( B , n = 19 gfap :GFP, 17 gfap :kras WT , and 38 gfap :kras G12V larvae; Chi-squared analysis performed). gfap :H2B-mCherry was mosaically co-expressed with gfap :kras plasmids to label nuclei of Kras-expressing cells ( C ). qRT-PCR was performed on whole-larvae isolates for EMT genes including vimentin , mmp9 , cdh1 ( e-cadherin ), and cdh2 ( n-cadherin ). Cq were normalized to housekeeping gene rps11 and then normalized to kras WT condition ( D , n = 5 kras WT and 5 kras G12V replicates). Larvae were fixed at 3 dpf or 6 dpf and immunostained for N-cadherin ( E ). N-cadherin co-localization was observed in gfap :kras G12V -expressing masses at 6 dpf (yellow asterisks). Kras-expressing larvae were fixed at 3 dpf and immunostained for proliferation marker phospho-Histone H3 (pH3, F ). pH3/GFP double positive cells were quantified using confocal optical sectioning (single optical slice from yellow dashed box shown in inset) and normalized to the total gfap -expressing area (G, n = 16 larvae/condition). *p

    Techniques Used: Expressing, Transformation Assay, Imaging, Injection, Quantitative RT-PCR, Marker

    37) Product Images from "Sustained Expression of Negative Regulators of Myelination Protects Schwann Cells from Dysmyelination in a Charcot–Marie–Tooth 1B Mouse Model"

    Article Title: Sustained Expression of Negative Regulators of Myelination Protects Schwann Cells from Dysmyelination in a Charcot–Marie–Tooth 1B Mouse Model

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0201-18.2018

    Ablation of Sox2 and Id2 increases P0 expression and exacerbates the UPR in S63del nerves. A , qRT-PCR for total P0 mRNA on P18–P21 sciatic nerves from WT control, S63del, and S63del/Sox2 SCKO /Id2 −/− mice. B – D , Allelic discrimination assay on RNA extracts from sciatic nerve of control, S63del, and S63del/Sox2 SCKO /Id2 −/− mice. Only the WT transcript is amplified in WT control extracts; S63del and S63del/Sox2 SCKO /Id2 −/− show comparable relative amounts of both WT and S63del transcript. E , qRT-PCR for CHOP, Bip, and spliced Xbp1. All analyzed UPR factors are strongly upregulated in S63del/Sox2 SCKO /Id2 −/− compared with S63del, suggesting exacerbation of the ER stress. n = 2 RT from independent pools of three sciatic nerves each.
    Figure Legend Snippet: Ablation of Sox2 and Id2 increases P0 expression and exacerbates the UPR in S63del nerves. A , qRT-PCR for total P0 mRNA on P18–P21 sciatic nerves from WT control, S63del, and S63del/Sox2 SCKO /Id2 −/− mice. B – D , Allelic discrimination assay on RNA extracts from sciatic nerve of control, S63del, and S63del/Sox2 SCKO /Id2 −/− mice. Only the WT transcript is amplified in WT control extracts; S63del and S63del/Sox2 SCKO /Id2 −/− show comparable relative amounts of both WT and S63del transcript. E , qRT-PCR for CHOP, Bip, and spliced Xbp1. All analyzed UPR factors are strongly upregulated in S63del/Sox2 SCKO /Id2 −/− compared with S63del, suggesting exacerbation of the ER stress. n = 2 RT from independent pools of three sciatic nerves each.

    Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay, Amplification

    S 63del nerves showing altered differentiation state. A , qRT-PCR analysis on P5 (top) and P28 (bottom) S63del and WT sciatic nerves to validate some of the early Schwann cell differentiation factors. S63del values are expressed as fold change compared with WT. Error bars indicate SEM; n = 3–5 RT from independent pools of nerves. * p
    Figure Legend Snippet: S 63del nerves showing altered differentiation state. A , qRT-PCR analysis on P5 (top) and P28 (bottom) S63del and WT sciatic nerves to validate some of the early Schwann cell differentiation factors. S63del values are expressed as fold change compared with WT. Error bars indicate SEM; n = 3–5 RT from independent pools of nerves. * p

    Techniques Used: Quantitative RT-PCR, Cell Differentiation

    38) Product Images from "MicroRNA Regulation of IFN? protein expression: Rapid and sensitive modulation of the Innate Immune Response 1"

    Article Title: MicroRNA Regulation of IFN? protein expression: Rapid and sensitive modulation of the Innate Immune Response 1

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

    doi: 10.4049/jimmunol.0902712

    IFNβ treatment of primary macrophages modulates three of four IFNβ-regulating miRNAs Primary macrophages from two pigtail macaque donors were treated with 100U/ml recombinant IFNβ, with RNA collected at 2, 8, and 24 hr post-treatment. miRNA levels for miRNAs -26a, -145, -34a, and let-7b were measured on all samples in triplicate by stem-loop qRT-PCR, including no reverse transcriptase and no template controls. The results were analyzed by ΔΔCt, with normalization to U6 snRNA levels and untreated controls. Error bars are standard deviation.
    Figure Legend Snippet: IFNβ treatment of primary macrophages modulates three of four IFNβ-regulating miRNAs Primary macrophages from two pigtail macaque donors were treated with 100U/ml recombinant IFNβ, with RNA collected at 2, 8, and 24 hr post-treatment. miRNA levels for miRNAs -26a, -145, -34a, and let-7b were measured on all samples in triplicate by stem-loop qRT-PCR, including no reverse transcriptase and no template controls. The results were analyzed by ΔΔCt, with normalization to U6 snRNA levels and untreated controls. Error bars are standard deviation.

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

    poly I:C treatment of macrophages induces IFNβ and miRNA production consistent with IFNβ-mediated miR upregulation Primary human macrophages were treated with 50 ug/ml poly I:C. IFNβ response (A) was detectable by ELISA by three hours and increased through 24 hours post-treatment. Limit of detection (about 1 IU/ml) is shown as a line. Results are from two donors, measured in duplicate. miRs -26a, -34a, and let-7b were quantitated by stem-loop qRT-PCR (B). Results are fold change of treated over untreated macrophages at each time point, normalized to U6 snRNA. Error bars indicate standard deviation.
    Figure Legend Snippet: poly I:C treatment of macrophages induces IFNβ and miRNA production consistent with IFNβ-mediated miR upregulation Primary human macrophages were treated with 50 ug/ml poly I:C. IFNβ response (A) was detectable by ELISA by three hours and increased through 24 hours post-treatment. Limit of detection (about 1 IU/ml) is shown as a line. Results are from two donors, measured in duplicate. miRs -26a, -34a, and let-7b were quantitated by stem-loop qRT-PCR (B). Results are fold change of treated over untreated macrophages at each time point, normalized to U6 snRNA. Error bars indicate standard deviation.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Standard Deviation

    39) Product Images from "An Argonaute phosphorylation cycle promotes microRNA-mediated silencing"

    Article Title: An Argonaute phosphorylation cycle promotes microRNA-mediated silencing

    Journal: Nature

    doi: 10.1038/nature21025

    Functional characterization of CSNK1A1 and AGO2 target binding mutants a, Western blot analysis confirms loss of CSNK1A1 expression in HCT116 ANKRD52 −/− ; CSNK1A1 −/− clonal knockout cells. All lanes came from the same blot but irrelevant lanes were removed. b, miR-19 expression normalized to U6 expression, assessed by qRT-PCR, in cells of the indicated genotypes ( N = 4 biological replicates, each assayed in triplicate). c, Co-immunoprecipitation of V5-CSNK1A1 with FH-AGO2, with or without RNase A treatment. d, miRNA association of FH-AGO2 assessed as in Fig. 3e ( N = 4 biological replicates, each assayed in triplicate). *p
    Figure Legend Snippet: Functional characterization of CSNK1A1 and AGO2 target binding mutants a, Western blot analysis confirms loss of CSNK1A1 expression in HCT116 ANKRD52 −/− ; CSNK1A1 −/− clonal knockout cells. All lanes came from the same blot but irrelevant lanes were removed. b, miR-19 expression normalized to U6 expression, assessed by qRT-PCR, in cells of the indicated genotypes ( N = 4 biological replicates, each assayed in triplicate). c, Co-immunoprecipitation of V5-CSNK1A1 with FH-AGO2, with or without RNase A treatment. d, miRNA association of FH-AGO2 assessed as in Fig. 3e ( N = 4 biological replicates, each assayed in triplicate). *p

    Techniques Used: Functional Assay, Binding Assay, Western Blot, Expressing, Knock-Out, Quantitative RT-PCR, Immunoprecipitation

    AGO2 phosphorylation impairs mRNA target association a, Measurement of AGO2-associated miRNA by qRT-PCR. N = 2 biological replicates each assayed in triplicate. Error bars indicate SD for this and all subsequent qRT-PCR data. b, AGO2-target association assessed as described in ( a ). *p
    Figure Legend Snippet: AGO2 phosphorylation impairs mRNA target association a, Measurement of AGO2-associated miRNA by qRT-PCR. N = 2 biological replicates each assayed in triplicate. Error bars indicate SD for this and all subsequent qRT-PCR data. b, AGO2-target association assessed as described in ( a ). *p

    Techniques Used: Quantitative RT-PCR

    BRD4 , CTNNB1 , and POU2F1 positively regulate miR-19 expression a, Model depicting how each gene may promote expression of the miR-17-92 cluster. b, c, d, Western blot analysis confirming loss of expression of the indicated gene in HCT116 knockout clones. Asterisk indicates non-specific band. For each protein, all lanes came from the same blot but irrelevant lanes were removed. e, f, g, qRT-PCR assays demonstrating reduced expression of MYC ( e ), pri-miR-17-92 ( f ), or mature miR-19a/b ( g ) in BRD4 −/− , CTNNB1 −/− , or POU2F1 −/− cells. For gel source data, see Supplementary Figure 1 .
    Figure Legend Snippet: BRD4 , CTNNB1 , and POU2F1 positively regulate miR-19 expression a, Model depicting how each gene may promote expression of the miR-17-92 cluster. b, c, d, Western blot analysis confirming loss of expression of the indicated gene in HCT116 knockout clones. Asterisk indicates non-specific band. For each protein, all lanes came from the same blot but irrelevant lanes were removed. e, f, g, qRT-PCR assays demonstrating reduced expression of MYC ( e ), pri-miR-17-92 ( f ), or mature miR-19a/b ( g ) in BRD4 −/− , CTNNB1 −/− , or POU2F1 −/− cells. For gel source data, see Supplementary Figure 1 .

    Techniques Used: Expressing, Western Blot, Knock-Out, Clone Assay, Quantitative RT-PCR

    General impairment of miRNA-mediated silencing in ANKRD52 −/− cells a, b, Flow cytometry analysis of EGFP expression in HCT116 cells stably expressing reporters for miR-16 ( a ) or miR-200 ( b ) after transduction with lentiCRISPR vectors targeting ANKRD52 or expressing a non-targeting sgRNA. c, qRT-PCR showing de-repression of established let-7 targets ( DICER1 or HMGA2 ) in AGO2 −/− or ANKRD52 −/− cells. *p
    Figure Legend Snippet: General impairment of miRNA-mediated silencing in ANKRD52 −/− cells a, b, Flow cytometry analysis of EGFP expression in HCT116 cells stably expressing reporters for miR-16 ( a ) or miR-200 ( b ) after transduction with lentiCRISPR vectors targeting ANKRD52 or expressing a non-targeting sgRNA. c, qRT-PCR showing de-repression of established let-7 targets ( DICER1 or HMGA2 ) in AGO2 −/− or ANKRD52 −/− cells. *p

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Stable Transfection, Transduction, Quantitative RT-PCR

    40) Product Images from "Molecular characterization and expression analysis of pitaya (Hylocereus polyrhizus) HpLRR genes in response to Neoscytalidium dimidiatum infection"

    Article Title: Molecular characterization and expression analysis of pitaya (Hylocereus polyrhizus) HpLRR genes in response to Neoscytalidium dimidiatum infection

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-020-02368-6

    Expression profiles of four Hp LRR genes in different pitaya tissues. The X-axis represents different pitaya tissues and the Y-axis represents the fold change (2 -ΔΔCт ) according to qRT-PCR. Annotations: CL445.contig4_All and Unigene2712_All: NBS-LRR; Unigene28_All and CL28.contig2_All: LRR-STK. LRR: leucine-rich repeat; NBS: nucleotide-binding site; STK: serine/threonine-protein kinase
    Figure Legend Snippet: Expression profiles of four Hp LRR genes in different pitaya tissues. The X-axis represents different pitaya tissues and the Y-axis represents the fold change (2 -ΔΔCт ) according to qRT-PCR. Annotations: CL445.contig4_All and Unigene2712_All: NBS-LRR; Unigene28_All and CL28.contig2_All: LRR-STK. LRR: leucine-rich repeat; NBS: nucleotide-binding site; STK: serine/threonine-protein kinase

    Techniques Used: Expressing, Quantitative RT-PCR, Binding Assay

    Verification of 12 differentially expressed LRR genes (DEGs) by qRT-PCR assays. Gene expression levels were measured by qRT-PCR and compared with RNA-Seq results. The histogram represents the fold changes of genes (diseased [D]/normal [N]) according to qRT-PCR, and the line chart represents gene expression according to RNA-Seq. All genes selected for qRT-PCR analysis were analyzed using three biological replicates. Error bars represent ±SD (2 -ΔΔCт ) based on three experiments
    Figure Legend Snippet: Verification of 12 differentially expressed LRR genes (DEGs) by qRT-PCR assays. Gene expression levels were measured by qRT-PCR and compared with RNA-Seq results. The histogram represents the fold changes of genes (diseased [D]/normal [N]) according to qRT-PCR, and the line chart represents gene expression according to RNA-Seq. All genes selected for qRT-PCR analysis were analyzed using three biological replicates. Error bars represent ±SD (2 -ΔΔCт ) based on three experiments

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

    Expression profiles of four Hp LRR transcriptional genes at different stages and different pitaya species of N. dimidiatum infection. ( a ): Hp LRR genesexpression profiles in red-fleshed pitaya ( Hylocereus polyrhizus ) infected by N. dimidiatum ; ( b ): Hp LRR genes expression profiles in white-fleshed pitaya( Hylocereus undatus ) infected by N. dimidiatum . The X-axis represents different stages of N. dimidiatum infection and the Y-axis represents the foldchange (2 -ΔΔCт ) according to qRT-PCR. Annotations: CL445.Contig4_All and Unigene2712_All: NBS-LRR; Unigene28_All and CL28.Contig2_All: LRRSTK. LRR: leucine-rich repeat; NBS: nucleotide-binding site; STK: serine/threonine-protein kinase
    Figure Legend Snippet: Expression profiles of four Hp LRR transcriptional genes at different stages and different pitaya species of N. dimidiatum infection. ( a ): Hp LRR genesexpression profiles in red-fleshed pitaya ( Hylocereus polyrhizus ) infected by N. dimidiatum ; ( b ): Hp LRR genes expression profiles in white-fleshed pitaya( Hylocereus undatus ) infected by N. dimidiatum . The X-axis represents different stages of N. dimidiatum infection and the Y-axis represents the foldchange (2 -ΔΔCт ) according to qRT-PCR. Annotations: CL445.Contig4_All and Unigene2712_All: NBS-LRR; Unigene28_All and CL28.Contig2_All: LRRSTK. LRR: leucine-rich repeat; NBS: nucleotide-binding site; STK: serine/threonine-protein kinase

    Techniques Used: Expressing, Infection, Quantitative RT-PCR, Binding Assay

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    Amplification:

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    Quantitative RT-PCR:

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    SYBR Green Assay:

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    Thermo Fisher quan titative reverse transcriptase polymerase chain reaction qrt pcr
    miR-138 up regulation leads to hTERT suppression in NB4 cells. A: total RNAs from NB4 cells that were transduced with GFP hsa-miR-138-expressing lentiviruses were extracted at 96 h after removal of the lentivirus-containing medium. hTERT mRNA expression was measured by <t>qRT-PCR.</t> Values were normalized to GAPDH . (n=3; *P
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    MARF1 binds the CCR4-NOT deadenylase complex and cyclin A mRNA. a Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1) and those from w 1118 negative control were tested. b Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-tagged MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1 full-length or fragments) and those from w 1118 negative control were tested. Black triangles indicate the detected 3xHA-tagged MARF1 proteins. c Co-immunoprecipitation using ovary lysates from w 1118 and anti-Not1 antibody or negative control IgG-bound protein G beads followed by Western blotting. d Fold enrichment of mRNAs relative to a control gapdh mRNA that were co-immunoprecipitated with MARF1 by anti-HA antibody-conjugated beads and were eluted from the beads normalized by w 1118 negative control, determined by <t>RNA-immunoprecipitation</t> followed by <t>qRT-PCR.</t> Mean ± SD ( n = 3). P -value
    Qrt Pcr Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative real time reverse transcription polymerase chain reaction qrt pcr
    Knockdown or suppression of p65 reduces GLS1 expression in HCC cells. ( A ) Western blot shows the knockdown of c-Myc without interfering with GLS1 expression in HepG2 and Hep3B cells. ( B ) Western blot shows that p65 knockdown reduces GLS1 protein levels in both HepG2 and Hep3B cells. ( C ) <t>qRT-PCR</t> indicates that p65 knockdown reduces GLS1 mRNA levels in both HepG2 and Hep3B cells. ** p
    Quantitative Real Time Reverse Transcription Polymerase Chain Reaction Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative real time pcr qrt pcr
    LINC01296 knockdown suppressed the migration of bladder cancer cells. Notes: ( A , B ) Transwell assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( C , D ) Wound healing assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( E ) <t>qRT-</t> <t>PCR</t> analyses were used to measure mRNA levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. ( F ) Western blot assays were used to measure protein levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. Data are presented as mean ± SD of three independent experiments. *P
    Quantitative Real Time Pcr Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1655 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    miR-138 up regulation leads to hTERT suppression in NB4 cells. A: total RNAs from NB4 cells that were transduced with GFP hsa-miR-138-expressing lentiviruses were extracted at 96 h after removal of the lentivirus-containing medium. hTERT mRNA expression was measured by qRT-PCR. Values were normalized to GAPDH . (n=3; *P

    Journal: International Journal of Molecular and Cellular Medicine

    Article Title: Overexpression of MiR-138 Inhibits Cell Growth and Induces Caspase-mediated Apoptosis in Acute Promyelocytic Leukemia Cell Line

    doi: 10.22088/IJMCM.BUMS.7.1.24

    Figure Lengend Snippet: miR-138 up regulation leads to hTERT suppression in NB4 cells. A: total RNAs from NB4 cells that were transduced with GFP hsa-miR-138-expressing lentiviruses were extracted at 96 h after removal of the lentivirus-containing medium. hTERT mRNA expression was measured by qRT-PCR. Values were normalized to GAPDH . (n=3; *P

    Article Snippet: The prepared cDNA was subjected to quan-titative reverse-transcriptase polymerase chain reaction (qRT-PCR), using Maxima SYBR Green Master mix (Thermo Scientific, Waltham, Massa-chusetts, USA) in the Rotor Gene 6000 Real Time PCR inst rument (Corbett Research, Hilden, Germany).

    Techniques: Transduction, Expressing, Quantitative RT-PCR

    MARF1 binds the CCR4-NOT deadenylase complex and cyclin A mRNA. a Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1) and those from w 1118 negative control were tested. b Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-tagged MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1 full-length or fragments) and those from w 1118 negative control were tested. Black triangles indicate the detected 3xHA-tagged MARF1 proteins. c Co-immunoprecipitation using ovary lysates from w 1118 and anti-Not1 antibody or negative control IgG-bound protein G beads followed by Western blotting. d Fold enrichment of mRNAs relative to a control gapdh mRNA that were co-immunoprecipitated with MARF1 by anti-HA antibody-conjugated beads and were eluted from the beads normalized by w 1118 negative control, determined by RNA-immunoprecipitation followed by qRT-PCR. Mean ± SD ( n = 3). P -value

    Journal: Nature Communications

    Article Title: LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes

    doi: 10.1038/s41467-018-06404-w

    Figure Lengend Snippet: MARF1 binds the CCR4-NOT deadenylase complex and cyclin A mRNA. a Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1) and those from w 1118 negative control were tested. b Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-tagged MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1 full-length or fragments) and those from w 1118 negative control were tested. Black triangles indicate the detected 3xHA-tagged MARF1 proteins. c Co-immunoprecipitation using ovary lysates from w 1118 and anti-Not1 antibody or negative control IgG-bound protein G beads followed by Western blotting. d Fold enrichment of mRNAs relative to a control gapdh mRNA that were co-immunoprecipitated with MARF1 by anti-HA antibody-conjugated beads and were eluted from the beads normalized by w 1118 negative control, determined by RNA-immunoprecipitation followed by qRT-PCR. Mean ± SD ( n = 3). P -value

    Article Snippet: qRT-PCR RNA from oocytes was prepared using miRVana (Thermo Fisher Scientific).

    Techniques: Immunoprecipitation, Western Blot, Expressing, Negative Control, Quantitative RT-PCR

    Tethered MARF1 shortens reporter mRNA poly-A tail and reduces reporter protein level. a GFP-5x BoxB reporter structure, harboring a ubiquitous tubulin promoter, GFP-coding sequence, and a 3′ UTR containing five BoxB hairpins. LambdaN-HA-fused control peptide, MARF1, GW182, and Piwi under a UASP promoter were expressed in germline cells using maternal-alpha-tubulin-Gal4 driver. b Confocal images of GFP signal in stage 14 oocytes. Scale bar is 100 μm. c Western blots using stage 14 oocyte lysates. Black triangles indicate the transgenic lambda-HA-fused proteins. d Quantification of band intensities in c . e ePAT assay measuring GFP reporter mRNA poly-A tail length in stage 14 oocytes. The amplified DNA sizes were analyzed on an agarose gel. f ePAT assay measuring poly-A tail lengths of GFP reporter mRNA and a negative control cdk1 mRNA in stage 14 oocytes. Amplified DNA sizes were analyzed by capillary fragment analysis. g Relative abundance of GFP-5xBoxB mRNA normalized by gapdh mRNA determined by qRT-PCR. Mean ± SD ( n = 6 for the control and n = 4 for all the others)

    Journal: Nature Communications

    Article Title: LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes

    doi: 10.1038/s41467-018-06404-w

    Figure Lengend Snippet: Tethered MARF1 shortens reporter mRNA poly-A tail and reduces reporter protein level. a GFP-5x BoxB reporter structure, harboring a ubiquitous tubulin promoter, GFP-coding sequence, and a 3′ UTR containing five BoxB hairpins. LambdaN-HA-fused control peptide, MARF1, GW182, and Piwi under a UASP promoter were expressed in germline cells using maternal-alpha-tubulin-Gal4 driver. b Confocal images of GFP signal in stage 14 oocytes. Scale bar is 100 μm. c Western blots using stage 14 oocyte lysates. Black triangles indicate the transgenic lambda-HA-fused proteins. d Quantification of band intensities in c . e ePAT assay measuring GFP reporter mRNA poly-A tail length in stage 14 oocytes. The amplified DNA sizes were analyzed on an agarose gel. f ePAT assay measuring poly-A tail lengths of GFP reporter mRNA and a negative control cdk1 mRNA in stage 14 oocytes. Amplified DNA sizes were analyzed by capillary fragment analysis. g Relative abundance of GFP-5xBoxB mRNA normalized by gapdh mRNA determined by qRT-PCR. Mean ± SD ( n = 6 for the control and n = 4 for all the others)

    Article Snippet: qRT-PCR RNA from oocytes was prepared using miRVana (Thermo Fisher Scientific).

    Techniques: Sequencing, Western Blot, Transgenic Assay, Amplification, Agarose Gel Electrophoresis, Negative Control, Quantitative RT-PCR

    Knockdown or suppression of p65 reduces GLS1 expression in HCC cells. ( A ) Western blot shows the knockdown of c-Myc without interfering with GLS1 expression in HepG2 and Hep3B cells. ( B ) Western blot shows that p65 knockdown reduces GLS1 protein levels in both HepG2 and Hep3B cells. ( C ) qRT-PCR indicates that p65 knockdown reduces GLS1 mRNA levels in both HepG2 and Hep3B cells. ** p

    Journal: OncoTargets and therapy

    Article Title: Nuclear factor-κB p65 regulates glutaminase 1 expression in human hepatocellular carcinoma

    doi: 10.2147/OTT.S167408

    Figure Lengend Snippet: Knockdown or suppression of p65 reduces GLS1 expression in HCC cells. ( A ) Western blot shows the knockdown of c-Myc without interfering with GLS1 expression in HepG2 and Hep3B cells. ( B ) Western blot shows that p65 knockdown reduces GLS1 protein levels in both HepG2 and Hep3B cells. ( C ) qRT-PCR indicates that p65 knockdown reduces GLS1 mRNA levels in both HepG2 and Hep3B cells. ** p

    Article Snippet: Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) Upon relative treatment, cells were lysed, and total RNA was isolated using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Expressing, Western Blot, Quantitative RT-PCR

    LINC01296 knockdown suppressed the migration of bladder cancer cells. Notes: ( A , B ) Transwell assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( C , D ) Wound healing assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( E ) qRT- PCR analyses were used to measure mRNA levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. ( F ) Western blot assays were used to measure protein levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. Data are presented as mean ± SD of three independent experiments. *P

    Journal: OncoTargets and therapy

    Article Title: Long noncoding RNA LINC01296 promotes cancer-cell proliferation and metastasis in urothelial carcinoma of the bladder

    doi: 10.2147/OTT.S192809

    Figure Lengend Snippet: LINC01296 knockdown suppressed the migration of bladder cancer cells. Notes: ( A , B ) Transwell assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( C , D ) Wound healing assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( E ) qRT- PCR analyses were used to measure mRNA levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. ( F ) Western blot assays were used to measure protein levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. Data are presented as mean ± SD of three independent experiments. *P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was performed on an ABI 7500 (Thermo Fisher Scientific) instrument using a KAPA SYBR Green FAST qPCR Kit (KAPA, Wilmington, MA, US) according to the manufacturer’s instructions.

    Techniques: Migration, Transfection, Quantitative RT-PCR, Western Blot

    LINC01296 knockdown inhibited the proliferation of bladder cancer cells. Notes: ( A ) Relative LINC01296 expressions in human bladder cancer cell lines (RT4, T24, and 5637). ( B , C ) MTT assays were performed to measure the proliferation viability of T24 and 5637 cells after transfection. ( D , E ) qRT-PCR results of LINC01296 expressions following the treatment of T24 and 5637 cells with anti-LINC01296 siRNA. ( F , G ) Colony formation assays were used to measure the number of clone formation of bladder cancer cells after transfection. ( H , I ) Flow cytometry assays were performed to analyze the cell cycle progression of T24 and 5637 cells after they were transfected with anti-LINC01296 siRNA. Data are presented as mean ± SD of three independent experiments. *P

    Journal: OncoTargets and therapy

    Article Title: Long noncoding RNA LINC01296 promotes cancer-cell proliferation and metastasis in urothelial carcinoma of the bladder

    doi: 10.2147/OTT.S192809

    Figure Lengend Snippet: LINC01296 knockdown inhibited the proliferation of bladder cancer cells. Notes: ( A ) Relative LINC01296 expressions in human bladder cancer cell lines (RT4, T24, and 5637). ( B , C ) MTT assays were performed to measure the proliferation viability of T24 and 5637 cells after transfection. ( D , E ) qRT-PCR results of LINC01296 expressions following the treatment of T24 and 5637 cells with anti-LINC01296 siRNA. ( F , G ) Colony formation assays were used to measure the number of clone formation of bladder cancer cells after transfection. ( H , I ) Flow cytometry assays were performed to analyze the cell cycle progression of T24 and 5637 cells after they were transfected with anti-LINC01296 siRNA. Data are presented as mean ± SD of three independent experiments. *P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was performed on an ABI 7500 (Thermo Fisher Scientific) instrument using a KAPA SYBR Green FAST qPCR Kit (KAPA, Wilmington, MA, US) according to the manufacturer’s instructions.

    Techniques: MTT Assay, Transfection, Quantitative RT-PCR, Flow Cytometry, Cytometry

    LINC01296 was upregulated in bladder cancer and the expression level of LINC01296 was correlated with clinicopathological features of bladder cancer patients. Notes: ( A ) Comparison of relative expressions of LINC01296 in cancer and normal tissues using the GEPIA database. ( B ) Comparison of relative expressions of LINC01296 in bladder cancer and adjacent normal tissues using the GEPIA database. ( C ) A microarray containing 37,000 lncRNA and 34,000 mRNA probes was used to screen differentially expressed lncRNAs in four pairs of bladder cancer patients. ( D ) Comparison of relative expressions of LINC01296 in 54 bladder cancer tissues and 24 normal bladder tissues using qRT-PCR. ( E ) Two groups were set up according to the mean expression of LINC01296 in tumor tissues. ( F ) The LINC01296 expression was significantly higher in patients with a higher tumor stage, lymph node metastasis and a higher pathologic grade. * P

    Journal: OncoTargets and therapy

    Article Title: Long noncoding RNA LINC01296 promotes cancer-cell proliferation and metastasis in urothelial carcinoma of the bladder

    doi: 10.2147/OTT.S192809

    Figure Lengend Snippet: LINC01296 was upregulated in bladder cancer and the expression level of LINC01296 was correlated with clinicopathological features of bladder cancer patients. Notes: ( A ) Comparison of relative expressions of LINC01296 in cancer and normal tissues using the GEPIA database. ( B ) Comparison of relative expressions of LINC01296 in bladder cancer and adjacent normal tissues using the GEPIA database. ( C ) A microarray containing 37,000 lncRNA and 34,000 mRNA probes was used to screen differentially expressed lncRNAs in four pairs of bladder cancer patients. ( D ) Comparison of relative expressions of LINC01296 in 54 bladder cancer tissues and 24 normal bladder tissues using qRT-PCR. ( E ) Two groups were set up according to the mean expression of LINC01296 in tumor tissues. ( F ) The LINC01296 expression was significantly higher in patients with a higher tumor stage, lymph node metastasis and a higher pathologic grade. * P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was performed on an ABI 7500 (Thermo Fisher Scientific) instrument using a KAPA SYBR Green FAST qPCR Kit (KAPA, Wilmington, MA, US) according to the manufacturer’s instructions.

    Techniques: Expressing, Microarray, Quantitative RT-PCR