qrt pcr assays  (Thermo Fisher)


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    Thermo Fisher qrt pcr assays
    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 <t>(qRT-PCR).</t> ( 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
    Qrt Pcr Assays, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 320 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) 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

    2) 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

    3) 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

    4) 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

    5) 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

    6) 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

    7) 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

    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 "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

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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

    15) 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

    16) 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

    17) 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

    18) 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

    19) 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

    20) 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

    21) 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

    22) 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

    23) 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

    24) Product Images from "One-step Quantitative RT-PCR Assays for Detecting, Genotyping and Differentiating Wild-Type Group a Rotaviruses and Vaccine (Rotarix® and RotaTeq®) Strains in Stool Samples"

    Article Title: One-step Quantitative RT-PCR Assays for Detecting, Genotyping and Differentiating Wild-Type Group a Rotaviruses and Vaccine (Rotarix® and RotaTeq®) Strains in Stool Samples

    Journal: Journal of vaccines & vaccination

    doi: 10.4172/2157-7560.1000341

    Flow chart showing qRT-PCR assays developed for RVA detection (NSP3 assay), vaccine strain detection in singleplex format (Rotarix ® NSP2-primary assay, Rotarix ® VP4-secondary assay; RotaTeq ® VP6- primary assay, RotaTeq ®
    Figure Legend Snippet: Flow chart showing qRT-PCR assays developed for RVA detection (NSP3 assay), vaccine strain detection in singleplex format (Rotarix ® NSP2-primary assay, Rotarix ® VP4-secondary assay; RotaTeq ® VP6- primary assay, RotaTeq ®

    Techniques Used: Flow Cytometry, Quantitative RT-PCR

    25) Product Images from "Two members of the velvet family, VmVeA and VmVelB, affect conidiation, virulence and pectinase expression in Valsa mali"

    Article Title: Two members of the velvet family, VmVeA and VmVelB, affect conidiation, virulence and pectinase expression in Valsa mali

    Journal: Molecular Plant Pathology

    doi: 10.1111/mpp.12645

    Transcript levels of pectinase, hemi‐cellulase, cellulase and ligninase genes determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) in the wild‐type (WT), VmVeA and VmVelB deletion mutants. RNA samples were isolated from the border of Valsa mali ‐colonized apple tree bark at 3 days post‐inoculation (dpi), and transcript levels of pectinase, hemi‐cellulase, cellulase and ligninase genes in the wild‐type, VmVeA and VmVelB deletion mutants were quantified by qRT‐PCR. The transcript level of the glyceraldehyde‐6‐phosphate dehydrogenase gene ( G6PDH ) was used to normalize different samples. Transcript levels of the wild‐type were set to unity. The mean and standard deviation were calculated with data from three independent biological replicates. Data from three replicates were analysed with Student's t ‐test. Asterisks represent a significant difference in transcript levels ( P
    Figure Legend Snippet: Transcript levels of pectinase, hemi‐cellulase, cellulase and ligninase genes determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) in the wild‐type (WT), VmVeA and VmVelB deletion mutants. RNA samples were isolated from the border of Valsa mali ‐colonized apple tree bark at 3 days post‐inoculation (dpi), and transcript levels of pectinase, hemi‐cellulase, cellulase and ligninase genes in the wild‐type, VmVeA and VmVelB deletion mutants were quantified by qRT‐PCR. The transcript level of the glyceraldehyde‐6‐phosphate dehydrogenase gene ( G6PDH ) was used to normalize different samples. Transcript levels of the wild‐type were set to unity. The mean and standard deviation were calculated with data from three independent biological replicates. Data from three replicates were analysed with Student's t ‐test. Asterisks represent a significant difference in transcript levels ( P

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

    26) Product Images from "MicroRNA-410 Suppresses Migration and Invasion by Targeting MDM2 in Gastric Cancer"

    Article Title: MicroRNA-410 Suppresses Migration and Invasion by Targeting MDM2 in Gastric Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0104510

    miR-410 negatively regulated MDM2 gene expression. (A) The relative MDM2 mRNA expression levels were determined by qRT-PCR in four cell lines derived from gastric cancer and one nonmalignant gastric cell line (GES). The expression of MDM2 was normalized to GAPDH. (B) Western blot analysis of MDM2 expression in four cell lines derived from gastric cancer and one nonmalignant gastric cell line (GES). GAPDH was also detected as a loading control. (C) qRT-PCR analysis of MDM2 expression in 40 pairs gastric cancer tissues and their corresponding adjacent normal tissues. The expression of MDM2 was normalized to GAPDH. The expression of MDM2 in gastric cancer tissues was significant higher than in the corresponding adjacent normal tissues. (D) Analysis of correlation of miR-410 and MDM2 expression in gastric cancer tissues. *p
    Figure Legend Snippet: miR-410 negatively regulated MDM2 gene expression. (A) The relative MDM2 mRNA expression levels were determined by qRT-PCR in four cell lines derived from gastric cancer and one nonmalignant gastric cell line (GES). The expression of MDM2 was normalized to GAPDH. (B) Western blot analysis of MDM2 expression in four cell lines derived from gastric cancer and one nonmalignant gastric cell line (GES). GAPDH was also detected as a loading control. (C) qRT-PCR analysis of MDM2 expression in 40 pairs gastric cancer tissues and their corresponding adjacent normal tissues. The expression of MDM2 was normalized to GAPDH. The expression of MDM2 in gastric cancer tissues was significant higher than in the corresponding adjacent normal tissues. (D) Analysis of correlation of miR-410 and MDM2 expression in gastric cancer tissues. *p

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

    miR-410 targets at MDM2 in GC cells. (A) The sequences of miR-410 binding sites within the human MDM2 3′UTRs and schematic reporter constructs, in this panel, c-MDM2-WT represent the reporter constructs containing the entire 3′UTR sequences of MDM2. C-MDM2-MUT represent the reporter constructs containing mutated nucleotides. (B) The analysis of the relative luciferase activities of MDM2-WT, MDM2-MUT in 293T cells. The error bars are derived from triplicate expriments. (C) qRT-PCR analysis of MDM2 mRNA expression in HGC-27 cells after treatment with miRNA mimics or scramble or no transfection. The expression of MDM2 was normalized to GAPDH. (D) Western blot analysis of MDM2 expression in HGC-27 cells transfected with miR-410 mimics or scramble or no transfection. GAPDH was also detected as a loading control.
    Figure Legend Snippet: miR-410 targets at MDM2 in GC cells. (A) The sequences of miR-410 binding sites within the human MDM2 3′UTRs and schematic reporter constructs, in this panel, c-MDM2-WT represent the reporter constructs containing the entire 3′UTR sequences of MDM2. C-MDM2-MUT represent the reporter constructs containing mutated nucleotides. (B) The analysis of the relative luciferase activities of MDM2-WT, MDM2-MUT in 293T cells. The error bars are derived from triplicate expriments. (C) qRT-PCR analysis of MDM2 mRNA expression in HGC-27 cells after treatment with miRNA mimics or scramble or no transfection. The expression of MDM2 was normalized to GAPDH. (D) Western blot analysis of MDM2 expression in HGC-27 cells transfected with miR-410 mimics or scramble or no transfection. GAPDH was also detected as a loading control.

    Techniques Used: Binding Assay, Construct, Luciferase, Derivative Assay, Quantitative RT-PCR, Expressing, Transfection, Western Blot

    miR-410 was downregulated in gastric cancer. A. qRT-PCR analysis of miR-410 expression in four cell lines derived from gastric cancer and one nonmalignant gastric cell line (GES). (B) qRT-PCR analysis of miR-410 expression in 60 pairs GC tissues and their corresponding adjacent nontumorous tissues. The expression of miR-410 was normalized to U6 snRNA. (C) Relative miR-410 expression levels in GC tissues and adjacent normal regions; (D) Statistical analysis of the association between miRNA level between pM stage (No metastasis and Metastasis, respectively); *p
    Figure Legend Snippet: miR-410 was downregulated in gastric cancer. A. qRT-PCR analysis of miR-410 expression in four cell lines derived from gastric cancer and one nonmalignant gastric cell line (GES). (B) qRT-PCR analysis of miR-410 expression in 60 pairs GC tissues and their corresponding adjacent nontumorous tissues. The expression of miR-410 was normalized to U6 snRNA. (C) Relative miR-410 expression levels in GC tissues and adjacent normal regions; (D) Statistical analysis of the association between miRNA level between pM stage (No metastasis and Metastasis, respectively); *p

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

    Overexpression of miR-410 inhibited the cell proliferation, migration, and invasion in gastric cancer cells (A) Expression levels of miR-410 were examined by qRT-PCR after transfection of 20 nmol/L of miR-410 mimics, inhibitors or sramble or no transfection. The expression of miR-410 was normalized to U6 snRNA. (B) The cells treated with miR-410 mimics, inhibitors or sramble or no transfection were measured by CCK8 assay at different time periods. (C) Transwell analysis of HGC-27 cells after treatment with miRNA mimics, inhibitors or scramble or no transfection; the relative ratio of migrated cells per field is shown below. (D) Transwell analysis of HGC-27 cells after treatment with miRNA mimics, inhibitors or scramble or no transfection; the relative ratio of invasive cells per field is shown below, *p
    Figure Legend Snippet: Overexpression of miR-410 inhibited the cell proliferation, migration, and invasion in gastric cancer cells (A) Expression levels of miR-410 were examined by qRT-PCR after transfection of 20 nmol/L of miR-410 mimics, inhibitors or sramble or no transfection. The expression of miR-410 was normalized to U6 snRNA. (B) The cells treated with miR-410 mimics, inhibitors or sramble or no transfection were measured by CCK8 assay at different time periods. (C) Transwell analysis of HGC-27 cells after treatment with miRNA mimics, inhibitors or scramble or no transfection; the relative ratio of migrated cells per field is shown below. (D) Transwell analysis of HGC-27 cells after treatment with miRNA mimics, inhibitors or scramble or no transfection; the relative ratio of invasive cells per field is shown below, *p

    Techniques Used: Over Expression, Migration, Expressing, Quantitative RT-PCR, Transfection, CCK-8 Assay

    27) Product Images from "Ectopic Expression of a WRKY Homolog from Glycine soja Alters Flowering Time in Arabidopsis"

    Article Title: Ectopic Expression of a WRKY Homolog from Glycine soja Alters Flowering Time in Arabidopsis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073295

    The effect of GsWRKY20 over-expression on the transcription of FLC , FT , SOC1 , CO , AP1 , SEP3, AG, PI and AP3 . Ten-day-old WT and GsWRKY20 ox seedlings were harvested every 4 h during LD condition, and mRNA expression level was determined by qRT-PCR. Each value is the mean ±SE of three independent measurements, error bars represent the standard deviation (n=3). White and black bars at the bottom represent light and dark phases, respectively. ZT, Zeitgeber.
    Figure Legend Snippet: The effect of GsWRKY20 over-expression on the transcription of FLC , FT , SOC1 , CO , AP1 , SEP3, AG, PI and AP3 . Ten-day-old WT and GsWRKY20 ox seedlings were harvested every 4 h during LD condition, and mRNA expression level was determined by qRT-PCR. Each value is the mean ±SE of three independent measurements, error bars represent the standard deviation (n=3). White and black bars at the bottom represent light and dark phases, respectively. ZT, Zeitgeber.

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

    Over-expression of GsWRKY20 in Arabidopsis accelerates plant flowering. ( a ) Flowering time of GsWRKY20 ox plants was accelerated under both LD and SD conditions. (b) qRT-PCR analysis of GsWRKY20 transcript levels in WT and the three homozygous 35S :: GsWRKY20 lines. Expression of ACTIN2 was used as an internal control. The experiment included three fully independent biological repeats, and three technical repeats and the mean value is shown. ( c ) Average flowering time of WT and GsWRKY20 ox plants at the time of flowering under LD conditions. ( d ) Average rosette leaf numbers of WT and GsWRKY20 ox plants at the time of flowering under LD conditions. ( e ) Average flowering time of WT and GsWRKY20 ox plants at the time of flowering under SD conditions. ( f ) Average rosette leaf numbers of WT and GsWRKY20 ox plants at the time of flowering under SD conditions. All values in (c, d, e, f) are means (±S.E.) from three independent experiments (At least 30 seedlings per experiment). Data were analyzed statistically using the t-test, Asterisk and double asterisks indicate significant differences from the corresponding WT at 0.01
    Figure Legend Snippet: Over-expression of GsWRKY20 in Arabidopsis accelerates plant flowering. ( a ) Flowering time of GsWRKY20 ox plants was accelerated under both LD and SD conditions. (b) qRT-PCR analysis of GsWRKY20 transcript levels in WT and the three homozygous 35S :: GsWRKY20 lines. Expression of ACTIN2 was used as an internal control. The experiment included three fully independent biological repeats, and three technical repeats and the mean value is shown. ( c ) Average flowering time of WT and GsWRKY20 ox plants at the time of flowering under LD conditions. ( d ) Average rosette leaf numbers of WT and GsWRKY20 ox plants at the time of flowering under LD conditions. ( e ) Average flowering time of WT and GsWRKY20 ox plants at the time of flowering under SD conditions. ( f ) Average rosette leaf numbers of WT and GsWRKY20 ox plants at the time of flowering under SD conditions. All values in (c, d, e, f) are means (±S.E.) from three independent experiments (At least 30 seedlings per experiment). Data were analyzed statistically using the t-test, Asterisk and double asterisks indicate significant differences from the corresponding WT at 0.01

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

    Sequence and expression analysis of GsWRKY20 . ( a ) Amino acid sequence alignment of WRKY domains among GsWRKY20 and other type Ⅲ WRKY TFs, Sequences were aligned using ClustalW, and gaps were introduced to maximize alignment, filled triangle marks the cystine and histidine in the C2HC-type zinc finger domain. ( b ) The phylogenetic tree of the WRKY TFs. The phylogenetic tree was constructed using MEGA 4.1. Total 24 WRKY proteins from Oryza , Arabidopsis, Gossypium, Medicago, Triticum aestivum, Glycine max, Thlaspi caerulescens and Solanum were selected to construct the phylogenetic tree. ( c ) Tissue-specific expression analysis of GsWRKY20 by real-time quantitative PCR (qRT-PCR). Tissues included trifoliate leaf (TL), root (RO), stem tip (ST), flower bud (FB), and pod (PD). Expression of GAPDH was used as an internal control. The experiment included three fully independent biological repeats, and three technical repeats and the mean value is shown. ( d ) qRT-PCR analysis of GsWRKY20 diurnal expression under SD and LD. Trifoliate leaves were sampled every 4 h at 21 DAE. White and black bars at the top represent light and dark phases, respectively. Relative transcript levels were analyzed by qRT-PCR and normalized by GAPDH . The experiment included three fully independent biological repeats, and three technical repeats and the mean value is shown.
    Figure Legend Snippet: Sequence and expression analysis of GsWRKY20 . ( a ) Amino acid sequence alignment of WRKY domains among GsWRKY20 and other type Ⅲ WRKY TFs, Sequences were aligned using ClustalW, and gaps were introduced to maximize alignment, filled triangle marks the cystine and histidine in the C2HC-type zinc finger domain. ( b ) The phylogenetic tree of the WRKY TFs. The phylogenetic tree was constructed using MEGA 4.1. Total 24 WRKY proteins from Oryza , Arabidopsis, Gossypium, Medicago, Triticum aestivum, Glycine max, Thlaspi caerulescens and Solanum were selected to construct the phylogenetic tree. ( c ) Tissue-specific expression analysis of GsWRKY20 by real-time quantitative PCR (qRT-PCR). Tissues included trifoliate leaf (TL), root (RO), stem tip (ST), flower bud (FB), and pod (PD). Expression of GAPDH was used as an internal control. The experiment included three fully independent biological repeats, and three technical repeats and the mean value is shown. ( d ) qRT-PCR analysis of GsWRKY20 diurnal expression under SD and LD. Trifoliate leaves were sampled every 4 h at 21 DAE. White and black bars at the top represent light and dark phases, respectively. Relative transcript levels were analyzed by qRT-PCR and normalized by GAPDH . The experiment included three fully independent biological repeats, and three technical repeats and the mean value is shown.

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

    28) Product Images from "HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells"

    Article Title: HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells

    Journal: eLife

    doi: 10.7554/eLife.08955

    The genes modulated by ectopic expression of Tat are also detected during a time-course HIV infection experiment. ( A ) Jurkat T cells were infected with HIV (NL4-3) and levels of p24/Capsid protein was quantified using ELISA at different time points post-infection (0, 3 and 7 hr; 1, 2, 4, 6, 8, 10, and 12 days). Values represent the average of three independent experiments (mean ± SEM; n = 3). Cells from panel ( A ) were used to isolate total RNA and the expression of three TSG: CD69 ( B ), FAM46C ( C ), and PPM1H ( D ); and three TDG: CD1E ( E ), EOMES ( F ) and FBLN2 ( G ) normalized to RPL19 was measured by qRT-PCR and plotted as fold RNA change over the GFP cell line arbitrarily set at 1 (mean ± SEM; n = 3). The points in the curve were fitted to a non-linear regression in GraphPad Prism. ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; HIV, human immunodeficiency virus; qRT-PCR, quantitative real time polymerase chain reaction; SEM, standard error of the mean; TSG, Tat stimulated genes; TDG, Tat downregulated genes. DOI: http://dx.doi.org/10.7554/eLife.08955.010
    Figure Legend Snippet: The genes modulated by ectopic expression of Tat are also detected during a time-course HIV infection experiment. ( A ) Jurkat T cells were infected with HIV (NL4-3) and levels of p24/Capsid protein was quantified using ELISA at different time points post-infection (0, 3 and 7 hr; 1, 2, 4, 6, 8, 10, and 12 days). Values represent the average of three independent experiments (mean ± SEM; n = 3). Cells from panel ( A ) were used to isolate total RNA and the expression of three TSG: CD69 ( B ), FAM46C ( C ), and PPM1H ( D ); and three TDG: CD1E ( E ), EOMES ( F ) and FBLN2 ( G ) normalized to RPL19 was measured by qRT-PCR and plotted as fold RNA change over the GFP cell line arbitrarily set at 1 (mean ± SEM; n = 3). The points in the curve were fitted to a non-linear regression in GraphPad Prism. ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; HIV, human immunodeficiency virus; qRT-PCR, quantitative real time polymerase chain reaction; SEM, standard error of the mean; TSG, Tat stimulated genes; TDG, Tat downregulated genes. DOI: http://dx.doi.org/10.7554/eLife.08955.010

    Techniques Used: Expressing, Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    The C22A non-functional Tat mutant fails to bind ETS1 and evidence that ETS1 is critical for transcription activation of Tat target genes. ( A ) Western blots showing interactions between Tat (but not GFP or the C22A non-functional Tat mutant) and ETS1. CDK9 was used as a positive control in the interaction. Strep-tagged GFP, Tat, and C22A were AP from the Jurkat-GFP and -Tat cell lines using Strep beads, and analyzed by western blot using the indicated antibodies. ( B ) qRT-PCR analysis of CD69 expression in the indicated cell lines. ( C ) qRT-PCR analysis of ADCYAP1 expression in the indicated cell lines. ( D ) qRT-PCR analysis of VAV3 expression in the indicated cell lines. ( E ) qRT-PCR analysis of FAM46C expression in the indicated cell lines. ( F ) qRT-PCR analysis of RPL19 expression in the indicated cell lines. ( G ) qRT-PCR analysis of 7SK expression in the indicated cell lines. Expression of the indicated genes (panels B–G) was normalized to ACTB . Values represent the average of three independent experiments (mean ± SEM; n = 3). AP, affinity purified; GFP, green fluorescent protein; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of the mean. DOI: http://dx.doi.org/10.7554/eLife.08955.039
    Figure Legend Snippet: The C22A non-functional Tat mutant fails to bind ETS1 and evidence that ETS1 is critical for transcription activation of Tat target genes. ( A ) Western blots showing interactions between Tat (but not GFP or the C22A non-functional Tat mutant) and ETS1. CDK9 was used as a positive control in the interaction. Strep-tagged GFP, Tat, and C22A were AP from the Jurkat-GFP and -Tat cell lines using Strep beads, and analyzed by western blot using the indicated antibodies. ( B ) qRT-PCR analysis of CD69 expression in the indicated cell lines. ( C ) qRT-PCR analysis of ADCYAP1 expression in the indicated cell lines. ( D ) qRT-PCR analysis of VAV3 expression in the indicated cell lines. ( E ) qRT-PCR analysis of FAM46C expression in the indicated cell lines. ( F ) qRT-PCR analysis of RPL19 expression in the indicated cell lines. ( G ) qRT-PCR analysis of 7SK expression in the indicated cell lines. Expression of the indicated genes (panels B–G) was normalized to ACTB . Values represent the average of three independent experiments (mean ± SEM; n = 3). AP, affinity purified; GFP, green fluorescent protein; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of the mean. DOI: http://dx.doi.org/10.7554/eLife.08955.039

    Techniques Used: Functional Assay, Mutagenesis, Activation Assay, Western Blot, Positive Control, Quantitative RT-PCR, Expressing, Affinity Purification, Real-time Polymerase Chain Reaction

    HIV infection of central memory CD4+ T cells triggers deregulation of TSG and TDG detected in the genome-wide approaches. ( A ) Scheme of the pipeline used to generate primary central memory T cells (T CM ) and infect with replication competent HIV to identify differentially expressed genes. ( B ) qRT-PCR analysis on the indicated class I and II TSG, TDG and non-target genes (mean ± SEM; n = 3). Cells from panel ( A ) were used to isolate total RNA and the expression of initiating (In) and elongating (El) transcripts for class I TSG ( C ), class II TSG ( D ), class I TDG ( E ) and class II TDG ( F ) was measured by qRT-PCR, normalized to RPL19 , and plotted as fold RNA change: HIV infection/mock infection (mean ± SEM; n = 3). HIV, human immunodeficiency virus; qRT-PCR, quantitative real time polymerase chain reaction; TDG, Tat downregulated genes; TSG, Tat stimulated genes; SEM, standard error of the mean. DOI: http://dx.doi.org/10.7554/eLife.08955.011
    Figure Legend Snippet: HIV infection of central memory CD4+ T cells triggers deregulation of TSG and TDG detected in the genome-wide approaches. ( A ) Scheme of the pipeline used to generate primary central memory T cells (T CM ) and infect with replication competent HIV to identify differentially expressed genes. ( B ) qRT-PCR analysis on the indicated class I and II TSG, TDG and non-target genes (mean ± SEM; n = 3). Cells from panel ( A ) were used to isolate total RNA and the expression of initiating (In) and elongating (El) transcripts for class I TSG ( C ), class II TSG ( D ), class I TDG ( E ) and class II TDG ( F ) was measured by qRT-PCR, normalized to RPL19 , and plotted as fold RNA change: HIV infection/mock infection (mean ± SEM; n = 3). HIV, human immunodeficiency virus; qRT-PCR, quantitative real time polymerase chain reaction; TDG, Tat downregulated genes; TSG, Tat stimulated genes; SEM, standard error of the mean. DOI: http://dx.doi.org/10.7554/eLife.08955.011

    Techniques Used: Infection, Genome Wide, Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction

    Non-functional Tat mutants have compromised chromatin interaction and modulation of cellular gene expression. ( A ) Scheme of Tat showing the position of its domains (AD, RBD and Ct) along with the location of the mutated residues. ( B ) Western blots of Jurkat CD4+ T cell lines expressing GFP, wild-type Tat or non-functional mutants (C22A, K50Q, R52R53K) ( D'Orso et al., 2012 ). Cells from panel ( B ) were used in ChIP assays to analyze the occupancy of GFP, Tat or the non-functional mutants at class I TSG promoters ( C ), class II TSG promoters ( D ), class I TDG promoters ( E ) and class II TDG promoters ( F ). Values representing the average of three independent experiments (mean ± SEM; n = 3). Cells from panel ( B ) were used to isolate total RNA and the expression of class I TSG ( G ), class II TSG ( H ), class I TDG ( I ) and class II TDG ( J ) was measured by qRT-PCR, normalized to RPL19 , and plotted as fold RNA change over the GFP line arbitrarily set at 1 (mean ± SEM; n = 3). AD, activation domain; ChIP-seq, chromatin immunoprecipitation sequencing; Ct, C-terminal domain; GFP, green fluorescent protein; qRT-PCR, quantitative real time polymerase chain reaction; RBD, RNA-binding domain; TDG, Tat downregulated genes; TSG, Tat stimulated genes. DOI: http://dx.doi.org/10.7554/eLife.08955.006
    Figure Legend Snippet: Non-functional Tat mutants have compromised chromatin interaction and modulation of cellular gene expression. ( A ) Scheme of Tat showing the position of its domains (AD, RBD and Ct) along with the location of the mutated residues. ( B ) Western blots of Jurkat CD4+ T cell lines expressing GFP, wild-type Tat or non-functional mutants (C22A, K50Q, R52R53K) ( D'Orso et al., 2012 ). Cells from panel ( B ) were used in ChIP assays to analyze the occupancy of GFP, Tat or the non-functional mutants at class I TSG promoters ( C ), class II TSG promoters ( D ), class I TDG promoters ( E ) and class II TDG promoters ( F ). Values representing the average of three independent experiments (mean ± SEM; n = 3). Cells from panel ( B ) were used to isolate total RNA and the expression of class I TSG ( G ), class II TSG ( H ), class I TDG ( I ) and class II TDG ( J ) was measured by qRT-PCR, normalized to RPL19 , and plotted as fold RNA change over the GFP line arbitrarily set at 1 (mean ± SEM; n = 3). AD, activation domain; ChIP-seq, chromatin immunoprecipitation sequencing; Ct, C-terminal domain; GFP, green fluorescent protein; qRT-PCR, quantitative real time polymerase chain reaction; RBD, RNA-binding domain; TDG, Tat downregulated genes; TSG, Tat stimulated genes. DOI: http://dx.doi.org/10.7554/eLife.08955.006

    Techniques Used: Functional Assay, Expressing, Western Blot, Chromatin Immunoprecipitation, Quantitative RT-PCR, Activation Assay, ChIP-sequencing, Real-time Polymerase Chain Reaction, RNA Binding Assay

    Tat recruits chromatin-modifying enzymes and elongation factors at selected target genes to promote transcription elongation. ( A ) Strep-tagged GFP and Tat were affinity purified from nuclear preparations of the Jurkat cell lines (1 × 10 9 total cells) and interacting partners were analyzed by western blot with the indicated antibodies. CDK9 was used as a positive protein interacting control. ( B ) ChIP assay to analyze the distribution of histone marks (H3K79me3 and H3K36me3) and chromatin-modifying enzymes (Dot1L and SetD2) at the CD69 locus of the GFP (green) and Tat (black) cell lines. The position of the amplicons used in ChIP-qPCR is shown with the schematic of the locus. Values represent the average of three independent experiments (mean ± SEM; n = 3). ( C ) Genome browsers of FLAG, Pol II, H3K4me3, H379me3 and H3K36me3 ChIP-seq tracks in the GFP and Tat cell lines along with the Refseq track for the CD69 locus. The position of the TSS is indicated with an arrow. ( D ) Knockdown of Dot1L, SetD2 and CDK9 impairs Tat-mediated CD69 transcription activation. qRT-PCR of CD69 in the Jurkat-GFP and -Tat cell lines expressing non-target (NT), Dot1L, SetD2 and CDK9 shRNAs (mean ± SEM; n = 3). ( E ) Knockdown of Dot1L, SetD2 and CDK9 does not alter RNA steady state levels of RPL19 . qRT-PCR of RPL19 in the Jurkat-GFP and -Tat cell lines expressing non-target (NT), Dot1L, SetD2 and CDK9 shRNAs (mean ± SEM; n = 3). ( F,G,H ) qRT-PCR validation of Dot1L, SetD2 and CDK9 knockdown in the Jurkat-GFP and -Tat cell lines using gene-specific primers and normalized to RPL19 (mean ± SEM; n = 3). ChIP-seq, chromatin immunoprecipitation sequencing; GFP, green fluorescent protein; NT, non-target; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of the mean; shRNA, small hairpin RNA; TSG, Tat stimulated genes. DOI: http://dx.doi.org/10.7554/eLife.08955.021
    Figure Legend Snippet: Tat recruits chromatin-modifying enzymes and elongation factors at selected target genes to promote transcription elongation. ( A ) Strep-tagged GFP and Tat were affinity purified from nuclear preparations of the Jurkat cell lines (1 × 10 9 total cells) and interacting partners were analyzed by western blot with the indicated antibodies. CDK9 was used as a positive protein interacting control. ( B ) ChIP assay to analyze the distribution of histone marks (H3K79me3 and H3K36me3) and chromatin-modifying enzymes (Dot1L and SetD2) at the CD69 locus of the GFP (green) and Tat (black) cell lines. The position of the amplicons used in ChIP-qPCR is shown with the schematic of the locus. Values represent the average of three independent experiments (mean ± SEM; n = 3). ( C ) Genome browsers of FLAG, Pol II, H3K4me3, H379me3 and H3K36me3 ChIP-seq tracks in the GFP and Tat cell lines along with the Refseq track for the CD69 locus. The position of the TSS is indicated with an arrow. ( D ) Knockdown of Dot1L, SetD2 and CDK9 impairs Tat-mediated CD69 transcription activation. qRT-PCR of CD69 in the Jurkat-GFP and -Tat cell lines expressing non-target (NT), Dot1L, SetD2 and CDK9 shRNAs (mean ± SEM; n = 3). ( E ) Knockdown of Dot1L, SetD2 and CDK9 does not alter RNA steady state levels of RPL19 . qRT-PCR of RPL19 in the Jurkat-GFP and -Tat cell lines expressing non-target (NT), Dot1L, SetD2 and CDK9 shRNAs (mean ± SEM; n = 3). ( F,G,H ) qRT-PCR validation of Dot1L, SetD2 and CDK9 knockdown in the Jurkat-GFP and -Tat cell lines using gene-specific primers and normalized to RPL19 (mean ± SEM; n = 3). ChIP-seq, chromatin immunoprecipitation sequencing; GFP, green fluorescent protein; NT, non-target; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of the mean; shRNA, small hairpin RNA; TSG, Tat stimulated genes. DOI: http://dx.doi.org/10.7554/eLife.08955.021

    Techniques Used: Affinity Purification, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Activation Assay, Quantitative RT-PCR, Expressing, ChIP-sequencing, shRNA

    29) Product Images from "Fnr and ArcA Regulate Lipid A Hydroxylation in Salmonella Enteritidis by Controlling lpxO Expression in Response to Oxygen Availability"

    Article Title: Fnr and ArcA Regulate Lipid A Hydroxylation in Salmonella Enteritidis by Controlling lpxO Expression in Response to Oxygen Availability

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01220

    Expression of lpxO in S . Enteritidis strains. Expression levels were determined by qRT-PCR and normalized using rpoD as housekeeping gene (A) or by measuring β-galactosidase activity using strains carrying an lpxO – lacZ transcriptional fusion (B) . Bars represent mean values from three independent replicates. Error bars denote standard deviation. Statistical significance of observed differences was determined using a two-tailed Student’s t -test ( ∗ p
    Figure Legend Snippet: Expression of lpxO in S . Enteritidis strains. Expression levels were determined by qRT-PCR and normalized using rpoD as housekeeping gene (A) or by measuring β-galactosidase activity using strains carrying an lpxO – lacZ transcriptional fusion (B) . Bars represent mean values from three independent replicates. Error bars denote standard deviation. Statistical significance of observed differences was determined using a two-tailed Student’s t -test ( ∗ p

    Techniques Used: Expressing, Quantitative RT-PCR, Activity Assay, Standard Deviation, Two Tailed Test

    30) Product Images from "microRNA-802/Rnd3 pathway imposes on carcinogenesis and metastasis of fine particulate matter exposure"

    Article Title: microRNA-802/Rnd3 pathway imposes on carcinogenesis and metastasis of fine particulate matter exposure

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9019

    Functional analysis of the potential targets and validation of the significantly modulated miRNAs A. The GO enrichment analysis of biological processes showed that most categories were involved in actin-dependent processes and apoptosis. B. Nine genes and their related miRNAs were involved in BP enrichment. C. The miRNA expression levels were validated by qRT-PCR in A549 cells. The expression of miR-1469, −1322, and −802 were modulated in a dose-dependent manner. * P
    Figure Legend Snippet: Functional analysis of the potential targets and validation of the significantly modulated miRNAs A. The GO enrichment analysis of biological processes showed that most categories were involved in actin-dependent processes and apoptosis. B. Nine genes and their related miRNAs were involved in BP enrichment. C. The miRNA expression levels were validated by qRT-PCR in A549 cells. The expression of miR-1469, −1322, and −802 were modulated in a dose-dependent manner. * P

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

    31) 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

    32) 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

    33) Product Images from "Persistence of Viral Reservoirs in Multiple Tissues after Antiretroviral Therapy Suppression in a Macaque RT-SHIV Model"

    Article Title: Persistence of Viral Reservoirs in Multiple Tissues after Antiretroviral Therapy Suppression in a Macaque RT-SHIV Model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0084275

    Plasma viremia was measured in all twelve macaques by qRT-PCR of RT-SHIV gag RNA. Animals were infected at week 0 and were (A) untreated or (B) treated with 3 or 4 antiretroviral drugs. Animals treated with 3 drugs (TFV, FTC, EFV) are denoted by closed symbols and treatment was initiated at week 10, denoted by the solid arrow. The animals treated with 4 drugs (TFV, FTC, EFV, and L-870812) are denoted by open symbols and treatment was initiated at week 13 (GV08 and GN19) or week 14 (GG45 and GV40), denoted by the open arrow. Treatment was continued daily until necropsy (week 30 or 31). The limit of detection of the assay was 30 vRNA copies/ml plasma.
    Figure Legend Snippet: Plasma viremia was measured in all twelve macaques by qRT-PCR of RT-SHIV gag RNA. Animals were infected at week 0 and were (A) untreated or (B) treated with 3 or 4 antiretroviral drugs. Animals treated with 3 drugs (TFV, FTC, EFV) are denoted by closed symbols and treatment was initiated at week 10, denoted by the solid arrow. The animals treated with 4 drugs (TFV, FTC, EFV, and L-870812) are denoted by open symbols and treatment was initiated at week 13 (GV08 and GN19) or week 14 (GG45 and GV40), denoted by the open arrow. Treatment was continued daily until necropsy (week 30 or 31). The limit of detection of the assay was 30 vRNA copies/ml plasma.

    Techniques Used: Quantitative RT-PCR, Infection

    34) Product Images from "Gene Profiling Studies in Skeletal Muscle by Quantitative Real-Time Polymerase Chain Reaction Assay"

    Article Title: Gene Profiling Studies in Skeletal Muscle by Quantitative Real-Time Polymerase Chain Reaction Assay

    Journal: Methods in Molecular Biology (Clifton, N.j.)

    doi: 10.1007/978-1-61779-343-1_18

    Dissociation curves in qRT-PCR assay
    Figure Legend Snippet: Dissociation curves in qRT-PCR assay

    Techniques Used: Quantitative RT-PCR

    35) Product Images from "Membrane-associated proteomics of chickpea identifies Sad1/UNC-84 protein (CaSUN1), a novel component of dehydration signaling"

    Article Title: Membrane-associated proteomics of chickpea identifies Sad1/UNC-84 protein (CaSUN1), a novel component of dehydration signaling

    Journal: Scientific Reports

    doi: 10.1038/srep04177

    Functional complementation of slp1 mutant by CaSUN1. Wild-type (WT), slp1 mutant and slp1 -CaSUN1 cells were cultured in SD-Ura and SD-Gal/Raf-Ura media and subjected to DTT treatment. OD 600 was determined every 3 h and growth curves were constructed for (a) non-induced and (b) induced conditions. (c) Relative abundance of KAR2 was analyzed by qRT-PCR in wild-type, slp1 and slp1 -CaSUN1 cells, subjected to DTT treatment for 24 h. The values are mean ± SD (n = 3). NT represents untreated condition. (d) Confocal micrographs displaying fusion proteins of transformed cells pAG426GPD-CaSUN1-EYFP (upper panel) and co-localization of pAG426GPD-CaSUN1-EGFP and pSM1960-Sec63p-RFP (lower panel). The right panel represents the merged images.
    Figure Legend Snippet: Functional complementation of slp1 mutant by CaSUN1. Wild-type (WT), slp1 mutant and slp1 -CaSUN1 cells were cultured in SD-Ura and SD-Gal/Raf-Ura media and subjected to DTT treatment. OD 600 was determined every 3 h and growth curves were constructed for (a) non-induced and (b) induced conditions. (c) Relative abundance of KAR2 was analyzed by qRT-PCR in wild-type, slp1 and slp1 -CaSUN1 cells, subjected to DTT treatment for 24 h. The values are mean ± SD (n = 3). NT represents untreated condition. (d) Confocal micrographs displaying fusion proteins of transformed cells pAG426GPD-CaSUN1-EYFP (upper panel) and co-localization of pAG426GPD-CaSUN1-EGFP and pSM1960-Sec63p-RFP (lower panel). The right panel represents the merged images.

    Techniques Used: Functional Assay, Mutagenesis, Cell Culture, Construct, Quantitative RT-PCR, Transformation Assay

    Transcript analysis of CaSUN1 . Determination of CaSUN1 transcript levels by qRT-PCR in response to (a) dehydration, (b) cold, (c) NaCl, (d) MV, (e) salicylic acid, and (f) ABA. Relative fold change in mRNA level is shown on Y-axis. (g) The tissue-specific expression of CaSUN1 was analyzed using high throughput transcriptomic data from Chickpea Transcriptome Database ( http://59.163.192.90:8080/ctdb/ ).
    Figure Legend Snippet: Transcript analysis of CaSUN1 . Determination of CaSUN1 transcript levels by qRT-PCR in response to (a) dehydration, (b) cold, (c) NaCl, (d) MV, (e) salicylic acid, and (f) ABA. Relative fold change in mRNA level is shown on Y-axis. (g) The tissue-specific expression of CaSUN1 was analyzed using high throughput transcriptomic data from Chickpea Transcriptome Database ( http://59.163.192.90:8080/ctdb/ ).

    Techniques Used: Quantitative RT-PCR, Expressing, High Throughput Screening Assay

    36) Product Images from "Gut-derived lipopolysaccharide augments adipose macrophage accumulation but is not essential for impaired glucose or insulin tolerance in mice"

    Article Title: Gut-derived lipopolysaccharide augments adipose macrophage accumulation but is not essential for impaired glucose or insulin tolerance in mice

    Journal: Gut

    doi: 10.1136/gutjnl-2011-301689

    Gut-derived lipopolysaccharide (LPS) increases crown-like structure (CLS) formation, M1/M2 ratio and expression of proinflammatory and anti-inflammatory cytokines in white adipose tissue (WAT). (A) Representative immunostaining of MAC-2 in WAT from germ-free (GF) mice and mice colonised for 4 weeks with W3110 or MLK1067. Arrowheads indicate CLS. Scale bars = 100 μm. (B) Quantification of CLS (n=4–5 mice per group). (C) qRT-PCR analysis of Emr1 (F4/80) in WAT (n=6–7 mice per group). (D) Ratio between M1 and M2 activated macrophages in WAT determined by flow cytometry with antibodies against CD11c and CD209 conjugated (n=4 (GF) and 9 (W3110 and MLK1067)). (E) Number of CD11c + (M1) and CD209 (M2) macrophages per gram WAT (n=4–5). (F–I) qRT-PCR analysis of Tnfα , Saa3 , Mgl1 and Il-10 expression in WAT (n=5–6 mice per group). (J) Total protein extracts from WAT were analysed by immunoblotting with antibodies against phospho-JNK and actin. (K) Relative quantification of the phospho-p46 form of JNK normalised to actin (n=6 mice per group). Mean values ± SEM are plotted; *p
    Figure Legend Snippet: Gut-derived lipopolysaccharide (LPS) increases crown-like structure (CLS) formation, M1/M2 ratio and expression of proinflammatory and anti-inflammatory cytokines in white adipose tissue (WAT). (A) Representative immunostaining of MAC-2 in WAT from germ-free (GF) mice and mice colonised for 4 weeks with W3110 or MLK1067. Arrowheads indicate CLS. Scale bars = 100 μm. (B) Quantification of CLS (n=4–5 mice per group). (C) qRT-PCR analysis of Emr1 (F4/80) in WAT (n=6–7 mice per group). (D) Ratio between M1 and M2 activated macrophages in WAT determined by flow cytometry with antibodies against CD11c and CD209 conjugated (n=4 (GF) and 9 (W3110 and MLK1067)). (E) Number of CD11c + (M1) and CD209 (M2) macrophages per gram WAT (n=4–5). (F–I) qRT-PCR analysis of Tnfα , Saa3 , Mgl1 and Il-10 expression in WAT (n=5–6 mice per group). (J) Total protein extracts from WAT were analysed by immunoblotting with antibodies against phospho-JNK and actin. (K) Relative quantification of the phospho-p46 form of JNK normalised to actin (n=6 mice per group). Mean values ± SEM are plotted; *p

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

    Gut microbiota increases crown-like structure (CLS) formation, M1/M2 ratio and expression of proinflammatory and anti-inflammatory cytokines in white adipose tissue (WAT). (A) Representative MAC-2 immunostaining of WAT from germ-free (GF) and conventionally raised (CONV-R) mice. Arrowheads indicate CLS. Scale bars = 100 μm. (B) Quantification of CLS (n=9 mice per group). (C) qRT-PCR analysis of Emr1 expression in WAT from GF and CONV-R mice (n=8–9 mice per group). (D) Ratio between WAT M1 and M2 macrophages determined by flow cytometry with antibodies against CD11c conjugated with phycoerythrin and CD209 conjugated antigen presenting cells, respectively (n=4 (GF) and 8 (CONV-R)). (E) Number of CD11c (M1) and CD209 (M2) macrophages per gram WAT (n=4). (F–I) qRT-PCR analysis of Tnfα , Saa3 , Mgl1 and Il-10 expression in WAT from GF and CONV-R mice (n=8–9 mice per group). (J) Lipopolysaccharide (LPS) levels in blood sampled from the portal vein of GF (n=9) and CONV-R (n=15) mice. Mean values ± SEM are plotted; *p
    Figure Legend Snippet: Gut microbiota increases crown-like structure (CLS) formation, M1/M2 ratio and expression of proinflammatory and anti-inflammatory cytokines in white adipose tissue (WAT). (A) Representative MAC-2 immunostaining of WAT from germ-free (GF) and conventionally raised (CONV-R) mice. Arrowheads indicate CLS. Scale bars = 100 μm. (B) Quantification of CLS (n=9 mice per group). (C) qRT-PCR analysis of Emr1 expression in WAT from GF and CONV-R mice (n=8–9 mice per group). (D) Ratio between WAT M1 and M2 macrophages determined by flow cytometry with antibodies against CD11c conjugated with phycoerythrin and CD209 conjugated antigen presenting cells, respectively (n=4 (GF) and 8 (CONV-R)). (E) Number of CD11c (M1) and CD209 (M2) macrophages per gram WAT (n=4). (F–I) qRT-PCR analysis of Tnfα , Saa3 , Mgl1 and Il-10 expression in WAT from GF and CONV-R mice (n=8–9 mice per group). (J) Lipopolysaccharide (LPS) levels in blood sampled from the portal vein of GF (n=9) and CONV-R (n=15) mice. Mean values ± SEM are plotted; *p

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

    37) Product Images from "Genes Involved in Radiation Therapy Response in Head and Neck Cancers"

    Article Title: Genes Involved in Radiation Therapy Response in Head and Neck Cancers

    Journal: The Laryngoscope

    doi: 10.1002/lary.20005

    QRT-PCR gene expression validation. Relative gene expression of the TNC, PTHLH, and LTB genes in the 14 HNSCC samples, measured by Affymetrix gene expression arrays (bar chart) and by TaqMan chemistry (line chart). The PPIA-normalized fold changes (Log2)
    Figure Legend Snippet: QRT-PCR gene expression validation. Relative gene expression of the TNC, PTHLH, and LTB genes in the 14 HNSCC samples, measured by Affymetrix gene expression arrays (bar chart) and by TaqMan chemistry (line chart). The PPIA-normalized fold changes (Log2)

    Techniques Used: Quantitative RT-PCR, Expressing

    38) Product Images from "Breaking through an epigenetic wall"

    Article Title: Breaking through an epigenetic wall

    Journal: Epigenetics

    doi: 10.4161/epi.23503

    Figure 3. KRAB-ZF-119 upregulates Oct4 expression in breast and ovarian cancer cell lines. (A-D) Quantification of Oct4, Sox2 and Nanog mRNA expression levels by qRT-PCR in MDA-MB-231 (A-B) , SUM159 (C) and OVCAR-3 cells (D). X-axis represents
    Figure Legend Snippet: Figure 3. KRAB-ZF-119 upregulates Oct4 expression in breast and ovarian cancer cell lines. (A-D) Quantification of Oct4, Sox2 and Nanog mRNA expression levels by qRT-PCR in MDA-MB-231 (A-B) , SUM159 (C) and OVCAR-3 cells (D). X-axis represents

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

    39) Product Images from "mRNA Expression and BRAF Mutation in Circulating Melanoma Cells Isolated from Peripheral Blood with High Molecular Weight Melanoma-Associated Antigen-Specific Monoclonal Antibody Beads"

    Article Title: mRNA Expression and BRAF Mutation in Circulating Melanoma Cells Isolated from Peripheral Blood with High Molecular Weight Melanoma-Associated Antigen-Specific Monoclonal Antibody Beads

    Journal: Clinical chemistry

    doi: 10.1373/clinchem.2008.116467

    qRT-PCR detection limit for melanoma cells (10 4 , 10 3 , 10 2 , 10, 1, and 0 cells) mixed with 5 × 10 6 PBCs from healthy donors.
    Figure Legend Snippet: qRT-PCR detection limit for melanoma cells (10 4 , 10 3 , 10 2 , 10, 1, and 0 cells) mixed with 5 × 10 6 PBCs from healthy donors.

    Techniques Used: Quantitative RT-PCR

    qRT-PCR DETECTION LIMIT FOR MELANOMA CELLS MIXED WITH PBCs
    Figure Legend Snippet: qRT-PCR DETECTION LIMIT FOR MELANOMA CELLS MIXED WITH PBCs

    Techniques Used: Quantitative RT-PCR

    40) Product Images from "miR-409-3p/-5p promotes tumorigenesis, epithelial to mesenchymal transition and bone metastasis of human prostate cancer"

    Article Title: miR-409-3p/-5p promotes tumorigenesis, epithelial to mesenchymal transition and bone metastasis of human prostate cancer

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-14-0305

    miR-409 inhibits tumor suppressor genes in prostate cancer. A, mRNA targets of miR-409-5p: STAG2, RBL2, RSU1 and NPRL2 and mRNA targets of miR-409-3p: RSU1, PHC3 and TUSC1, assayed by triplicate wells in qRT-PCR of ARCaP E and ARCaP M cells. The representative
    Figure Legend Snippet: miR-409 inhibits tumor suppressor genes in prostate cancer. A, mRNA targets of miR-409-5p: STAG2, RBL2, RSU1 and NPRL2 and mRNA targets of miR-409-3p: RSU1, PHC3 and TUSC1, assayed by triplicate wells in qRT-PCR of ARCaP E and ARCaP M cells. The representative

    Techniques Used: Quantitative RT-PCR

    Ectopic expression of miR-409 leads to increased invasiveness and aggressiveness of prostate cancer cells and conversely inhibition of miR-409 results in increased cell death in PCa cells. A, miR-409-5p and -3p expression by qRT-PCR in ARCaP E -C and ARCaP
    Figure Legend Snippet: Ectopic expression of miR-409 leads to increased invasiveness and aggressiveness of prostate cancer cells and conversely inhibition of miR-409 results in increased cell death in PCa cells. A, miR-409-5p and -3p expression by qRT-PCR in ARCaP E -C and ARCaP

    Techniques Used: Expressing, Inhibition, Quantitative RT-PCR

    41) Product Images from "Long noncoding AGAP2-AS1 is activated by SP1 and promotes cell proliferation and invasion in gastric cancer"

    Article Title: Long noncoding AGAP2-AS1 is activated by SP1 and promotes cell proliferation and invasion in gastric cancer

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-017-0420-4

    AGAP2-AS1 epigenetically suppresses P21 and E-cadherin by interacting with EZH2 and LSD1. a , b qRT-PCR analysis of the expression levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with EZH2, LSD1, or negative control siRNAs. c , d ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the P21 and E-cadherin promoters in the BGC823 and AGS cells after transfection with AGAP2-AS1 or NC siRNA. IgG was used as a negative control. e The relationship between AGAP2-AS1 expression and P21/E-cadherin in the GC tissues was analyzed. * P
    Figure Legend Snippet: AGAP2-AS1 epigenetically suppresses P21 and E-cadherin by interacting with EZH2 and LSD1. a , b qRT-PCR analysis of the expression levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with EZH2, LSD1, or negative control siRNAs. c , d ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the P21 and E-cadherin promoters in the BGC823 and AGS cells after transfection with AGAP2-AS1 or NC siRNA. IgG was used as a negative control. e The relationship between AGAP2-AS1 expression and P21/E-cadherin in the GC tissues was analyzed. * P

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection, Negative Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    AGAP2-AS1 knockdown inhibits GC cell tumor growth in vivo. a The BGC823 cells with stable knockdown of AGAP2-AS1 were used for the in vivo tumorigenesis assays. The tumors formed from the BGC823 cells with AGAP2-AS1 knockdown and the control cells in nude mice are shown. b The tumor growth curves were measured 3 days after the injection of the BGC823 cells once the tumor had formed, and the volume was calculated every 3 days. c Tumor weights in the sh-AGAP2-AS1 and control groups are presented. d qRT-PCR analysis of AGAP2-AS1 expression levels in the tumor tissues formed from the AGAP2-AS1-downregulated cells or control cells. e Tumors formed from sh-AGAP2-AS1-transfected BGC823 cells showed lower Ki67-positive signals than tumors formed from the control cells. Upper ; hematoxylin eosin staining, Lower Ki67 immunostaining. * P
    Figure Legend Snippet: AGAP2-AS1 knockdown inhibits GC cell tumor growth in vivo. a The BGC823 cells with stable knockdown of AGAP2-AS1 were used for the in vivo tumorigenesis assays. The tumors formed from the BGC823 cells with AGAP2-AS1 knockdown and the control cells in nude mice are shown. b The tumor growth curves were measured 3 days after the injection of the BGC823 cells once the tumor had formed, and the volume was calculated every 3 days. c Tumor weights in the sh-AGAP2-AS1 and control groups are presented. d qRT-PCR analysis of AGAP2-AS1 expression levels in the tumor tissues formed from the AGAP2-AS1-downregulated cells or control cells. e Tumors formed from sh-AGAP2-AS1-transfected BGC823 cells showed lower Ki67-positive signals than tumors formed from the control cells. Upper ; hematoxylin eosin staining, Lower Ki67 immunostaining. * P

    Techniques Used: In Vivo, Mouse Assay, Injection, Quantitative RT-PCR, Expressing, Transfection, Staining, Immunostaining

    AGAP2-AS1 interacting with EZH2 and LSD1 in the GC cells. a The distribution of AGAP2-AS1 levels in the cytoplasmic or nuclear fraction of GC cell lines was determined by qRT-PCR. U1 was used as a nuclear control; GAPDH was used as a cytoplasmic control. b The AGAP2-AS1 RNA levels in EZH2, LSD1, SUZ12, CoREST, and HuR immunoprecipitates were determined by qRT-PCR, and data are presented as fold enrichment relative to IgG immunoprecipitates. c EZH2 and LSD1 protein levels in immunoprecipitates with AGAP2-AS1 RNA were determined by Western blot. HuR protein immunoprecipitates with AR RNA were used as a positive control. d qRT-PCR analysis of the expression levels of KLF2, LATS1, and LATS2, and others in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. e Western blot analysis of the protein levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. f Immunofluorescence staining analysis of E-cadherin in BGC823 cells after transfection with AGAP2-AS1 or negative control siRNAs. * P
    Figure Legend Snippet: AGAP2-AS1 interacting with EZH2 and LSD1 in the GC cells. a The distribution of AGAP2-AS1 levels in the cytoplasmic or nuclear fraction of GC cell lines was determined by qRT-PCR. U1 was used as a nuclear control; GAPDH was used as a cytoplasmic control. b The AGAP2-AS1 RNA levels in EZH2, LSD1, SUZ12, CoREST, and HuR immunoprecipitates were determined by qRT-PCR, and data are presented as fold enrichment relative to IgG immunoprecipitates. c EZH2 and LSD1 protein levels in immunoprecipitates with AGAP2-AS1 RNA were determined by Western blot. HuR protein immunoprecipitates with AR RNA were used as a positive control. d qRT-PCR analysis of the expression levels of KLF2, LATS1, and LATS2, and others in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. e Western blot analysis of the protein levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. f Immunofluorescence staining analysis of E-cadherin in BGC823 cells after transfection with AGAP2-AS1 or negative control siRNAs. * P

    Techniques Used: Quantitative RT-PCR, Western Blot, Positive Control, Expressing, Transfection, Negative Control, Immunofluorescence, Staining

    AGAP2-AS1 is overexpressed in the human GC tissues and cells. a Data mining of AGAP2-AS1 expression levels in the GC tissue samples from gene profiling (GSE51575 and GSE65801). b qRT-PCR analysis of AGAP2-AS1 level in the 50 paired GC tissues and adjacent nontumor tissues. AGAP2-AS1 level was normalized to GAPDH expression. c qRT-PCR analysis of AGAP2-AS1 expression in the GC cell lines BGC823, MGC803, SGC7901, AGS, and MKN45 and the normal gastric cell line GSE1. AGAP2-AS1 level was normalized to the GAPDH level. d GC patients were divided into two groups according to AGAP2-AS1 expression profiles. The median fold change was used as the threshold. e Kaplan–Meier overall and disease-free survival analyses were used to investigate the relationship between AGAP2-AS1 expression and GC patient survival. * P
    Figure Legend Snippet: AGAP2-AS1 is overexpressed in the human GC tissues and cells. a Data mining of AGAP2-AS1 expression levels in the GC tissue samples from gene profiling (GSE51575 and GSE65801). b qRT-PCR analysis of AGAP2-AS1 level in the 50 paired GC tissues and adjacent nontumor tissues. AGAP2-AS1 level was normalized to GAPDH expression. c qRT-PCR analysis of AGAP2-AS1 expression in the GC cell lines BGC823, MGC803, SGC7901, AGS, and MKN45 and the normal gastric cell line GSE1. AGAP2-AS1 level was normalized to the GAPDH level. d GC patients were divided into two groups according to AGAP2-AS1 expression profiles. The median fold change was used as the threshold. e Kaplan–Meier overall and disease-free survival analyses were used to investigate the relationship between AGAP2-AS1 expression and GC patient survival. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    SP1 activates AGAP2-AS1 expression in GC cells. a JASPR online prediction of SP1 binding sites in the AGAP2-AS1 promoter regions. b Western blot analysis of SP1 protein levels in the AGS and BGC823 cells after transfection with SP1 siRNA. c qRT-PCR analysis of SP1 and AGAP2-AS1 expression in AGS and BGC823 cells after transfection with SP1 siRNA. d Western blot and qRT-PCR analyses of SP1 and AGAP2-AS1 expression in the AGS and BGC823 cells after transfection with SP1 vector or empty vector. e ChIP-qPCR analysis of SP1 occupancy in the AGAP2-AS1 promoter regions in the BGC823 and AGS cells. IgG was used as a negative control. f Luciferase reporter analysis of luciferase activity in the HEK293 cells cotransfected with pGL3-AGAP2-AS1 and SP1 vector or an empty vector. * P
    Figure Legend Snippet: SP1 activates AGAP2-AS1 expression in GC cells. a JASPR online prediction of SP1 binding sites in the AGAP2-AS1 promoter regions. b Western blot analysis of SP1 protein levels in the AGS and BGC823 cells after transfection with SP1 siRNA. c qRT-PCR analysis of SP1 and AGAP2-AS1 expression in AGS and BGC823 cells after transfection with SP1 siRNA. d Western blot and qRT-PCR analyses of SP1 and AGAP2-AS1 expression in the AGS and BGC823 cells after transfection with SP1 vector or empty vector. e ChIP-qPCR analysis of SP1 occupancy in the AGAP2-AS1 promoter regions in the BGC823 and AGS cells. IgG was used as a negative control. f Luciferase reporter analysis of luciferase activity in the HEK293 cells cotransfected with pGL3-AGAP2-AS1 and SP1 vector or an empty vector. * P

    Techniques Used: Expressing, Binding Assay, Western Blot, Transfection, Quantitative RT-PCR, Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Luciferase, Activity Assay

    42) Product Images from "Methaneseleninic acid and γ-Tocopherol combination inhibits prostate tumor growth in Vivo in a xenograft mouse model"

    Article Title: Methaneseleninic acid and γ-Tocopherol combination inhibits prostate tumor growth in Vivo in a xenograft mouse model

    Journal: Oncotarget

    doi:

    Effects of MSA and/or γT on Bad protein and mRNA The effects of MSA and γT on pro-apoptosis Bad was analyzed at protein as well as mRNA levels. For immunoblot analysis, 40 μg protein was separated on SDS-PAGE and immunobloted as detailed in ‘Materials and Methods’. The blots were reprobed for β-actin for loading control. Relative protein expression of Bad was calculated and plotted (A). Relative mRNA levels of Bad were assessed using qRT-PCR assay as detailed in ‘Materials and Methods’ (B). The data represented are mean ± 2 standard error of three replicates (representing 6 mice) (*P
    Figure Legend Snippet: Effects of MSA and/or γT on Bad protein and mRNA The effects of MSA and γT on pro-apoptosis Bad was analyzed at protein as well as mRNA levels. For immunoblot analysis, 40 μg protein was separated on SDS-PAGE and immunobloted as detailed in ‘Materials and Methods’. The blots were reprobed for β-actin for loading control. Relative protein expression of Bad was calculated and plotted (A). Relative mRNA levels of Bad were assessed using qRT-PCR assay as detailed in ‘Materials and Methods’ (B). The data represented are mean ± 2 standard error of three replicates (representing 6 mice) (*P

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

    Effects of MSA and/or γT on Bax and Bcl2 mRNA At the termination of xenograft experiment, effects of treatments on Bax and Bcl2 transcription were assessed by qRT-PCR analyses. RNA from tumor tissue samples was isolated and cDNA was made. qRT-PCR was run for Bax and Bcl2 as detailed in ‘Materials and Methods’. GAPDH was used as endogenous control. The qRT-PCR data are represented as relative mRNA levels for Bax (A) and Bcl2 (B). Ratio of Bax/Bcl2 mRNA was calculated and plotted (C). The data represented are mean ± 2 standard error of three replicates (representing 6 mice) (*P
    Figure Legend Snippet: Effects of MSA and/or γT on Bax and Bcl2 mRNA At the termination of xenograft experiment, effects of treatments on Bax and Bcl2 transcription were assessed by qRT-PCR analyses. RNA from tumor tissue samples was isolated and cDNA was made. qRT-PCR was run for Bax and Bcl2 as detailed in ‘Materials and Methods’. GAPDH was used as endogenous control. The qRT-PCR data are represented as relative mRNA levels for Bax (A) and Bcl2 (B). Ratio of Bax/Bcl2 mRNA was calculated and plotted (C). The data represented are mean ± 2 standard error of three replicates (representing 6 mice) (*P

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

    Effects of MSA and/or γT on oxidative stress markers To assess the effects of MSA and/or γT on modulation of oxidative stress, ApoE, SepP and Nrf2 mRNA level were analyzed. RNA isolation from tumor tissue followed by cDNA synthesis and then qRT-PCR was performed as detailed in ‘Materials and Methods’. The qRT-PCR data are represented as relative quantity (normalized to GAPDH) for ApoE (A), SepP (B) and Nrf2 (C) transcriptional levels. Representative data are expressed as mean ± 2 standard error of three replicates (representing 6 mice) (*P
    Figure Legend Snippet: Effects of MSA and/or γT on oxidative stress markers To assess the effects of MSA and/or γT on modulation of oxidative stress, ApoE, SepP and Nrf2 mRNA level were analyzed. RNA isolation from tumor tissue followed by cDNA synthesis and then qRT-PCR was performed as detailed in ‘Materials and Methods’. The qRT-PCR data are represented as relative quantity (normalized to GAPDH) for ApoE (A), SepP (B) and Nrf2 (C) transcriptional levels. Representative data are expressed as mean ± 2 standard error of three replicates (representing 6 mice) (*P

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

    43) Product Images from "MLL2, not MLL1, Plays a Major Role in Sustaining MLL-rearranged Acute Myeloid Leukemia"

    Article Title: MLL2, not MLL1, Plays a Major Role in Sustaining MLL-rearranged Acute Myeloid Leukemia

    Journal: Cancer cell

    doi: 10.1016/j.ccell.2017.05.002

    Reduced expression of MLL2 target genes is exacerbated by loss of MLL1, but through mechanisms other than H3K4 methylation A) Validation of RNA-seq results using qRT-PCR focusing on direct MLL2 target genes. Three independent clones randomly picked from each leukemia pool were tested for expression of the indicated genes 72 hours post-gene deletion. Bars represent PCR data from 3 independent clones ± SD; **, p
    Figure Legend Snippet: Reduced expression of MLL2 target genes is exacerbated by loss of MLL1, but through mechanisms other than H3K4 methylation A) Validation of RNA-seq results using qRT-PCR focusing on direct MLL2 target genes. Three independent clones randomly picked from each leukemia pool were tested for expression of the indicated genes 72 hours post-gene deletion. Bars represent PCR data from 3 independent clones ± SD; **, p

    Techniques Used: Expressing, Methylation, RNA Sequencing Assay, Quantitative RT-PCR, Clone Assay, Polymerase Chain Reaction

    Two distinct Mll1 targeting strategies corroborate lack of requirement for MLL1 A) Serial replating of MLL-AF9-transformed leukemia cells after deletion of Mll1 using either the Mll1 E (generated in Ernst laboratory) or Mll1 S (generated in Stewart laboratory) conditional knockout alleles. One thousand cells were replated and CFUs were scored in triplicate every 5 days. CFUs were normalized to ethanol (EtOH)-treated cultures for each genotype. Bars indicate averages of triplicate cultures ± SD. Representative results of three independent experiments are shown. B) Representative colony images at the end of the 5th replating. C) Expression of Mll1 Hoxa9 and Meis1 at the end of the 1st and 4th serial replating as measured by qRT-PCR. Bars indicate triplicate PCR reactions ± SD. Representative results of two independent experiments are shown. D) Sequence of 5–6 independent genomic clones from each of 3 single cell line clones after CRISPR/Cas9 targeting (day 26) of human MLL1 ( KMT2A ) in MOLM-14 cells. The relevant region is shown with dashes representing deleted nucleotides. E) MLL1-C terminal western blot from cell clones shown in D after sequencing. F) .
    Figure Legend Snippet: Two distinct Mll1 targeting strategies corroborate lack of requirement for MLL1 A) Serial replating of MLL-AF9-transformed leukemia cells after deletion of Mll1 using either the Mll1 E (generated in Ernst laboratory) or Mll1 S (generated in Stewart laboratory) conditional knockout alleles. One thousand cells were replated and CFUs were scored in triplicate every 5 days. CFUs were normalized to ethanol (EtOH)-treated cultures for each genotype. Bars indicate averages of triplicate cultures ± SD. Representative results of three independent experiments are shown. B) Representative colony images at the end of the 5th replating. C) Expression of Mll1 Hoxa9 and Meis1 at the end of the 1st and 4th serial replating as measured by qRT-PCR. Bars indicate triplicate PCR reactions ± SD. Representative results of two independent experiments are shown. D) Sequence of 5–6 independent genomic clones from each of 3 single cell line clones after CRISPR/Cas9 targeting (day 26) of human MLL1 ( KMT2A ) in MOLM-14 cells. The relevant region is shown with dashes representing deleted nucleotides. E) MLL1-C terminal western blot from cell clones shown in D after sequencing. F) .

    Techniques Used: Transformation Assay, Generated, Knock-Out, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Sequencing, Clone Assay, CRISPR, Western Blot

    44) Product Images from "Cryptochrome 2 extensively regulates transcription of the chloroplast genome in tomato"

    Article Title: Cryptochrome 2 extensively regulates transcription of the chloroplast genome in tomato

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12082

    Expression of CRY 2 gene in WT and CRY 2‐ OX (line 52.3) tomato plants analyzed by QRT ‐ PCR . Results are presented as a ratio after normalization with b‐actin. Data shown are the average of two biological replicates, with error bars representing SEM . ***Student's t test with P
    Figure Legend Snippet: Expression of CRY 2 gene in WT and CRY 2‐ OX (line 52.3) tomato plants analyzed by QRT ‐ PCR . Results are presented as a ratio after normalization with b‐actin. Data shown are the average of two biological replicates, with error bars representing SEM . ***Student's t test with P

    Techniques Used: Expressing, Quantitative RT-PCR

    45) Product Images from "Genomic organization of the crested ibis MHC provides new insight into ancestral avian MHC structure"

    Article Title: Genomic organization of the crested ibis MHC provides new insight into ancestral avian MHC structure

    Journal: Scientific Reports

    doi: 10.1038/srep07963

    Amino acid alignments (a) and expression levels (b) of Nini -MHC class I genes. (a) Dots and dashes indicate identities and gaps within the first sequence. Conserved features along sequences are represented as follows: intra- and interdomain contacts (light grey shading), intradomain disulfide bridges (wide bracket), N-glycosylation site (“n”), CD8 binding sites (“8”), essential CD8 co-receptor sites (*), phosphorylated sites in cytoplasmic (cyt)-tail (“p”), antigen-peptide main chain-binding sites (i.e., antigen-binding sites anchoring two ends of peptides) (dark grey shading; “a” or “f” for A or F pocket, respectively) 36 37 38 39 41 , antigen-peptide non-main chain-binding sites (“c”) (i.e., antigen-binding sites binding the middle segment of peptides) 37 . (b) Expression levels of the five genes in nine different tissues were examined by qRT-PCR and normalized to the housekeeping gene GAPDH . The relative expression level of the Nini -I genes was calculated using the 2 −ΔΔCT method 54 . The two nearly identical loci, UCA1 and UCA2 , were simultaneously amplified using a single set of primers, and the average values were presented herein as the potential expression levels of each gene.
    Figure Legend Snippet: Amino acid alignments (a) and expression levels (b) of Nini -MHC class I genes. (a) Dots and dashes indicate identities and gaps within the first sequence. Conserved features along sequences are represented as follows: intra- and interdomain contacts (light grey shading), intradomain disulfide bridges (wide bracket), N-glycosylation site (“n”), CD8 binding sites (“8”), essential CD8 co-receptor sites (*), phosphorylated sites in cytoplasmic (cyt)-tail (“p”), antigen-peptide main chain-binding sites (i.e., antigen-binding sites anchoring two ends of peptides) (dark grey shading; “a” or “f” for A or F pocket, respectively) 36 37 38 39 41 , antigen-peptide non-main chain-binding sites (“c”) (i.e., antigen-binding sites binding the middle segment of peptides) 37 . (b) Expression levels of the five genes in nine different tissues were examined by qRT-PCR and normalized to the housekeeping gene GAPDH . The relative expression level of the Nini -I genes was calculated using the 2 −ΔΔCT method 54 . The two nearly identical loci, UCA1 and UCA2 , were simultaneously amplified using a single set of primers, and the average values were presented herein as the potential expression levels of each gene.

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

    46) Product Images from "RNA editing of the AMD1 gene is important for ascus maturation and ascospore discharge in Fusarium graminearum"

    Article Title: RNA editing of the AMD1 gene is important for ascus maturation and ascospore discharge in Fusarium graminearum

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-04960-7

    Expression of AMD1 in late stages of sexual development. (A) The expression level of AMD1 transcripts was assayed by qRT-PCR with RNA isolated from 12 h YEPD cultures (Hyp; arbitrarily set to 1) and 8 days post-fertilization (dpf) perithecia (Peri). Mean and standard deviation were calculated with data from three independent replicates. ( B ) The abundance of AMD1 transcripts in different sexual stages based on RNA-seq data of mating cultures collected at 1–2 dpf and perithecia sampled at 3–8 dpf. FPKM: Fragments Per Kilobase of exon per Million fragments mapped.
    Figure Legend Snippet: Expression of AMD1 in late stages of sexual development. (A) The expression level of AMD1 transcripts was assayed by qRT-PCR with RNA isolated from 12 h YEPD cultures (Hyp; arbitrarily set to 1) and 8 days post-fertilization (dpf) perithecia (Peri). Mean and standard deviation were calculated with data from three independent replicates. ( B ) The abundance of AMD1 transcripts in different sexual stages based on RNA-seq data of mating cultures collected at 1–2 dpf and perithecia sampled at 3–8 dpf. FPKM: Fragments Per Kilobase of exon per Million fragments mapped.

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

    Similarity between Fgkin1 and amd1 mutants and reduced AMD1 expression in Fgkin1 . (A) The Fgkin1 and amd1 mutants had similar defects in ascospore discharge, ascus wall disintegration, and ascospore germination inside perithecia. Bar = 20 μm. ( B) The expression level of AMD1 was assayed by qRT-PCR with RNA isolated from 7 dpf perithecia of the wild-type PH-1 and Fgkin1 mutant. The expression level of AMD1 in PH-1 was arbitrarily set to 1. Mean and standard deviation were calculated with data from three independent replicates.
    Figure Legend Snippet: Similarity between Fgkin1 and amd1 mutants and reduced AMD1 expression in Fgkin1 . (A) The Fgkin1 and amd1 mutants had similar defects in ascospore discharge, ascus wall disintegration, and ascospore germination inside perithecia. Bar = 20 μm. ( B) The expression level of AMD1 was assayed by qRT-PCR with RNA isolated from 7 dpf perithecia of the wild-type PH-1 and Fgkin1 mutant. The expression level of AMD1 in PH-1 was arbitrarily set to 1. Mean and standard deviation were calculated with data from three independent replicates.

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

    47) Product Images from "The Lnc RNA SPRY4-IT1 Modulates Trophoblast Cell Invasion and Migration by Affecting the Epithelial-Mesenchymal Transition"

    Article Title: The Lnc RNA SPRY4-IT1 Modulates Trophoblast Cell Invasion and Migration by Affecting the Epithelial-Mesenchymal Transition

    Journal: Scientific Reports

    doi: 10.1038/srep37183

    SPRY4-IT1 modulates the WNT/β-catenin pathway. The relative expression of Wnt3 and Wnt5b mRNA increased 4.6-fold and 4.5-fold, respectively after overexpression of SPRY4-IT1 ( B ), whereas it decreased 58% and 39% after SPRY4-IT1 knockdown ( A ), as determined by qRT-PCR. ( C ) Western blot assay of Wnt3 and Wnt5b expression after SPRY4-IT1 was overexpressed or knocked down in HTR-8/SVneo cells. GAPDH protein is an internal control. The columns indicate the relative expression of Wnt3 and Wnt5b in HTR-8/SVneo cells transfected with si-SPRY4-IT1 ( D ) and pEGFP-SPRY4-IT1 ( E ) in the western blot assay. (Values are means ± SD *P
    Figure Legend Snippet: SPRY4-IT1 modulates the WNT/β-catenin pathway. The relative expression of Wnt3 and Wnt5b mRNA increased 4.6-fold and 4.5-fold, respectively after overexpression of SPRY4-IT1 ( B ), whereas it decreased 58% and 39% after SPRY4-IT1 knockdown ( A ), as determined by qRT-PCR. ( C ) Western blot assay of Wnt3 and Wnt5b expression after SPRY4-IT1 was overexpressed or knocked down in HTR-8/SVneo cells. GAPDH protein is an internal control. The columns indicate the relative expression of Wnt3 and Wnt5b in HTR-8/SVneo cells transfected with si-SPRY4-IT1 ( D ) and pEGFP-SPRY4-IT1 ( E ) in the western blot assay. (Values are means ± SD *P

    Techniques Used: Expressing, Over Expression, Quantitative RT-PCR, Western Blot, Transfection

    Inc RNA SPRY4-IT1 expression is increased in PE placentas. The relative expression of lncRNA SPRY4-IT1 was assessed by qRT-PCR using SYBR green and normalized to GAPDH. The levels of SPRY4-IT1 were lower in preeclamptic placentas (n = 50) than that in normal placentas (n = 50).
    Figure Legend Snippet: Inc RNA SPRY4-IT1 expression is increased in PE placentas. The relative expression of lncRNA SPRY4-IT1 was assessed by qRT-PCR using SYBR green and normalized to GAPDH. The levels of SPRY4-IT1 were lower in preeclamptic placentas (n = 50) than that in normal placentas (n = 50).

    Techniques Used: Expressing, Quantitative RT-PCR, SYBR Green Assay

    SPRY4-IT1 mediates expression of the key downstream target β-catenin through binding to HuR. ( A ) SPRY4-IT1 expression levels in different subcellular fractions in HTR-8/SVneo cells were detected by qRT-PCR. White range indicates the nuclear fraction, and the grey indicates the cytoplasmic fraction. ( B ) The relative expression of HuR decreased 65% and 81% after transfection of si-HuR. ( C ) The relative expression of β-catenin was significantly decreased in HTR-8/SVneo cells treated with HuR siRNA. ( D ) Western blot assay of β-catenin expression after HuR was knocked down in HTR-8/SVneo cells. GAPDH protein was used as an internal control. ( E ) The columns indicate the relative expression of β-catenin in HTR-8/SVneo cells transfected with si-HuR in the western blot assay. (Values are means ± SD *P
    Figure Legend Snippet: SPRY4-IT1 mediates expression of the key downstream target β-catenin through binding to HuR. ( A ) SPRY4-IT1 expression levels in different subcellular fractions in HTR-8/SVneo cells were detected by qRT-PCR. White range indicates the nuclear fraction, and the grey indicates the cytoplasmic fraction. ( B ) The relative expression of HuR decreased 65% and 81% after transfection of si-HuR. ( C ) The relative expression of β-catenin was significantly decreased in HTR-8/SVneo cells treated with HuR siRNA. ( D ) Western blot assay of β-catenin expression after HuR was knocked down in HTR-8/SVneo cells. GAPDH protein was used as an internal control. ( E ) The columns indicate the relative expression of β-catenin in HTR-8/SVneo cells transfected with si-HuR in the western blot assay. (Values are means ± SD *P

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

    48) Product Images from "Long non-coding RNA LOC100507600 functions as a competitive endogenous RNA to regulate BMI1 expression by sponging miR128-1-3p in Hirschsprung's disease"

    Article Title: Long non-coding RNA LOC100507600 functions as a competitive endogenous RNA to regulate BMI1 expression by sponging miR128-1-3p in Hirschsprung's disease

    Journal: Cell Cycle

    doi: 10.1080/15384101.2017.1403688

    Cytobiology change after treating cells with LOC100507600 siRNA. (A) Human 293T and SH-SY5Y cell lines were transfected with LOC100507600 siRNA and then qRT-PCR is repeated three times to determine the efficiency of transfection. (B) Human 293T and SH-SY5Y cell lines were transfected with LOC100507600 siRNA to regulate its expression levels and cell proliferation was detected using the CCK8 assay. Knockdown of LOC100507600 suppressed cell proliferation. (C) Transwell assay was performed as described in method and indicated that down-regulation of LOC100507600 delayed cell migration. Pictures were captured under a light microscope with the magnification, × 10. (D and E)Flow cytometry demonstrated that the down-regulation of LOC100507600 had no effect on cell cycle progression and apoptosis.
    Figure Legend Snippet: Cytobiology change after treating cells with LOC100507600 siRNA. (A) Human 293T and SH-SY5Y cell lines were transfected with LOC100507600 siRNA and then qRT-PCR is repeated three times to determine the efficiency of transfection. (B) Human 293T and SH-SY5Y cell lines were transfected with LOC100507600 siRNA to regulate its expression levels and cell proliferation was detected using the CCK8 assay. Knockdown of LOC100507600 suppressed cell proliferation. (C) Transwell assay was performed as described in method and indicated that down-regulation of LOC100507600 delayed cell migration. Pictures were captured under a light microscope with the magnification, × 10. (D and E)Flow cytometry demonstrated that the down-regulation of LOC100507600 had no effect on cell cycle progression and apoptosis.

    Techniques Used: Transfection, Quantitative RT-PCR, Expressing, CCK-8 Assay, Transwell Assay, Migration, Light Microscopy, Cytometry

    LOC100507600 serves as a sponge for miR128–1-3p. (A) The levels of nuclear control transcript (U6), cytoplasmic control transcript (GAPDH), and LOC100507600 were assessed by qRT-PCR in nuclear and cytoplasmic fractions. (B) The expression of miR128–1-3p in HSCR tissues and normal tissues. miR128–1-3p was significantly rose in patient tissues compared with normal tissues. (C) Superstratum:sequence alignment of human miR128–1-3p with LOC100507600. Bottom: mutations in the LOC100507600 sequence to create the mutant luciferase reporter constructs. (D) Luciferase reporter assay in 293T and SH-SY5Y cells after transfected with negative control or miR128–1-3p mimics, renilla luciferase vector pRL-SV40 and the reporter constructs. Both frefly and renilla luciferase activities are measured in the same sample. Firefly luciferase signals were normalized with renilla luciferase signals.
    Figure Legend Snippet: LOC100507600 serves as a sponge for miR128–1-3p. (A) The levels of nuclear control transcript (U6), cytoplasmic control transcript (GAPDH), and LOC100507600 were assessed by qRT-PCR in nuclear and cytoplasmic fractions. (B) The expression of miR128–1-3p in HSCR tissues and normal tissues. miR128–1-3p was significantly rose in patient tissues compared with normal tissues. (C) Superstratum:sequence alignment of human miR128–1-3p with LOC100507600. Bottom: mutations in the LOC100507600 sequence to create the mutant luciferase reporter constructs. (D) Luciferase reporter assay in 293T and SH-SY5Y cells after transfected with negative control or miR128–1-3p mimics, renilla luciferase vector pRL-SV40 and the reporter constructs. Both frefly and renilla luciferase activities are measured in the same sample. Firefly luciferase signals were normalized with renilla luciferase signals.

    Techniques Used: Quantitative RT-PCR, Expressing, Sequencing, Mutagenesis, Luciferase, Construct, Reporter Assay, Transfection, Negative Control, Plasmid Preparation

    49) Product Images from "OsRAMOSA2 Shapes Panicle Architecture through Regulating Pedicel Length"

    Article Title: OsRAMOSA2 Shapes Panicle Architecture through Regulating Pedicel Length

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2017.01538

    Possible relationships between OsRA2 and other genes. (A) qRT-PCR analyses of LAX1 in the ∼2 cm young panicles of pUbi::OsRA2 and dsRNAiOsRA2 plants. (B) qRT-PCR analyses of OsRA2 in the lax1 mutant. (C,D) PBs and pedicels of the dsRNAiOsRA2 plant, lax1 mutant, lax1 /dsRNAiOsRA2 plant, and ZH11. (E,F) Statistical analysis of the pedicel length and number of SBs in dsRNAiOsRA2 plants, lax1 mutants, lax1 /dsRNAiOsRA2 plants and ZH11. Values are means ± SE n = 15 panicles. Single and double asterisks represent significant difference determined by the Student’s t -test at ∗ P
    Figure Legend Snippet: Possible relationships between OsRA2 and other genes. (A) qRT-PCR analyses of LAX1 in the ∼2 cm young panicles of pUbi::OsRA2 and dsRNAiOsRA2 plants. (B) qRT-PCR analyses of OsRA2 in the lax1 mutant. (C,D) PBs and pedicels of the dsRNAiOsRA2 plant, lax1 mutant, lax1 /dsRNAiOsRA2 plant, and ZH11. (E,F) Statistical analysis of the pedicel length and number of SBs in dsRNAiOsRA2 plants, lax1 mutants, lax1 /dsRNAiOsRA2 plants and ZH11. Values are means ± SE n = 15 panicles. Single and double asterisks represent significant difference determined by the Student’s t -test at ∗ P

    Techniques Used: Quantitative RT-PCR, Mutagenesis

    50) Product Images from "Transcriptome Analysis Identifies Candidate Genes Related to Triacylglycerol and Pigment Biosynthesis and Photoperiodic Flowering in the Ornamental and Oil-Producing Plant, Camellia reticulata (Theaceae)"

    Article Title: Transcriptome Analysis Identifies Candidate Genes Related to Triacylglycerol and Pigment Biosynthesis and Photoperiodic Flowering in the Ornamental and Oil-Producing Plant, Camellia reticulata (Theaceae)

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2016.00163

    qRT-PCR validations of 11 putative genes involved in TAG biosynthesis . The histograms show the qRT-PCR results of accC (A) , ACC_Ho (B) , KAS II (C) , SAD_a (D) , FAD2_a (E) , FAD6 (F) , FAD8_a (G) , FATA (H) , DGAT1 (I) , DGAT2_a (J) , and PDAT_a (K) ; the line charts show the FPKM values of these genes. qRT-PCR results represent the mean (±SD) of three biological replicates. Gene abbreviations can be referenced in Supplementary Table S12 . LB, leaf buds; ML, mature leaves; FB, flower buds; FL, flowers; FR, immature fruits; SE, seeds.
    Figure Legend Snippet: qRT-PCR validations of 11 putative genes involved in TAG biosynthesis . The histograms show the qRT-PCR results of accC (A) , ACC_Ho (B) , KAS II (C) , SAD_a (D) , FAD2_a (E) , FAD6 (F) , FAD8_a (G) , FATA (H) , DGAT1 (I) , DGAT2_a (J) , and PDAT_a (K) ; the line charts show the FPKM values of these genes. qRT-PCR results represent the mean (±SD) of three biological replicates. Gene abbreviations can be referenced in Supplementary Table S12 . LB, leaf buds; ML, mature leaves; FB, flower buds; FL, flowers; FR, immature fruits; SE, seeds.

    Techniques Used: Quantitative RT-PCR

    qRT-PCR validations of 11 putative genes involved in flavonoid biosynthesis . The histograms show the qRT-PCR results of CHS_a (A) , ANS_a (B) , FLS_a (C) , FLS_b (D) , LAR_a (E) , LAR_b (F) , ANR_a (G) , UF3GT_a (H) , MYBPA1_a (I) , MYBF1 (J) , and MYBA1_a (K) ; the line charts show the FPKM values of these genes. qRT-PCR results represent the mean (± SD) of three biological replicates. Gene abbreviations can be referenced in Supplementary Table S13 . LB, leaf buds; ML, mature leaves; FB, flower buds; FL, flowers; FR, immature fruits; SE, seeds.
    Figure Legend Snippet: qRT-PCR validations of 11 putative genes involved in flavonoid biosynthesis . The histograms show the qRT-PCR results of CHS_a (A) , ANS_a (B) , FLS_a (C) , FLS_b (D) , LAR_a (E) , LAR_b (F) , ANR_a (G) , UF3GT_a (H) , MYBPA1_a (I) , MYBF1 (J) , and MYBA1_a (K) ; the line charts show the FPKM values of these genes. qRT-PCR results represent the mean (± SD) of three biological replicates. Gene abbreviations can be referenced in Supplementary Table S13 . LB, leaf buds; ML, mature leaves; FB, flower buds; FL, flowers; FR, immature fruits; SE, seeds.

    Techniques Used: Quantitative RT-PCR

    51) Product Images from "The ceramide pathway is involved in the survival, apoptosis and exosome functions of human multiple myeloma cells in vitro"

    Article Title: The ceramide pathway is involved in the survival, apoptosis and exosome functions of human multiple myeloma cells in vitro

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2017.118

    Ceramide pathway modulates the levels of several tumor-suppressive miRs in MM cells and their released exosomes. Samples of cell pellets and exosomes after C6-cer treated (A-D) and GW4869 treated (E-H) were prepared, RNA was extracted, and qRT-PCR analyses were performed. U6 small nuclear RNA (snRNA) was used as internal controls. Data were shown as the mean±SEM. * P
    Figure Legend Snippet: Ceramide pathway modulates the levels of several tumor-suppressive miRs in MM cells and their released exosomes. Samples of cell pellets and exosomes after C6-cer treated (A-D) and GW4869 treated (E-H) were prepared, RNA was extracted, and qRT-PCR analyses were performed. U6 small nuclear RNA (snRNA) was used as internal controls. Data were shown as the mean±SEM. * P

    Techniques Used: Quantitative RT-PCR

    The effect of ceramide pathway on the exosome functions and miR levels of targeting MM cells. (A) Markers for MM exosomes that were examined by using Western blot. (B) The effects of exosomes C6-cer and exosomes GW4869 on MM cell growth; (C) The effect of exosomes C6-cer or exosomes GW4869 on MM apoptosis by using Hoechst 33342 stain assay; (D) Expressions of caspase pathway after the treatment of exosomes C6-cer or exosomes GW4869 ; (E, F) The levels of miR 202, miR 16, miR 29b and miR 15a were measured by qRT-PCR in MM cells treated by exosomes C6-cer or exosomes GW4869 . * P
    Figure Legend Snippet: The effect of ceramide pathway on the exosome functions and miR levels of targeting MM cells. (A) Markers for MM exosomes that were examined by using Western blot. (B) The effects of exosomes C6-cer and exosomes GW4869 on MM cell growth; (C) The effect of exosomes C6-cer or exosomes GW4869 on MM apoptosis by using Hoechst 33342 stain assay; (D) Expressions of caspase pathway after the treatment of exosomes C6-cer or exosomes GW4869 ; (E, F) The levels of miR 202, miR 16, miR 29b and miR 15a were measured by qRT-PCR in MM cells treated by exosomes C6-cer or exosomes GW4869 . * P

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

    52) Product Images from "Intestine-Specific Mttp Deletion Decreases Mortality and Prevents Sepsis-Induced Intestinal Injury in a Murine Model of Pseudomonas aeruginosa Pneumonia"

    Article Title: Intestine-Specific Mttp Deletion Decreases Mortality and Prevents Sepsis-Induced Intestinal Injury in a Murine Model of Pseudomonas aeruginosa Pneumonia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0049159

    Effect of impaired intestinal lipid transport on serum lipoproteins. Lipoprotein distribution measured by fast protein liquid chromatography in control (A) and Mttp-IKO (B) mice. Pooled samples of serum from n = 5–10 mice per genotype were analyzed in sham animals and both before and 24 hr after induction of pneumonia in the experimental groups. Cholesterol was assayed enzymatically and peaks corresponding to fractions 10–16 indicate particles in the low density lipoprotein (LDL) range while fractions 19–26 correspond to high density lipoprotein (HDL). (C) Expression of genes implicated in hepatic cholesterol efflux were analyzed by qRT-PCR on samples of RNA from the indicated groups (n = 4 mice per genotype and treatment) (D) and (E). Expression of hepatic Abca1, apoA1 and apoA4 protein by SDS-PAGE and western blot. Gapdh was used as loading control. Panel D shows representative Western blotting results. Panel E shows densitometric scanning from groups of 4 mice per genotype and treatment. (F) Expression of genes implicated in intestinal lipid metabolism were analyzed by qRT-PCR on samples of small intestinal RNA from the indicated groups (n = 4 mice per genotype and treatment). *p
    Figure Legend Snippet: Effect of impaired intestinal lipid transport on serum lipoproteins. Lipoprotein distribution measured by fast protein liquid chromatography in control (A) and Mttp-IKO (B) mice. Pooled samples of serum from n = 5–10 mice per genotype were analyzed in sham animals and both before and 24 hr after induction of pneumonia in the experimental groups. Cholesterol was assayed enzymatically and peaks corresponding to fractions 10–16 indicate particles in the low density lipoprotein (LDL) range while fractions 19–26 correspond to high density lipoprotein (HDL). (C) Expression of genes implicated in hepatic cholesterol efflux were analyzed by qRT-PCR on samples of RNA from the indicated groups (n = 4 mice per genotype and treatment) (D) and (E). Expression of hepatic Abca1, apoA1 and apoA4 protein by SDS-PAGE and western blot. Gapdh was used as loading control. Panel D shows representative Western blotting results. Panel E shows densitometric scanning from groups of 4 mice per genotype and treatment. (F) Expression of genes implicated in intestinal lipid metabolism were analyzed by qRT-PCR on samples of small intestinal RNA from the indicated groups (n = 4 mice per genotype and treatment). *p

    Techniques Used: Fast Protein Liquid Chromatography, Mouse Assay, Expressing, Quantitative RT-PCR, SDS Page, Western Blot

    53) Product Images from "Transcriptomics of Maternal and Fetal Membranes Can Discriminate between Gestational-Age Matched Preterm Neonates with and without Cognitive Impairment Diagnosed at 18–24 Months"

    Article Title: Transcriptomics of Maternal and Fetal Membranes Can Discriminate between Gestational-Age Matched Preterm Neonates with and without Cognitive Impairment Diagnosed at 18–24 Months

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0118573

    qRT-PCR study flow diagram. Differentially expressed genes identified from the microarray study were profiled using qRT-PCR for validation and construction of a multi-gene disease classifier using an extended set of 33 cases and 33 controls. Set A was used to build the prediction model and then this model was tested on set B, consisting of 19 cases and 19 controls.
    Figure Legend Snippet: qRT-PCR study flow diagram. Differentially expressed genes identified from the microarray study were profiled using qRT-PCR for validation and construction of a multi-gene disease classifier using an extended set of 33 cases and 33 controls. Set A was used to build the prediction model and then this model was tested on set B, consisting of 19 cases and 19 controls.

    Techniques Used: Quantitative RT-PCR, Flow Cytometry, Microarray

    Gene expression-based classifier for neurocognitive impairment using OSR1/VWF and HAND1/VWF expression ratios. The Fig. shows the linear discriminant model (see oblique black line) built using qRT-PCR measured expression data from the training set. Since −Ct values are surrogate for log2 gene abundance, differences in −Ct values of two genes is equivalent to their log 2 expression ratios. Data are represented as log2 expression ratios (y-axis: −Ct HAND1 +Ct VWF ; x-axis: −Ct OSR1 +Ct VWF ). The dots represent data from patients from the test set. The model was tuned on the training data to yield a specificity of ∼85%. The actual performance on the test set was sensitivity of 74%, at specificity of 83%.
    Figure Legend Snippet: Gene expression-based classifier for neurocognitive impairment using OSR1/VWF and HAND1/VWF expression ratios. The Fig. shows the linear discriminant model (see oblique black line) built using qRT-PCR measured expression data from the training set. Since −Ct values are surrogate for log2 gene abundance, differences in −Ct values of two genes is equivalent to their log 2 expression ratios. Data are represented as log2 expression ratios (y-axis: −Ct HAND1 +Ct VWF ; x-axis: −Ct OSR1 +Ct VWF ). The dots represent data from patients from the test set. The model was tuned on the training data to yield a specificity of ∼85%. The actual performance on the test set was sensitivity of 74%, at specificity of 83%.

    Techniques Used: Expressing, Quantitative RT-PCR

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky279

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

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

    55) Product Images from "IL-22 Is Expressed by the Invasive Trophoblast of the Equine (Equus caballus) Chorionic Girdle"

    Article Title: IL-22 Is Expressed by the Invasive Trophoblast of the Equine (Equus caballus) Chorionic Girdle

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

    doi: 10.4049/jimmunol.1103509

    IL-22R1 expression in equine tissues. IL-22RA1 expression assessed by qRT-PCR in 3-d 34 equine conceptuses ( A ) and in other equine epithelial tissues ( B ). Epithelial samples included endometrium from days 32 ( n = 2) and 34 ( n = 8) of pregnancy, skin (
    Figure Legend Snippet: IL-22R1 expression in equine tissues. IL-22RA1 expression assessed by qRT-PCR in 3-d 34 equine conceptuses ( A ) and in other equine epithelial tissues ( B ). Epithelial samples included endometrium from days 32 ( n = 2) and 34 ( n = 8) of pregnancy, skin (

    Techniques Used: Expressing, Quantitative RT-PCR

    qRT-PCR assays
    Figure Legend Snippet: qRT-PCR assays

    Techniques Used: Quantitative RT-PCR

    56) Product Images from "The lncRNA CRNDE promotes colorectal cancer cell proliferation and chemoresistance via miR-181a-5p-mediated regulation of Wnt/β-catenin signaling"

    Article Title: The lncRNA CRNDE promotes colorectal cancer cell proliferation and chemoresistance via miR-181a-5p-mediated regulation of Wnt/β-catenin signaling

    Journal: Molecular Cancer

    doi: 10.1186/s12943-017-0583-1

    CRNDE knockdown and miR-181a-5p overexpression repress CRC cell chemoresistance. a MTT cell proliferation assay performed in HCT116 and SW480 cells transfected with siRNA targeting CRNDE or a control siRNA and treated with the indicated concentrations of 5-Fu. b MTT cell proliferation assay performed in HCT116 and SW480 cells transfected with miR-181a-5p or a control microRNA and treated with the indicated concentrations of 5-Fu. c MTT cell proliferation assay performed in HCT116 and SW480 cells transfected with siR-CRNDE or a control siRNA and treated with the indicated concentrations of Oxa. d MTT cell proliferation assay performed in HCT116 and SW480 cells transfected with miR-181a-5p or miR-control and treated with the indicated concentrations of Oxa. e Expression levels of CRNDE and f miR-181a-5p as determined by qRT-PCR in 5-Fu resistant HCT116 and SW480 cells. * P
    Figure Legend Snippet: CRNDE knockdown and miR-181a-5p overexpression repress CRC cell chemoresistance. a MTT cell proliferation assay performed in HCT116 and SW480 cells transfected with siRNA targeting CRNDE or a control siRNA and treated with the indicated concentrations of 5-Fu. b MTT cell proliferation assay performed in HCT116 and SW480 cells transfected with miR-181a-5p or a control microRNA and treated with the indicated concentrations of 5-Fu. c MTT cell proliferation assay performed in HCT116 and SW480 cells transfected with siR-CRNDE or a control siRNA and treated with the indicated concentrations of Oxa. d MTT cell proliferation assay performed in HCT116 and SW480 cells transfected with miR-181a-5p or miR-control and treated with the indicated concentrations of Oxa. e Expression levels of CRNDE and f miR-181a-5p as determined by qRT-PCR in 5-Fu resistant HCT116 and SW480 cells. * P

    Techniques Used: Over Expression, MTT Assay, Proliferation Assay, Transfection, Expressing, Quantitative RT-PCR

    57) Product Images from "Short-term retinoic acid treatment sustains pluripotency and suppresses differentiation of human induced pluripotent stem cells"

    Article Title: Short-term retinoic acid treatment sustains pluripotency and suppresses differentiation of human induced pluripotent stem cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0028-1

    Effects of short-term retinoic acid treatment on pluripotency a hiPSCs were cultured in regular medium (mTeSR1) for 5 days before ready for passaging, while hiPSCs-RA were treated with RA for 24 h 2 days after splitting. b qRT-PCR analysis of pluripotency and differentiation markers in hiPSCs (-F and -TL) cultured in regular medium and in medium supplemented with 0.5 µM RA at day 4. All expression values are normalized to GAPDH and relative to untreated hiPSCs. Data are mean ± SEM from three independent experiments. A statistical comparison was made between hiPSCs-RA and hiPSCs by Student’s t -test (* p
    Figure Legend Snippet: Effects of short-term retinoic acid treatment on pluripotency a hiPSCs were cultured in regular medium (mTeSR1) for 5 days before ready for passaging, while hiPSCs-RA were treated with RA for 24 h 2 days after splitting. b qRT-PCR analysis of pluripotency and differentiation markers in hiPSCs (-F and -TL) cultured in regular medium and in medium supplemented with 0.5 µM RA at day 4. All expression values are normalized to GAPDH and relative to untreated hiPSCs. Data are mean ± SEM from three independent experiments. A statistical comparison was made between hiPSCs-RA and hiPSCs by Student’s t -test (* p

    Techniques Used: Cell Culture, Passaging, Quantitative RT-PCR, Expressing

    In vitro differentiation capacity of RA 8 -treated hPSCs a Representative image of EBs formation capability of RA-treated and untreated hiPSCs-F and -TL. Scale bar, 500 μm. b Diagram indicating the average diameter of the EBs, 100 μm. c The efficiency of EBs formation with diameter range of 100–300 µm is higher in hiPSCs-RA 8 compared to untreated cells, while the percentage of EBs with diameter range of 50–100 μm is comparable between hiPSCs-RA 8 and hiPSCs. d The potential of hiPSCs-RA 8 to differentiate into cell of all three germ layers by expression analysis of HAND1 , NESTIN , and SOX17 , the gene expression levels in EBs hiPSCs and EBs hiPSCs-RA 8 were relative to hiPSCs. qRT-PCR data are represented as the mean ± SEM from three independent experiments. A statistical comparison was made between EBs hiPSCs-RA 8 and EBs hiPSCs-RA by Student’s t -test (** p
    Figure Legend Snippet: In vitro differentiation capacity of RA 8 -treated hPSCs a Representative image of EBs formation capability of RA-treated and untreated hiPSCs-F and -TL. Scale bar, 500 μm. b Diagram indicating the average diameter of the EBs, 100 μm. c The efficiency of EBs formation with diameter range of 100–300 µm is higher in hiPSCs-RA 8 compared to untreated cells, while the percentage of EBs with diameter range of 50–100 μm is comparable between hiPSCs-RA 8 and hiPSCs. d The potential of hiPSCs-RA 8 to differentiate into cell of all three germ layers by expression analysis of HAND1 , NESTIN , and SOX17 , the gene expression levels in EBs hiPSCs and EBs hiPSCs-RA 8 were relative to hiPSCs. qRT-PCR data are represented as the mean ± SEM from three independent experiments. A statistical comparison was made between EBs hiPSCs-RA 8 and EBs hiPSCs-RA by Student’s t -test (** p

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

    58) Product Images from "Fnr and ArcA Regulate Lipid A Hydroxylation in Salmonella Enteritidis by Controlling lpxO Expression in Response to Oxygen Availability"

    Article Title: Fnr and ArcA Regulate Lipid A Hydroxylation in Salmonella Enteritidis by Controlling lpxO Expression in Response to Oxygen Availability

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01220

    Expression of lpxO in S . Enteritidis strains. Expression levels were determined by qRT-PCR and normalized using rpoD as housekeeping gene (A) or by measuring β-galactosidase activity using strains carrying an lpxO – lacZ transcriptional fusion (B) . Bars represent mean values from three independent replicates. Error bars denote standard deviation. Statistical significance of observed differences was determined using a two-tailed Student’s t -test ( ∗ p
    Figure Legend Snippet: Expression of lpxO in S . Enteritidis strains. Expression levels were determined by qRT-PCR and normalized using rpoD as housekeeping gene (A) or by measuring β-galactosidase activity using strains carrying an lpxO – lacZ transcriptional fusion (B) . Bars represent mean values from three independent replicates. Error bars denote standard deviation. Statistical significance of observed differences was determined using a two-tailed Student’s t -test ( ∗ p

    Techniques Used: Expressing, Quantitative RT-PCR, Activity Assay, Standard Deviation, Two Tailed Test

    59) Product Images from "Pax3 Gene Regulated Melanin Synthesis by Tyrosinase Pathway in Pteria penguin"

    Article Title: Pax3 Gene Regulated Melanin Synthesis by Tyrosinase Pathway in Pteria penguin

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19123700

    Relative expression of PpPax3 in various tissues of P. penguin estimated by qRT-PCR. Each bar was a mean of 6 pearl oysters. Error bars were the SD. Different letters (a, b, c and d) meaned significant difference ( P
    Figure Legend Snippet: Relative expression of PpPax3 in various tissues of P. penguin estimated by qRT-PCR. Each bar was a mean of 6 pearl oysters. Error bars were the SD. Different letters (a, b, c and d) meaned significant difference ( P

    Techniques Used: Expressing, Quantitative RT-PCR

    Expression of PpPax3, PpMitf, PpTyr, PpCreb2, PpBcl2 and PpCdk2 after PpPax3 RNA interference (RNAi). ( A ) Expression of PpPax3 . ( B ) Expression of PpMitf, PpTyr, PpCreb2, PpBcl2 and PpCdk2 . The qRT-PCR was done with RNA samples from blank group (RNase-free water), NC group (GFP-siRNA), PpPax3 -siRNA1 group and PpPax3 -siRNA2 group. The β-actin of P. penguin was used as an internal control. Each bar was a mean of 6 individuals. Significant difference was indicated by * ( P
    Figure Legend Snippet: Expression of PpPax3, PpMitf, PpTyr, PpCreb2, PpBcl2 and PpCdk2 after PpPax3 RNA interference (RNAi). ( A ) Expression of PpPax3 . ( B ) Expression of PpMitf, PpTyr, PpCreb2, PpBcl2 and PpCdk2 . The qRT-PCR was done with RNA samples from blank group (RNase-free water), NC group (GFP-siRNA), PpPax3 -siRNA1 group and PpPax3 -siRNA2 group. The β-actin of P. penguin was used as an internal control. Each bar was a mean of 6 individuals. Significant difference was indicated by * ( P

    Techniques Used: Expressing, Quantitative RT-PCR

    60) Product Images from "Effects of yeast trehalose-6-phosphate synthase 1 on gene expression and carbohydrate contents of potato leaves under drought stress conditions"

    Article Title: Effects of yeast trehalose-6-phosphate synthase 1 on gene expression and carbohydrate contents of potato leaves under drought stress conditions

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-12-74

    Linear regression analysis between qRT-PCR data obtained from the independent TPS1 transgenic lines T1 and T2. Log2 ratios are presented on both axes. The following genes were analysed: ribulose bisphosphate carboxylase small chain 2B, fructose bisphosphate aldolase, ETHYLENE-INSENSITIVE3-like 1 transcription factor, a bZIP transcription factor family protein, EMBRYO DEFECTIVE 2220 transcription factor, a plant homeodomain finger family protein, a universal stress protein, and StDS2 , a drought-inducible potato gene.
    Figure Legend Snippet: Linear regression analysis between qRT-PCR data obtained from the independent TPS1 transgenic lines T1 and T2. Log2 ratios are presented on both axes. The following genes were analysed: ribulose bisphosphate carboxylase small chain 2B, fructose bisphosphate aldolase, ETHYLENE-INSENSITIVE3-like 1 transcription factor, a bZIP transcription factor family protein, EMBRYO DEFECTIVE 2220 transcription factor, a plant homeodomain finger family protein, a universal stress protein, and StDS2 , a drought-inducible potato gene.

    Techniques Used: Quantitative RT-PCR, Transgenic Assay

    61) Product Images from "FOXC1 modulates MYOC secretion through regulation of the exocytic proteins RAB3GAP1, RAB3GAP2 and SNAP25"

    Article Title: FOXC1 modulates MYOC secretion through regulation of the exocytic proteins RAB3GAP1, RAB3GAP2 and SNAP25

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0178518

    FOXC1 knockdown decreases RNA levels of RAB3GAP1 , RAB3GAP2 and SNAP25 . qRT-PCR experiments using RNA isolated from HeLa cells transfected with scrambled siRNA. qPCR was used to evaluate changes in RNA levels of A) FOXC1 B) RAB3GAP1 C) RAB3GAP2 , D) Total SNAP25 ( SNAP25ab ) E) SNAP25 isoform a ( SNAP25a ), F) SNAP25 isoform b ( SNAP25b ) and HPRT1 (housekeeping gene control). Fold change in RNA levels was calculated using the ΔΔCt method normalized to HPRT1 and scaled to siRNA scrambled control. Experiments were performed three times in triplicate. Error bars represent standard error. * P ˂0.05.
    Figure Legend Snippet: FOXC1 knockdown decreases RNA levels of RAB3GAP1 , RAB3GAP2 and SNAP25 . qRT-PCR experiments using RNA isolated from HeLa cells transfected with scrambled siRNA. qPCR was used to evaluate changes in RNA levels of A) FOXC1 B) RAB3GAP1 C) RAB3GAP2 , D) Total SNAP25 ( SNAP25ab ) E) SNAP25 isoform a ( SNAP25a ), F) SNAP25 isoform b ( SNAP25b ) and HPRT1 (housekeeping gene control). Fold change in RNA levels was calculated using the ΔΔCt method normalized to HPRT1 and scaled to siRNA scrambled control. Experiments were performed three times in triplicate. Error bars represent standard error. * P ˂0.05.

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

    62) Product Images from "The tomato RLK superfamily: phylogeny and functional predictions about the role of the LRRII-RLK subfamily in antiviral defense"

    Article Title: The tomato RLK superfamily: phylogeny and functional predictions about the role of the LRRII-RLK subfamily in antiviral defense

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-12-229

    Phylogenetic and expression analysis of LRRII subfamily members. ( A ) Phylogenetic tree reconstructed by the maximum likelihood method (JTT+G+I, bootstrap replicates = 1000) of the LRRII subfamily. Members of this subfamily can be separated in three well-supported clades, referred to here as SERK, NIK and LRRIIc clades. Expression analysis of LRRII subfamily members in ( B ) tomato and ( C ) Arabidopsis in different plant tissues. The expression data of tomato and Arabidopsis members were obtained by qRT-PCR and from normalized data from the AtGenExpress database [ 55 ], respectively. No expression data were obtained for AtSERK5 (At2g13800). ( D ) Tissues with high mean-expression are summarized for each gene. Orthologous genes that had similar expression profiles are delimited by brackets.
    Figure Legend Snippet: Phylogenetic and expression analysis of LRRII subfamily members. ( A ) Phylogenetic tree reconstructed by the maximum likelihood method (JTT+G+I, bootstrap replicates = 1000) of the LRRII subfamily. Members of this subfamily can be separated in three well-supported clades, referred to here as SERK, NIK and LRRIIc clades. Expression analysis of LRRII subfamily members in ( B ) tomato and ( C ) Arabidopsis in different plant tissues. The expression data of tomato and Arabidopsis members were obtained by qRT-PCR and from normalized data from the AtGenExpress database [ 55 ], respectively. No expression data were obtained for AtSERK5 (At2g13800). ( D ) Tissues with high mean-expression are summarized for each gene. Orthologous genes that had similar expression profiles are delimited by brackets.

    Techniques Used: Expressing, Quantitative RT-PCR

    Expression analyses of tomato members of the LRRII subfamily in various plant organs by qRT-PCR. Expression of each gene was quantified using SlAPT1 as an endogenous control. Bars represent the mean expression from three biological samples and two replicates, except for the flower and hypocotyl samples, for which two biological samples and two replicates were used. Error bars represent a confidence interval of 95%.
    Figure Legend Snippet: Expression analyses of tomato members of the LRRII subfamily in various plant organs by qRT-PCR. Expression of each gene was quantified using SlAPT1 as an endogenous control. Bars represent the mean expression from three biological samples and two replicates, except for the flower and hypocotyl samples, for which two biological samples and two replicates were used. Error bars represent a confidence interval of 95%.

    Techniques Used: Expressing, Quantitative RT-PCR

    63) Product Images from "Transcriptomic de novo analysis of pitaya (Hylocereus polyrhizus) canker disease caused by Neoscytalidium dimidiatum"

    Article Title: Transcriptomic de novo analysis of pitaya (Hylocereus polyrhizus) canker disease caused by Neoscytalidium dimidiatum

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-5343-0

    Validation of gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Gene expression levels were measured by qRT-PCR and compared with RNA-Seq results. Histograms represent the fold changes of genes (D3/N3) by qRT-PCR, whereas line charts represent gene expression according to the log2 ratio (fragments per kilobase of transcript per million mapped reads (FPKM) of D/FPKM of N) in RNA-Seq. All genes selected for qRT-PCR analysis were analyzed in three biological replicates. Bars represent the ± SEΔCт of three experiments. Details of the genes selected for qRT-PCR are provided in Table 3
    Figure Legend Snippet: Validation of gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Gene expression levels were measured by qRT-PCR and compared with RNA-Seq results. Histograms represent the fold changes of genes (D3/N3) by qRT-PCR, whereas line charts represent gene expression according to the log2 ratio (fragments per kilobase of transcript per million mapped reads (FPKM) of D/FPKM of N) in RNA-Seq. All genes selected for qRT-PCR analysis were analyzed in three biological replicates. Bars represent the ± SEΔCт of three experiments. Details of the genes selected for qRT-PCR are provided in Table 3

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

    64) Product Images from "SLC3A2 is upregulated in human osteosarcoma and promotes tumor growth through the PI3K/Akt signaling pathway"

    Article Title: SLC3A2 is upregulated in human osteosarcoma and promotes tumor growth through the PI3K/Akt signaling pathway

    Journal: Oncology Reports

    doi: 10.3892/or.2017.5530

    SLC3A2 expression is upregulated in OS clinical samples and cell lines. (A and B) The mRNA and protein expression levels of SLC3A2 were measured by TaqMan real-time PCR and western blot assays in OS cell lines (including MNNG/HOS, MG63 and U2OS) and a human osteoblastic cell line (hFOB). (C and D) Relative expression of SLC3A2 was detected using qRT-PCR in 20 pairs of OS samples and their corresponding non-cancerous samples. Statistical analysis was performed using paired t-test in C.
    Figure Legend Snippet: SLC3A2 expression is upregulated in OS clinical samples and cell lines. (A and B) The mRNA and protein expression levels of SLC3A2 were measured by TaqMan real-time PCR and western blot assays in OS cell lines (including MNNG/HOS, MG63 and U2OS) and a human osteoblastic cell line (hFOB). (C and D) Relative expression of SLC3A2 was detected using qRT-PCR in 20 pairs of OS samples and their corresponding non-cancerous samples. Statistical analysis was performed using paired t-test in C.

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

    65) Product Images from "A FoxO-Smad synexpression group in human keratinocytes"

    Article Title: A FoxO-Smad synexpression group in human keratinocytes

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

    doi: 10.1073/pnas.0605333103

    Variability in promoter configuration and regulation in the FoxO–Smad synexpression group. ( A ) A graphic comparison of the promoter regions of the FoxO–Smad synexpression genes is shown. The conserved FHBEs (green) surrounded by SBEs (orange) within 100 nt and the C/EBPβ binding elements (C/EBPβBE, blue) identified by computational sequence analysis are indicated. The track (black) indicates mouse sequence segments that are similar to its human ortholog. Percent identities of the segments ranged from 75% to 100%. Shaded boxes (orange) identify the functionally validated TGF-β responsive promoter regions. ( B ) Venn diagram depicting the overlap between FoxO-dependent and C/EBPβ-dependent TGF-β gene responses in HaCaT keratinocytes. ( C ) Effect of TGF-β on FoxO–Smad responsive genes in control and C/EBPβ-depleted HaCaT cells. Cells were incubated with or without TGF-β for 3 h. qRT-PCR was used to measure mRNA levels of the indicated genes. The fold increase of these levels by TGF-β is indicated. Data are mean ± SD ( n = 3) of three independent experiments.
    Figure Legend Snippet: Variability in promoter configuration and regulation in the FoxO–Smad synexpression group. ( A ) A graphic comparison of the promoter regions of the FoxO–Smad synexpression genes is shown. The conserved FHBEs (green) surrounded by SBEs (orange) within 100 nt and the C/EBPβ binding elements (C/EBPβBE, blue) identified by computational sequence analysis are indicated. The track (black) indicates mouse sequence segments that are similar to its human ortholog. Percent identities of the segments ranged from 75% to 100%. Shaded boxes (orange) identify the functionally validated TGF-β responsive promoter regions. ( B ) Venn diagram depicting the overlap between FoxO-dependent and C/EBPβ-dependent TGF-β gene responses in HaCaT keratinocytes. ( C ) Effect of TGF-β on FoxO–Smad responsive genes in control and C/EBPβ-depleted HaCaT cells. Cells were incubated with or without TGF-β for 3 h. qRT-PCR was used to measure mRNA levels of the indicated genes. The fold increase of these levels by TGF-β is indicated. Data are mean ± SD ( n = 3) of three independent experiments.

    Techniques Used: Binding Assay, Sequencing, Incubation, Quantitative RT-PCR

    FoxO–Smad-dependent TGF-β gene responses. ( A ) Control and Smad4-depleted HaCaT cells were incubated with TGF-β for 3 h, and then total RNA was subjected to Affymetrix analysis with the U133-A microarray. The heat map plot represents the expression level of genes identified as members of the FoxO–Smad synexpression group in response to TGF-β and the levels of Smads, FoxOs, and C/EBPβ. ( B ) Control and Smad4-depleted HaCaT cells were incubated with or without TGF-β for 3 h. qRT-PCR was used to measure RNA levels of the indicated genes. The fold increase in mRNA level is indicated. Data are mean ± SD of three independent experiments.
    Figure Legend Snippet: FoxO–Smad-dependent TGF-β gene responses. ( A ) Control and Smad4-depleted HaCaT cells were incubated with TGF-β for 3 h, and then total RNA was subjected to Affymetrix analysis with the U133-A microarray. The heat map plot represents the expression level of genes identified as members of the FoxO–Smad synexpression group in response to TGF-β and the levels of Smads, FoxOs, and C/EBPβ. ( B ) Control and Smad4-depleted HaCaT cells were incubated with or without TGF-β for 3 h. qRT-PCR was used to measure RNA levels of the indicated genes. The fold increase in mRNA level is indicated. Data are mean ± SD of three independent experiments.

    Techniques Used: Incubation, Microarray, Expressing, Quantitative RT-PCR

    66) Product Images from "Carbon dioxide receptor genes and their expression profile in Diabrotica virgifera virgifera"

    Article Title: Carbon dioxide receptor genes and their expression profile in Diabrotica virgifera virgifera

    Journal: BMC Research Notes

    doi: 10.1186/s13104-015-1794-4

    Expression of CO 2 receptors ( a Dvv_Gr1, b Dvv_Gr3, c Dvv_Gr2) in different development stages of Diabrotica v. virgifera . For qRT-PCR, relative expression of Dvv_Gr genes in different stages was measured and normalized to an endogenous control (actin) as described in the “ Methods ” section. Values represent the means and the standard deviation of three analytical replicates on samples that contain tissue from five 3rd instar larvae. Different letters above the bars reflect significantly different expression levels (ANOVA of Tukey Test, P
    Figure Legend Snippet: Expression of CO 2 receptors ( a Dvv_Gr1, b Dvv_Gr3, c Dvv_Gr2) in different development stages of Diabrotica v. virgifera . For qRT-PCR, relative expression of Dvv_Gr genes in different stages was measured and normalized to an endogenous control (actin) as described in the “ Methods ” section. Values represent the means and the standard deviation of three analytical replicates on samples that contain tissue from five 3rd instar larvae. Different letters above the bars reflect significantly different expression levels (ANOVA of Tukey Test, P

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

    Expression of CO 2 receptors ( a Dvv_Gr1, b Dvv_Gr3, c Dvv_Gr2) in different tissues of Diabrotica v. virgifera . For qRT-PCR, relative expression of Dvv_Gr genes in different tissues was measured and normalized to an endogenous control (EF1a) as described in the “ Methods ” section. Values represent the means and the standard deviation of three analytical replicates on samples that contain tissue from five 3rd instar larvae. Different letters above the bars reflect significantly different expression levels (ANOVA of Tukey Test, P
    Figure Legend Snippet: Expression of CO 2 receptors ( a Dvv_Gr1, b Dvv_Gr3, c Dvv_Gr2) in different tissues of Diabrotica v. virgifera . For qRT-PCR, relative expression of Dvv_Gr genes in different tissues was measured and normalized to an endogenous control (EF1a) as described in the “ Methods ” section. Values represent the means and the standard deviation of three analytical replicates on samples that contain tissue from five 3rd instar larvae. Different letters above the bars reflect significantly different expression levels (ANOVA of Tukey Test, P

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

    67) Product Images from "The Rescue of miR-148a Expression in Pancreatic Cancer: An Inappropriate Therapeutic Tool"

    Article Title: The Rescue of miR-148a Expression in Pancreatic Cancer: An Inappropriate Therapeutic Tool

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0055513

    Gemcitabine sensitivity of PDAC cell lines over-expressing miR-148a. ( A ) miR-148a endogenous expression level was measured by qRT-PCR in several PDAC cell lines and in hPDE and hPNE normal pancreatic cell lines. ( B ) Cell sensitivity to gemcitabine was measured after a 72 h-treatment in several PDAC cell lines and in normal hPNE pancreatic cells. Surviving cell fraction represents the number of treated cells during 72 h compared to the number of untreated cells (represented as 100%). The lethal concentration 50 (LC50) represents the dose of gemcitabine sufficient to obtain 50% of surviving cells compared to untreated cells. The results are the mean of three independent experiments (±SEM). ( C ) Cell sensitivity to gemcitabine was measured in MIA PaCa-2 cells stably over-expressing miR-148a (LV-TO-miR-148a) or a control miR (LV-TO-miR-CT) to gemcitabine in presence of doxycycline after a 72 h-treatment. Surviving cell fraction represents the number of treated cells during 72 h compared to the number of untreated cells (represented as 100%). The results are the mean of three independent experiments (±SEM).
    Figure Legend Snippet: Gemcitabine sensitivity of PDAC cell lines over-expressing miR-148a. ( A ) miR-148a endogenous expression level was measured by qRT-PCR in several PDAC cell lines and in hPDE and hPNE normal pancreatic cell lines. ( B ) Cell sensitivity to gemcitabine was measured after a 72 h-treatment in several PDAC cell lines and in normal hPNE pancreatic cells. Surviving cell fraction represents the number of treated cells during 72 h compared to the number of untreated cells (represented as 100%). The lethal concentration 50 (LC50) represents the dose of gemcitabine sufficient to obtain 50% of surviving cells compared to untreated cells. The results are the mean of three independent experiments (±SEM). ( C ) Cell sensitivity to gemcitabine was measured in MIA PaCa-2 cells stably over-expressing miR-148a (LV-TO-miR-148a) or a control miR (LV-TO-miR-CT) to gemcitabine in presence of doxycycline after a 72 h-treatment. Surviving cell fraction represents the number of treated cells during 72 h compared to the number of untreated cells (represented as 100%). The results are the mean of three independent experiments (±SEM).

    Techniques Used: Expressing, Quantitative RT-PCR, Concentration Assay, Stable Transfection

    68) Product Images from "Epigenetic Downregulation and Growth Inhibition of IGFBP7 in Gastric Cancer"

    Article Title: Epigenetic Downregulation and Growth Inhibition of IGFBP7 in Gastric Cancer

    Journal: Asian Pacific Journal of Cancer Prevention : APJCP

    doi: 10.22034/APJCP.2018.19.3.667

    Inhibition of IGFBP7 Expression Promotes Gastric Cancer Cell Growth. (A) Knock-down of IGFBP7 was determined using western blot and qRT-PCR. (B) Inhibition of IGFBP7 expression increased cell proliferation. (C) Colony formation was significantly promoted in IGFBP7 suppressed cells. (D) Western blot analysis was performed with the indicated antibodies on shRNA transduced cells. (E) Quantification of apoptosis by flow cytometry. Data presented mean ± SD and experiments were performed three times in triplicate. *p
    Figure Legend Snippet: Inhibition of IGFBP7 Expression Promotes Gastric Cancer Cell Growth. (A) Knock-down of IGFBP7 was determined using western blot and qRT-PCR. (B) Inhibition of IGFBP7 expression increased cell proliferation. (C) Colony formation was significantly promoted in IGFBP7 suppressed cells. (D) Western blot analysis was performed with the indicated antibodies on shRNA transduced cells. (E) Quantification of apoptosis by flow cytometry. Data presented mean ± SD and experiments were performed three times in triplicate. *p

    Techniques Used: Inhibition, Expressing, Western Blot, Quantitative RT-PCR, shRNA, Flow Cytometry, Cytometry

    Expression and Methylation of IGFBP7 in Gastric Cancer Cells and Tissues. (A) Expression of IGFBP7 mRNA was detected by qRT-PCR. (B) The methylation status of IGFBP7 exon 1 was examined by MSP. Distilled water (DW), negative loading control; UM, unmethylated DNA band; M, methylated DNA band. (C) By qRT-PCR, treatment with 5-aza-dc, TSA, or combination restored IGFBP7 mRNA expression. (D) IGFBP7 protein expression in gastric cancer (C) tissues compared to paired normal (N) gastric tissues was assessed by western blot. (E) A representative result of methylation was measured using MSP. (F) Immunohistochemical expression of IGFBP7. Left, negative staining for IGFBP7. Middle, weakly positive staining for IGFBP7. Right, strongly positive staining for IGFBP7. Original magnification, 100X. (G) Kaplan-Meier analysis of overall survival for different IGFBP7 expression in gastric cancer patients.
    Figure Legend Snippet: Expression and Methylation of IGFBP7 in Gastric Cancer Cells and Tissues. (A) Expression of IGFBP7 mRNA was detected by qRT-PCR. (B) The methylation status of IGFBP7 exon 1 was examined by MSP. Distilled water (DW), negative loading control; UM, unmethylated DNA band; M, methylated DNA band. (C) By qRT-PCR, treatment with 5-aza-dc, TSA, or combination restored IGFBP7 mRNA expression. (D) IGFBP7 protein expression in gastric cancer (C) tissues compared to paired normal (N) gastric tissues was assessed by western blot. (E) A representative result of methylation was measured using MSP. (F) Immunohistochemical expression of IGFBP7. Left, negative staining for IGFBP7. Middle, weakly positive staining for IGFBP7. Right, strongly positive staining for IGFBP7. Original magnification, 100X. (G) Kaplan-Meier analysis of overall survival for different IGFBP7 expression in gastric cancer patients.

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

    Effect of IGFBP7 Overexpression on Gastric Cancer Cells. (A) Upregulation of IGFBP7 was proved by western blot and qRT-PCR. (B) Overexpression of IGFBP7 inhibited cell proliferation. (C) Colonies in transfected cells were visualized by staining and counted. (D) Western blot analysis was performed with the indicated antibodies on IGFBP7 overexpressed cells. (E) Apoptosis was detected by flow cytometry. (F) Detection of p-IGF-1R after IGF-1 ligand stimulation by western blot. (G) Cell growth rate was measured using proliferation assay. Data presented mean ± SD and experiments were performed three times in triplicate. *p
    Figure Legend Snippet: Effect of IGFBP7 Overexpression on Gastric Cancer Cells. (A) Upregulation of IGFBP7 was proved by western blot and qRT-PCR. (B) Overexpression of IGFBP7 inhibited cell proliferation. (C) Colonies in transfected cells were visualized by staining and counted. (D) Western blot analysis was performed with the indicated antibodies on IGFBP7 overexpressed cells. (E) Apoptosis was detected by flow cytometry. (F) Detection of p-IGF-1R after IGF-1 ligand stimulation by western blot. (G) Cell growth rate was measured using proliferation assay. Data presented mean ± SD and experiments were performed three times in triplicate. *p

    Techniques Used: Over Expression, Western Blot, Quantitative RT-PCR, Transfection, Staining, Flow Cytometry, Cytometry, Proliferation Assay

    69) Product Images from "Expression of human ARGONAUTE 2 inhibits endogenous microRNA activity in Arabidopsis"

    Article Title: Expression of human ARGONAUTE 2 inhibits endogenous microRNA activity in Arabidopsis

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2013.00096

    Overexpression of HsAGO2 or AtAGO1 results in similar morphological and molecular phenotypes. (A) 24-day old primary transformants for the 35S:HsAGO2 , 35S:AtAGO1 , and 35S:4mAGO1 constructs, grown in parallel, were categorized as exhibiting an obvious abnormal morphological phenotype, characterized by broad, flattened, serrated leaves, or no/mild abnormal phenotype. Wild type (WT) and ago1–27 plants were grown in parallel as comparators. Scale bars represent 10 mm. (B) The abundances of HsAGO2 mRNA and un-cleaved mRNA for AtAGO1, PHB, MYB33 , CUC2 , and DCL1 were measured in total RNA from sample pools composed of 4–8 transformants, 24-days old, from each morphological category for each construct. Wild type (WT) and ago1–27 plants, grown in parallel, were included as controls. Measurements of “un-cleaved” mRNA are obtained by using a qRT-PCR amplicon spanning the cleavage site for each transcript, such that cleaved mRNA does not contribute to the recorded abundance. All measurements are normalized to CYCLOPHILIN mRNA. Data is averaged from two technical cDNA replicates, each of which comprised triplicate measurements, and error bars depict standard error of the mean. Values marked with * are significantly larger ( P
    Figure Legend Snippet: Overexpression of HsAGO2 or AtAGO1 results in similar morphological and molecular phenotypes. (A) 24-day old primary transformants for the 35S:HsAGO2 , 35S:AtAGO1 , and 35S:4mAGO1 constructs, grown in parallel, were categorized as exhibiting an obvious abnormal morphological phenotype, characterized by broad, flattened, serrated leaves, or no/mild abnormal phenotype. Wild type (WT) and ago1–27 plants were grown in parallel as comparators. Scale bars represent 10 mm. (B) The abundances of HsAGO2 mRNA and un-cleaved mRNA for AtAGO1, PHB, MYB33 , CUC2 , and DCL1 were measured in total RNA from sample pools composed of 4–8 transformants, 24-days old, from each morphological category for each construct. Wild type (WT) and ago1–27 plants, grown in parallel, were included as controls. Measurements of “un-cleaved” mRNA are obtained by using a qRT-PCR amplicon spanning the cleavage site for each transcript, such that cleaved mRNA does not contribute to the recorded abundance. All measurements are normalized to CYCLOPHILIN mRNA. Data is averaged from two technical cDNA replicates, each of which comprised triplicate measurements, and error bars depict standard error of the mean. Values marked with * are significantly larger ( P

    Techniques Used: Over Expression, Construct, Quantitative RT-PCR, Amplification

    70) Product Images from "Sex Modulates Lactobacillus johnsonii N6.2 and Phytophenol Effectiveness in Reducing High Fat Diet Induced mTOR Activation in Sprague-Dawley Rats"

    Article Title: Sex Modulates Lactobacillus johnsonii N6.2 and Phytophenol Effectiveness in Reducing High Fat Diet Induced mTOR Activation in Sprague-Dawley Rats

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02649

    HFD induces morphological changes in the liver. (A) Representative liver histology sections at 400× magnification of REDD-fed male (top left), HFD-fed male (top right), REDD-fed female (bottom left), and HFD-fed female (bottom right) rats. Arrows identify fat deposits. (B) Hepatic Srebp1c gene expression quantified by qRT-PCR between male (top) and female (bottom). Rplp0 expression was used as an internal standard. Results are expressed as Log 2 (Fold Induction) with repression values (i.e.,
    Figure Legend Snippet: HFD induces morphological changes in the liver. (A) Representative liver histology sections at 400× magnification of REDD-fed male (top left), HFD-fed male (top right), REDD-fed female (bottom left), and HFD-fed female (bottom right) rats. Arrows identify fat deposits. (B) Hepatic Srebp1c gene expression quantified by qRT-PCR between male (top) and female (bottom). Rplp0 expression was used as an internal standard. Results are expressed as Log 2 (Fold Induction) with repression values (i.e.,

    Techniques Used: Expressing, Quantitative RT-PCR

    HFD alters insulin signaling. (A) Quantification of serum adiponectin levels via ELISA for male (top) and female (bottom). (B) Hepatic Ceacam1 gene expression quantified by qRT-PCR for male (top) and female (bottom). Rplp0 expression was used as an internal standard. Results are expressed as Log 2 (Fold Induction) with repression values (i.e.,
    Figure Legend Snippet: HFD alters insulin signaling. (A) Quantification of serum adiponectin levels via ELISA for male (top) and female (bottom). (B) Hepatic Ceacam1 gene expression quantified by qRT-PCR for male (top) and female (bottom). Rplp0 expression was used as an internal standard. Results are expressed as Log 2 (Fold Induction) with repression values (i.e.,

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

    Treatments affect the transcription of mTORC1-activated genes in the stomach. (A) Pgd gene (B) Pfk gene expression quantified by qRT-PCR in male (top) and female (bottom). Rplp0 was used as an internal standard. Results are expressed as Log 2 (Fold Induction) with repression values (i.e.,
    Figure Legend Snippet: Treatments affect the transcription of mTORC1-activated genes in the stomach. (A) Pgd gene (B) Pfk gene expression quantified by qRT-PCR in male (top) and female (bottom). Rplp0 was used as an internal standard. Results are expressed as Log 2 (Fold Induction) with repression values (i.e.,

    Techniques Used: Expressing, Quantitative RT-PCR

    71) Product Images from "Myb proteins regulate expression of histone variant H2A.Z during thymocyte development"

    Article Title: Myb proteins regulate expression of histone variant H2A.Z during thymocyte development

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2007.02697.x

    Elevated histone variant H2A.Z mRNA in vMyb4 T-cell populations in vivo . (a) Flow cytometry of thymocytes (top and centre panels) and splenocytes (bottom panels) from vMyb4 transgenic mice and wild-type littermate controls (WT), stained with anti-CD4 and anti-CD8 antibodies. (b) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of H2A.Z mRNA expression using RNA from sorted T lymphocytes. Data are presented as means from triplicate qRT-PCR reactions performed from each of three independent sorts. Error bars represent standard error. DP, double positive; HPRT, hypoxanthine-guanine phosphoribosyltransferase.
    Figure Legend Snippet: Elevated histone variant H2A.Z mRNA in vMyb4 T-cell populations in vivo . (a) Flow cytometry of thymocytes (top and centre panels) and splenocytes (bottom panels) from vMyb4 transgenic mice and wild-type littermate controls (WT), stained with anti-CD4 and anti-CD8 antibodies. (b) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of H2A.Z mRNA expression using RNA from sorted T lymphocytes. Data are presented as means from triplicate qRT-PCR reactions performed from each of three independent sorts. Error bars represent standard error. DP, double positive; HPRT, hypoxanthine-guanine phosphoribosyltransferase.

    Techniques Used: Variant Assay, In Vivo, Flow Cytometry, Cytometry, Transgenic Assay, Mouse Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

    Deletion of c-myb at the double negative 4 (DN4) stage reduces histone variant H2A.Z expression in thymic double positive (DP) subsets. (a) Fluorescence-activated cell sorting (FACS) profiles of thymocytes stained with antibodies against CD4 ( y -axis) and CD8 ( x -axis) from Myb F/F mice in the absence or presence of the CD4Cre transgene. (b) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of H2A.Z mRNA expression using RNA isolated from sorted T lymphocytes of Myb F/F CD4Cre mice. Data are presented as means from triplicate qRT-PCR reactions performed from each of three independent sorts. Error bars represent standard error. HPRT, hypoxanthine guanine phosphoribosyltransferase.
    Figure Legend Snippet: Deletion of c-myb at the double negative 4 (DN4) stage reduces histone variant H2A.Z expression in thymic double positive (DP) subsets. (a) Fluorescence-activated cell sorting (FACS) profiles of thymocytes stained with antibodies against CD4 ( y -axis) and CD8 ( x -axis) from Myb F/F mice in the absence or presence of the CD4Cre transgene. (b) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of H2A.Z mRNA expression using RNA isolated from sorted T lymphocytes of Myb F/F CD4Cre mice. Data are presented as means from triplicate qRT-PCR reactions performed from each of three independent sorts. Error bars represent standard error. HPRT, hypoxanthine guanine phosphoribosyltransferase.

    Techniques Used: Variant Assay, Expressing, Fluorescence, FACS, Staining, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Isolation

    Deletion of c-myb at the double negative 2 (DN2) stage reduces histone variant H2A.Z expression in DN3 thymocytes. (a) Fluorescence-activated cell sorting (FACS) profiles of thymocytes. Upper panel: DN thymocytes stained with antibodies against CD44 ( y -axis) and CD25 ( x -axis) from Myb F/F mice in the absence or presence of the CD2Cre transgene. A recombination activating gene (RAG)-2 –/– profile is also shown. DN2 (grey boxes) and DN3 (black boxes) populations used for mRNA preparation are indicated. Lower panel: FACS profiles of total thymocytes stained with antibodies against CD4 ( y -axis) and CD8 ( x -axis) from Myb F/F mice in the absence or presence of the CD2Cre transgene. Percentages of cells in each quadrant are shown. (b) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of H2A.Z mRNA expression using RNA isolated from sorted DN2 and DN3 cells of the indicated genotypes. Data are presented as means from triplicate qRT-PCR reactions. Error bars represent standard deviation.
    Figure Legend Snippet: Deletion of c-myb at the double negative 2 (DN2) stage reduces histone variant H2A.Z expression in DN3 thymocytes. (a) Fluorescence-activated cell sorting (FACS) profiles of thymocytes. Upper panel: DN thymocytes stained with antibodies against CD44 ( y -axis) and CD25 ( x -axis) from Myb F/F mice in the absence or presence of the CD2Cre transgene. A recombination activating gene (RAG)-2 –/– profile is also shown. DN2 (grey boxes) and DN3 (black boxes) populations used for mRNA preparation are indicated. Lower panel: FACS profiles of total thymocytes stained with antibodies against CD4 ( y -axis) and CD8 ( x -axis) from Myb F/F mice in the absence or presence of the CD2Cre transgene. Percentages of cells in each quadrant are shown. (b) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of H2A.Z mRNA expression using RNA isolated from sorted DN2 and DN3 cells of the indicated genotypes. Data are presented as means from triplicate qRT-PCR reactions. Error bars represent standard deviation.

    Techniques Used: Variant Assay, Expressing, Fluorescence, FACS, Staining, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Isolation, Standard Deviation

    72) Product Images from "Comparative liver transcriptome analysis in ducklings infected with duck hepatitis A virus 3 (DHAV-3) at 12 and 48 hours post-infection through RNA-seq"

    Article Title: Comparative liver transcriptome analysis in ducklings infected with duck hepatitis A virus 3 (DHAV-3) at 12 and 48 hours post-infection through RNA-seq

    Journal: Veterinary Research

    doi: 10.1186/s13567-018-0545-7

    Pathological changes in the livers of DHAV-3-infected ducklings at different time points. A Histopathological examination of duckling livers in the control group. B Histopathological changes were not observed in the liver at 12 hpi. C Many cells have dissolved and disappeared, hepatocyte necrosis with hemorrhage at 48 hpi. D DHAV-3 replication in the livers of ducklings with DHAV-3 infection at 12, 48, and 72 hpi. The data are expressed as mean ± standard deviation. Three dead ducklings were selected for detecting the viral RNA load using the qRT-PCR method.
    Figure Legend Snippet: Pathological changes in the livers of DHAV-3-infected ducklings at different time points. A Histopathological examination of duckling livers in the control group. B Histopathological changes were not observed in the liver at 12 hpi. C Many cells have dissolved and disappeared, hepatocyte necrosis with hemorrhage at 48 hpi. D DHAV-3 replication in the livers of ducklings with DHAV-3 infection at 12, 48, and 72 hpi. The data are expressed as mean ± standard deviation. Three dead ducklings were selected for detecting the viral RNA load using the qRT-PCR method.

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

    73) Product Images from "Postnatal lymphatic partitioning from the blood vasculature in the small intestine requires fasting-induced adipose factor"

    Article Title: Postnatal lymphatic partitioning from the blood vasculature in the small intestine requires fasting-induced adipose factor

    Journal:

    doi: 10.1073/pnas.0605957104

    Prox1 expression is regulated in the small intestine by Fiaf . ( A ) qRT-PCR analysis of the relative expression of Fiaf in the small intestine of wild-type E18–P24 mice ( n = 4 per group, each intestine analyzed individually; mean values ±
    Figure Legend Snippet: Prox1 expression is regulated in the small intestine by Fiaf . ( A ) qRT-PCR analysis of the relative expression of Fiaf in the small intestine of wild-type E18–P24 mice ( n = 4 per group, each intestine analyzed individually; mean values ±

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

    74) Product Images from "Loss of p53 induces cell proliferation via Ras-independent activation of the Raf/Mek/Erk signaling pathway"

    Article Title: Loss of p53 induces cell proliferation via Ras-independent activation of the Raf/Mek/Erk signaling pathway

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

    doi: 10.1073/pnas.1417549111

    Activation of p53 target genes in Rasless cells. ( A and B ) qRT-PCR of the indicated p53 target genes in Rasless vs. K-Raslox cells. Genes have been grouped by function, including apoptosis (black bars), cell cycle arrest (green bars), tumor suppression
    Figure Legend Snippet: Activation of p53 target genes in Rasless cells. ( A and B ) qRT-PCR of the indicated p53 target genes in Rasless vs. K-Raslox cells. Genes have been grouped by function, including apoptosis (black bars), cell cycle arrest (green bars), tumor suppression

    Techniques Used: Activation Assay, Quantitative RT-PCR

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky279

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

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

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    Quantitative RT-PCR:

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    SYBR Green Assay:

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    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
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    Expressing:

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

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    Cell Culture:

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    Generated:

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    Polymerase Chain Reaction:

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    Article Snippet: .. The qRT-PCR assays were performed using Power SYBR Green PCR Master Mix (Life Technologies, Carlsbad, CA, USA), using an ABI Prism 7900HT instrument (Applied Biosystems, Carlsbad, CA, USA) with the primers listed in Additional file : Table S3 and normalized to the relative glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level. .. For the miRNA analyses, a TaqMan® MicroRNA RT kit and TaqMan® Universal Master Mix II no UNG (Applied Biosystems, Carlsbad, CA, USA) were used to perform the RT and PCR, respectively.

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    Article Snippet: Proper edgeR normalization was verified by quantitative reverse transcriptase PCR (qRT-PCR) quantification of 23 mRNAs representing high- and low-abundance transcripts with large, small, or no changes in levels post-PTF1A depletion (see Fig. S2). edgeR analyses used the default TMM settings of 0.3 for trim of log ratios (M values), 0.05 for trim of combined absolute levels (A values), and an false discovery rate (FDR) cutoff of < 0.05 ( ). .. The hierarchical cluster analysis of transcriptomes was performed using the R Stats package ( ). qRT-PCR assays were performed with SYBR green and an Applied Biosystems 2500 fast instrument.

    Sequencing:

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    RNA Sequencing Assay:

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    Article Snippet: Paragraph title: RNA-Seq and data analysis. ... The hierarchical cluster analysis of transcriptomes was performed using the R Stats package ( ). qRT-PCR assays were performed with SYBR green and an Applied Biosystems 2500 fast instrument.

    Fluorescence:

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    Isolation:

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    Article Snippet: .. RNA extraction and quantitative real time PCR Total cellular RNA was isolated with TRIzol® Reagent (Vazyme) and reverse transcribed with the RevertAid™ First Strand cDNA Synthesis Kit (Takara). qRT-PCR assays were performed to analyze relative mRNA levels using a SYBR Green-based system (Applied Biosystems). ..

    Article Title: Curcumin Inhibits Rift Valley Fever Virus Replication in Human Cells *
    Article Snippet: Viral RNA from cell culture supernatants was extracted using Ambion's MagMAXTM -96 viral RNA isolation kit, and RNA was analyzed by qRT-PCR. .. The primers and probe used for amplification of viral RNA were originally described by Drosten et al. ( ). qRT-PCR assays were performed using the ABI Prism 7000 and Invitrogen's RNA UltraSenseTM one-step quantitative RT-PCR system.

    Article Title: A Calpain-Like Protein Is Involved in the Execution Phase of Programmed Cell Death of Entamoeba histolytica
    Article Snippet: Total RNAs from trophozoites without treatment or treated with 10 μg/ml G418 for 0.5, 1.5, 3, 6, and 9 h were isolated using the Trizol reagent (Invitrogen) according to the manufacturer's protocol. cDNA was synthesized using an oligo(dT) primer and the Superscript II reverse transcriptase (Invitrogen). .. For qRT-PCR assays, specific primers for the calpain-like gene ( ) were designed by the Primer Express Software for Real-Time PCR version 3.0 (Applied Biosystems), (sense primer: 5′-GTTTCAATATCACAACCT CGTTGTG-3′ and antisense primer: 5′-AAAGTCTCTCCAGAATCACCTCCA-3′).

    Article Title: Regulation of Serum Amyloid A3 (SAA3) in Mouse Colonic Epithelium and Adipose Tissue by the Intestinal Microbiota
    Article Snippet: All homogenates were processed for RNA isolation using an RNeasy kit (Qiagen) with on-column DNase I (Qiagen) treatment according to the manufacturer's instructions. .. Random hexamer-primed cDNA templates were synthesized from purified RNAs using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. qRT-PCR assays were performed in 25-µl reactions containing 1× SYBR Green Master Mix buffer (Thermo Scientific), and 900 nM gene-specific primers (300 nM primer concentrations were used to assess L32 transcripts).

    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
    Article Snippet: Paragraph title: RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR) ... The SYBR®Green Real-time PCR Kit (Takara, Liaoning, China) and the miRcute Plus miRNA qPCR Detection Kit (TIANGEN, China) were used for qRT-PCR assays using the ABI 7500 Real-Time PCR System (Applied Biosystems, CA, USA).

    Purification:

    Article Title: Regulation of Serum Amyloid A3 (SAA3) in Mouse Colonic Epithelium and Adipose Tissue by the Intestinal Microbiota
    Article Snippet: .. Random hexamer-primed cDNA templates were synthesized from purified RNAs using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. qRT-PCR assays were performed in 25-µl reactions containing 1× SYBR Green Master Mix buffer (Thermo Scientific), and 900 nM gene-specific primers (300 nM primer concentrations were used to assess L32 transcripts). .. A melting curve was performed for each primer pair to identify a temperature where only amplicon, and not primer dimers, accounted for SYBR Green-bound fluorescence.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Transcriptional Maintenance of Pancreatic Acinar Identity, Differentiation, and Homeostasis by PTF1A
    Article Snippet: The hierarchical cluster analysis of transcriptomes was performed using the R Stats package ( ). qRT-PCR assays were performed with SYBR green and an Applied Biosystems 2500 fast instrument. .. Measurement of the ratio of spliced to unspliced Xbp1 mRNA was performed by RT-PCR as described previously ( ).

    Plasmid Preparation:

    Article Title: Evaluation of Two Triplex One-Step qRT-PCR Assays for the Quantification of Human Enteric Viruses in Environmental Samples
    Article Snippet: qRT-PCR Assay All qRT-PCR assays were carried out in a QuantStudio™ Flex 6 Real-Time PCR System (Applied Biosystems, USA). .. SaV and MgV standards were derived from cloning qRT-PCR amplicons into pGem-T Easy vector (Promega, USA).

    Software:

    Article Title: A Single Agent Dual Specificity Targeting of FOLR1 and DR5 as an Effective Strategy for Ovarian Cancer
    Article Snippet: For qRT-PCR assays, RNA was extracted using the Trizol Reagent (Invitrogen). cDNA was prepared by amplifying 500 ng of RNA by the SuperScript-II cDNA Synthesis Kit (Life Technologies). .. Data was analyzed using StepOneV2.0 software (Applied Biosystems).

    Article Title: A Calpain-Like Protein Is Involved in the Execution Phase of Programmed Cell Death of Entamoeba histolytica
    Article Snippet: .. For qRT-PCR assays, specific primers for the calpain-like gene ( ) were designed by the Primer Express Software for Real-Time PCR version 3.0 (Applied Biosystems), (sense primer: 5′-GTTTCAATATCACAACCT CGTTGTG-3′ and antisense primer: 5′-AAAGTCTCTCCAGAATCACCTCCA-3′). ..

    Real-time Polymerase Chain Reaction:

    Article Title: Evaluation of Two Triplex One-Step qRT-PCR Assays for the Quantification of Human Enteric Viruses in Environmental Samples
    Article Snippet: .. qRT-PCR Assay All qRT-PCR assays were carried out in a QuantStudio™ Flex 6 Real-Time PCR System (Applied Biosystems, USA). ..

    Article Title: Identification and characterization of miRNAs in two closely related C4 and C3 species of Cleome by high-throughput sequencing
    Article Snippet: .. Fourteen miRNAs were randomly selected for the qRT-PCR assays in the samples of C. gynandra and C. hassleriana by Platinum SYBR Green-based qPCR (Invitrogen, USA) with the High-Specificity miRNA QuantiMir RT Kit (RA610A-1, System Biosciences) on the ViiA™ 7 Dx platform (ABI, USA). .. Amplified primers for all miRNAs were designed according to Varkonyi-Gasic et al .

    Article Title: A Single Agent Dual Specificity Targeting of FOLR1 and DR5 as an Effective Strategy for Ovarian Cancer
    Article Snippet: For qRT-PCR assays, RNA was extracted using the Trizol Reagent (Invitrogen). cDNA was prepared by amplifying 500 ng of RNA by the SuperScript-II cDNA Synthesis Kit (Life Technologies). .. Quantitative PCR was performed using PowerUp SYBR Green Master mix (Applied Biosystems) following manufacturer’s instructions.

    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
    Article Snippet: .. The qRT-PCR assays were run in a 7900HT Fast Real Time PCR system (Life Technologies) once cDNA templates and TaqMan® Universal Master Mix (Life Technologies) were added. ..

    Article Title: Adipocyte-derived IL-6 and leptin promote breast Cancer metastasis via upregulation of Lysyl Hydroxylase-2 expression
    Article Snippet: .. RNA extraction and quantitative real time PCR Total cellular RNA was isolated with TRIzol® Reagent (Vazyme) and reverse transcribed with the RevertAid™ First Strand cDNA Synthesis Kit (Takara). qRT-PCR assays were performed to analyze relative mRNA levels using a SYBR Green-based system (Applied Biosystems). ..

    Article Title: A Calpain-Like Protein Is Involved in the Execution Phase of Programmed Cell Death of Entamoeba histolytica
    Article Snippet: .. For qRT-PCR assays, specific primers for the calpain-like gene ( ) were designed by the Primer Express Software for Real-Time PCR version 3.0 (Applied Biosystems), (sense primer: 5′-GTTTCAATATCACAACCT CGTTGTG-3′ and antisense primer: 5′-AAAGTCTCTCCAGAATCACCTCCA-3′). ..

    Article Title: Regulation of Serum Amyloid A3 (SAA3) in Mouse Colonic Epithelium and Adipose Tissue by the Intestinal Microbiota
    Article Snippet: Paragraph title: Quantitative Real-Time PCR ... Random hexamer-primed cDNA templates were synthesized from purified RNAs using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. qRT-PCR assays were performed in 25-µl reactions containing 1× SYBR Green Master Mix buffer (Thermo Scientific), and 900 nM gene-specific primers (300 nM primer concentrations were used to assess L32 transcripts).

    Article Title: The MADS transcription factor CmANR1 positively modulates root system development by directly regulating CmPIN2 in chrysanthemum
    Article Snippet: .. The qRT-PCR assays were carried out according to the StepOne real-time PCR system (Applied Biosystems). ..

    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
    Article Snippet: .. The SYBR®Green Real-time PCR Kit (Takara, Liaoning, China) and the miRcute Plus miRNA qPCR Detection Kit (TIANGEN, China) were used for qRT-PCR assays using the ABI 7500 Real-Time PCR System (Applied Biosystems, CA, USA). .. Relative expression was normalized to that of endogenous controls using the comparative cycle threshold method, and the fold change in gene expression was calculated using the 2−ΔΔCt method.

    RNA Extraction:

    Article Title: Adipocyte-derived IL-6 and leptin promote breast Cancer metastasis via upregulation of Lysyl Hydroxylase-2 expression
    Article Snippet: .. RNA extraction and quantitative real time PCR Total cellular RNA was isolated with TRIzol® Reagent (Vazyme) and reverse transcribed with the RevertAid™ First Strand cDNA Synthesis Kit (Takara). qRT-PCR assays were performed to analyze relative mRNA levels using a SYBR Green-based system (Applied Biosystems). ..

    In Situ:

    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
    Article Snippet: Real time quantitative RT-PCR (qRT-PCR) Synthesis of unlabeled single stranded cDNA was performed as indicated above except for the dNTP mixture (10 mM each dATP, dCTP, dGTP and dTTP in this case) The design of primers and FAM-MGB probes (Additional file : Table S1), configuration of 384-well plates and in situ synthesis was managed by Custom TaqMan® Assays-by-Design (Life Technologies). .. The qRT-PCR assays were run in a 7900HT Fast Real Time PCR system (Life Technologies) once cDNA templates and TaqMan® Universal Master Mix (Life Technologies) were added.

    Transgenic Assay:

    Article Title: The MADS transcription factor CmANR1 positively modulates root system development by directly regulating CmPIN2 in chrysanthemum
    Article Snippet: Quantitative real-time (qRT)-PCR analysis Total RNA was extracted from the roots of 40-day-old hydroponic-cultured transgenic and WT chrysanthemum using the RNA plant plus Reagent (Tiangen, Beijing, China).Then, cDNA was synthesized using the PrimeScript first-strand cDNA synthesis kit (TaKaRa, Dalian, China). .. The qRT-PCR assays were carried out according to the StepOne real-time PCR system (Applied Biosystems).

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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
    Quan Titative Reverse Transcriptase Polymerase Chain Reaction Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher qrt pcr rna
    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: 99/100, based on 285 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: 99/100, based on 44 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: 99/100, based on 1152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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