high capacity cdna reverse transcription kit  (Thermo Fisher)


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    Name:
    High Capacity cDNA Reverse Transcription Kit
    Description:
    The High Capacity cDNA Reverse Transcription Kit contains all components necessary for the quantitative conversion of up to 2 µg of total RNA to single stranded cDNA in a single 20 µL reaction The High Capacity cDNA Reverse Transcription Kit delivers extremely high quality single stranded cDNA from 0 02 to 2 µg total RNA Reactions can be scaled up to 100 µL to generate 10 µg of cDNA from a single reaction Downstream applications include real time PCR standard PCR and microarrays The kit is ideal for generating cDNA archives Features of this kit include • Linear target amplification for real time PCR• Higher yields and precision than other cDNA synthesis kits at a fraction of the cost• 10 fold greater dynamic range than other kitsExtensively tested with a variety of templates Quantitative first strand synthesis of all RNA species is achieved with the use of the random primers The kit has been extensively tested with a variety of RNA templates including GC and AU rich targets and RNAs expressed at low levels cRNA can also be efficiently generated from in vitro transcription of cDNA
    Catalog Number:
    4368813
    Price:
    None
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
    Category:
    Kits and Assays
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    Structured Review

    Thermo Fisher high capacity cdna reverse transcription kit
    Dexamethasone synergizes with venetoclax through the induction of Bcl-2 and Bim A. MM.1s and KMS18 were treated with the indicated concentrations of venetoclax (Ven) and dexamethasone alone or in combination for 24 h. Real-time <t>PCR</t> was performed on <t>cDNA</t> generated from RNA isolated from control and treated samples. The data are presented as the means (±SE) of 3 independent experiments. B. Protein lysates were obtained from MM.1s and KMS18 6 and 24 h following treatment in A. Twenty μg of proteins were subjected to Western blotting and membranes probed with anti-Mcl-1, anti-Bcl-x L , anti-Bcl-2, anti-Bim, anti-Noxa, and anti-β-Actin antibodies. Blots are representative of two experiments.
    The High Capacity cDNA Reverse Transcription Kit contains all components necessary for the quantitative conversion of up to 2 µg of total RNA to single stranded cDNA in a single 20 µL reaction The High Capacity cDNA Reverse Transcription Kit delivers extremely high quality single stranded cDNA from 0 02 to 2 µg total RNA Reactions can be scaled up to 100 µL to generate 10 µg of cDNA from a single reaction Downstream applications include real time PCR standard PCR and microarrays The kit is ideal for generating cDNA archives Features of this kit include • Linear target amplification for real time PCR• Higher yields and precision than other cDNA synthesis kits at a fraction of the cost• 10 fold greater dynamic range than other kitsExtensively tested with a variety of templates Quantitative first strand synthesis of all RNA species is achieved with the use of the random primers The kit has been extensively tested with a variety of RNA templates including GC and AU rich targets and RNAs expressed at low levels cRNA can also be efficiently generated from in vitro transcription of cDNA
    https://www.bioz.com/result/high capacity cdna reverse transcription kit/product/Thermo Fisher
    Average 99 stars, based on 10121 article reviews
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    high capacity cdna reverse transcription kit - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "Dexamethasone treatment promotes Bcl-2-dependence in multiple myeloma resulting in sensitivity to Venetoclax"

    Article Title: Dexamethasone treatment promotes Bcl-2-dependence in multiple myeloma resulting in sensitivity to Venetoclax

    Journal: Leukemia

    doi: 10.1038/leu.2015.350

    Dexamethasone synergizes with venetoclax through the induction of Bcl-2 and Bim A. MM.1s and KMS18 were treated with the indicated concentrations of venetoclax (Ven) and dexamethasone alone or in combination for 24 h. Real-time PCR was performed on cDNA generated from RNA isolated from control and treated samples. The data are presented as the means (±SE) of 3 independent experiments. B. Protein lysates were obtained from MM.1s and KMS18 6 and 24 h following treatment in A. Twenty μg of proteins were subjected to Western blotting and membranes probed with anti-Mcl-1, anti-Bcl-x L , anti-Bcl-2, anti-Bim, anti-Noxa, and anti-β-Actin antibodies. Blots are representative of two experiments.
    Figure Legend Snippet: Dexamethasone synergizes with venetoclax through the induction of Bcl-2 and Bim A. MM.1s and KMS18 were treated with the indicated concentrations of venetoclax (Ven) and dexamethasone alone or in combination for 24 h. Real-time PCR was performed on cDNA generated from RNA isolated from control and treated samples. The data are presented as the means (±SE) of 3 independent experiments. B. Protein lysates were obtained from MM.1s and KMS18 6 and 24 h following treatment in A. Twenty μg of proteins were subjected to Western blotting and membranes probed with anti-Mcl-1, anti-Bcl-x L , anti-Bcl-2, anti-Bim, anti-Noxa, and anti-β-Actin antibodies. Blots are representative of two experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Generated, Isolation, Western Blot

    2) Product Images from "Complete restoration of multiple dystrophin isoforms in genetically corrected Duchenne muscular dystrophy patient–derived cardiomyocytes"

    Article Title: Complete restoration of multiple dystrophin isoforms in genetically corrected Duchenne muscular dystrophy patient–derived cardiomyocytes

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1038/mtm.2013.1

    HAC-driven expression of dystrophin sequences originally deleted in DMD patients. ( a ) The genomic organization of the dystrophin gene: the gray vertical bars represent the exons; the green arrows indicate the promoters driving the expression of the different dystrophin isoforms within the gene 1 , 2 ; primer pairs used to amplify exon–exon junctions inside the deleted region are indicated by red arrowheads, and those used to amplify specific isoforms are indicated by blue arrowheads. Exon number here reported refers to the muscle dystrophin isoform Dp427m. ( b ) RT-PCR of specific dystrophin sequences localized inside the patient with deletion of exons (Ex) 4–43. Primers were constructed to spam five different exon–exon junctions (Ex. J.). cDNA from human heart and skeletal muscle was used as positive control. Analyses were performed at three different stages of the differentiation process: undifferentiated colonies, differentiated EBs cultured in suspension (day 20), and adhered cells (day 24), for each hiPS cell lines (DMD, healthy, and DYS-HAC hiPS cells). cDNA from human tissues was used as positive control. cDNA, complementary DNA; DMD, Duchenne muscular dystrophy; DYS-HAC, human artificial chromosome carrying the whole dystrophin genomic sequence; EBs, embryoid bodies; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HAC, human artificial chromosome; hiPS cells, human induced pluripotent stem cells; RT-PCR, reverse transcriptase–polymerase chain reaction.
    Figure Legend Snippet: HAC-driven expression of dystrophin sequences originally deleted in DMD patients. ( a ) The genomic organization of the dystrophin gene: the gray vertical bars represent the exons; the green arrows indicate the promoters driving the expression of the different dystrophin isoforms within the gene 1 , 2 ; primer pairs used to amplify exon–exon junctions inside the deleted region are indicated by red arrowheads, and those used to amplify specific isoforms are indicated by blue arrowheads. Exon number here reported refers to the muscle dystrophin isoform Dp427m. ( b ) RT-PCR of specific dystrophin sequences localized inside the patient with deletion of exons (Ex) 4–43. Primers were constructed to spam five different exon–exon junctions (Ex. J.). cDNA from human heart and skeletal muscle was used as positive control. Analyses were performed at three different stages of the differentiation process: undifferentiated colonies, differentiated EBs cultured in suspension (day 20), and adhered cells (day 24), for each hiPS cell lines (DMD, healthy, and DYS-HAC hiPS cells). cDNA from human tissues was used as positive control. cDNA, complementary DNA; DMD, Duchenne muscular dystrophy; DYS-HAC, human artificial chromosome carrying the whole dystrophin genomic sequence; EBs, embryoid bodies; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HAC, human artificial chromosome; hiPS cells, human induced pluripotent stem cells; RT-PCR, reverse transcriptase–polymerase chain reaction.

    Techniques Used: HAC Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Construct, Positive Control, Cell Culture, Sequencing, Polymerase Chain Reaction

    HAC-driven expression of multiple dystrophin isoforms. ( a ) RT-PCR of dystrophin isoforms Dp427m, Dp260, Dp140, and Dp71 at three different stages of the differentiation process: undifferentiated colonies, differentiated EBs cultured in suspension (day 20), and adhered cells (day 24), for each hiPS cell lines (DMD, healthy, and DYS-HAC hiPS cells). cDNA from human tissues was used as positive control. ( b ) Real-time PCR for dystrophin isoforms present in cardiac tissue. Muscle-specific Dp427m is double checked with Dp427m-specific primer set spanning exons 1–3 and an all Dp427 isoform-specific primer set spanning exons 25–26. Data are presented as mean ± SD. cDNA, complementary DNA; DYS-HAC, human artificial chromosome carrying the whole dystrophin genomic sequence; EBs, embryoid bodies; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HAC, human artificial chromosome; hiPS cells, human induced pluripotent stem cells; RT-PCR, reverse transcriptase–polymerase chain reaction; DMD, Duchenne muscular dystrophy.
    Figure Legend Snippet: HAC-driven expression of multiple dystrophin isoforms. ( a ) RT-PCR of dystrophin isoforms Dp427m, Dp260, Dp140, and Dp71 at three different stages of the differentiation process: undifferentiated colonies, differentiated EBs cultured in suspension (day 20), and adhered cells (day 24), for each hiPS cell lines (DMD, healthy, and DYS-HAC hiPS cells). cDNA from human tissues was used as positive control. ( b ) Real-time PCR for dystrophin isoforms present in cardiac tissue. Muscle-specific Dp427m is double checked with Dp427m-specific primer set spanning exons 1–3 and an all Dp427 isoform-specific primer set spanning exons 25–26. Data are presented as mean ± SD. cDNA, complementary DNA; DYS-HAC, human artificial chromosome carrying the whole dystrophin genomic sequence; EBs, embryoid bodies; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HAC, human artificial chromosome; hiPS cells, human induced pluripotent stem cells; RT-PCR, reverse transcriptase–polymerase chain reaction; DMD, Duchenne muscular dystrophy.

    Techniques Used: HAC Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Positive Control, Real-time Polymerase Chain Reaction, Sequencing, Polymerase Chain Reaction

    3) Product Images from "Reconstitution of the Ataxia-Telangiectasia Cellular Phenotype With Lentiviral Vectors"

    Article Title: Reconstitution of the Ataxia-Telangiectasia Cellular Phenotype With Lentiviral Vectors

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02703

    Construction and expression of a lentiviral vector containing a normal ATM cDNA. (A) Map of the lentiviral vector containing the full-length cDNA insert of the ATM gene WPRE: Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element. (B) Integration of the therapeutic gene into the genomic DNA of healthy (WT) transduced (TD) and untransduced (UTD) cells determined by PCR using vector-specific sequences. ThATM plasmid DNA was used as positive control. (C) ATM mRNA expression in transduced cells at indicated MOI (5, 1, and 0.5). Negative controls (right-hand gel) used equivalent amounts of non-retrotranscribed templates and positive control used the pThATM plasmid as amplification template. Neg: no template. (D) ATM expression in nuclear extracts of native ATM (top) or Ser1981 p-ATM (bottom) after cell irradiation. The nuclear protein NBS1 was used as loading control. WT, HIFs derived from healthy subjects; UTD, untransduced A-T HIFs; TD, A-T HIFs cells transduced with the ThATM lentiviral vector.
    Figure Legend Snippet: Construction and expression of a lentiviral vector containing a normal ATM cDNA. (A) Map of the lentiviral vector containing the full-length cDNA insert of the ATM gene WPRE: Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element. (B) Integration of the therapeutic gene into the genomic DNA of healthy (WT) transduced (TD) and untransduced (UTD) cells determined by PCR using vector-specific sequences. ThATM plasmid DNA was used as positive control. (C) ATM mRNA expression in transduced cells at indicated MOI (5, 1, and 0.5). Negative controls (right-hand gel) used equivalent amounts of non-retrotranscribed templates and positive control used the pThATM plasmid as amplification template. Neg: no template. (D) ATM expression in nuclear extracts of native ATM (top) or Ser1981 p-ATM (bottom) after cell irradiation. The nuclear protein NBS1 was used as loading control. WT, HIFs derived from healthy subjects; UTD, untransduced A-T HIFs; TD, A-T HIFs cells transduced with the ThATM lentiviral vector.

    Techniques Used: Expressing, Plasmid Preparation, Polymerase Chain Reaction, Positive Control, Amplification, Irradiation, Derivative Assay, Transduction

    4) Product Images from "Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime"

    Article Title: Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1259

    Correction of endogenous gDNA with ValidPrime. Comparison of results obtained with two assays targeting Serpine1 (left) or Chi3l3 (right) in cDNA prepared from mouse peritoneal macrophages and measured in the BioMark qPCR system. The ‘gDNA-sensitive’ assays amplify both gDNA and cDNA, while the ‘gDNA-insensitive’ assays only recognize the transcript. ( A ) Scatter plots showing the correlation between the %DNA [as defined in Equation (6) ] and Cq NA data obtained with the gDNA-sensitive assays in each of 81 independent RNA preparations (means of duplicates). The positive correlation between %DNA and Cq illustrates the increasing impact of the gDNA contamination with decreasing total signal. Mean and median values refer to %DNA levels. ( B ) Cq NA data measured with the gDNA-insensitive assays plotted against the corresponding Cq NA data (dark blue) or ValidPrime-estimated Cq RNA (light blue and orange), obtained with the gDNA-sensitive assays. Samples with a DNA contribution of 60–90 % are shown in orange and those with
    Figure Legend Snippet: Correction of endogenous gDNA with ValidPrime. Comparison of results obtained with two assays targeting Serpine1 (left) or Chi3l3 (right) in cDNA prepared from mouse peritoneal macrophages and measured in the BioMark qPCR system. The ‘gDNA-sensitive’ assays amplify both gDNA and cDNA, while the ‘gDNA-insensitive’ assays only recognize the transcript. ( A ) Scatter plots showing the correlation between the %DNA [as defined in Equation (6) ] and Cq NA data obtained with the gDNA-sensitive assays in each of 81 independent RNA preparations (means of duplicates). The positive correlation between %DNA and Cq illustrates the increasing impact of the gDNA contamination with decreasing total signal. Mean and median values refer to %DNA levels. ( B ) Cq NA data measured with the gDNA-insensitive assays plotted against the corresponding Cq NA data (dark blue) or ValidPrime-estimated Cq RNA (light blue and orange), obtained with the gDNA-sensitive assays. Samples with a DNA contribution of 60–90 % are shown in orange and those with

    Techniques Used: Real-time Polymerase Chain Reaction

    ValidPrime flowchart. ValidPrime GOI assay validation. ValidPrime can be used as a reliable, cost-efficient alternative to RT(−) controls to survey gDNA background in RT–qPCR, and as a tool to determine the RNA-derived signal ( Cq RNA ) in RT(+)–qPCR reactions. To optimize its accuracy when Cq RNA calculation is desired, validation of GOI assays in gDNA samples is recommended, as outlined in ( A ). Asterisks indicates the efficiency evaluation and melting curve/electrophoresis-based analysis. This includes an evaluation of the gDNA sensitivity of GOI assays using dilution series with gDNA samples spanning at least three log 10 in copy number. GOI assays that do not amplify gDNA are attributed the grade A+. The amplification of gDNA by high-confidence assays should be specific and with an efficiency similar to that of the VPA (see ‘Discussion’ section and Supplementary Figure S3 ). For GOI assays with suboptimal, but confidently determined ( 17 , 21 ) efficiency, Equation (7) could be applied to adjust Cq NA data. To optimize specificity, there should also be consistency between the melting curves of PCR products in gDNA and cDNA samples. ( B ) Cq RNA calculation with ValidPrime-validated GOI assays. High confidence and A+ assays can be used with less gDNA samples for Cq RNA determination. It is recommended to confirm the absence of gDNA amplification at least once for A+ assays. Samples that do not contain sufficient gDNA to generate a signal with the VPA are attributed A*. As for gDNA insensitive A+ assays, Cq RNA equals Cq NA (i.e. output = input) in A* samples, since the DNA-derived signal is negligible [see Equations ( 2 and 4 )]. For gDNA-sensitive GOI assays, Cq RNA is calculated by a Cq DNA -based correction of Cq NA using Equations ( 4 and 5 ). To minimize the risk of jeopardizing the accuracy of the Cq RNA estimation, it is not advisable to perform correction on samples where the DNA-derived signal exceeds 60%. The calculations are facilitated using the ValidPrime software. Details on additional assay/sample grading and data output formats employed by the software are provided in Supplementary Figure S7 . The Cq RNA output data can be used for downstream data processing, such as normalization against reference genes.
    Figure Legend Snippet: ValidPrime flowchart. ValidPrime GOI assay validation. ValidPrime can be used as a reliable, cost-efficient alternative to RT(−) controls to survey gDNA background in RT–qPCR, and as a tool to determine the RNA-derived signal ( Cq RNA ) in RT(+)–qPCR reactions. To optimize its accuracy when Cq RNA calculation is desired, validation of GOI assays in gDNA samples is recommended, as outlined in ( A ). Asterisks indicates the efficiency evaluation and melting curve/electrophoresis-based analysis. This includes an evaluation of the gDNA sensitivity of GOI assays using dilution series with gDNA samples spanning at least three log 10 in copy number. GOI assays that do not amplify gDNA are attributed the grade A+. The amplification of gDNA by high-confidence assays should be specific and with an efficiency similar to that of the VPA (see ‘Discussion’ section and Supplementary Figure S3 ). For GOI assays with suboptimal, but confidently determined ( 17 , 21 ) efficiency, Equation (7) could be applied to adjust Cq NA data. To optimize specificity, there should also be consistency between the melting curves of PCR products in gDNA and cDNA samples. ( B ) Cq RNA calculation with ValidPrime-validated GOI assays. High confidence and A+ assays can be used with less gDNA samples for Cq RNA determination. It is recommended to confirm the absence of gDNA amplification at least once for A+ assays. Samples that do not contain sufficient gDNA to generate a signal with the VPA are attributed A*. As for gDNA insensitive A+ assays, Cq RNA equals Cq NA (i.e. output = input) in A* samples, since the DNA-derived signal is negligible [see Equations ( 2 and 4 )]. For gDNA-sensitive GOI assays, Cq RNA is calculated by a Cq DNA -based correction of Cq NA using Equations ( 4 and 5 ). To minimize the risk of jeopardizing the accuracy of the Cq RNA estimation, it is not advisable to perform correction on samples where the DNA-derived signal exceeds 60%. The calculations are facilitated using the ValidPrime software. Details on additional assay/sample grading and data output formats employed by the software are provided in Supplementary Figure S7 . The Cq RNA output data can be used for downstream data processing, such as normalization against reference genes.

    Techniques Used: Quantitative RT-PCR, Derivative Assay, Electrophoresis, Amplification, Polymerase Chain Reaction, Software

    5) Product Images from "Validation of Tuba1a as Appropriate Internal Control for Normalization of Gene Expression Analysis during Mouse Lung Development"

    Article Title: Validation of Tuba1a as Appropriate Internal Control for Normalization of Gene Expression Analysis during Mouse Lung Development

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms16034492

    Characterization of primer pairs designed for expression analysis of the internal control during mouse lung development. ( A ) Amplification efficiency for each primer pair was calculated using 10-fold serial dilutions of a cDNA template. Primer amplification efficiency was assessed by plotting the cycle threshold ( C T ) value for each concentration against the logarithm (base 10) of the fold dilution (log 10 (Quantity)). Efficiency was calculated using the slope of the linear function; ( B ) Dissociation curve analysis of primer specific products was performed by constantly monitoring the fluorescence with increasing temperatures from 60 to 95 °C. Melt curves were generated by plotting the negative first derivative of the fluorescence (−d/d T (Fluorescence) 520 nm) versus temperature (degree Celsius, °C); ( C ) Agarose gel electrophoresis after qRT-PCR indicates a specific product for each of the primer pairs. +RT, template is cDNA; −RT, template is RNA without reverse transcriptase, W, water control.
    Figure Legend Snippet: Characterization of primer pairs designed for expression analysis of the internal control during mouse lung development. ( A ) Amplification efficiency for each primer pair was calculated using 10-fold serial dilutions of a cDNA template. Primer amplification efficiency was assessed by plotting the cycle threshold ( C T ) value for each concentration against the logarithm (base 10) of the fold dilution (log 10 (Quantity)). Efficiency was calculated using the slope of the linear function; ( B ) Dissociation curve analysis of primer specific products was performed by constantly monitoring the fluorescence with increasing temperatures from 60 to 95 °C. Melt curves were generated by plotting the negative first derivative of the fluorescence (−d/d T (Fluorescence) 520 nm) versus temperature (degree Celsius, °C); ( C ) Agarose gel electrophoresis after qRT-PCR indicates a specific product for each of the primer pairs. +RT, template is cDNA; −RT, template is RNA without reverse transcriptase, W, water control.

    Techniques Used: Expressing, Amplification, Concentration Assay, Fluorescence, Generated, Agarose Gel Electrophoresis, Quantitative RT-PCR

    6) Product Images from "A parental requirement for dual-specificity phosphatase 6 in zebrafish"

    Article Title: A parental requirement for dual-specificity phosphatase 6 in zebrafish

    Journal: BMC Developmental Biology

    doi: 10.1186/s12861-018-0164-6

    Loss of dusp6 and dusp2 does not impact early development. a Offspring from crosses between double heterozygous dusp2 um287/+ ;dusp6 um286/+ males and females were raised to adulthood and genotyped in order to determine the percentage of each possible genotype in surviving fish. A chi-square test indicates no significant statistical difference between the actual and expected Mendelian ratios. b Outline of RNA-seq library production from wildtype and dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males) 18hpf embryos. c Diagrams representing the number of differentially expressed up-regulated (left circles) and down-regulated (right circles) genes in dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males) versus wildtype embryos. Inner circles indicate the subset of genes annotated in ZFIN. d 23 genes identified as differentially expressed by RNA-seq were re-examined by qPCR on cDNA derived from wildtype versus dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males) embryos. e-h 48hpf wildtype ( e, f ) and dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males; g, h ) embryos were assayed for changes in otx5 ( e, g ) and six7 ( f, h ) expression by in situ hybridization. i-q Uninjected ( i-k ) or MO-injected ( l-q ) dusp6 mutant (derived from crosses between dusp6 um286/um286 females and dusp6 um286/um286 males; i ), dusp2 mutant (derived from crosses between dusp2 um287/um287 females and dusp2 um287/um287 males; j ), and dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males; k ) embryos were assayed by immunostaining with 3A10 antibody to detect the Mauthner neurons at 48hpf. r-w 12hpf wildtype ( r, s, t ) and dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males; u, v, w ) embryos were assayed by immunostaining for pERK (green in r, u ; red counterstain detects the Valentino transcription factor), as well as by in situ hybridization for expression of pea3 ( s, v ), and erm ( t, w ). x-aa Wildtype ( x, y ) and dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males; z, aa ) embryos were injected with fgf8 mRNA and the expression pattern of pea3 visualized by in situ hybridization. All embryos are in dorsal view with anterior to the top. Numbers in top right corner of each panel indicate the total number of embryos assayed for that condition. Numbers in bottom right corner indicate percent of embryos with the phenotype shown
    Figure Legend Snippet: Loss of dusp6 and dusp2 does not impact early development. a Offspring from crosses between double heterozygous dusp2 um287/+ ;dusp6 um286/+ males and females were raised to adulthood and genotyped in order to determine the percentage of each possible genotype in surviving fish. A chi-square test indicates no significant statistical difference between the actual and expected Mendelian ratios. b Outline of RNA-seq library production from wildtype and dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males) 18hpf embryos. c Diagrams representing the number of differentially expressed up-regulated (left circles) and down-regulated (right circles) genes in dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males) versus wildtype embryos. Inner circles indicate the subset of genes annotated in ZFIN. d 23 genes identified as differentially expressed by RNA-seq were re-examined by qPCR on cDNA derived from wildtype versus dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males) embryos. e-h 48hpf wildtype ( e, f ) and dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males; g, h ) embryos were assayed for changes in otx5 ( e, g ) and six7 ( f, h ) expression by in situ hybridization. i-q Uninjected ( i-k ) or MO-injected ( l-q ) dusp6 mutant (derived from crosses between dusp6 um286/um286 females and dusp6 um286/um286 males; i ), dusp2 mutant (derived from crosses between dusp2 um287/um287 females and dusp2 um287/um287 males; j ), and dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males; k ) embryos were assayed by immunostaining with 3A10 antibody to detect the Mauthner neurons at 48hpf. r-w 12hpf wildtype ( r, s, t ) and dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males; u, v, w ) embryos were assayed by immunostaining for pERK (green in r, u ; red counterstain detects the Valentino transcription factor), as well as by in situ hybridization for expression of pea3 ( s, v ), and erm ( t, w ). x-aa Wildtype ( x, y ) and dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males; z, aa ) embryos were injected with fgf8 mRNA and the expression pattern of pea3 visualized by in situ hybridization. All embryos are in dorsal view with anterior to the top. Numbers in top right corner of each panel indicate the total number of embryos assayed for that condition. Numbers in bottom right corner indicate percent of embryos with the phenotype shown

    Techniques Used: Fluorescence In Situ Hybridization, RNA Sequencing Assay, Mutagenesis, Derivative Assay, Real-time Polymerase Chain Reaction, Expressing, In Situ Hybridization, Injection, Immunostaining

    7) Product Images from "The role of G-protein-coupled membrane estrogen receptor in mouse Leydig cell function—in vivo and in vitro evaluation"

    Article Title: The role of G-protein-coupled membrane estrogen receptor in mouse Leydig cell function—in vivo and in vitro evaluation

    Journal: Cell and Tissue Research

    doi: 10.1007/s00441-018-2861-7

    GPER mRNA level and protein localization in mouse testes and Leydig cells. ( a – f ’).  a  Representative gel electrophoresis of qualitative expression, (line N1-negative control without complementary DNA template, line N2-negative control without nonreverse transcribed RNA),  b  and relative quantification (RQ) of mRNA for GPER in mouse testes; immature, mature, aged [control and G-15 (50 μg/kg bw)-treated] and mouse MA-10 Leydig cells [control and G-15 (10 nM)-treated]. RQ is expressed as means ± SD. Asterisks show significant differences between control and G-15-treated testes/cells. Values are denoted as * p
    Figure Legend Snippet: GPER mRNA level and protein localization in mouse testes and Leydig cells. ( a – f ’). a Representative gel electrophoresis of qualitative expression, (line N1-negative control without complementary DNA template, line N2-negative control without nonreverse transcribed RNA), b and relative quantification (RQ) of mRNA for GPER in mouse testes; immature, mature, aged [control and G-15 (50 μg/kg bw)-treated] and mouse MA-10 Leydig cells [control and G-15 (10 nM)-treated]. RQ is expressed as means ± SD. Asterisks show significant differences between control and G-15-treated testes/cells. Values are denoted as * p

    Techniques Used: Nucleic Acid Electrophoresis, Expressing, Negative Control

    8) Product Images from "A Gammaherpesvirus Complement Regulatory Protein Promotes Initiation of Infection by Activation of Protein Kinase Akt/PKB"

    Article Title: A Gammaherpesvirus Complement Regulatory Protein Promotes Initiation of Infection by Activation of Protein Kinase Akt/PKB

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011672

    Delayed infection kinetics of the ORF4 − Tet + and ORF4.STOP mutants as determined by quantitative RT-PCR. The expression of the mRNA of the MHV-68 immediate-early gene Rta (ORF50) was examined by quantitative RT-PCR. Cells were infected at a multiplicity of infection of 10 for 1h at 37°C. Then, the inoculum was removed and fresh medium with PAA was added. Total RNA was isolated from infected cells 18 hours after infection and reverse transcribed. The resulting cDNA was used as template for PCR amplification of L8 and MHV-68 ORF50, respectively. Data are presented as relative expression of ORF50 after normalization to the corresponding L8 levels using the delta-delta C t method. 18 hours after infection, the expression of the ORF50 mRNA was significantly reduced (*, p = 0,002; **, p = 0,025; Student's t-test) in cells infected with the ORF4 mutants when compared with the parental or revertant virus. Data shown are means ± SD of two independent experiments, each determined in duplicates.
    Figure Legend Snippet: Delayed infection kinetics of the ORF4 − Tet + and ORF4.STOP mutants as determined by quantitative RT-PCR. The expression of the mRNA of the MHV-68 immediate-early gene Rta (ORF50) was examined by quantitative RT-PCR. Cells were infected at a multiplicity of infection of 10 for 1h at 37°C. Then, the inoculum was removed and fresh medium with PAA was added. Total RNA was isolated from infected cells 18 hours after infection and reverse transcribed. The resulting cDNA was used as template for PCR amplification of L8 and MHV-68 ORF50, respectively. Data are presented as relative expression of ORF50 after normalization to the corresponding L8 levels using the delta-delta C t method. 18 hours after infection, the expression of the ORF50 mRNA was significantly reduced (*, p = 0,002; **, p = 0,025; Student's t-test) in cells infected with the ORF4 mutants when compared with the parental or revertant virus. Data shown are means ± SD of two independent experiments, each determined in duplicates.

    Techniques Used: Infection, Quantitative RT-PCR, Expressing, Isolation, Polymerase Chain Reaction, Amplification

    9) Product Images from "LESR2 is a lymphatic endothelial-specific lncRNA that governs cell proliferation and migration through KLF4 and SEMA3C"

    Article Title: LESR2 is a lymphatic endothelial-specific lncRNA that governs cell proliferation and migration through KLF4 and SEMA3C

    Journal: bioRxiv

    doi: 10.1101/2020.05.25.114546

    Knockdown of LESR2 reduces cell growth, cell cycle progression, and migration of LECs in vitro . (a, b) Cell growth profiles and cell growth rates of neonatal LECs over 48h after ASOKD (a) or CRISPRi-KD (b) of LESR2 using IncuCyte. Sample’s confluences were normalized to T 0 . Growth rates were calculated as the slope of linear regression and normalized to control ASO/sgRNA. (c) Representative flow cytometry plots of neonatal LECs after 24h LESR2-ASOKD. Cells were firstly gated with live/dead Zombie staining (upper plots). Resulting living cells were further gated for non-proliferating stages subG0 and G0, and proliferating stages G1, S, G2, and M, using propidium iodide (IP) and Ki-67 (lower plots). (d) Quantification of the cell cycle progression analysis of neonatal LECs after 24h LESR2-ASOKD. Bars represent percentages of gated living cells in subG0, G0, G1, S, G2, and M. Statistical analysis was performed on G0 populations. (e) Representative images of the wound closure assay (9h) in neonatal LECs after LESR2-ASOKD. Confluence mask is shown for all time points. Before scratch, cells were incubated for 2h with 2 μ g/mL Mitomycin C (proliferation inhibitor) at 37°C. Scale bar represents 200 μ m. (f) Quantification of the wound closure assay (up to 9h) of neonatal LECs after LESR2-ASOKD. Percentages were determined for each time point using TScratch 98 . (g) Schematic representation of 3’ RACE results depicting the three LESR2 transcripts expressed in LECs: LESR2-1 (approx. 1,100bp), LESR2-2 (approx. 1,200bp), LESR2-3 (approx. 600bp). RNA-Seq signal was visualized through the Zenbu genome browser 106 . LESR2 transcript sequences are listed in Supplementary Table 7. (h) Comparison of qPCR levels of GAPDH (polyA+), H2BK (polyA-), LESR2-1, LESR2-2, LESR2-3 after cDNA synthesis with either oligodT or random hexamers primers in neonatal LECs derived from 3 donors. (i) Expression of LESR2-1, LESR2-2, and LESR2-3 relative to housekeeping gene GAPDH in neonatal LECs derived from 3 donors. (j) Quantification of the cell cycle progression analysis of pCDH-empty vector (pCDH-EV) and pCDH-LESR2 infected neonatal LECs after 24h LESR2-ASOKD. Statistical analysis was performed on G0 populations. (k) Quantification of the wound closure assay (up to 9h) of pCDH-EV and pCDH-LESR2 infected neonatal LECs after LESR2-ASOKD. Data are displayed as mean + SD (n = 10 in a, f, and k; n = 5 in b; n = 3 in h, i, and j; n = 2 in d). Percentages represent LESR2 knockdown efficiencies after the experiments. **P
    Figure Legend Snippet: Knockdown of LESR2 reduces cell growth, cell cycle progression, and migration of LECs in vitro . (a, b) Cell growth profiles and cell growth rates of neonatal LECs over 48h after ASOKD (a) or CRISPRi-KD (b) of LESR2 using IncuCyte. Sample’s confluences were normalized to T 0 . Growth rates were calculated as the slope of linear regression and normalized to control ASO/sgRNA. (c) Representative flow cytometry plots of neonatal LECs after 24h LESR2-ASOKD. Cells were firstly gated with live/dead Zombie staining (upper plots). Resulting living cells were further gated for non-proliferating stages subG0 and G0, and proliferating stages G1, S, G2, and M, using propidium iodide (IP) and Ki-67 (lower plots). (d) Quantification of the cell cycle progression analysis of neonatal LECs after 24h LESR2-ASOKD. Bars represent percentages of gated living cells in subG0, G0, G1, S, G2, and M. Statistical analysis was performed on G0 populations. (e) Representative images of the wound closure assay (9h) in neonatal LECs after LESR2-ASOKD. Confluence mask is shown for all time points. Before scratch, cells were incubated for 2h with 2 μ g/mL Mitomycin C (proliferation inhibitor) at 37°C. Scale bar represents 200 μ m. (f) Quantification of the wound closure assay (up to 9h) of neonatal LECs after LESR2-ASOKD. Percentages were determined for each time point using TScratch 98 . (g) Schematic representation of 3’ RACE results depicting the three LESR2 transcripts expressed in LECs: LESR2-1 (approx. 1,100bp), LESR2-2 (approx. 1,200bp), LESR2-3 (approx. 600bp). RNA-Seq signal was visualized through the Zenbu genome browser 106 . LESR2 transcript sequences are listed in Supplementary Table 7. (h) Comparison of qPCR levels of GAPDH (polyA+), H2BK (polyA-), LESR2-1, LESR2-2, LESR2-3 after cDNA synthesis with either oligodT or random hexamers primers in neonatal LECs derived from 3 donors. (i) Expression of LESR2-1, LESR2-2, and LESR2-3 relative to housekeeping gene GAPDH in neonatal LECs derived from 3 donors. (j) Quantification of the cell cycle progression analysis of pCDH-empty vector (pCDH-EV) and pCDH-LESR2 infected neonatal LECs after 24h LESR2-ASOKD. Statistical analysis was performed on G0 populations. (k) Quantification of the wound closure assay (up to 9h) of pCDH-EV and pCDH-LESR2 infected neonatal LECs after LESR2-ASOKD. Data are displayed as mean + SD (n = 10 in a, f, and k; n = 5 in b; n = 3 in h, i, and j; n = 2 in d). Percentages represent LESR2 knockdown efficiencies after the experiments. **P

    Techniques Used: Migration, In Vitro, Allele-specific Oligonucleotide, Flow Cytometry, Staining, Wound Closure Assay, Incubation, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Derivative Assay, Expressing, Plasmid Preparation, Infection

    10) Product Images from "Analysis of the nucleocytoplasmic shuttling RNA-binding protein HNRNPU using optimized HITS-CLIP method"

    Article Title: Analysis of the nucleocytoplasmic shuttling RNA-binding protein HNRNPU using optimized HITS-CLIP method

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0231450

    Schematic workflow of optimized CLIP. Cells are irradiated with 254 nm UV light on ice to form crosslinked RNA-RBP complexes, followed by partial RNase digestion and immunoprecipitation with RBP-specific antibodies. RNA crosslinked with RBPs is dephosphorylated for linker ligation, phosphorylated with [γ- 32 P]- ATP if necessary, separated by NuPAGE, and then transferred to a membrane. Isolated RNA is ligated with a 5'-App linker at the 3'-end and is subjected to BrdU-labeled cDNA synthesis by reverse transcription using a primer with a barcode and cleavage site. The cDNA is purified with the anti-BrdU antibody-coupled magnetic beads, circularized, digested with APE I, and amplified by PCR. Lastly, the CLIP cDNA library is subjected to size purification by PAGE. Steps modified from the original BrdU-CLIP protocol are indicated in red.
    Figure Legend Snippet: Schematic workflow of optimized CLIP. Cells are irradiated with 254 nm UV light on ice to form crosslinked RNA-RBP complexes, followed by partial RNase digestion and immunoprecipitation with RBP-specific antibodies. RNA crosslinked with RBPs is dephosphorylated for linker ligation, phosphorylated with [γ- 32 P]- ATP if necessary, separated by NuPAGE, and then transferred to a membrane. Isolated RNA is ligated with a 5'-App linker at the 3'-end and is subjected to BrdU-labeled cDNA synthesis by reverse transcription using a primer with a barcode and cleavage site. The cDNA is purified with the anti-BrdU antibody-coupled magnetic beads, circularized, digested with APE I, and amplified by PCR. Lastly, the CLIP cDNA library is subjected to size purification by PAGE. Steps modified from the original BrdU-CLIP protocol are indicated in red.

    Techniques Used: Cross-linking Immunoprecipitation, Irradiation, Immunoprecipitation, Ligation, Isolation, Labeling, Purification, Magnetic Beads, Amplification, Polymerase Chain Reaction, cDNA Library Assay, Polyacrylamide Gel Electrophoresis, Modification

    Optimization of dephosphorylation, linker ligation and circularization. (A–B) Quantification of reverse transcribed cDNA to evaluate the yield of linker-ligated RNA. Template RNA was isolated from HNRNPU-RNA, dephosphorylated with PNK or CIAP, and subsequently ligated using linkers with phosphorylated or adenylated 5’-ends. Linkers were phosphorylated or adenylated prior to polyacrylmide gel electrophoresis (PAGE) or after isolation from the membrane. cDNA that was reverse transcribed in the presence of [α- 32 P]-dCTP was purified with anti-BrdU beads and separated in a 10% TBE-urea gel (A). Signal density was quantified from the autoradiograph (B). (C) Schematics for optimization of elution during anti-BrdU purification and during circularization using RNA ladders. (D–E) Reverse transcribed cDNAs from linker-ligated RNA ladders were eluted from anti-BrdU beads as indicated in (C), and were subsequently circularized and re-linearized in tubes (EL2-1, -2, -3) or on beads (EL2-4).
    Figure Legend Snippet: Optimization of dephosphorylation, linker ligation and circularization. (A–B) Quantification of reverse transcribed cDNA to evaluate the yield of linker-ligated RNA. Template RNA was isolated from HNRNPU-RNA, dephosphorylated with PNK or CIAP, and subsequently ligated using linkers with phosphorylated or adenylated 5’-ends. Linkers were phosphorylated or adenylated prior to polyacrylmide gel electrophoresis (PAGE) or after isolation from the membrane. cDNA that was reverse transcribed in the presence of [α- 32 P]-dCTP was purified with anti-BrdU beads and separated in a 10% TBE-urea gel (A). Signal density was quantified from the autoradiograph (B). (C) Schematics for optimization of elution during anti-BrdU purification and during circularization using RNA ladders. (D–E) Reverse transcribed cDNAs from linker-ligated RNA ladders were eluted from anti-BrdU beads as indicated in (C), and were subsequently circularized and re-linearized in tubes (EL2-1, -2, -3) or on beads (EL2-4).

    Techniques Used: De-Phosphorylation Assay, Ligation, Isolation, Nucleic Acid Electrophoresis, Polyacrylamide Gel Electrophoresis, Purification, Autoradiography

    11) Product Images from "Activated α2-macroglobulin binding to cell surface GRP78 induces trophoblastic cell fusion"

    Article Title: Activated α2-macroglobulin binding to cell surface GRP78 induces trophoblastic cell fusion

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-66554-0

    α 2 M expression in primary villous cytotrophoblastic cells and BeWo cells. vCTB were purified from early (8 weeks of gestation) and late first-trimester (11 weeks of gestation) trophoblast and term placenta and seeded for 24, 48, 72 and 96 h. BeWo cells were seeded and treated or not with 20 µM Forskolin (FSK) for 48 h. RNA was retrotranscribed, and 50 ng of cDNA was used to perform α 2 M and GAPDH PCR. n = 3.
    Figure Legend Snippet: α 2 M expression in primary villous cytotrophoblastic cells and BeWo cells. vCTB were purified from early (8 weeks of gestation) and late first-trimester (11 weeks of gestation) trophoblast and term placenta and seeded for 24, 48, 72 and 96 h. BeWo cells were seeded and treated or not with 20 µM Forskolin (FSK) for 48 h. RNA was retrotranscribed, and 50 ng of cDNA was used to perform α 2 M and GAPDH PCR. n = 3.

    Techniques Used: Expressing, Purification, Polymerase Chain Reaction

    α 2 M* induced BeWo cell fusion through p-CREB, p-ERK1/2 and p-JNK activation, without affecting syncytin expression. ( A,B ) BeWo cells were seeded for 24 h prior to 24 h of starvation. Subsequently, cells were treated with 5 μM KT5720, 10 μM SP600125 or 10 μM UO126 for 1 h, and 100 pM of α 2 M* was added or not for 30 min. ( A ) Western blotting was performed. p-CREB, CREB, p-ERK1/2, ERK1/2, p-JNK and JNK levels were quantified using the ImageJ software, and data are expressed as the fold change relative to the control. n = 3. The images of bands for the target proteins were taken from the same gel, and each image was cropped, as delineated by black dividing lines, as well as adjusted for image intensity for optimal visualisation. ( B ) Nuclei and syncytia were counted, and a fusion index was calculated. n = 3. Data represented as mean±SEM. ns (not significant), *P ≤ 0.05, **P ≤ 0.01, *** P ≤ 0.005; ANOVA comparison test. ( C , D ) BeWo cells were seeded for 24 h prior to treatment with or without 100 pM of α 2 M*. ( C ) RNA was retrotranscribed, and 10 ng of cDNA was used to perform qPCR using syncytin-1 and syncytin-2 primers. n = 3. Data represented as mean±SEM. **P ≤ 0.01; t-test comparison test. ( D ) BeWo cells were seeded for 24 h prior to treatment with or without 100 pM of α 2 M* for 48 h. Western blotting was performed. Syncytin-1 and GAPDH levels were quantified using the ImageJ software, and data are expressed as the fold change relative to the control. n = 3. The images of bands for the target proteins were taken from the same gel, and each image was cropped, as delineated by black dividing lines, as well as adjusted for image intensity for optimal visualisation.
    Figure Legend Snippet: α 2 M* induced BeWo cell fusion through p-CREB, p-ERK1/2 and p-JNK activation, without affecting syncytin expression. ( A,B ) BeWo cells were seeded for 24 h prior to 24 h of starvation. Subsequently, cells were treated with 5 μM KT5720, 10 μM SP600125 or 10 μM UO126 for 1 h, and 100 pM of α 2 M* was added or not for 30 min. ( A ) Western blotting was performed. p-CREB, CREB, p-ERK1/2, ERK1/2, p-JNK and JNK levels were quantified using the ImageJ software, and data are expressed as the fold change relative to the control. n = 3. The images of bands for the target proteins were taken from the same gel, and each image was cropped, as delineated by black dividing lines, as well as adjusted for image intensity for optimal visualisation. ( B ) Nuclei and syncytia were counted, and a fusion index was calculated. n = 3. Data represented as mean±SEM. ns (not significant), *P ≤ 0.05, **P ≤ 0.01, *** P ≤ 0.005; ANOVA comparison test. ( C , D ) BeWo cells were seeded for 24 h prior to treatment with or without 100 pM of α 2 M*. ( C ) RNA was retrotranscribed, and 10 ng of cDNA was used to perform qPCR using syncytin-1 and syncytin-2 primers. n = 3. Data represented as mean±SEM. **P ≤ 0.01; t-test comparison test. ( D ) BeWo cells were seeded for 24 h prior to treatment with or without 100 pM of α 2 M* for 48 h. Western blotting was performed. Syncytin-1 and GAPDH levels were quantified using the ImageJ software, and data are expressed as the fold change relative to the control. n = 3. The images of bands for the target proteins were taken from the same gel, and each image was cropped, as delineated by black dividing lines, as well as adjusted for image intensity for optimal visualisation.

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

    12) Product Images from "Mouse Hepatitis Virus Infection Remodels Connexin43-Mediated Gap Junction Intercellular Communication In Vitro and In Vivo"

    Article Title: Mouse Hepatitis Virus Infection Remodels Connexin43-Mediated Gap Junction Intercellular Communication In Vitro and In Vivo

    Journal: Journal of Virology

    doi: 10.1128/JVI.02420-15

    Alteration of Cx43 in mouse brain due to MHV-A59 infection. Mice were infected intracranially with MHV-A59 at 50% of the LD 50 or with PBS-BSA for mock-infected mice. Mice were sacrificed at day 5 p.i., after which their liver and brain tissues were processed for RNA extraction. cDNA was synthesized from RNA of the brains and livers. To confirm infection, cDNAs from liver were amplified for virus-specific antinucleocapsid primers (IZJ5 and IZJ6). (A) Intracranial injection of mice with the virus showed the presence of nucleocapsid-specific amplicons (Infected lanes 1, 2, and 3) in liver. As expected, no such amplification was noted from mock-inoculated mice (Mock lanes 1 and 2). (B) Real-time qPCR analysis of the RNA samples from brain showed a significant 3.13- ± 0.06-fold reduction in relative abundance of Cx43 mRNA after MHV-A59 infection. The mean ± SEM incidences from three different mice are shown. (****, P
    Figure Legend Snippet: Alteration of Cx43 in mouse brain due to MHV-A59 infection. Mice were infected intracranially with MHV-A59 at 50% of the LD 50 or with PBS-BSA for mock-infected mice. Mice were sacrificed at day 5 p.i., after which their liver and brain tissues were processed for RNA extraction. cDNA was synthesized from RNA of the brains and livers. To confirm infection, cDNAs from liver were amplified for virus-specific antinucleocapsid primers (IZJ5 and IZJ6). (A) Intracranial injection of mice with the virus showed the presence of nucleocapsid-specific amplicons (Infected lanes 1, 2, and 3) in liver. As expected, no such amplification was noted from mock-inoculated mice (Mock lanes 1 and 2). (B) Real-time qPCR analysis of the RNA samples from brain showed a significant 3.13- ± 0.06-fold reduction in relative abundance of Cx43 mRNA after MHV-A59 infection. The mean ± SEM incidences from three different mice are shown. (****, P

    Techniques Used: Infection, Mouse Assay, RNA Extraction, Synthesized, Amplification, Injection, Real-time Polymerase Chain Reaction

    Reduction of Cx43 mRNA abundance in viral infection. Primary astrocytes were infected with MHV-A59 at an MOI of 2, and mock-infected cells were maintained in parallel. RNA was extracted at 24 h p.i., and subsequently cDNA was synthesized. Equal amounts of cDNA template were used for qPCR analysis. The relative expression of Cx43 mRNA was obtained using the ΔΔ C T method. (A) Compared to that of mock-infected cells, Cx43 expression was 3.49- ± 0.25-fold downregulated after MHV-A59 infection. The end products of the qPCR of Cx43 and an internal control, β-actin, were resolved in a 4% agarose gel and are shown in panel B. Mean ± SEM incidences from three different experiments are shown (***, P
    Figure Legend Snippet: Reduction of Cx43 mRNA abundance in viral infection. Primary astrocytes were infected with MHV-A59 at an MOI of 2, and mock-infected cells were maintained in parallel. RNA was extracted at 24 h p.i., and subsequently cDNA was synthesized. Equal amounts of cDNA template were used for qPCR analysis. The relative expression of Cx43 mRNA was obtained using the ΔΔ C T method. (A) Compared to that of mock-infected cells, Cx43 expression was 3.49- ± 0.25-fold downregulated after MHV-A59 infection. The end products of the qPCR of Cx43 and an internal control, β-actin, were resolved in a 4% agarose gel and are shown in panel B. Mean ± SEM incidences from three different experiments are shown (***, P

    Techniques Used: Infection, Synthesized, Real-time Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis

    13) Product Images from "Na+/K+ ATPase activity promotes invasion of endocrine resistant breast cancer cells"

    Article Title: Na+/K+ ATPase activity promotes invasion of endocrine resistant breast cancer cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0193779

    NKP expression and activity in normal and breast cancer cell lines. Panel A: NKP mRNA expression in ER+ (YS 1.2) and ER-ve (pII and MDA-MB-231) breast cancer cells. RNA was extracted from cells, converted to cDNA and PCR amplified. Ct values were converted to ratios relative to actin levels as described in Methods. Panel B: NKP protein expression level in the membranous compartment of the tested cell lines was determined by western blotting. Membranous fraction lysate protein (3 μg) was electrophoresed on 10% SDS polyacrylamide gel, blotted onto nitrocellulose membrane and probed with antisera to T-NKP and actin (loading control). This blot represents one of 3 independent determinations. NKP activity in the membranous fraction of all cell lines (panel C), and in pII cells in response to ouabain/TTX treatment (panel D) was determined by a colorimetric NKP activity kit. Histobars represent means ± SEM of at least 3 independent determinations. *denotes significant difference from MCF10A (panel C) or pII control (panel D) with p
    Figure Legend Snippet: NKP expression and activity in normal and breast cancer cell lines. Panel A: NKP mRNA expression in ER+ (YS 1.2) and ER-ve (pII and MDA-MB-231) breast cancer cells. RNA was extracted from cells, converted to cDNA and PCR amplified. Ct values were converted to ratios relative to actin levels as described in Methods. Panel B: NKP protein expression level in the membranous compartment of the tested cell lines was determined by western blotting. Membranous fraction lysate protein (3 μg) was electrophoresed on 10% SDS polyacrylamide gel, blotted onto nitrocellulose membrane and probed with antisera to T-NKP and actin (loading control). This blot represents one of 3 independent determinations. NKP activity in the membranous fraction of all cell lines (panel C), and in pII cells in response to ouabain/TTX treatment (panel D) was determined by a colorimetric NKP activity kit. Histobars represent means ± SEM of at least 3 independent determinations. *denotes significant difference from MCF10A (panel C) or pII control (panel D) with p

    Techniques Used: Expressing, Activity Assay, Multiple Displacement Amplification, Polymerase Chain Reaction, Amplification, Western Blot

    Effect of siRNA-mediated knockdown of NKP on pII cell motility. Panel A: NKP mRNA expression in pII cells transfected with a scrambled sequence (control, open bar) or NKP siRNA (48 h, solid bar). RNA was extracted from cells, converted to cDNA and amplified by PCR. Ct values were converted to ratios relative to actin. Panel B: mean distance moved (in pixels) as a measurement of cell motility for pII cell transfected with a scrambled sequence (control, open bar, taken as 100%) or NKP siRNA (48 h, solid bar) determined after 24 h of incubation. Histobars represent means ± SEM of at least 3 independent determinations. Asterisk denotes significant difference from control with p
    Figure Legend Snippet: Effect of siRNA-mediated knockdown of NKP on pII cell motility. Panel A: NKP mRNA expression in pII cells transfected with a scrambled sequence (control, open bar) or NKP siRNA (48 h, solid bar). RNA was extracted from cells, converted to cDNA and amplified by PCR. Ct values were converted to ratios relative to actin. Panel B: mean distance moved (in pixels) as a measurement of cell motility for pII cell transfected with a scrambled sequence (control, open bar, taken as 100%) or NKP siRNA (48 h, solid bar) determined after 24 h of incubation. Histobars represent means ± SEM of at least 3 independent determinations. Asterisk denotes significant difference from control with p

    Techniques Used: Expressing, Transfection, Sequencing, Amplification, Polymerase Chain Reaction, Incubation

    14) Product Images from "Induction of the unfolded protein response by cigarette smoke is primarily an activating transcription factor 4-C/EBP homologous protein mediated process"

    Article Title: Induction of the unfolded protein response by cigarette smoke is primarily an activating transcription factor 4-C/EBP homologous protein mediated process

    Journal: International Journal of Chronic Obstructive Pulmonary Disease

    doi: 10.2147/COPD.S19599

    Cigarette smoke extract exposure appears to induce CHOP expression in an ATF4 signaling cascades in SAE cells. A ) AFT6 and B ) XBP-1 levels were examined in SAE cells by qPCR, following 5% CSE exposure for 24 hours. C ) XBP1 PCR samples were examined on 3% agarose gel uncut or following Pst1 digestion. SAE cells were exposed to CSE (5 or 10%), thapsigargin or tunicamycin for 3 hours. XBP1 splicing was examined on uncut and Pst1 digested XBP1 amplified cDNA. D ) Phosphorylation of PERK was examined by Western blot following small airway epithelial cells exposed to 5% cigarette smoke extract for 1 hour. Densitometry confirmed an increase in phosphorylation of PERK. E ) ATF4 levels were examined in SAE cells by qPCR, following 5% and 10% CSE exposure for 24 hours. Notes: Each assay was performed on 4 independent days and P -values shown, comparing CSE exposed cells to control. Abbreviations: CHOP, C/EBP homologous protein; CSE, cigarette smoke extract; SAE, small airway epithelial.
    Figure Legend Snippet: Cigarette smoke extract exposure appears to induce CHOP expression in an ATF4 signaling cascades in SAE cells. A ) AFT6 and B ) XBP-1 levels were examined in SAE cells by qPCR, following 5% CSE exposure for 24 hours. C ) XBP1 PCR samples were examined on 3% agarose gel uncut or following Pst1 digestion. SAE cells were exposed to CSE (5 or 10%), thapsigargin or tunicamycin for 3 hours. XBP1 splicing was examined on uncut and Pst1 digested XBP1 amplified cDNA. D ) Phosphorylation of PERK was examined by Western blot following small airway epithelial cells exposed to 5% cigarette smoke extract for 1 hour. Densitometry confirmed an increase in phosphorylation of PERK. E ) ATF4 levels were examined in SAE cells by qPCR, following 5% and 10% CSE exposure for 24 hours. Notes: Each assay was performed on 4 independent days and P -values shown, comparing CSE exposed cells to control. Abbreviations: CHOP, C/EBP homologous protein; CSE, cigarette smoke extract; SAE, small airway epithelial.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Western Blot

    15) Product Images from "Upregulation of Serotonin Transporter by Alcohol in Human Dendritic Cells: Possible Implication in Neuroimmune Deregulation"

    Article Title: Upregulation of Serotonin Transporter by Alcohol in Human Dendritic Cells: Possible Implication in Neuroimmune Deregulation

    Journal: Alcoholism, clinical and experimental research

    doi: 10.1111/j.1530-0277.2009.01010.x

    Monoamine oxidase-A (MAO-A) gene is upregulated by alcohol. After 24 hours treatment with 0.1% alcohol, the dendritic cells were harvested, cytoplasmic RNA from the cell pellet was extracted, reverse transcribed to cDNA, and the cDNA was used for the
    Figure Legend Snippet: Monoamine oxidase-A (MAO-A) gene is upregulated by alcohol. After 24 hours treatment with 0.1% alcohol, the dendritic cells were harvested, cytoplasmic RNA from the cell pellet was extracted, reverse transcribed to cDNA, and the cDNA was used for the

    Techniques Used:

    16) Product Images from "What Kaplan-Meier Survival Curves Don’t Tell Us About CNS Disease"

    Article Title: What Kaplan-Meier Survival Curves Don’t Tell Us About CNS Disease

    Journal: Journal of neuroimmunology

    doi: 10.1016/j.jneuroim.2017.01.020

    Immune deficient mice maintain increased levels of viral RNA after survival of a neurotropic infection NSE-CD46 + and NSE-CD46 + mice of the indicated immune KO background challenged intracerebrally with 1×10 4 PFU MV-Edmonston. RT-qPCR data were generated using random hexamer priming for cDNA generation followed by qPCR using primers specific for the MV nucleoprotein and cyclophilin B as a standard. Data analyzed using the ΔΔCT method. # P
    Figure Legend Snippet: Immune deficient mice maintain increased levels of viral RNA after survival of a neurotropic infection NSE-CD46 + and NSE-CD46 + mice of the indicated immune KO background challenged intracerebrally with 1×10 4 PFU MV-Edmonston. RT-qPCR data were generated using random hexamer priming for cDNA generation followed by qPCR using primers specific for the MV nucleoprotein and cyclophilin B as a standard. Data analyzed using the ΔΔCT method. # P

    Techniques Used: Mouse Assay, Infection, Quantitative RT-PCR, Generated, Random Hexamer Labeling, Real-time Polymerase Chain Reaction

    17) Product Images from "Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis"

    Article Title: Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2019.10.006

    HFD-fed iRhom2 KO mice have increased thermogenesis and browning of the white adipose tissue. A-C Thermal images (A), and BAT (B) or body (C) temperature of iRhom2 KO and WT mice fed with SD and HFD for 19 weeks. One experiment with 8 mice per group. D RT-PCR analysis of UCP1, PGC1α, Cidea, PRDM16, and Cox8b expression in SD-fed iRhom2 KO and HFD-fed WT and iRhom2 KO mice BAT samples compared to WT SD-fed control samples. Two experiments with 3–4 replicates in each. E, G Representative photographs of eWAT (E) and sWAT (G) UCP1 immunohistochemistry derived from iRhom2 KO and WT mice fed with HFD for 20 weeks. Scale bar = 100 μm. F, H Graphics showing the UCP1 percentage of area stained in the eWAT (F) and sWAT (H) of the mice described above. One experiment with 8 mice per group (with 2 photographs analyzed per mouse). I RT-PCR analysis of UCP1 in brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT or iRhom2 KO mice. Three independent experiments. J RT-PCR analysis of UCP1 in immortalized WT brown preadipocytes transduced with empty vector or iRhom2-HA, differentiated in vitro and stimulated with norepinephrine for 6 h. Two independent experiments. K HA expression in differentiated immortalized WT brown preadipocytes transduced with retrovirus containing iRhom2 cDNA fused to C-terminal HA tag (iR2-HA). As a negative control, we used the same cells transduced with retrovirus containing the empty vector (EV), and as a loading control we measured p97 protein level. Two independent experiments. L Mitochondrial oxygen consumption rate (OCR) of brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT and iRhom2 KO mice and stimulated or not with norepinephrine. The results were normalized to the protein content. Two experiments with one or two independent samples per genotype, respectively. M Mitochondrial proton leak of the cells described in L normalized to the protein content. Error bars represent SEM; * represents p
    Figure Legend Snippet: HFD-fed iRhom2 KO mice have increased thermogenesis and browning of the white adipose tissue. A-C Thermal images (A), and BAT (B) or body (C) temperature of iRhom2 KO and WT mice fed with SD and HFD for 19 weeks. One experiment with 8 mice per group. D RT-PCR analysis of UCP1, PGC1α, Cidea, PRDM16, and Cox8b expression in SD-fed iRhom2 KO and HFD-fed WT and iRhom2 KO mice BAT samples compared to WT SD-fed control samples. Two experiments with 3–4 replicates in each. E, G Representative photographs of eWAT (E) and sWAT (G) UCP1 immunohistochemistry derived from iRhom2 KO and WT mice fed with HFD for 20 weeks. Scale bar = 100 μm. F, H Graphics showing the UCP1 percentage of area stained in the eWAT (F) and sWAT (H) of the mice described above. One experiment with 8 mice per group (with 2 photographs analyzed per mouse). I RT-PCR analysis of UCP1 in brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT or iRhom2 KO mice. Three independent experiments. J RT-PCR analysis of UCP1 in immortalized WT brown preadipocytes transduced with empty vector or iRhom2-HA, differentiated in vitro and stimulated with norepinephrine for 6 h. Two independent experiments. K HA expression in differentiated immortalized WT brown preadipocytes transduced with retrovirus containing iRhom2 cDNA fused to C-terminal HA tag (iR2-HA). As a negative control, we used the same cells transduced with retrovirus containing the empty vector (EV), and as a loading control we measured p97 protein level. Two independent experiments. L Mitochondrial oxygen consumption rate (OCR) of brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT and iRhom2 KO mice and stimulated or not with norepinephrine. The results were normalized to the protein content. Two experiments with one or two independent samples per genotype, respectively. M Mitochondrial proton leak of the cells described in L normalized to the protein content. Error bars represent SEM; * represents p

    Techniques Used: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemistry, Derivative Assay, Staining, In Vitro, Transduction, Plasmid Preparation, Negative Control

    18) Product Images from "TLR-4-Dependent and -Independent Mechanisms of Fetal Brain Injury in the Setting of Preterm Birth"

    Article Title: TLR-4-Dependent and -Independent Mechanisms of Fetal Brain Injury in the Setting of Preterm Birth

    Journal: Reproductive Sciences

    doi: 10.1177/1933719112438439

    Toll-like receptor 4 (TLR-4) messenger RNA (mRNA) expression at day 2 and day 7 in culture (cortical) as determined by polymerase chain reaction (PCR). Fetal brains harvested at embryonic day 15 (E15) (A) or E18 (B) have low levels of TLR-4 expression at both 2 and 7 days in culture. Intensity increases at higher cycle numbers (indicated on right). Lane M is base marker. Lanes C1 to C3 represent TLR-4 expression in cortical cultures harvested from fetuses exposed to intrauterine saline (controls). Lanes L1 to L3 represent TLR-4 mRNA expression in cortical cultures from fetuses exposed to intrauterine inflammation (lipopolysaccharide [LPS]). Positive controls for TLR-4 mRNA expression were E18 whole fetal brains (E18), postnatal brains from P21 (PND) and from pregnant mouse uterus, which is known to highly express TLR-4. The last lane represents the negative control with water instead of complementary DNA (cDNA).
    Figure Legend Snippet: Toll-like receptor 4 (TLR-4) messenger RNA (mRNA) expression at day 2 and day 7 in culture (cortical) as determined by polymerase chain reaction (PCR). Fetal brains harvested at embryonic day 15 (E15) (A) or E18 (B) have low levels of TLR-4 expression at both 2 and 7 days in culture. Intensity increases at higher cycle numbers (indicated on right). Lane M is base marker. Lanes C1 to C3 represent TLR-4 expression in cortical cultures harvested from fetuses exposed to intrauterine saline (controls). Lanes L1 to L3 represent TLR-4 mRNA expression in cortical cultures from fetuses exposed to intrauterine inflammation (lipopolysaccharide [LPS]). Positive controls for TLR-4 mRNA expression were E18 whole fetal brains (E18), postnatal brains from P21 (PND) and from pregnant mouse uterus, which is known to highly express TLR-4. The last lane represents the negative control with water instead of complementary DNA (cDNA).

    Techniques Used: Expressing, Polymerase Chain Reaction, Marker, Negative Control

    19) Product Images from "Tissue, developmental, and caste-specific expression of odorant binding proteins in a eusocial insect, the red imported fire ant, Solenopsis invicta"

    Article Title: Tissue, developmental, and caste-specific expression of odorant binding proteins in a eusocial insect, the red imported fire ant, Solenopsis invicta

    Journal: Scientific Reports

    doi: 10.1038/srep35452

    Expression of SiOBP s in S. invicta workers. ( A ) qRT-PCR was performed on RNA isolated from fire ant worker antennae, head, thorax, and abdomen. Absolute quantification values are shown. Color scale shows transcripts/μg total RNA used for cDNA synthesis. ( B ) Data in ( A ) normalized to EF1α expression levels.
    Figure Legend Snippet: Expression of SiOBP s in S. invicta workers. ( A ) qRT-PCR was performed on RNA isolated from fire ant worker antennae, head, thorax, and abdomen. Absolute quantification values are shown. Color scale shows transcripts/μg total RNA used for cDNA synthesis. ( B ) Data in ( A ) normalized to EF1α expression levels.

    Techniques Used: Expressing, Quantitative RT-PCR, Isolation

    Expression of SiOBP s in S. invicta female alates. ( A ) qRT-PCR was performed on RNA isolated from fire ant female alate antennae, head, thorax, and abdomen. Absolute quantification values are shown. Color scale shows transcripts/μg total RNA used for cDNA synthesis. ( B ) Data in ( A ) normalized to EF1α expression levels.
    Figure Legend Snippet: Expression of SiOBP s in S. invicta female alates. ( A ) qRT-PCR was performed on RNA isolated from fire ant female alate antennae, head, thorax, and abdomen. Absolute quantification values are shown. Color scale shows transcripts/μg total RNA used for cDNA synthesis. ( B ) Data in ( A ) normalized to EF1α expression levels.

    Techniques Used: Expressing, Quantitative RT-PCR, Isolation

    Expression of SiOBP s in S. invicta male alates. ( A ) qRT-PCR was performed on RNA isolated from fire ant male alate antennae, head, thorax, and abdomen. Absolute quantification values are shown. Color scale shows transcripts/μg total RNA used for cDNA synthesis. (B) Data in ( A ) normalized to EF1α expression levels.
    Figure Legend Snippet: Expression of SiOBP s in S. invicta male alates. ( A ) qRT-PCR was performed on RNA isolated from fire ant male alate antennae, head, thorax, and abdomen. Absolute quantification values are shown. Color scale shows transcripts/μg total RNA used for cDNA synthesis. (B) Data in ( A ) normalized to EF1α expression levels.

    Techniques Used: Expressing, Quantitative RT-PCR, Isolation

    Developmental expression of SiOBP s. ( A ) qRT-PCR was performed on RNA isolated from fire ant eggs, early and late instar and pupae. Absolute quantification values are shown. Color scale shows transcripts/μg total RNA used for cDNA synthesis. ( B ) Data in ( A ) normalized to EF1α expression levels.
    Figure Legend Snippet: Developmental expression of SiOBP s. ( A ) qRT-PCR was performed on RNA isolated from fire ant eggs, early and late instar and pupae. Absolute quantification values are shown. Color scale shows transcripts/μg total RNA used for cDNA synthesis. ( B ) Data in ( A ) normalized to EF1α expression levels.

    Techniques Used: Expressing, Quantitative RT-PCR, Isolation

    20) Product Images from "Altered hepatic glucose homeostasis in AnxA6-KO mice fed a high-fat diet"

    Article Title: Altered hepatic glucose homeostasis in AnxA6-KO mice fed a high-fat diet

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201310

    Expression of nuclear transcription factors during the PTT of HFD-fed WT and AnxA6-KO mice. (A) Nuclear fractions from liver samples from WT and AnxA6-KO (AnxA6 -/- ) mice before (0 min; WT lane 1–4, AnxA6-KO lane 5–8) and 120 min after pyruvate administration (120 min; WT lane 9–12, AnxA6-KO lane 13–16; n = 4 per group) were prepared and analyzed by western blotting for the transcription factors FoxO1, PPARα, SREBP1 and LXR. β-actin served as loading control. Molecular weight markers are shown. Arrowhead points at LXR. (B) Relative levels of FoxO1, PPARα, SREBP1 and LXR were quantified and normalized to β-actin expression. The mean values (± SEM) relative to WT at t = 0 min are shown. (C) RNA from HFD-fed WT and AnxA6-KO livers before (0 min) and 120 min after pyruvate administration was isolated (n = 4 per group). cDNA was generated and RT-PCR for AnxA6, fibroblast growth factor 21 (Fgf21), glucose-6 phosphatase (G6P) and insulin induced gene 1 (Insig1) was performed as described in Material and Methods. Relative mRNA expression was normalised to the housekeeper Tbp levels using the ΔΔCT method. The expression relative to the WT at t = 0 min is shown. * P
    Figure Legend Snippet: Expression of nuclear transcription factors during the PTT of HFD-fed WT and AnxA6-KO mice. (A) Nuclear fractions from liver samples from WT and AnxA6-KO (AnxA6 -/- ) mice before (0 min; WT lane 1–4, AnxA6-KO lane 5–8) and 120 min after pyruvate administration (120 min; WT lane 9–12, AnxA6-KO lane 13–16; n = 4 per group) were prepared and analyzed by western blotting for the transcription factors FoxO1, PPARα, SREBP1 and LXR. β-actin served as loading control. Molecular weight markers are shown. Arrowhead points at LXR. (B) Relative levels of FoxO1, PPARα, SREBP1 and LXR were quantified and normalized to β-actin expression. The mean values (± SEM) relative to WT at t = 0 min are shown. (C) RNA from HFD-fed WT and AnxA6-KO livers before (0 min) and 120 min after pyruvate administration was isolated (n = 4 per group). cDNA was generated and RT-PCR for AnxA6, fibroblast growth factor 21 (Fgf21), glucose-6 phosphatase (G6P) and insulin induced gene 1 (Insig1) was performed as described in Material and Methods. Relative mRNA expression was normalised to the housekeeper Tbp levels using the ΔΔCT method. The expression relative to the WT at t = 0 min is shown. * P

    Techniques Used: Expressing, Mouse Assay, Western Blot, Molecular Weight, Isolation, Generated, Reverse Transcription Polymerase Chain Reaction

    21) Product Images from "Comprehensive evaluation of blood-brain barrier-forming micro-vasculatures: Reference and marker genes with cellular composition"

    Article Title: Comprehensive evaluation of blood-brain barrier-forming micro-vasculatures: Reference and marker genes with cellular composition

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0197379

    The expression stability of RG candidates among endothelial cell lines and brain samples. Total RNA was extracted and converted into cDNA by reverse-transcription at 25 ng/ul, and followed by real-time qPCR with 25 ng/reaction. (A) Distribution of cycle threshold (Ct) values for five RG candidates by quantitative RT-PCR in BMEC and bEnd3 cell lines. The experiments were repeated 3 times in duplicate reactions. Boxes showed the range of Ct values for each candidate gene. The central line indicated the median Ct; the extended upper and lower indicate 75 and 25 percentiles. (B) The average expression stability (M value) of RG candidates in two endothelial cell lines analyzed by geNorm. RG candidates were ranked from the least stable to the most stable (left to right). (C) Ct values for four RG candidates in BrMV and CDB. Data were derived from 10 BrMV isolation experiments with 4 from WT mice, 2 from Het and 4 from Idua knock-out mice. Each sample was tested 3 times in duplicate. **, p
    Figure Legend Snippet: The expression stability of RG candidates among endothelial cell lines and brain samples. Total RNA was extracted and converted into cDNA by reverse-transcription at 25 ng/ul, and followed by real-time qPCR with 25 ng/reaction. (A) Distribution of cycle threshold (Ct) values for five RG candidates by quantitative RT-PCR in BMEC and bEnd3 cell lines. The experiments were repeated 3 times in duplicate reactions. Boxes showed the range of Ct values for each candidate gene. The central line indicated the median Ct; the extended upper and lower indicate 75 and 25 percentiles. (B) The average expression stability (M value) of RG candidates in two endothelial cell lines analyzed by geNorm. RG candidates were ranked from the least stable to the most stable (left to right). (C) Ct values for four RG candidates in BrMV and CDB. Data were derived from 10 BrMV isolation experiments with 4 from WT mice, 2 from Het and 4 from Idua knock-out mice. Each sample was tested 3 times in duplicate. **, p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Derivative Assay, Isolation, Mouse Assay, Knock-Out

    Verification of Actb as the best reference gene by Idua expression in BrMV of WT mice and Het mice. (A) Standard curve for absolute quantification of Idua mRNA. A plasmid containing Idua cDNA was used for generating standard curve with copy numbers by qPCR. Data was derived from 2 sets of standard samples, each amplified three times in duplicate. Error bars, standard deviation. (B, C) Idua expression in BrMV isolated from either wild-type C57/Bl6 mice (WT) or littermates of heterozygous for Idua knock-out (Het) with normalization by RG candidates. Total RNA from 4 WT and 4 Het samples were examined by RT-qPCR and calculated either by absolute Idua standard curve for copy numbers per ng RNA (B), or by ΔΔCt method for relative Idua fold changes (C).
    Figure Legend Snippet: Verification of Actb as the best reference gene by Idua expression in BrMV of WT mice and Het mice. (A) Standard curve for absolute quantification of Idua mRNA. A plasmid containing Idua cDNA was used for generating standard curve with copy numbers by qPCR. Data was derived from 2 sets of standard samples, each amplified three times in duplicate. Error bars, standard deviation. (B, C) Idua expression in BrMV isolated from either wild-type C57/Bl6 mice (WT) or littermates of heterozygous for Idua knock-out (Het) with normalization by RG candidates. Total RNA from 4 WT and 4 Het samples were examined by RT-qPCR and calculated either by absolute Idua standard curve for copy numbers per ng RNA (B), or by ΔΔCt method for relative Idua fold changes (C).

    Techniques Used: Expressing, Mouse Assay, Plasmid Preparation, Real-time Polymerase Chain Reaction, Derivative Assay, Amplification, Standard Deviation, Isolation, Knock-Out, Quantitative RT-PCR

    22) Product Images from "The gut microbiota profile is associated with insulin action in humans"

    Article Title: The gut microbiota profile is associated with insulin action in humans

    Journal: Acta Diabetologica

    doi: 10.1007/s00592-012-0410-5

    RNA gut microbial profile from ceca of insulin-sensitive and insulin-resistant obese patients. Total RNA from both luminal and mucosal cecum (appendix) was extracted from obese insulin-resistant and obese insulin-sensitive patients. The 16S rRNA was retrotranscripted, then cDNA amplified, and the amplicons were separated by electrophoresis on a gel with a denaturating gradient (DGGE). Each band was referred to as a microbial marker. The figure shows a the RNA-DGGE gel, with an internal marker ( M ) for electrophoresis control and b the cluster analysis showing the Pearson’s evolutionary tree ( left - side )
    Figure Legend Snippet: RNA gut microbial profile from ceca of insulin-sensitive and insulin-resistant obese patients. Total RNA from both luminal and mucosal cecum (appendix) was extracted from obese insulin-resistant and obese insulin-sensitive patients. The 16S rRNA was retrotranscripted, then cDNA amplified, and the amplicons were separated by electrophoresis on a gel with a denaturating gradient (DGGE). Each band was referred to as a microbial marker. The figure shows a the RNA-DGGE gel, with an internal marker ( M ) for electrophoresis control and b the cluster analysis showing the Pearson’s evolutionary tree ( left - side )

    Techniques Used: Amplification, Electrophoresis, Denaturing Gradient Gel Electrophoresis, Marker

    23) Product Images from "Ndr kinases regulate retinal interneuron proliferation and homeostasis"

    Article Title: Ndr kinases regulate retinal interneuron proliferation and homeostasis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30492-9

    Mouse Ndr1 and Ndr2 knockout strategy and confirmation. ( A ) The conditional-ready Ndr2/Stk38l deletion allele obtained from KOMP. Ndr2/Stk38l exon 7 (green box) is flanked by LoxP sites (red triangles) and excised by the cre recombinase under control of the actinB promoter to produce Ndr2 KO mice. LacZ is indicated by the blue box, Neo cassette is indicated by the orange box. RT-qPCR primers for Exons 13–14 are indicated by red arrows. ( B ) RT-qPCR data confirms Ndr2 deletion. cDNA was isolated from brain and eye tissue from P28 wild type (WT) and Ndr2 KO mice. Data are from 4 sets of RT-qPCRs, targeting exons 13 to 14, with each sample run in duplicate (p
    Figure Legend Snippet: Mouse Ndr1 and Ndr2 knockout strategy and confirmation. ( A ) The conditional-ready Ndr2/Stk38l deletion allele obtained from KOMP. Ndr2/Stk38l exon 7 (green box) is flanked by LoxP sites (red triangles) and excised by the cre recombinase under control of the actinB promoter to produce Ndr2 KO mice. LacZ is indicated by the blue box, Neo cassette is indicated by the orange box. RT-qPCR primers for Exons 13–14 are indicated by red arrows. ( B ) RT-qPCR data confirms Ndr2 deletion. cDNA was isolated from brain and eye tissue from P28 wild type (WT) and Ndr2 KO mice. Data are from 4 sets of RT-qPCRs, targeting exons 13 to 14, with each sample run in duplicate (p

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

    24) Product Images from "Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels"

    Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3070-16.2017

    RNF138 reduces Ca V 2.1 protein stability. A , Lack of effect of RNF138/RNF138-H36E overexpression on human Ca V 2.1 mRNA level in HEK293T cells subject to the indicated transfection condition ( p > 0.05; n = 3). To rule out the potential contamination arising from human Ca V 2.1 plasmid in RNA prepared from transfected cells, RT-PCR was performed in the absence (left) or presence (right) of DNase I treatment before reverse transcription reaction. Also shown is the blank control that involves identical PCR in the absence of cDNA template (vertical arrows). The signals of Ca V 2.1 were standardized as the ratio to those of cognate GAPDH, followed by normalization to the corresponding Myc vector control. B , RNF138 knock-down does not significantly change rat Ca V 2.1 mRNA level in neurons ( p > 0.05; n = 3). RT-PCR analyses were based on RNA extracted from cultured cortical neurons subject to the indicated shRNA infection. Standardized Ca V 2.1 signals were normalized to the shGFP infection control. C , Representative immunoblots showing the effect of RNF128, RNF138, or RNF138-H36E coexpression on protein stability of human Ca V 2.1 subunit. Ca V 2.1 protein turnover kinetics in HEK293T cells was analyzed by applying cycloheximide (CHX) with the indicated treatment durations (h). Coexpression with the Myc vector was used as the control experiment. D , Quantification of Ca V 2.1 protein half-life in the presence of Myc vector (black), RNF128 (green), RNF138 (blue), or RNF138-H36E (red). Left, Normalized Ca V 2.1 protein densities with respect to cycloheximide treatment durations. Data points represent the average of 7–8 independent experiments. Center, Same data points were transformed into a semilogarithmic plot, which is subject to single linear-regression analyses (solid lines; top) or double linear-regression analyses (solid lines; bottom with RNF138 only). Right, Comparison of Ca V 2.1 protein half-life values derived from linear-regression analyses. The estimated Ca V 2.1 protein half-life values based on single linear-regression analyses (top right) are ∼8.1 ± 0.3 (with vector; n = 8), 8.7 ± 1.3 (with RNF128; n = 7), 3.1 ± 0.4 (with RNF138; n = 8), and 10.9 ± 0.7 (with RNF138-H36E; n = 8) h. Based on double linear-regression analyses (bottom right), the estimated Ca V 2.1 protein half-life values in the presence of RNF138 are ∼1.3 ± 0.3 h (fast component) and 5.5 ± 0.6 h (slow component). E , Representative immunoblots showing the effect of shRNA knock-down of endogenous RNF13 8 on Ca V 2.1 protein turnover kinetics in HEK293T cells. shGFP infection was used as the control experiment. F , Quantification and comparison of Ca V 2.1 protein half-life values derived from different shRNA infection conditions. The estimated Ca V 2.1 protein half-life values are ∼6.4 ± 1.0 h (with shGFP; n = 9; black) and 10.3 ± 1.4 h (with shRNF138–1; n = 9; red). The protein half-life value of Ca V 2.1 in the presence of shGFP is not statistically different ( p > 0.05) from that of Ca V 2.1 with vector in D . Asterisks denote significant difference from the control (* p
    Figure Legend Snippet: RNF138 reduces Ca V 2.1 protein stability. A , Lack of effect of RNF138/RNF138-H36E overexpression on human Ca V 2.1 mRNA level in HEK293T cells subject to the indicated transfection condition ( p > 0.05; n = 3). To rule out the potential contamination arising from human Ca V 2.1 plasmid in RNA prepared from transfected cells, RT-PCR was performed in the absence (left) or presence (right) of DNase I treatment before reverse transcription reaction. Also shown is the blank control that involves identical PCR in the absence of cDNA template (vertical arrows). The signals of Ca V 2.1 were standardized as the ratio to those of cognate GAPDH, followed by normalization to the corresponding Myc vector control. B , RNF138 knock-down does not significantly change rat Ca V 2.1 mRNA level in neurons ( p > 0.05; n = 3). RT-PCR analyses were based on RNA extracted from cultured cortical neurons subject to the indicated shRNA infection. Standardized Ca V 2.1 signals were normalized to the shGFP infection control. C , Representative immunoblots showing the effect of RNF128, RNF138, or RNF138-H36E coexpression on protein stability of human Ca V 2.1 subunit. Ca V 2.1 protein turnover kinetics in HEK293T cells was analyzed by applying cycloheximide (CHX) with the indicated treatment durations (h). Coexpression with the Myc vector was used as the control experiment. D , Quantification of Ca V 2.1 protein half-life in the presence of Myc vector (black), RNF128 (green), RNF138 (blue), or RNF138-H36E (red). Left, Normalized Ca V 2.1 protein densities with respect to cycloheximide treatment durations. Data points represent the average of 7–8 independent experiments. Center, Same data points were transformed into a semilogarithmic plot, which is subject to single linear-regression analyses (solid lines; top) or double linear-regression analyses (solid lines; bottom with RNF138 only). Right, Comparison of Ca V 2.1 protein half-life values derived from linear-regression analyses. The estimated Ca V 2.1 protein half-life values based on single linear-regression analyses (top right) are ∼8.1 ± 0.3 (with vector; n = 8), 8.7 ± 1.3 (with RNF128; n = 7), 3.1 ± 0.4 (with RNF138; n = 8), and 10.9 ± 0.7 (with RNF138-H36E; n = 8) h. Based on double linear-regression analyses (bottom right), the estimated Ca V 2.1 protein half-life values in the presence of RNF138 are ∼1.3 ± 0.3 h (fast component) and 5.5 ± 0.6 h (slow component). E , Representative immunoblots showing the effect of shRNA knock-down of endogenous RNF13 8 on Ca V 2.1 protein turnover kinetics in HEK293T cells. shGFP infection was used as the control experiment. F , Quantification and comparison of Ca V 2.1 protein half-life values derived from different shRNA infection conditions. The estimated Ca V 2.1 protein half-life values are ∼6.4 ± 1.0 h (with shGFP; n = 9; black) and 10.3 ± 1.4 h (with shRNF138–1; n = 9; red). The protein half-life value of Ca V 2.1 in the presence of shGFP is not statistically different ( p > 0.05) from that of Ca V 2.1 with vector in D . Asterisks denote significant difference from the control (* p

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Cell Culture, shRNA, Infection, Western Blot, Transformation Assay, Derivative Assay

    25) Product Images from "Induced Pluripotent Stem Cell Modeling of Multisystemic, Hereditary Transthyretin Amyloidosis"

    Article Title: Induced Pluripotent Stem Cell Modeling of Multisystemic, Hereditary Transthyretin Amyloidosis

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2013.10.003

    Derivation of Neuronal-Lineage Cells from ATTR L55P iPSCs that Are Capable of TTR Internalization (A) Phase-contrast image shows neuronal cells derived from ATTR L55P iPSCs. (B) Immunofluorescence staining presents ATTR L55P iPSC-derived neuronal cells for the neuronal marker TUJ1 (red). Nuclei were stained with DAPI (blue). (C) Immunofluorescence staining shows ATTR L55P iPSC-derived neuronal cells for the MAP2 (red). Nuclei were stained with DAPI (blue). (D) Quantitative PCR analysis of derived neuronal-lineage cells compared to undifferentiated iPSCs and human brain cDNA is shown. Neuronal cells exhibit upregulated levels of TUJ1 and motor neuron homeobox transcription factor HB9. All independent biological samples (n = 3) were normalized to GAPDH. Error bars, SD. (E) ATTR L55P iPSC neuronal-lineage cells are capable of TTR (both WT and L55P) internalization as demonstrated by exposure to AF488-labeled human recombinant proteins. Foci of labeled proteins (green fluorescence) are visible in the cells. Nuclei were stained with DAPI (blue).
    Figure Legend Snippet: Derivation of Neuronal-Lineage Cells from ATTR L55P iPSCs that Are Capable of TTR Internalization (A) Phase-contrast image shows neuronal cells derived from ATTR L55P iPSCs. (B) Immunofluorescence staining presents ATTR L55P iPSC-derived neuronal cells for the neuronal marker TUJ1 (red). Nuclei were stained with DAPI (blue). (C) Immunofluorescence staining shows ATTR L55P iPSC-derived neuronal cells for the MAP2 (red). Nuclei were stained with DAPI (blue). (D) Quantitative PCR analysis of derived neuronal-lineage cells compared to undifferentiated iPSCs and human brain cDNA is shown. Neuronal cells exhibit upregulated levels of TUJ1 and motor neuron homeobox transcription factor HB9. All independent biological samples (n = 3) were normalized to GAPDH. Error bars, SD. (E) ATTR L55P iPSC neuronal-lineage cells are capable of TTR (both WT and L55P) internalization as demonstrated by exposure to AF488-labeled human recombinant proteins. Foci of labeled proteins (green fluorescence) are visible in the cells. Nuclei were stained with DAPI (blue).

    Techniques Used: Derivative Assay, Immunofluorescence, Staining, Marker, Real-time Polymerase Chain Reaction, Labeling, Recombinant, Fluorescence

    26) Product Images from "A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation"

    Article Title: A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-017-0906-6

    Kv1.3 channels regulate TAP1, EHD1, GABPA, and IL1B expression in primary murine microglia. a Experimental plan for in vivo studies: adult C57BL/6 mice received four daily IP doses of PBS, ShK-223, LPS, or LPS+ShK-223, and brain mononuclear cells were isolated for flow cytometry from one hemisphere and qRT-PCR studies from the other hemisphere ( n = 3, mice/group). b Results from flow cytometric studies measuring intracellular EHD1 protein expression in freshly isolated CNS MPs (gated first on live cells, followed by CD11b + CD45 low and CD11b + CD45 high populations). At least 10,000 live CNS MPs were counted per sample. Left: Example of flow cytometric histograms comparing EHD1 expression in CD11b + CD45 low microglia from one mouse from each treatment group. Right: Quantitative analysis of EHD1 + cells in CD11b + CD45 low and CD11b + CD45 high populations. c Results from pRT-PCR studies measuring mRNA expression of TAP1, EHD1, GABPA, and IL1B in CNS MPs. For these studies, all CNS MPs isolated from one hemisphere were used for RNA extraction in Trizol, followed by cDNA preparation followed by quantitative PCR. HPRT was used as the housekeeping gene. Data normalized to HPRT were then normalized to PBS-treated control samples (* p
    Figure Legend Snippet: Kv1.3 channels regulate TAP1, EHD1, GABPA, and IL1B expression in primary murine microglia. a Experimental plan for in vivo studies: adult C57BL/6 mice received four daily IP doses of PBS, ShK-223, LPS, or LPS+ShK-223, and brain mononuclear cells were isolated for flow cytometry from one hemisphere and qRT-PCR studies from the other hemisphere ( n = 3, mice/group). b Results from flow cytometric studies measuring intracellular EHD1 protein expression in freshly isolated CNS MPs (gated first on live cells, followed by CD11b + CD45 low and CD11b + CD45 high populations). At least 10,000 live CNS MPs were counted per sample. Left: Example of flow cytometric histograms comparing EHD1 expression in CD11b + CD45 low microglia from one mouse from each treatment group. Right: Quantitative analysis of EHD1 + cells in CD11b + CD45 low and CD11b + CD45 high populations. c Results from pRT-PCR studies measuring mRNA expression of TAP1, EHD1, GABPA, and IL1B in CNS MPs. For these studies, all CNS MPs isolated from one hemisphere were used for RNA extraction in Trizol, followed by cDNA preparation followed by quantitative PCR. HPRT was used as the housekeeping gene. Data normalized to HPRT were then normalized to PBS-treated control samples (* p

    Techniques Used: Expressing, In Vivo, Mouse Assay, Isolation, Flow Cytometry, Cytometry, Quantitative RT-PCR, Polymerase Chain Reaction, RNA Extraction, Real-time Polymerase Chain Reaction

    27) Product Images from "Combined transcriptome studies identify AFF3 as a mediator of the oncogenic effects of β-catenin in adrenocortical carcinoma"

    Article Title: Combined transcriptome studies identify AFF3 as a mediator of the oncogenic effects of β-catenin in adrenocortical carcinoma

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2015.20

    AFF3 is a β-catenin target gene in adrenocortical carcinoma cells. ( a ) Genomic structure of the AFF3 gene with the two putative transcription start sites (TSS) at the beginning of exons 1 and 3 indicated by two arrows. Each black rectangle represents an exon. Y axis values show the read depth of RNA-seq analysis on adrenocortical cells H295R. Finer details of the AFF3 TSS regions are shown and demonstrate that only the TSS at exon 3 is used. Promoter analysis (nucleotide positions –1500 to +500) by Genomatix software identified one Wnt Response Element (WRE) shown in the lower left box. The AFF3 variant mRNAs produced were analyzed by producing cDNA from total mRNA from H295R cells, then PCR using oligonucleotides corresponding to sequences in exon 3 and exon 5, followed by agarose gel electrophoresis; 167 bp: isoform 1 and 242 bp: isoform 2. ( b ) The upper histograms represent the effects in H295R cells of ΔN-TCF4 (an inhibitor of the β-catenin/TCF pathway) and LEF1 silencing on the β-catenin-LEF/TCF-dependent luciferase reporter construct Top-flash (left panel) and expression of other genes as indicated (right panel). The lower histograms represent the effects of β-catenin silencing by dox treatment of the shβ line on the expression of Top-Flash and luciferase constructs, with two copies of the putative AFF3 WRE wild-type sequence (2xWT) or mutant sequence (2xMut). ( c ) Western blot analysis of AFF3 protein accumulation in H295R cells (cytosol/nucleus) after CTNNB1 (siCTNNB1) or AFF3 (siAFF3-1 and 2) silencing. Histograms represent the quantification of AFF3 proteins from one experiment representative of three independent experiments.
    Figure Legend Snippet: AFF3 is a β-catenin target gene in adrenocortical carcinoma cells. ( a ) Genomic structure of the AFF3 gene with the two putative transcription start sites (TSS) at the beginning of exons 1 and 3 indicated by two arrows. Each black rectangle represents an exon. Y axis values show the read depth of RNA-seq analysis on adrenocortical cells H295R. Finer details of the AFF3 TSS regions are shown and demonstrate that only the TSS at exon 3 is used. Promoter analysis (nucleotide positions –1500 to +500) by Genomatix software identified one Wnt Response Element (WRE) shown in the lower left box. The AFF3 variant mRNAs produced were analyzed by producing cDNA from total mRNA from H295R cells, then PCR using oligonucleotides corresponding to sequences in exon 3 and exon 5, followed by agarose gel electrophoresis; 167 bp: isoform 1 and 242 bp: isoform 2. ( b ) The upper histograms represent the effects in H295R cells of ΔN-TCF4 (an inhibitor of the β-catenin/TCF pathway) and LEF1 silencing on the β-catenin-LEF/TCF-dependent luciferase reporter construct Top-flash (left panel) and expression of other genes as indicated (right panel). The lower histograms represent the effects of β-catenin silencing by dox treatment of the shβ line on the expression of Top-Flash and luciferase constructs, with two copies of the putative AFF3 WRE wild-type sequence (2xWT) or mutant sequence (2xMut). ( c ) Western blot analysis of AFF3 protein accumulation in H295R cells (cytosol/nucleus) after CTNNB1 (siCTNNB1) or AFF3 (siAFF3-1 and 2) silencing. Histograms represent the quantification of AFF3 proteins from one experiment representative of three independent experiments.

    Techniques Used: RNA Sequencing Assay, Software, Variant Assay, Produced, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Luciferase, Construct, Expressing, Sequencing, Mutagenesis, Western Blot

    28) Product Images from "The Tyrphostin Agent AG490 Prevents and Reverses Type 1 Diabetes in NOD Mice"

    Article Title: The Tyrphostin Agent AG490 Prevents and Reverses Type 1 Diabetes in NOD Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036079

    AG490 modulates phenotype and function of DC. A - Dendritic cells were generated from bone marrow of NOD mice treated either with AG490 or DMSO as described in materials and methods and stained directly with CD11c, MHC II, CD80 and CD86. Data shown represents CD11c gated population of pool fresh purified immature CD11c of AG490 and DMSO treated mice. B- Histograms represent expression of CD11c, CD86, CD80 and CD62L on gated CD11c population of mice treated with AG490 or DMSO in vivo . C- Bone marrow cells were isolated from NOD mice treated with AG490/DMSO for 5 weeks and then differentiated into DC in vitro as described. Total RNA (200 ng) of DC was converted into cDNA and then expression of Tgf-β1 was measured by Real-Time TaqMan Gene expression assays (Applied Biosystems). Data represents the mean (fold change) of two real-time RT-PCR experiments and the bars represent standard deviation of the mean. Fold change of Tgf-β1expression is shown (n = 5 mice/group, * p = 0.02). D- BMDCs were generated from BALB/c mice treated in vitro with 20 µM AG490 or DMSO for 12 hrs and then co-cultured with constant numbers of CD4+CD25− T-cells (1×10 5 ) with soluble anti-CD3 (2.5 ng/ml) for 72 hrs. Incorporation of 3 H-thymidine was measured in the last 16 hr of cell culture. Experiment was performed in triplicate format and repeated at least twice. Data analysis was performed using the student t -test and p value≤0.05 considered significant. Bars represent deviation of the mean (* p = 0.002, **p = 0.0002; ***p = 0.001). [Figures A B are intended to be in color online and black and white in print].
    Figure Legend Snippet: AG490 modulates phenotype and function of DC. A - Dendritic cells were generated from bone marrow of NOD mice treated either with AG490 or DMSO as described in materials and methods and stained directly with CD11c, MHC II, CD80 and CD86. Data shown represents CD11c gated population of pool fresh purified immature CD11c of AG490 and DMSO treated mice. B- Histograms represent expression of CD11c, CD86, CD80 and CD62L on gated CD11c population of mice treated with AG490 or DMSO in vivo . C- Bone marrow cells were isolated from NOD mice treated with AG490/DMSO for 5 weeks and then differentiated into DC in vitro as described. Total RNA (200 ng) of DC was converted into cDNA and then expression of Tgf-β1 was measured by Real-Time TaqMan Gene expression assays (Applied Biosystems). Data represents the mean (fold change) of two real-time RT-PCR experiments and the bars represent standard deviation of the mean. Fold change of Tgf-β1expression is shown (n = 5 mice/group, * p = 0.02). D- BMDCs were generated from BALB/c mice treated in vitro with 20 µM AG490 or DMSO for 12 hrs and then co-cultured with constant numbers of CD4+CD25− T-cells (1×10 5 ) with soluble anti-CD3 (2.5 ng/ml) for 72 hrs. Incorporation of 3 H-thymidine was measured in the last 16 hr of cell culture. Experiment was performed in triplicate format and repeated at least twice. Data analysis was performed using the student t -test and p value≤0.05 considered significant. Bars represent deviation of the mean (* p = 0.002, **p = 0.0002; ***p = 0.001). [Figures A B are intended to be in color online and black and white in print].

    Techniques Used: Generated, Mouse Assay, Staining, Purification, Expressing, In Vivo, Isolation, In Vitro, Quantitative RT-PCR, Standard Deviation, Cell Culture

    29) Product Images from "Altered hepatic glucose homeostasis in AnxA6-KO mice fed a high-fat diet"

    Article Title: Altered hepatic glucose homeostasis in AnxA6-KO mice fed a high-fat diet

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201310

    Expression of nuclear transcription factors during the PTT of HFD-fed WT and AnxA6-KO mice. (A) Nuclear fractions from liver samples from WT and AnxA6-KO (AnxA6 -/- ) mice before (0 min; WT lane 1–4, AnxA6-KO lane 5–8) and 120 min after pyruvate administration (120 min; WT lane 9–12, AnxA6-KO lane 13–16; n = 4 per group) were prepared and analyzed by western blotting for the transcription factors FoxO1, PPARα, SREBP1 and LXR. β-actin served as loading control. Molecular weight markers are shown. Arrowhead points at LXR. (B) Relative levels of FoxO1, PPARα, SREBP1 and LXR were quantified and normalized to β-actin expression. The mean values (± SEM) relative to WT at t = 0 min are shown. (C) RNA from HFD-fed WT and AnxA6-KO livers before (0 min) and 120 min after pyruvate administration was isolated (n = 4 per group). cDNA was generated and RT-PCR for AnxA6, fibroblast growth factor 21 (Fgf21), glucose-6 phosphatase (G6P) and insulin induced gene 1 (Insig1) was performed as described in Material and Methods. Relative mRNA expression was normalised to the housekeeper Tbp levels using the ΔΔCT method. The expression relative to the WT at t = 0 min is shown. * P
    Figure Legend Snippet: Expression of nuclear transcription factors during the PTT of HFD-fed WT and AnxA6-KO mice. (A) Nuclear fractions from liver samples from WT and AnxA6-KO (AnxA6 -/- ) mice before (0 min; WT lane 1–4, AnxA6-KO lane 5–8) and 120 min after pyruvate administration (120 min; WT lane 9–12, AnxA6-KO lane 13–16; n = 4 per group) were prepared and analyzed by western blotting for the transcription factors FoxO1, PPARα, SREBP1 and LXR. β-actin served as loading control. Molecular weight markers are shown. Arrowhead points at LXR. (B) Relative levels of FoxO1, PPARα, SREBP1 and LXR were quantified and normalized to β-actin expression. The mean values (± SEM) relative to WT at t = 0 min are shown. (C) RNA from HFD-fed WT and AnxA6-KO livers before (0 min) and 120 min after pyruvate administration was isolated (n = 4 per group). cDNA was generated and RT-PCR for AnxA6, fibroblast growth factor 21 (Fgf21), glucose-6 phosphatase (G6P) and insulin induced gene 1 (Insig1) was performed as described in Material and Methods. Relative mRNA expression was normalised to the housekeeper Tbp levels using the ΔΔCT method. The expression relative to the WT at t = 0 min is shown. * P

    Techniques Used: Expressing, Mouse Assay, Western Blot, Molecular Weight, Isolation, Generated, Reverse Transcription Polymerase Chain Reaction

    30) Product Images from "Functional conservation of EXA1 among diverse plant species for the infection by a family of plant viruses"

    Article Title: Functional conservation of EXA1 among diverse plant species for the infection by a family of plant viruses

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-42400-w

    Cloning of EXA1 homologs in Nicotiana benthamiana and their functional analysis in potexvirus infection. ( a ) Schematic image of the cDNA structure of NbEXA1a . GYF domain- and eIF4E-binding motif-encoding regions are depicted by stripes. Target regions for virus-induced gene silencing (VIGS) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) are indicated by bars under the image. ( b ) Morphological phenotypes of NbEXA1 -silenced and control plants. Photographs were taken from the top (upper) and side (bottom) of plants at 27 days post-inoculation (dpi). Bars = 5 cm. ( c ) Relative accumulation of NbEXA1 mRNA in NbEXA1 -silenced and control plants. Total RNA was extracted at 27 dpi and analyzed using qRT-PCR. The mean level of NbEXA1 transcript in control plants was used as the standard (1.0), and that in NbEXA1 -silenced plants is shown above the bar. Error bars indicate standard deviation (SD) of 10 samples. ** P
    Figure Legend Snippet: Cloning of EXA1 homologs in Nicotiana benthamiana and their functional analysis in potexvirus infection. ( a ) Schematic image of the cDNA structure of NbEXA1a . GYF domain- and eIF4E-binding motif-encoding regions are depicted by stripes. Target regions for virus-induced gene silencing (VIGS) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) are indicated by bars under the image. ( b ) Morphological phenotypes of NbEXA1 -silenced and control plants. Photographs were taken from the top (upper) and side (bottom) of plants at 27 days post-inoculation (dpi). Bars = 5 cm. ( c ) Relative accumulation of NbEXA1 mRNA in NbEXA1 -silenced and control plants. Total RNA was extracted at 27 dpi and analyzed using qRT-PCR. The mean level of NbEXA1 transcript in control plants was used as the standard (1.0), and that in NbEXA1 -silenced plants is shown above the bar. Error bars indicate standard deviation (SD) of 10 samples. ** P

    Techniques Used: Clone Assay, Functional Assay, Infection, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation

    31) Product Images from "Knockdown and replacement therapy mediated by artificial mirtrons in spinocerebellar ataxia 7"

    Article Title: Knockdown and replacement therapy mediated by artificial mirtrons in spinocerebellar ataxia 7

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx483

    Mirtrons were developed to silence ataxin 7. Forty eight hours transfections were carried out and the measurement obtained with each control (nontargeting NAD intron or nonspecific shRNA) was set as 1. ( A ) Mirtrons were designed against various target sites within the ataxin 7 cDNA sequence, utilizing either the 5΄ or 3΄ mirtron arm as indicated (black/red respectively). Target sequences were incorporated into the 3΄ UTR of Renilla luciferase of a dual-luciferase construct (Tar1/Tar2, dashed boxes). Relative normalized Renilla-to-firefly luciferase ratios are shown for each mirtron against the appropriate target, co-transfected in HEK-293 cells. Values are the mean±standard deviation (SD) of N = 3. ( B ) Sequence and predicted secondary structure of mirt-18 and mirt-1cN, with intended mature miRNA in red uppercase letters. Lines indicate Watson–Crick base pairs, circles indicate weak (G-U) base pairs. ( C ) Relative mCherry fluorescence, indicating silencing activity of mirtrons and shRNAs against full-length mCherry-tagged mutant ataxin 7 containing an expansion of 100 glutamines (ataxin 7-Q100-mCherry), co-transfected in HEK-293 cells. Values are mean ± SD of N = 6, except † N = 5. ( D ) qPCR analysis of endogenous ataxin 7 mRNA in patient-derived fibroblasts, normalised to 18S. Values are mean ± SD of N = 4. ( E ) qPCR analysis of endogenous ataxin 7 mRNA, miR-124 and miR-16 in SH-SY5Y cells, normalised to 18S. Values are mean ± SD of N = 4. * P
    Figure Legend Snippet: Mirtrons were developed to silence ataxin 7. Forty eight hours transfections were carried out and the measurement obtained with each control (nontargeting NAD intron or nonspecific shRNA) was set as 1. ( A ) Mirtrons were designed against various target sites within the ataxin 7 cDNA sequence, utilizing either the 5΄ or 3΄ mirtron arm as indicated (black/red respectively). Target sequences were incorporated into the 3΄ UTR of Renilla luciferase of a dual-luciferase construct (Tar1/Tar2, dashed boxes). Relative normalized Renilla-to-firefly luciferase ratios are shown for each mirtron against the appropriate target, co-transfected in HEK-293 cells. Values are the mean±standard deviation (SD) of N = 3. ( B ) Sequence and predicted secondary structure of mirt-18 and mirt-1cN, with intended mature miRNA in red uppercase letters. Lines indicate Watson–Crick base pairs, circles indicate weak (G-U) base pairs. ( C ) Relative mCherry fluorescence, indicating silencing activity of mirtrons and shRNAs against full-length mCherry-tagged mutant ataxin 7 containing an expansion of 100 glutamines (ataxin 7-Q100-mCherry), co-transfected in HEK-293 cells. Values are mean ± SD of N = 6, except † N = 5. ( D ) qPCR analysis of endogenous ataxin 7 mRNA in patient-derived fibroblasts, normalised to 18S. Values are mean ± SD of N = 4. ( E ) qPCR analysis of endogenous ataxin 7 mRNA, miR-124 and miR-16 in SH-SY5Y cells, normalised to 18S. Values are mean ± SD of N = 4. * P

    Techniques Used: Transfection, shRNA, Sequencing, Luciferase, Construct, Standard Deviation, Fluorescence, Activity Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Derivative Assay

    32) Product Images from "Peripheral Blood Mitochondrial DNA Damage as a Potential Noninvasive Biomarker of Diabetic Retinopathy"

    Article Title: Peripheral Blood Mitochondrial DNA Damage as a Potential Noninvasive Biomarker of Diabetic Retinopathy

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.16-19073

    Copy number of mtDNA and its transcription in the peripheral blood from diabetic subjects. ( a ) Copy number was quantified in the genomic DNA by qPCR using CYTB as mtDNA-encoded and β-ACTIN as a nuclear DNA-encoded gene. ( b ) Transcripts of mtDNA-encoded CYTB were quantified in the blood cDNA by qPCR using β-ACTIN as a housekeeping gene. The values are represented as mean ± SD obtained from five to six diabetic patients each with retinopathy (Diab-Ret) or without retinopathy (Diab-No Ret), and 7 to 10 nondiabetic subjects (Norm). * P
    Figure Legend Snippet: Copy number of mtDNA and its transcription in the peripheral blood from diabetic subjects. ( a ) Copy number was quantified in the genomic DNA by qPCR using CYTB as mtDNA-encoded and β-ACTIN as a nuclear DNA-encoded gene. ( b ) Transcripts of mtDNA-encoded CYTB were quantified in the blood cDNA by qPCR using β-ACTIN as a housekeeping gene. The values are represented as mean ± SD obtained from five to six diabetic patients each with retinopathy (Diab-Ret) or without retinopathy (Diab-No Ret), and 7 to 10 nondiabetic subjects (Norm). * P

    Techniques Used: Real-time Polymerase Chain Reaction

    33) Product Images from "Transcriptional targeting of sphingosine-1-phosphate receptor S1P2 by epigallocatechin-3-gallate prevents sphingosine-1-phosphate-mediated signaling in macrophage-differentiated HL-60 promyelomonocytic leukemia cells"

    Article Title: Transcriptional targeting of sphingosine-1-phosphate receptor S1P2 by epigallocatechin-3-gallate prevents sphingosine-1-phosphate-mediated signaling in macrophage-differentiated HL-60 promyelomonocytic leukemia cells

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S62717

    Macrophage differentiation corr elates with significant increases in S1P1 , S1P2 , and S1P5 gene expression. Notes: Serum-starved HL-60 cells were treated for 18 hours with 3 μM PMA to induce HL-60 cell differentiation and adhesion. Total RNA was then isolated from adherent cells, transcribed into cDNA, and qRT-PCR was performed to assess the expression of the S1P receptors S1P1, S1P2, S1P3, S1P4, and S1P5. ( A ) A representative agarose gel electrophoresis is shown to confirm the generation of single amplicons. ( B ) The qRT-PCR gene expression assay for each of the five genes assessed was performed and expressed as the mean of triplicates. This experiment is a representative quantification of one out of four PMA treatments performed at different HL-60 cell passages. Abbreviations: cDNA, complementary deoxyribonucleic acid; HL, human leukemia; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MW stds, molecular weight standard deviations; PMA, phorbol-12-myristate-13-acetate; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; RNA, ribonucleic acid; S1P, sphingosine-1-phosphate.
    Figure Legend Snippet: Macrophage differentiation corr elates with significant increases in S1P1 , S1P2 , and S1P5 gene expression. Notes: Serum-starved HL-60 cells were treated for 18 hours with 3 μM PMA to induce HL-60 cell differentiation and adhesion. Total RNA was then isolated from adherent cells, transcribed into cDNA, and qRT-PCR was performed to assess the expression of the S1P receptors S1P1, S1P2, S1P3, S1P4, and S1P5. ( A ) A representative agarose gel electrophoresis is shown to confirm the generation of single amplicons. ( B ) The qRT-PCR gene expression assay for each of the five genes assessed was performed and expressed as the mean of triplicates. This experiment is a representative quantification of one out of four PMA treatments performed at different HL-60 cell passages. Abbreviations: cDNA, complementary deoxyribonucleic acid; HL, human leukemia; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MW stds, molecular weight standard deviations; PMA, phorbol-12-myristate-13-acetate; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; RNA, ribonucleic acid; S1P, sphingosine-1-phosphate.

    Techniques Used: Expressing, Cell Differentiation, Isolation, Quantitative RT-PCR, Agarose Gel Electrophoresis, Molecular Weight, Reverse Transcription Polymerase Chain Reaction

    34) Product Images from "Argonaute proteins regulate HIV-1 multiply spliced RNA and viral production in a Dicer independent manner"

    Article Title: Argonaute proteins regulate HIV-1 multiply spliced RNA and viral production in a Dicer independent manner

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1289

    Knockdown of Ago1 in Ago2 knockout cells increases the level of multiply spliced HIV-1 transcripts independently on the miRNA pathway. ( A ) pNL4-3 provirus was transfected in Ago2 knockout and Ago1 knockdown (Ago2 KO/ Ago1 KD) HeLa cells and in Dicer knockout (Dicer KO) HeLa-P4.2 cells or their respective parental cells. Seventy-two hours later, RNA was extracted, reverse transcribed and total viral cDNA was quantified by qPCR using specific primers. Semi-quantitative PCR was performed on the same amount of total viral cDNA using primers specific for total, MS and SS HIV-1 mRNAs (see Supplementary Table S3 for primer sequences). Products of amplification were visualized on 1% agarose gel. A representative gel of five independent experiments is presented. ( B, C and D) (US) (B), SS (C) or MS (D) mRNA isolated from Ago2 KO/ Ago1 KD cells were quantified by qPCR and their ratio to total viral RNA are indicated. Values are presented as percentage of the ratio in parental cells. Data are presented as mean of five replicates ± SD. ( E ) US, ( F ) SS or ( G ) MS mRNAs isolated from Dicer KO cells were quantified by qPCR and their ratio to total viral RNA are indicated. Values are presented as percentage of the ratio in parental cells. Data are presented as mean of four replicates ± SD. P- values were calculated using a Student's t- test (** P
    Figure Legend Snippet: Knockdown of Ago1 in Ago2 knockout cells increases the level of multiply spliced HIV-1 transcripts independently on the miRNA pathway. ( A ) pNL4-3 provirus was transfected in Ago2 knockout and Ago1 knockdown (Ago2 KO/ Ago1 KD) HeLa cells and in Dicer knockout (Dicer KO) HeLa-P4.2 cells or their respective parental cells. Seventy-two hours later, RNA was extracted, reverse transcribed and total viral cDNA was quantified by qPCR using specific primers. Semi-quantitative PCR was performed on the same amount of total viral cDNA using primers specific for total, MS and SS HIV-1 mRNAs (see Supplementary Table S3 for primer sequences). Products of amplification were visualized on 1% agarose gel. A representative gel of five independent experiments is presented. ( B, C and D) (US) (B), SS (C) or MS (D) mRNA isolated from Ago2 KO/ Ago1 KD cells were quantified by qPCR and their ratio to total viral RNA are indicated. Values are presented as percentage of the ratio in parental cells. Data are presented as mean of five replicates ± SD. ( E ) US, ( F ) SS or ( G ) MS mRNAs isolated from Dicer KO cells were quantified by qPCR and their ratio to total viral RNA are indicated. Values are presented as percentage of the ratio in parental cells. Data are presented as mean of four replicates ± SD. P- values were calculated using a Student's t- test (** P

    Techniques Used: Knock-Out, Transfection, Real-time Polymerase Chain Reaction, Mass Spectrometry, Amplification, Agarose Gel Electrophoresis, Isolation

    HITS-CLIP reveals HIV-1 regions bound by Ago2 in HIV-1 infected cells. ( A ) HEK293T cells were transfected with plasmid expressing either GFP-Ago2 or control GFP. Twenty-four hours later, they were infected with VSVg-pseudotyped HIV-1 NL4-3 at a M.O.I. of 1 or mock infected. Twenty-four hours later, cells were subjected to UV cross-linking and RNA–protein complexes were subjected to immunoprecipitation using an anti-GFP antibody. IPed RNA was radiolabeled with [γ- 32 P]ATP and samples resolved on SDS-PAGE. Radiolabeled RNA was visualized by autoradiography. RNAs migrating at a higher molecular mass than the protein of interest (GFP-Ago2:RNA, between 125 and 225 kDa) were extracted from the membrane, reverse transcribed into cDNA and subjected to high throughput sequencing. ( B ) Reads obtained from non-infected (NI) and infected (INF) samples were uploaded to CLIPZ software. Reads that aligned to the human genome were sorted into categories: messenger RNA (mRNA), microRNA (miRNA), ribosomal RNA (rRNA), repeat, small non-coding RNA (snRNA), small nucleolar RNA (snoRNA), transfer RNA (tRNA) and other. Pie charts correspond to the distribution of the mean value of three biological replicates. ( C ) Reads obtained from infected samples were aligned to the HIV-1 NL4-3 sequence. Only reads ≥50 nucleotides length that aligned once without mismatch were considered for this analysis. Reads obtained from three independent HITS-CLIP experiments are presented in shades of orange and those obtained from the corresponding inputs in shades of blue. The map of the HIV-1 genome is presented below. Splice donor (D) and acceptor (A) sites are indicated. Peaks corresponding to clusters 2, 13, 16, 21, 25 and 30 are indicated by arrows.
    Figure Legend Snippet: HITS-CLIP reveals HIV-1 regions bound by Ago2 in HIV-1 infected cells. ( A ) HEK293T cells were transfected with plasmid expressing either GFP-Ago2 or control GFP. Twenty-four hours later, they were infected with VSVg-pseudotyped HIV-1 NL4-3 at a M.O.I. of 1 or mock infected. Twenty-four hours later, cells were subjected to UV cross-linking and RNA–protein complexes were subjected to immunoprecipitation using an anti-GFP antibody. IPed RNA was radiolabeled with [γ- 32 P]ATP and samples resolved on SDS-PAGE. Radiolabeled RNA was visualized by autoradiography. RNAs migrating at a higher molecular mass than the protein of interest (GFP-Ago2:RNA, between 125 and 225 kDa) were extracted from the membrane, reverse transcribed into cDNA and subjected to high throughput sequencing. ( B ) Reads obtained from non-infected (NI) and infected (INF) samples were uploaded to CLIPZ software. Reads that aligned to the human genome were sorted into categories: messenger RNA (mRNA), microRNA (miRNA), ribosomal RNA (rRNA), repeat, small non-coding RNA (snRNA), small nucleolar RNA (snoRNA), transfer RNA (tRNA) and other. Pie charts correspond to the distribution of the mean value of three biological replicates. ( C ) Reads obtained from infected samples were aligned to the HIV-1 NL4-3 sequence. Only reads ≥50 nucleotides length that aligned once without mismatch were considered for this analysis. Reads obtained from three independent HITS-CLIP experiments are presented in shades of orange and those obtained from the corresponding inputs in shades of blue. The map of the HIV-1 genome is presented below. Splice donor (D) and acceptor (A) sites are indicated. Peaks corresponding to clusters 2, 13, 16, 21, 25 and 30 are indicated by arrows.

    Techniques Used: Cross-linking Immunoprecipitation, Infection, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, SDS Page, Autoradiography, Next-Generation Sequencing, Software, Sequencing

    35) Product Images from "A small amount of cyclooxygenase 2 (COX2) is constitutively expressed in platelets"

    Article Title: A small amount of cyclooxygenase 2 (COX2) is constitutively expressed in platelets

    Journal: Platelets

    doi: 10.1080/09537104.2016.1203406

    COX2 mRNA is detected in human platelets (A–B) Establishment of linear regressional equations for output variable Ct of RT-PCR and input variable logged cDNA copy number of COX2 and COX1, respectively. (C) Estimate of the COX2 mRNA level relative to that of COX1 in platelets and ovarian cell lines by RT-PCR. Total RNA samples from platelets and ovarian cell lines were assayed by RT-PCR to obtain corresponding Ct values. COX2 and COX1 mRNA copy numbers were calculated by using the above mentioned linear regressional equations and subsequent exponential transformation.
    Figure Legend Snippet: COX2 mRNA is detected in human platelets (A–B) Establishment of linear regressional equations for output variable Ct of RT-PCR and input variable logged cDNA copy number of COX2 and COX1, respectively. (C) Estimate of the COX2 mRNA level relative to that of COX1 in platelets and ovarian cell lines by RT-PCR. Total RNA samples from platelets and ovarian cell lines were assayed by RT-PCR to obtain corresponding Ct values. COX2 and COX1 mRNA copy numbers were calculated by using the above mentioned linear regressional equations and subsequent exponential transformation.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Transformation Assay

    36) Product Images from "Redifferentiation of Adult Human β Cells Expanded In Vitro by Inhibition of the WNT Pathway"

    Article Title: Redifferentiation of Adult Human β Cells Expanded In Vitro by Inhibition of the WNT Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0112914

    Global changes in gene expression in BCD cells infected with β-catenin shRNA. A , Heat map of cDNA microarray analysis of RNA extracted from sorted eGFP + BCD cells at passages 4–5, 7 days following infection with β-catenin or NT shRNA viruses. n = 4 donors for β-catenin shRNA; n = 3 donors for NT shRNA. B , Gene ontology analysis of cDNA microarray results. C , Validation of candidate genes from the microarray analysis by qPCR analysis of RNA extracted from eGFP + BCD cells at passages 5–6, 7 days following infection with β-catenin or NT shRNA viruses. Data are mean±SE (n = 3–4 donors). *p
    Figure Legend Snippet: Global changes in gene expression in BCD cells infected with β-catenin shRNA. A , Heat map of cDNA microarray analysis of RNA extracted from sorted eGFP + BCD cells at passages 4–5, 7 days following infection with β-catenin or NT shRNA viruses. n = 4 donors for β-catenin shRNA; n = 3 donors for NT shRNA. B , Gene ontology analysis of cDNA microarray results. C , Validation of candidate genes from the microarray analysis by qPCR analysis of RNA extracted from eGFP + BCD cells at passages 5–6, 7 days following infection with β-catenin or NT shRNA viruses. Data are mean±SE (n = 3–4 donors). *p

    Techniques Used: Expressing, Infection, shRNA, Microarray, Real-time Polymerase Chain Reaction

    37) Product Images from "Endoplasmic reticulum stress response in an INS-1 pancreatic ?-cell line with inducible expression of a folding-deficient proinsulin"

    Article Title: Endoplasmic reticulum stress response in an INS-1 pancreatic ?-cell line with inducible expression of a folding-deficient proinsulin

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-11-59

    ER stress signaling pathways are activated by insulin 2 (C96Y)-EGFP expression . Clone #4S2 cells were untreated (-Dox) or treated with 2 μg/ml doxycycline (+Dox) for the times indicated. Control INS-1 cells exposed to thapsigargin (Tg) or dithiothreitol (DTT) were used as a positive control. A . RNA was isolated from the cells and XBP-1 cDNA was amplified by RT-PCR. The unspliced form of XBP-1 (uXBP-1, 480 bp) and spliced form of XBP-1 (sXBP-1, 454 bp) are indicated. B . Clone #4S2 cells were treated as in (A), washed in PBS and lysed. Equal amounts of protein were resolved by SDS-PAGE and immunoblotted with anti-phospho-eIF2α and GM130 antibodies. C . Clone #4S2 cells were treated with 1 mM dithiothreitol (DTT) for 30 min. or doxycycline as indicated and fixed for immunofluorescence labeling with anti-ATF6 antibody. Nuclear fluorescence of ATF6 was quantified as described in the methods.
    Figure Legend Snippet: ER stress signaling pathways are activated by insulin 2 (C96Y)-EGFP expression . Clone #4S2 cells were untreated (-Dox) or treated with 2 μg/ml doxycycline (+Dox) for the times indicated. Control INS-1 cells exposed to thapsigargin (Tg) or dithiothreitol (DTT) were used as a positive control. A . RNA was isolated from the cells and XBP-1 cDNA was amplified by RT-PCR. The unspliced form of XBP-1 (uXBP-1, 480 bp) and spliced form of XBP-1 (sXBP-1, 454 bp) are indicated. B . Clone #4S2 cells were treated as in (A), washed in PBS and lysed. Equal amounts of protein were resolved by SDS-PAGE and immunoblotted with anti-phospho-eIF2α and GM130 antibodies. C . Clone #4S2 cells were treated with 1 mM dithiothreitol (DTT) for 30 min. or doxycycline as indicated and fixed for immunofluorescence labeling with anti-ATF6 antibody. Nuclear fluorescence of ATF6 was quantified as described in the methods.

    Techniques Used: Expressing, Positive Control, Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, SDS Page, Immunofluorescence, Labeling, Fluorescence

    38) Product Images from "Single-cell transcriptome analysis of avian neural crest migration reveals signatures of invasion and molecular transitions"

    Article Title: Single-cell transcriptome analysis of avian neural crest migration reveals signatures of invasion and molecular transitions

    Journal: eLife

    doi: 10.7554/eLife.28415

    Single-cell RNA-seq quality control analyses. ( A ) Mapped read percentages for all single cells sequenced (n = 500). Two cells of the 502 cells harvested (0.4%) failed cDNA quality control measures and were not subsequently sequenced. ( B ) Box plots of log2 counts for 500 sequenced single cells. ( C ) Scatter plot of median log2 read count versus number of genes detected for each single cell sequenced. 31 (6.2%) of the 500 single cells sequenced were removed from downstream analysis due to distinctly lower counts ( B–C ). ( D ) Heatmap of log2 RPKM obtained for all External RNA Controls Consortium (ERCC) spike-ins across all single cells sequenced.
    Figure Legend Snippet: Single-cell RNA-seq quality control analyses. ( A ) Mapped read percentages for all single cells sequenced (n = 500). Two cells of the 502 cells harvested (0.4%) failed cDNA quality control measures and were not subsequently sequenced. ( B ) Box plots of log2 counts for 500 sequenced single cells. ( C ) Scatter plot of median log2 read count versus number of genes detected for each single cell sequenced. 31 (6.2%) of the 500 single cells sequenced were removed from downstream analysis due to distinctly lower counts ( B–C ). ( D ) Heatmap of log2 RPKM obtained for all External RNA Controls Consortium (ERCC) spike-ins across all single cells sequenced.

    Techniques Used: RNA Sequencing Assay

    39) Product Images from "Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA"

    Article Title: Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq684

    Quantification of genomic hmC and Tet transcripts in mouse tissues, undifferentiated ESCs and EBs. ( A and C ) hmC glucosylation assays. The percentage of hmC per total cytosine was calculated from the incorporation of [ 3 H]glucose using a calibration curve from the reference fragment (see Figure 1 C). Shown are average values and error bars from two (A) or one (C) biological replicates, each measured in two independent assays, with the exception of hippocampus that was measured only once. In every assay, each sample was measured in duplicate. The dashed line in (A) indicates the estimated limit of detection. ( B and D ) Real-time RT–PCR analysis for Tet transcript levels. Expression levels are all relative to Tet1 in kidney (set to 1), so that values in b and d are directly comparable. Shown are average values and error bars from two (B) and one (D) biological replicates, each measured from two independent cDNA synthesis reactions. In every real-time PCR reaction, each sample was measured in triplicate. Genomic DNA and RNA samples used in A/C and B/C, respectively, were isolated from the very same cell and tissue lysates.
    Figure Legend Snippet: Quantification of genomic hmC and Tet transcripts in mouse tissues, undifferentiated ESCs and EBs. ( A and C ) hmC glucosylation assays. The percentage of hmC per total cytosine was calculated from the incorporation of [ 3 H]glucose using a calibration curve from the reference fragment (see Figure 1 C). Shown are average values and error bars from two (A) or one (C) biological replicates, each measured in two independent assays, with the exception of hippocampus that was measured only once. In every assay, each sample was measured in duplicate. The dashed line in (A) indicates the estimated limit of detection. ( B and D ) Real-time RT–PCR analysis for Tet transcript levels. Expression levels are all relative to Tet1 in kidney (set to 1), so that values in b and d are directly comparable. Shown are average values and error bars from two (B) and one (D) biological replicates, each measured from two independent cDNA synthesis reactions. In every real-time PCR reaction, each sample was measured in triplicate. Genomic DNA and RNA samples used in A/C and B/C, respectively, were isolated from the very same cell and tissue lysates.

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

    40) Product Images from "Modulation of LINE-1 and Alu/SVA Retrotransposition by Aicardi-Goutières Syndrome-Related SAMHD1"

    Article Title: Modulation of LINE-1 and Alu/SVA Retrotransposition by Aicardi-Goutières Syndrome-Related SAMHD1

    Journal: Cell reports

    doi: 10.1016/j.celrep.2013.08.019

    SAMHD1 Inhibits LINE-1 ORF2p-Mediated Endogenous Reverse Transcription in LINE-1 RNP (A) SAMHD1 did not affect the expression of the LINE-1 ORF1 protein. HEK293T cells were transfected with the pc-L1-1FH vector plus the empty vector VR1012 or the expression vector for SAMHD1, HnRNPL, or MOV10. ImageJ software (NIH) was used to quantitate ORF1 band intensities, and their absolute readings are indicated above the immunoblot. (B) A diagram of the LEAP assay. The LINE-1 RNP (from pc-L1-1FH) was produced from transfected HEK293T cells and purified by ultracentrifugation through a sucrose cushion as previously described. The LEAP primer, containing a linker region (dashed line), was used to precisely target onto LINE-1 mRNA, and the reverse transcription occurred with the assistance of the ORF2 protein. Synthesized cDNA was then amplified through standard PCR with two primers (dash arrows) targeting to LINE-1 and the linker. (C) SAMHD1 reduced the reverse transcription efficiency mediated by ORF2p in LINE-1 RNPs. The amount of ORF1 proteins in isolated LINE-1 RNPs in the absence or presence of SAMHD1 was determined by immunoblotting using an anti-HA antibody. LINE-1 RNA was examined by using the LEAP primer and MuLV reverse transcriptase for cDNA synthesis, followed by PCR amplification. (D) Quantitative real-time PCR analysis of LEAP products and LINE-1 RNA RT-PCR products. (E) ORF2p level was lowered by 62% in average, with the presence of exogenous SAMDH1 protein. The bar chart was based on five independent experiments. The error bars indicated the SD. See also Figure S4 .
    Figure Legend Snippet: SAMHD1 Inhibits LINE-1 ORF2p-Mediated Endogenous Reverse Transcription in LINE-1 RNP (A) SAMHD1 did not affect the expression of the LINE-1 ORF1 protein. HEK293T cells were transfected with the pc-L1-1FH vector plus the empty vector VR1012 or the expression vector for SAMHD1, HnRNPL, or MOV10. ImageJ software (NIH) was used to quantitate ORF1 band intensities, and their absolute readings are indicated above the immunoblot. (B) A diagram of the LEAP assay. The LINE-1 RNP (from pc-L1-1FH) was produced from transfected HEK293T cells and purified by ultracentrifugation through a sucrose cushion as previously described. The LEAP primer, containing a linker region (dashed line), was used to precisely target onto LINE-1 mRNA, and the reverse transcription occurred with the assistance of the ORF2 protein. Synthesized cDNA was then amplified through standard PCR with two primers (dash arrows) targeting to LINE-1 and the linker. (C) SAMHD1 reduced the reverse transcription efficiency mediated by ORF2p in LINE-1 RNPs. The amount of ORF1 proteins in isolated LINE-1 RNPs in the absence or presence of SAMHD1 was determined by immunoblotting using an anti-HA antibody. LINE-1 RNA was examined by using the LEAP primer and MuLV reverse transcriptase for cDNA synthesis, followed by PCR amplification. (D) Quantitative real-time PCR analysis of LEAP products and LINE-1 RNA RT-PCR products. (E) ORF2p level was lowered by 62% in average, with the presence of exogenous SAMDH1 protein. The bar chart was based on five independent experiments. The error bars indicated the SD. See also Figure S4 .

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Software, Produced, Purification, Synthesized, Amplification, Polymerase Chain Reaction, Isolation, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

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    HFD-fed iRhom2 KO mice have increased thermogenesis and browning of the white adipose tissue. A-C Thermal images (A), and BAT (B) or body (C) temperature of iRhom2 KO and WT mice fed with SD and HFD for 19 weeks. One experiment with 8 mice per group. D RT-PCR analysis of UCP1, PGC1α, Cidea, PRDM16, and Cox8b expression in SD-fed iRhom2 KO and HFD-fed WT and iRhom2 KO mice BAT samples compared to WT SD-fed control samples. Two experiments with 3–4 replicates in each. E, G Representative photographs of eWAT (E) and sWAT (G) UCP1 immunohistochemistry derived from iRhom2 KO and WT mice fed with HFD for 20 weeks. Scale bar = 100 μm. F, H Graphics showing the UCP1 percentage of area stained in the eWAT (F) and sWAT (H) of the mice described above. One experiment with 8 mice per group (with 2 photographs analyzed per mouse). I RT-PCR analysis of UCP1 in brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT or iRhom2 KO mice. Three independent experiments. J RT-PCR analysis of UCP1 in immortalized WT brown preadipocytes transduced with empty vector or iRhom2-HA, differentiated in vitro and stimulated with norepinephrine for 6 h. Two independent experiments. K HA expression in differentiated immortalized WT brown preadipocytes transduced with retrovirus containing iRhom2 <t>cDNA</t> fused to C-terminal HA tag (iR2-HA). As a negative control, we used the same cells transduced with retrovirus containing the empty vector (EV), and as a loading control we measured p97 protein level. Two independent experiments. L Mitochondrial oxygen consumption rate (OCR) of brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT and iRhom2 KO mice and stimulated or not with norepinephrine. The results were normalized to the protein content. Two experiments with one or two independent samples per genotype, respectively. M Mitochondrial proton leak of the cells described in L normalized to the protein content. Error bars represent SEM; * represents p
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    HFD-fed iRhom2 KO mice have increased thermogenesis and browning of the white adipose tissue. A-C Thermal images (A), and BAT (B) or body (C) temperature of iRhom2 KO and WT mice fed with SD and HFD for 19 weeks. One experiment with 8 mice per group. D RT-PCR analysis of UCP1, PGC1α, Cidea, PRDM16, and Cox8b expression in SD-fed iRhom2 KO and HFD-fed WT and iRhom2 KO mice BAT samples compared to WT SD-fed control samples. Two experiments with 3–4 replicates in each. E, G Representative photographs of eWAT (E) and sWAT (G) UCP1 immunohistochemistry derived from iRhom2 KO and WT mice fed with HFD for 20 weeks. Scale bar = 100 μm. F, H Graphics showing the UCP1 percentage of area stained in the eWAT (F) and sWAT (H) of the mice described above. One experiment with 8 mice per group (with 2 photographs analyzed per mouse). I RT-PCR analysis of UCP1 in brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT or iRhom2 KO mice. Three independent experiments. J RT-PCR analysis of UCP1 in immortalized WT brown preadipocytes transduced with empty vector or iRhom2-HA, differentiated in vitro and stimulated with norepinephrine for 6 h. Two independent experiments. K HA expression in differentiated immortalized WT brown preadipocytes transduced with retrovirus containing iRhom2 cDNA fused to C-terminal HA tag (iR2-HA). As a negative control, we used the same cells transduced with retrovirus containing the empty vector (EV), and as a loading control we measured p97 protein level. Two independent experiments. L Mitochondrial oxygen consumption rate (OCR) of brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT and iRhom2 KO mice and stimulated or not with norepinephrine. The results were normalized to the protein content. Two experiments with one or two independent samples per genotype, respectively. M Mitochondrial proton leak of the cells described in L normalized to the protein content. Error bars represent SEM; * represents p

    Journal: Molecular Metabolism

    Article Title: Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis

    doi: 10.1016/j.molmet.2019.10.006

    Figure Lengend Snippet: HFD-fed iRhom2 KO mice have increased thermogenesis and browning of the white adipose tissue. A-C Thermal images (A), and BAT (B) or body (C) temperature of iRhom2 KO and WT mice fed with SD and HFD for 19 weeks. One experiment with 8 mice per group. D RT-PCR analysis of UCP1, PGC1α, Cidea, PRDM16, and Cox8b expression in SD-fed iRhom2 KO and HFD-fed WT and iRhom2 KO mice BAT samples compared to WT SD-fed control samples. Two experiments with 3–4 replicates in each. E, G Representative photographs of eWAT (E) and sWAT (G) UCP1 immunohistochemistry derived from iRhom2 KO and WT mice fed with HFD for 20 weeks. Scale bar = 100 μm. F, H Graphics showing the UCP1 percentage of area stained in the eWAT (F) and sWAT (H) of the mice described above. One experiment with 8 mice per group (with 2 photographs analyzed per mouse). I RT-PCR analysis of UCP1 in brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT or iRhom2 KO mice. Three independent experiments. J RT-PCR analysis of UCP1 in immortalized WT brown preadipocytes transduced with empty vector or iRhom2-HA, differentiated in vitro and stimulated with norepinephrine for 6 h. Two independent experiments. K HA expression in differentiated immortalized WT brown preadipocytes transduced with retrovirus containing iRhom2 cDNA fused to C-terminal HA tag (iR2-HA). As a negative control, we used the same cells transduced with retrovirus containing the empty vector (EV), and as a loading control we measured p97 protein level. Two independent experiments. L Mitochondrial oxygen consumption rate (OCR) of brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT and iRhom2 KO mice and stimulated or not with norepinephrine. The results were normalized to the protein content. Two experiments with one or two independent samples per genotype, respectively. M Mitochondrial proton leak of the cells described in L normalized to the protein content. Error bars represent SEM; * represents p

    Article Snippet: First-strand cDNA was synthesized from total RNA using the SuperScript® III First-Strand Synthesis SuperMix or the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific).

    Techniques: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemistry, Derivative Assay, Staining, In Vitro, Transduction, Plasmid Preparation, Negative Control

    RNF138 reduces Ca V 2.1 protein stability. A , Lack of effect of RNF138/RNF138-H36E overexpression on human Ca V 2.1 mRNA level in HEK293T cells subject to the indicated transfection condition ( p > 0.05; n = 3). To rule out the potential contamination arising from human Ca V 2.1 plasmid in RNA prepared from transfected cells, RT-PCR was performed in the absence (left) or presence (right) of DNase I treatment before reverse transcription reaction. Also shown is the blank control that involves identical PCR in the absence of cDNA template (vertical arrows). The signals of Ca V 2.1 were standardized as the ratio to those of cognate GAPDH, followed by normalization to the corresponding Myc vector control. B , RNF138 knock-down does not significantly change rat Ca V 2.1 mRNA level in neurons ( p > 0.05; n = 3). RT-PCR analyses were based on RNA extracted from cultured cortical neurons subject to the indicated shRNA infection. Standardized Ca V 2.1 signals were normalized to the shGFP infection control. C , Representative immunoblots showing the effect of RNF128, RNF138, or RNF138-H36E coexpression on protein stability of human Ca V 2.1 subunit. Ca V 2.1 protein turnover kinetics in HEK293T cells was analyzed by applying cycloheximide (CHX) with the indicated treatment durations (h). Coexpression with the Myc vector was used as the control experiment. D , Quantification of Ca V 2.1 protein half-life in the presence of Myc vector (black), RNF128 (green), RNF138 (blue), or RNF138-H36E (red). Left, Normalized Ca V 2.1 protein densities with respect to cycloheximide treatment durations. Data points represent the average of 7–8 independent experiments. Center, Same data points were transformed into a semilogarithmic plot, which is subject to single linear-regression analyses (solid lines; top) or double linear-regression analyses (solid lines; bottom with RNF138 only). Right, Comparison of Ca V 2.1 protein half-life values derived from linear-regression analyses. The estimated Ca V 2.1 protein half-life values based on single linear-regression analyses (top right) are ∼8.1 ± 0.3 (with vector; n = 8), 8.7 ± 1.3 (with RNF128; n = 7), 3.1 ± 0.4 (with RNF138; n = 8), and 10.9 ± 0.7 (with RNF138-H36E; n = 8) h. Based on double linear-regression analyses (bottom right), the estimated Ca V 2.1 protein half-life values in the presence of RNF138 are ∼1.3 ± 0.3 h (fast component) and 5.5 ± 0.6 h (slow component). E , Representative immunoblots showing the effect of shRNA knock-down of endogenous RNF13 8 on Ca V 2.1 protein turnover kinetics in HEK293T cells. shGFP infection was used as the control experiment. F , Quantification and comparison of Ca V 2.1 protein half-life values derived from different shRNA infection conditions. The estimated Ca V 2.1 protein half-life values are ∼6.4 ± 1.0 h (with shGFP; n = 9; black) and 10.3 ± 1.4 h (with shRNF138–1; n = 9; red). The protein half-life value of Ca V 2.1 in the presence of shGFP is not statistically different ( p > 0.05) from that of Ca V 2.1 with vector in D . Asterisks denote significant difference from the control (* p

    Journal: The Journal of Neuroscience

    Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels

    doi: 10.1523/JNEUROSCI.3070-16.2017

    Figure Lengend Snippet: RNF138 reduces Ca V 2.1 protein stability. A , Lack of effect of RNF138/RNF138-H36E overexpression on human Ca V 2.1 mRNA level in HEK293T cells subject to the indicated transfection condition ( p > 0.05; n = 3). To rule out the potential contamination arising from human Ca V 2.1 plasmid in RNA prepared from transfected cells, RT-PCR was performed in the absence (left) or presence (right) of DNase I treatment before reverse transcription reaction. Also shown is the blank control that involves identical PCR in the absence of cDNA template (vertical arrows). The signals of Ca V 2.1 were standardized as the ratio to those of cognate GAPDH, followed by normalization to the corresponding Myc vector control. B , RNF138 knock-down does not significantly change rat Ca V 2.1 mRNA level in neurons ( p > 0.05; n = 3). RT-PCR analyses were based on RNA extracted from cultured cortical neurons subject to the indicated shRNA infection. Standardized Ca V 2.1 signals were normalized to the shGFP infection control. C , Representative immunoblots showing the effect of RNF128, RNF138, or RNF138-H36E coexpression on protein stability of human Ca V 2.1 subunit. Ca V 2.1 protein turnover kinetics in HEK293T cells was analyzed by applying cycloheximide (CHX) with the indicated treatment durations (h). Coexpression with the Myc vector was used as the control experiment. D , Quantification of Ca V 2.1 protein half-life in the presence of Myc vector (black), RNF128 (green), RNF138 (blue), or RNF138-H36E (red). Left, Normalized Ca V 2.1 protein densities with respect to cycloheximide treatment durations. Data points represent the average of 7–8 independent experiments. Center, Same data points were transformed into a semilogarithmic plot, which is subject to single linear-regression analyses (solid lines; top) or double linear-regression analyses (solid lines; bottom with RNF138 only). Right, Comparison of Ca V 2.1 protein half-life values derived from linear-regression analyses. The estimated Ca V 2.1 protein half-life values based on single linear-regression analyses (top right) are ∼8.1 ± 0.3 (with vector; n = 8), 8.7 ± 1.3 (with RNF128; n = 7), 3.1 ± 0.4 (with RNF138; n = 8), and 10.9 ± 0.7 (with RNF138-H36E; n = 8) h. Based on double linear-regression analyses (bottom right), the estimated Ca V 2.1 protein half-life values in the presence of RNF138 are ∼1.3 ± 0.3 h (fast component) and 5.5 ± 0.6 h (slow component). E , Representative immunoblots showing the effect of shRNA knock-down of endogenous RNF13 8 on Ca V 2.1 protein turnover kinetics in HEK293T cells. shGFP infection was used as the control experiment. F , Quantification and comparison of Ca V 2.1 protein half-life values derived from different shRNA infection conditions. The estimated Ca V 2.1 protein half-life values are ∼6.4 ± 1.0 h (with shGFP; n = 9; black) and 10.3 ± 1.4 h (with shRNF138–1; n = 9; red). The protein half-life value of Ca V 2.1 in the presence of shGFP is not statistically different ( p > 0.05) from that of Ca V 2.1 with vector in D . Asterisks denote significant difference from the control (* p

    Article Snippet: RNA was reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Scientific).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Cell Culture, shRNA, Infection, Western Blot, Transformation Assay, Derivative Assay

    Cloning of EXA1 homologs in Nicotiana benthamiana and their functional analysis in potexvirus infection. ( a ) Schematic image of the cDNA structure of NbEXA1a . GYF domain- and eIF4E-binding motif-encoding regions are depicted by stripes. Target regions for virus-induced gene silencing (VIGS) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) are indicated by bars under the image. ( b ) Morphological phenotypes of NbEXA1 -silenced and control plants. Photographs were taken from the top (upper) and side (bottom) of plants at 27 days post-inoculation (dpi). Bars = 5 cm. ( c ) Relative accumulation of NbEXA1 mRNA in NbEXA1 -silenced and control plants. Total RNA was extracted at 27 dpi and analyzed using qRT-PCR. The mean level of NbEXA1 transcript in control plants was used as the standard (1.0), and that in NbEXA1 -silenced plants is shown above the bar. Error bars indicate standard deviation (SD) of 10 samples. ** P

    Journal: Scientific Reports

    Article Title: Functional conservation of EXA1 among diverse plant species for the infection by a family of plant viruses

    doi: 10.1038/s41598-019-42400-w

    Figure Lengend Snippet: Cloning of EXA1 homologs in Nicotiana benthamiana and their functional analysis in potexvirus infection. ( a ) Schematic image of the cDNA structure of NbEXA1a . GYF domain- and eIF4E-binding motif-encoding regions are depicted by stripes. Target regions for virus-induced gene silencing (VIGS) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) are indicated by bars under the image. ( b ) Morphological phenotypes of NbEXA1 -silenced and control plants. Photographs were taken from the top (upper) and side (bottom) of plants at 27 days post-inoculation (dpi). Bars = 5 cm. ( c ) Relative accumulation of NbEXA1 mRNA in NbEXA1 -silenced and control plants. Total RNA was extracted at 27 dpi and analyzed using qRT-PCR. The mean level of NbEXA1 transcript in control plants was used as the standard (1.0), and that in NbEXA1 -silenced plants is shown above the bar. Error bars indicate standard deviation (SD) of 10 samples. ** P

    Article Snippet: RNA isolation, RT-PCR, and qRT-PCR Total RNA was extracted from the plants using ISOGEN (Nippon Gene) and treated with DNase I (Takara Bio, Shiga, Japan), or using the ISOSPIN Plant RNA Kit (Nippongene) following the manufacturer’s instructions. cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Clone Assay, Functional Assay, Infection, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation