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    Structured Review

    Thermo Fisher rna levels
    Determination of norA mRNA stability. (A) Agarose gel with <t>RT-PCR,</t> which were performed using total cellular <t>RNA</t> extracted from strains ISP794 (wild type) and MT23142 ( flqB mutant) after addition of rifampin (200 μg/ml) at time zero (cells grown to OD 600 of 0.6) for both strains. Primers for norA mRNA (5′-AGA AAC TTT TTA CGA ATA TT-3′ and 5′-TGA CAC TGA AAA CAA AAT TA-3′) generated a 250-bp fragment. We used 0.2 μM each primer, 2 U of reverse transcriptase- Taq mix, and 4 μg of total RNA for ISP794 or 2 μg of total RNA for MT23142 for norA ). (B) Semilogarithmic graph of mRNA concentration as a function of time (expressed as percent of amount at time of addition of rifampin). Densitometric analysis was performed by scanning the gel pictures and performing analysis using the public-domain National Institutes of Health Image program (version 6.1). (C) Northern hybridization of norA mRNA. Total RNA was extracted using the SNAP Total RNA kit (Invitrogen). Times indicate time after the addition of rifampin at time 0. The norA ).
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    Images

    1) Product Images from "A Mutation in the 5? Untranslated Region Increases Stability of norA mRNA, Encoding a Multidrug Resistance Transporter of Staphylococcus aureus"

    Article Title: A Mutation in the 5? Untranslated Region Increases Stability of norA mRNA, Encoding a Multidrug Resistance Transporter of Staphylococcus aureus

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.183.7.2367-2371.2001

    Determination of norA mRNA stability. (A) Agarose gel with RT-PCR, which were performed using total cellular RNA extracted from strains ISP794 (wild type) and MT23142 ( flqB mutant) after addition of rifampin (200 μg/ml) at time zero (cells grown to OD 600 of 0.6) for both strains. Primers for norA mRNA (5′-AGA AAC TTT TTA CGA ATA TT-3′ and 5′-TGA CAC TGA AAA CAA AAT TA-3′) generated a 250-bp fragment. We used 0.2 μM each primer, 2 U of reverse transcriptase- Taq mix, and 4 μg of total RNA for ISP794 or 2 μg of total RNA for MT23142 for norA ). (B) Semilogarithmic graph of mRNA concentration as a function of time (expressed as percent of amount at time of addition of rifampin). Densitometric analysis was performed by scanning the gel pictures and performing analysis using the public-domain National Institutes of Health Image program (version 6.1). (C) Northern hybridization of norA mRNA. Total RNA was extracted using the SNAP Total RNA kit (Invitrogen). Times indicate time after the addition of rifampin at time 0. The norA ).
    Figure Legend Snippet: Determination of norA mRNA stability. (A) Agarose gel with RT-PCR, which were performed using total cellular RNA extracted from strains ISP794 (wild type) and MT23142 ( flqB mutant) after addition of rifampin (200 μg/ml) at time zero (cells grown to OD 600 of 0.6) for both strains. Primers for norA mRNA (5′-AGA AAC TTT TTA CGA ATA TT-3′ and 5′-TGA CAC TGA AAA CAA AAT TA-3′) generated a 250-bp fragment. We used 0.2 μM each primer, 2 U of reverse transcriptase- Taq mix, and 4 μg of total RNA for ISP794 or 2 μg of total RNA for MT23142 for norA ). (B) Semilogarithmic graph of mRNA concentration as a function of time (expressed as percent of amount at time of addition of rifampin). Densitometric analysis was performed by scanning the gel pictures and performing analysis using the public-domain National Institutes of Health Image program (version 6.1). (C) Northern hybridization of norA mRNA. Total RNA was extracted using the SNAP Total RNA kit (Invitrogen). Times indicate time after the addition of rifampin at time 0. The norA ).

    Techniques Used: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Cellular Antioxidant Activity Assay, Generated, Concentration Assay, Northern Blot, Hybridization

    2) Product Images from "Reactive Oxygen Species (ROS)-Inducing Triterpenoid Inhibits Rhabdomyosarcoma Cell and Tumor Growth Through Targeting Sp Transcription Factors"

    Article Title: Reactive Oxygen Species (ROS)-Inducing Triterpenoid Inhibits Rhabdomyosarcoma Cell and Tumor Growth Through Targeting Sp Transcription Factors

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-18-1071

    CF 3 DODA-Me modifies miRNA–ZBTB interactions. (A) RD and Rh30 cells were treated with 0, 1 and 2.5 μM of CF 3 DODA-Me for 6 h; (B) RD and Rh30 cells were pretreated with 5 mM of GSH for 3 h and then treated with 2.5 μM CF 3 DODA-Me alone, or in combination with GSH for 6 h, and total RNA was extracted and expression of miR-17, miR-20a and miR-27a was determined by real-time PCR. RNU6 was used as endogenous control. (C) RD and Rh30 cells were treated with 2.5 μM of CF 3 DODA-Me for the indicated times, and ZBTB4, ZBTB10 and ZBTB34 proteins were analyzed by western blots. (D) Cells were treated with DMSO (control), CF 3 DODA-Me and 5 mM GSH alone and in combination for 12 h, and whole cell lysates were analyzed by western blots. β-Actin was used as a loading control. (E) RD and Rh30 cells were treated with 2.5 μM of CF 3 DODA-Me for 3 h, and a ChIP assay was performed with control (IgG), polymerase II, and Myc antibodies to determine their interactions on the miR-23a/27a and miR-17/92 cluster promoter regions. The signals were quantitated by ImageJ software. Results shown are expressed as mean ± SE for replicate determinants, and significant ( P
    Figure Legend Snippet: CF 3 DODA-Me modifies miRNA–ZBTB interactions. (A) RD and Rh30 cells were treated with 0, 1 and 2.5 μM of CF 3 DODA-Me for 6 h; (B) RD and Rh30 cells were pretreated with 5 mM of GSH for 3 h and then treated with 2.5 μM CF 3 DODA-Me alone, or in combination with GSH for 6 h, and total RNA was extracted and expression of miR-17, miR-20a and miR-27a was determined by real-time PCR. RNU6 was used as endogenous control. (C) RD and Rh30 cells were treated with 2.5 μM of CF 3 DODA-Me for the indicated times, and ZBTB4, ZBTB10 and ZBTB34 proteins were analyzed by western blots. (D) Cells were treated with DMSO (control), CF 3 DODA-Me and 5 mM GSH alone and in combination for 12 h, and whole cell lysates were analyzed by western blots. β-Actin was used as a loading control. (E) RD and Rh30 cells were treated with 2.5 μM of CF 3 DODA-Me for 3 h, and a ChIP assay was performed with control (IgG), polymerase II, and Myc antibodies to determine their interactions on the miR-23a/27a and miR-17/92 cluster promoter regions. The signals were quantitated by ImageJ software. Results shown are expressed as mean ± SE for replicate determinants, and significant ( P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Chromatin Immunoprecipitation, Software

    3) Product Images from "NeuroD6 Genomic Signature Bridging Neuronal Differentiation to Survival via the Molecular Chaperone Network"

    Article Title: NeuroD6 Genomic Signature Bridging Neuronal Differentiation to Survival via the Molecular Chaperone Network

    Journal: Journal of neuroscience research

    doi: 10.1002/jnr.22182

    Unbiased genome-wide microarray analysis upon NeuroD6 overexpression. ( A ) Scatterplot analysis showing differentially expressed probe sets upon NeuroD6 overexpression. Total RNA from control PC12 and PC12-ND6 cells were isolated in six replicates and
    Figure Legend Snippet: Unbiased genome-wide microarray analysis upon NeuroD6 overexpression. ( A ) Scatterplot analysis showing differentially expressed probe sets upon NeuroD6 overexpression. Total RNA from control PC12 and PC12-ND6 cells were isolated in six replicates and

    Techniques Used: Genome Wide, Microarray, Over Expression, Isolation

    4) Product Images from "Multipotent mesenchymal stem cells in lung fibrosis"

    Article Title: Multipotent mesenchymal stem cells in lung fibrosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0181946

    Epithelial differentiation of mesenchymal stem cells. Primary human mesenchymal stem cells were cultured in an epithelial growth medium (Cnt-17, CellnTEC Advanced Cell System AB) for 3–4 days, cells were fixed, and permeabilized. Cells were immunostained for E-cadherin (B). The primary antibody was detected by addition of a fluorescein-labelled (green) secondary antibody. (A) Corresponding phase contrast picture. Visualisation by fluorescence microscopy. Magnification x 20. (C) E-cadherin RNA expression in mesenchymal stem cells before (grey bar) and after (black bar) epithelial differentiation. RNA expression was assessed by quantitative real time RT-PCR. Data are presented as mean ± SEM of independent experiments performed in three different cell lines.
    Figure Legend Snippet: Epithelial differentiation of mesenchymal stem cells. Primary human mesenchymal stem cells were cultured in an epithelial growth medium (Cnt-17, CellnTEC Advanced Cell System AB) for 3–4 days, cells were fixed, and permeabilized. Cells were immunostained for E-cadherin (B). The primary antibody was detected by addition of a fluorescein-labelled (green) secondary antibody. (A) Corresponding phase contrast picture. Visualisation by fluorescence microscopy. Magnification x 20. (C) E-cadherin RNA expression in mesenchymal stem cells before (grey bar) and after (black bar) epithelial differentiation. RNA expression was assessed by quantitative real time RT-PCR. Data are presented as mean ± SEM of independent experiments performed in three different cell lines.

    Techniques Used: Cell Culture, Fluorescence, Microscopy, RNA Expression, Quantitative RT-PCR

    Characterization of mesenchymal stem cells. Primary human mesenchymal stem cells immunostained for Oct3/4 (A), and Nanog (B). Cells were fixed and permeabilized. Primary antibodies were detected by addition of fluorescein-labelled (FITC) (green) and Alexa Fluor 594-labelled (red) secondary antibodies. E, G-I: Double immunofluorescence staining for CD90 (E), CD105 (G, H, I) and C-X-C-chemokine receptor type 4 (CXCR4) (panels E, G, H, I) in lung tissue sections from patients with histologically confirmed IPF/UIP and chronic fibrotic hypersensitivity pneumonitis. Primary antibodies were detected by addition of secondary antibodies labelled with fluorescein FITC (green) for CD105 and CXCR4 detection or Cy3-labelled (red) for CD90 and CXCR4 detection. Magnification x40. Images were acquired using LSM 510 confocal microscope. Panel H and I is a 3D reconstruction to show more clear co stainings on the same cell. F: Co-staining with CD44 (pink) and CXCR4 (dark red) (Scale bar 2μm, Magnification x100 (oil)). (C, D) Oct3/4 (C) and Nanog (D) mRNA expression in mesenchymal stem cells (black bars) and in fibroblasts (grey bars). RNA expression was assessed by quantitative real time RT-PCR. Data are presented as mean ± SEM of independent experiments performed in three different cell lines.
    Figure Legend Snippet: Characterization of mesenchymal stem cells. Primary human mesenchymal stem cells immunostained for Oct3/4 (A), and Nanog (B). Cells were fixed and permeabilized. Primary antibodies were detected by addition of fluorescein-labelled (FITC) (green) and Alexa Fluor 594-labelled (red) secondary antibodies. E, G-I: Double immunofluorescence staining for CD90 (E), CD105 (G, H, I) and C-X-C-chemokine receptor type 4 (CXCR4) (panels E, G, H, I) in lung tissue sections from patients with histologically confirmed IPF/UIP and chronic fibrotic hypersensitivity pneumonitis. Primary antibodies were detected by addition of secondary antibodies labelled with fluorescein FITC (green) for CD105 and CXCR4 detection or Cy3-labelled (red) for CD90 and CXCR4 detection. Magnification x40. Images were acquired using LSM 510 confocal microscope. Panel H and I is a 3D reconstruction to show more clear co stainings on the same cell. F: Co-staining with CD44 (pink) and CXCR4 (dark red) (Scale bar 2μm, Magnification x100 (oil)). (C, D) Oct3/4 (C) and Nanog (D) mRNA expression in mesenchymal stem cells (black bars) and in fibroblasts (grey bars). RNA expression was assessed by quantitative real time RT-PCR. Data are presented as mean ± SEM of independent experiments performed in three different cell lines.

    Techniques Used: Double Immunofluorescence Staining, Microscopy, Staining, Expressing, RNA Expression, Quantitative RT-PCR

    5) Product Images from "The mammalian ribo-interactome reveals ribosome functional diversity and heterogeneity"

    Article Title: The mammalian ribo-interactome reveals ribosome functional diversity and heterogeneity

    Journal: Cell

    doi: 10.1016/j.cell.2017.05.022

    The ufmylation enzyme UFL1 interacts with ribosomes and modifies key components of the translation machinery. A, Schematic of the ufmylation cascade. B, UFL1 is tagged endogenously with HA at its N terminus. The UFL1 antibody recognizes the C-terminal portion of human UFL1 protein. FLAG IPs for both control GFP-FLAG and eL36-FLAG cells are performed. Both the GFP- FLAG input and IP as well as the eL36-FLAG input and IP are blotted with HA, Ufl1, and Ufm1-specific antibodies. C, Sucrose gradient fractionation is performed and fractions are blotted for either the Ufm1 modification or the E3 ligase enzyme, UFL1. UV signal at 260 detects RNA and indicates rRNA abundance across fractions. . 80S human ribosome structure with the positions of uS3 (green), uS20 (orange), uL16 (dark blue), mRNA (red), E-site tRNA (dark grey), and EEF2 (black) are indicated. The ribosomal RNAs are shown in light blue (60S) or yellow (40S). PDB: 4V6X with mRNA superimposed are from PDB: 4KZZ.
    Figure Legend Snippet: The ufmylation enzyme UFL1 interacts with ribosomes and modifies key components of the translation machinery. A, Schematic of the ufmylation cascade. B, UFL1 is tagged endogenously with HA at its N terminus. The UFL1 antibody recognizes the C-terminal portion of human UFL1 protein. FLAG IPs for both control GFP-FLAG and eL36-FLAG cells are performed. Both the GFP- FLAG input and IP as well as the eL36-FLAG input and IP are blotted with HA, Ufl1, and Ufm1-specific antibodies. C, Sucrose gradient fractionation is performed and fractions are blotted for either the Ufm1 modification or the E3 ligase enzyme, UFL1. UV signal at 260 detects RNA and indicates rRNA abundance across fractions. . 80S human ribosome structure with the positions of uS3 (green), uS20 (orange), uL16 (dark blue), mRNA (red), E-site tRNA (dark grey), and EEF2 (black) are indicated. The ribosomal RNAs are shown in light blue (60S) or yellow (40S). PDB: 4V6X with mRNA superimposed are from PDB: 4KZZ.

    Techniques Used: Fractionation, Modification

    6) Product Images from "Dynamic interactions and cooperative functions of PGC-1? and MED1 in TR?-mediated activation of the brown fat-specific UCP-1 gene"

    Article Title: Dynamic interactions and cooperative functions of PGC-1? and MED1 in TR?-mediated activation of the brown fat-specific UCP-1 gene

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2009.09.015

    Role of MED1 in UCP-1 induction (A) . Requirement of the MED1 C-Terminus for UCP-1 induction in differentiated MEFs. WT /PPARγ MEFs, Med1 −/− /PPARγ MEFs and Med1 −/− /PPARγ MEFs that stably express MED1(1–530) ( Med1 −/− /PPARγ/MED1–530 MEFs) or full length MED1( Med1 −/− /PPARγ/MED1 MEFs) were differentiated to adipocytes, treated to induce UCP-1 gene expression and analyzed for UCP-1 RNA levels by real time RT-PCR as described in Experimental Procedures. Open bars show control non-treated preadipocytes (Pre) and solid bars show UCP-1 induced adipocytes (Induced). Error bars indicate standard deviation of three independent experiments. (B ). Structure and expression levels of MED1 proteins in differentiated MEFs. Top: Schematic representation of Flag-tagged MED1 proteins that were stably expressed in Med1 −/− /PPARγ MEFs. Bottom: Analysis of ectopic and endogenous MED1 expression levels by immunoblot with anti-MED1 (lanes 1–3) and anti-Flag (lanes 4 and 5) antibodies. (C). UCP-1 induction by PPARγ and TR ligands in differentiated MEFs. WT /PPARγ MEFs were first cultured under conditions leading to differentiation to adipocytes and then in complete UCP-1 induction medium (Normal induction, lane 2), induction medium minus T 3 (−T 3 induction, lane 3) or induction medium minus BRL49653 (−BRL49653 induction, lane 4). UCP-1 expression was monitored by real-time RT-PCR. Non-treated preadipocytes (No induction) are shown in lane 1. (D) Induction of UCP-1 in brown HIB1B cells detected by RT-PCR. (E) . Induced binding of factors to the UCP-1 enhancer in HIB1B cells. Top: Schematic representation of the UCP-1 enhancer and primers used for ChIP assays. Bottom: ChIP analysis of HIB1B preadipocytes and UCP-1 -induced adipocytes with the indicated antibodies. (F). Induced binding of factors to the UCP-1 enhancer in HIB1B cells. Real time PCR ChIP analyses of preadipocytes and UCP-1 induced adipocytes with the indicated antibodies. Error bars indicate standard deviation of two independent experiments.
    Figure Legend Snippet: Role of MED1 in UCP-1 induction (A) . Requirement of the MED1 C-Terminus for UCP-1 induction in differentiated MEFs. WT /PPARγ MEFs, Med1 −/− /PPARγ MEFs and Med1 −/− /PPARγ MEFs that stably express MED1(1–530) ( Med1 −/− /PPARγ/MED1–530 MEFs) or full length MED1( Med1 −/− /PPARγ/MED1 MEFs) were differentiated to adipocytes, treated to induce UCP-1 gene expression and analyzed for UCP-1 RNA levels by real time RT-PCR as described in Experimental Procedures. Open bars show control non-treated preadipocytes (Pre) and solid bars show UCP-1 induced adipocytes (Induced). Error bars indicate standard deviation of three independent experiments. (B ). Structure and expression levels of MED1 proteins in differentiated MEFs. Top: Schematic representation of Flag-tagged MED1 proteins that were stably expressed in Med1 −/− /PPARγ MEFs. Bottom: Analysis of ectopic and endogenous MED1 expression levels by immunoblot with anti-MED1 (lanes 1–3) and anti-Flag (lanes 4 and 5) antibodies. (C). UCP-1 induction by PPARγ and TR ligands in differentiated MEFs. WT /PPARγ MEFs were first cultured under conditions leading to differentiation to adipocytes and then in complete UCP-1 induction medium (Normal induction, lane 2), induction medium minus T 3 (−T 3 induction, lane 3) or induction medium minus BRL49653 (−BRL49653 induction, lane 4). UCP-1 expression was monitored by real-time RT-PCR. Non-treated preadipocytes (No induction) are shown in lane 1. (D) Induction of UCP-1 in brown HIB1B cells detected by RT-PCR. (E) . Induced binding of factors to the UCP-1 enhancer in HIB1B cells. Top: Schematic representation of the UCP-1 enhancer and primers used for ChIP assays. Bottom: ChIP analysis of HIB1B preadipocytes and UCP-1 -induced adipocytes with the indicated antibodies. (F). Induced binding of factors to the UCP-1 enhancer in HIB1B cells. Real time PCR ChIP analyses of preadipocytes and UCP-1 induced adipocytes with the indicated antibodies. Error bars indicate standard deviation of two independent experiments.

    Techniques Used: Stable Transfection, Expressing, Quantitative RT-PCR, Standard Deviation, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    7) Product Images from "Accumulation of 8,9-unsaturated sterols drives oligodendrocyte formation and remyelination"

    Article Title: Accumulation of 8,9-unsaturated sterols drives oligodendrocyte formation and remyelination

    Journal: Nature

    doi: 10.1038/s41586-018-0360-3

    Effect of independent chemical-genetic and genetic modulators of CYP51, sterol 14 reductase and EBP on oligodendrocyte formation and cholesterol biosynthesis. a, d, g) Percentage of MBP + oligodendrocytes generated from two independent derivation of OPCs at 72 h following treatment with the indicated concentrations of medroxyprogesterone acetate ( a ), 2-methyl ketoconazole ( d ) or TASIN-449 ( g ). n = 4 wells per condition, except DMSO, n = 12 in a and d . In g , for OPC-5, n = 4 except DMSO, n = 7; for OPC-1, n = 3 except DMSO, n = 6. b, e, h) GC/MS-based quantitation of sterol levels in two independent derivations of OPCs treated 24 h with medroxyprogesterone acetate at 10 μM ( a ), 2-methyl ketoconazole at 2.5 μM ( e ) and TASIN-449 at the indicated concentrations ( h ). n = 2 wells per condition. c, f) Rat CYP51 enzymatic activity following treatment with varying concentrations of medroxyprogesterone acetate ( c ) and 2-methyl ketoconazole ( f ) as measured by LC/MS-based quantitation of the CYP51 product FF-MAS. n = 2 independent enzymatic assays. i) Percentage of MBP + oligodendrocytes generated from OPCs (OPC-5) infected with lentivirus expressing Cas9 and an independent guide RNA targeting EBP (cf. Fig. 2c ). 8 wells per condition, with > 1,000 cells analyzed per well. Two-tailed Student’s t -test, * P = 0.0009. j) Functional validation of CRISPR-based targeting of EBP with a second sgRNA using GC/MS-based quantitation of zymostenol levels. n = 2 wells per condition. k) EBP mRNA levels measured by RT-qPCR in OPCs (OPC-5) infected with lentivirus expressing Cas9 and either of two guide RNAs targeting EBP. One well per condition, with results validated in an independent experiment. l ) Representative images of the oligodendrocyte formation assay shown in Main Figure 2c . Nuclei are labeled with DAPI (blue), and oligodendrocytes are indicated by immunostaining for myelin basic protein (green). Scale bar, 100 μm. All bar graphs indicate mean +/− standard deviation, and panels a, d, g , i and k are representative of two independent experiments.
    Figure Legend Snippet: Effect of independent chemical-genetic and genetic modulators of CYP51, sterol 14 reductase and EBP on oligodendrocyte formation and cholesterol biosynthesis. a, d, g) Percentage of MBP + oligodendrocytes generated from two independent derivation of OPCs at 72 h following treatment with the indicated concentrations of medroxyprogesterone acetate ( a ), 2-methyl ketoconazole ( d ) or TASIN-449 ( g ). n = 4 wells per condition, except DMSO, n = 12 in a and d . In g , for OPC-5, n = 4 except DMSO, n = 7; for OPC-1, n = 3 except DMSO, n = 6. b, e, h) GC/MS-based quantitation of sterol levels in two independent derivations of OPCs treated 24 h with medroxyprogesterone acetate at 10 μM ( a ), 2-methyl ketoconazole at 2.5 μM ( e ) and TASIN-449 at the indicated concentrations ( h ). n = 2 wells per condition. c, f) Rat CYP51 enzymatic activity following treatment with varying concentrations of medroxyprogesterone acetate ( c ) and 2-methyl ketoconazole ( f ) as measured by LC/MS-based quantitation of the CYP51 product FF-MAS. n = 2 independent enzymatic assays. i) Percentage of MBP + oligodendrocytes generated from OPCs (OPC-5) infected with lentivirus expressing Cas9 and an independent guide RNA targeting EBP (cf. Fig. 2c ). 8 wells per condition, with > 1,000 cells analyzed per well. Two-tailed Student’s t -test, * P = 0.0009. j) Functional validation of CRISPR-based targeting of EBP with a second sgRNA using GC/MS-based quantitation of zymostenol levels. n = 2 wells per condition. k) EBP mRNA levels measured by RT-qPCR in OPCs (OPC-5) infected with lentivirus expressing Cas9 and either of two guide RNAs targeting EBP. One well per condition, with results validated in an independent experiment. l ) Representative images of the oligodendrocyte formation assay shown in Main Figure 2c . Nuclei are labeled with DAPI (blue), and oligodendrocytes are indicated by immunostaining for myelin basic protein (green). Scale bar, 100 μm. All bar graphs indicate mean +/− standard deviation, and panels a, d, g , i and k are representative of two independent experiments.

    Techniques Used: Generated, Gas Chromatography-Mass Spectrometry, Quantitation Assay, Activity Assay, Liquid Chromatography with Mass Spectroscopy, Infection, Expressing, Two Tailed Test, Functional Assay, CRISPR, Quantitative RT-PCR, Tube Formation Assay, Labeling, Immunostaining, Standard Deviation

    Small-molecule inhibition of CYP51, TM7SF2, or EBP enhances oligodendrocyte formation via accumulation of 8,9-unsaturated sterols. a) Abbreviated cholesterol biosynthesis pathway. For greater detail, see Extended Data Figure 1 . b) Percentage of MBP + oligodendrocytes generated from OPCs treated with the indicated pathway inhibitors. n ≥ 4 wells per condition. c) Percentage of MBP + oligodendrocytes generated from OPCs expressing Cas9 and guide RNA targeting EBP. n ≥ 3 wells per condition. d) Functional validation of Cas9-based targeting of EBP using GC/MS-based quantitation of zymostenol levels. n = 2 wells per condition. e, f, g) Percentage of MBP + oligodendrocytes generated from OPCs with the indicated purified sterols. n ≥ 4 wells per condition. h) Structures of various sterols. All bar graphs indicate mean ± standard deviation. See Statistics and Reproducibility for exact well counts. Experiments in b-g are representative of two or more independent experiments using OPC-5 cells; for validation in an independent derivation of OPCs, see Extended Data Fig. 3 – 5 .
    Figure Legend Snippet: Small-molecule inhibition of CYP51, TM7SF2, or EBP enhances oligodendrocyte formation via accumulation of 8,9-unsaturated sterols. a) Abbreviated cholesterol biosynthesis pathway. For greater detail, see Extended Data Figure 1 . b) Percentage of MBP + oligodendrocytes generated from OPCs treated with the indicated pathway inhibitors. n ≥ 4 wells per condition. c) Percentage of MBP + oligodendrocytes generated from OPCs expressing Cas9 and guide RNA targeting EBP. n ≥ 3 wells per condition. d) Functional validation of Cas9-based targeting of EBP using GC/MS-based quantitation of zymostenol levels. n = 2 wells per condition. e, f, g) Percentage of MBP + oligodendrocytes generated from OPCs with the indicated purified sterols. n ≥ 4 wells per condition. h) Structures of various sterols. All bar graphs indicate mean ± standard deviation. See Statistics and Reproducibility for exact well counts. Experiments in b-g are representative of two or more independent experiments using OPC-5 cells; for validation in an independent derivation of OPCs, see Extended Data Fig. 3 – 5 .

    Techniques Used: Inhibition, Generated, Expressing, Functional Assay, Gas Chromatography-Mass Spectrometry, Quantitation Assay, Purification, Standard Deviation

    8) Product Images from "High-throughput, Efficient, and Unbiased Capture of Small RNAs from Low-input Samples for Sequencing"

    Article Title: High-throughput, Efficient, and Unbiased Capture of Small RNAs from Low-input Samples for Sequencing

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-38458-7

    Nucleotide biases seen at ligation sites and varies between individual miRNAs. ( A ) SeqLogo representation of the base composition of the degenerate Ns over all miRNAs and select miRs representing high (miR-21) mid (Let-7i, miR-96) and low (miR-151) expression. The height of the letter representing the base is proportional to its probability. Bases 1–4 are at the 3′ end of 5′ adapter. Bases 5–8 are at the 5′ end of 3′ adapter. Bases 9–12 follow barcode in 3′ adapter. ( B ) Cumulative divergence from expected probability of nucleotide composition at each base across all miRNAs, and the same select miRs as ( A ). Data shown is from a 500 ng cellular RNA input sample.
    Figure Legend Snippet: Nucleotide biases seen at ligation sites and varies between individual miRNAs. ( A ) SeqLogo representation of the base composition of the degenerate Ns over all miRNAs and select miRs representing high (miR-21) mid (Let-7i, miR-96) and low (miR-151) expression. The height of the letter representing the base is proportional to its probability. Bases 1–4 are at the 3′ end of 5′ adapter. Bases 5–8 are at the 5′ end of 3′ adapter. Bases 9–12 follow barcode in 3′ adapter. ( B ) Cumulative divergence from expected probability of nucleotide composition at each base across all miRNAs, and the same select miRs as ( A ). Data shown is from a 500 ng cellular RNA input sample.

    Techniques Used: Ligation, Expressing

    Unique molecular identifiers (UMIs) collapse duplicate reads and reveal linear relationship at low PCR cycles between total and collapsed counts, but drop out at high PCR cycles. ( A-C ) Effect of increasing length of UMI on number of miRNA counts following collapsing of miRNA + UMI, compared to total “raw” count. Number to left of + sign represents Ns on the 5′ adapter while numbers to right represent 3′ adapter. Insert represent collapse on miRNAs alone (i.e. without and UMI). Raw represents uncollapsed read count. Analysis shown for all miRNAs (A), a highly expressed miRNA (miR-21, B) and intermediately expressed miRNA (miR-96, C ). ( D ) Correlation plot of log10 counts per million for each miRNA comparing collapsed versus uncollapsed (total) reads for a library amplified for 14 cycles. All 12 random nucleotides were used for collapsing. ( E ) Same as D, but amplified for 24 cycles showing reduction in correlation for low expressed miRNAs. ( F ) Same as D, but comparing only collapsed reads between library amplified for 14 versus 24 cycles, showing much poorer correlation for low to intermediate expressed miRNAs. ( G-I ) Direct comparison of read counts for each miRNA from libraries differing in the number of PCR amplification cycles. miRNAs are ordered from high to low expression in 14 cycle library. ( G ) Uncollapsed (total) counts per million. ( H ) Collapsed counts per million. ( I ) Percent unique reads (i.e. collapsed counts/total counts *100). Note noise created by high PCR cycle number on the lowly to intermediately expressed miRNAs. All libraries were made from one cellular input RNA.
    Figure Legend Snippet: Unique molecular identifiers (UMIs) collapse duplicate reads and reveal linear relationship at low PCR cycles between total and collapsed counts, but drop out at high PCR cycles. ( A-C ) Effect of increasing length of UMI on number of miRNA counts following collapsing of miRNA + UMI, compared to total “raw” count. Number to left of + sign represents Ns on the 5′ adapter while numbers to right represent 3′ adapter. Insert represent collapse on miRNAs alone (i.e. without and UMI). Raw represents uncollapsed read count. Analysis shown for all miRNAs (A), a highly expressed miRNA (miR-21, B) and intermediately expressed miRNA (miR-96, C ). ( D ) Correlation plot of log10 counts per million for each miRNA comparing collapsed versus uncollapsed (total) reads for a library amplified for 14 cycles. All 12 random nucleotides were used for collapsing. ( E ) Same as D, but amplified for 24 cycles showing reduction in correlation for low expressed miRNAs. ( F ) Same as D, but comparing only collapsed reads between library amplified for 14 versus 24 cycles, showing much poorer correlation for low to intermediate expressed miRNAs. ( G-I ) Direct comparison of read counts for each miRNA from libraries differing in the number of PCR amplification cycles. miRNAs are ordered from high to low expression in 14 cycle library. ( G ) Uncollapsed (total) counts per million. ( H ) Collapsed counts per million. ( I ) Percent unique reads (i.e. collapsed counts/total counts *100). Note noise created by high PCR cycle number on the lowly to intermediately expressed miRNAs. All libraries were made from one cellular input RNA.

    Techniques Used: Polymerase Chain Reaction, Amplification, Expressing

    Modifications to high-throughput sequencing method improves capture of miRNAs. ( A ) Schematic of protocol to prepare miRNA libraries for sequencing. Modifications from original protocol noted in bold. ( B ) Percentage of different classes of RNAs captured from a plasma sample using the original conditions (0.85 μM 3′ adapter, 3.3 μM unmodified 5′ adapter). Ligations were performed in triplicate from the same RNA. Each replicate is shown as an individual bar. Note the low percentage of reads mapping to miRNAs (red). ( C ) Percentage of mature miRNAs captured using the optimized conditions (0.05 μΜ 3′ adapter, 0.33 μΜ amino-modified 5′ adapter) were compared to original conditions in three independent biological samples. The ligation reactions were performed in triplicate for each sample and protocol. Each replicate is shown as a black dot. Red dot represents average. ( D ) The average percentage of reads mapping to different classes of RNA for each sample and condition shown in C.
    Figure Legend Snippet: Modifications to high-throughput sequencing method improves capture of miRNAs. ( A ) Schematic of protocol to prepare miRNA libraries for sequencing. Modifications from original protocol noted in bold. ( B ) Percentage of different classes of RNAs captured from a plasma sample using the original conditions (0.85 μM 3′ adapter, 3.3 μM unmodified 5′ adapter). Ligations were performed in triplicate from the same RNA. Each replicate is shown as an individual bar. Note the low percentage of reads mapping to miRNAs (red). ( C ) Percentage of mature miRNAs captured using the optimized conditions (0.05 μΜ 3′ adapter, 0.33 μΜ amino-modified 5′ adapter) were compared to original conditions in three independent biological samples. The ligation reactions were performed in triplicate for each sample and protocol. Each replicate is shown as a black dot. Red dot represents average. ( D ) The average percentage of reads mapping to different classes of RNA for each sample and condition shown in C.

    Techniques Used: Next-Generation Sequencing, Sequencing, Modification, Ligation

    Improved miRNA capture also seen at low concentrations of input RNA. ( A ) The percentage of reads mapping to miRNAs at the indicated input following either the optimized (0.05 μΜ 3′ adapter, 0.33 μΜ amino-modified 5′adapter) or the original (0.85 μM 3′ adapter, 3.3 μM unmodified 5′ adapter) protocol. Black dots represent the three replicates from each input RNA for each sample, protocol, and concentration. Red dots represent average. ( B ) The average percentage of reads mapping to different RNA classes for each sample, protocol, and concentration.
    Figure Legend Snippet: Improved miRNA capture also seen at low concentrations of input RNA. ( A ) The percentage of reads mapping to miRNAs at the indicated input following either the optimized (0.05 μΜ 3′ adapter, 0.33 μΜ amino-modified 5′adapter) or the original (0.85 μM 3′ adapter, 3.3 μM unmodified 5′ adapter) protocol. Black dots represent the three replicates from each input RNA for each sample, protocol, and concentration. Red dots represent average. ( B ) The average percentage of reads mapping to different RNA classes for each sample, protocol, and concentration.

    Techniques Used: Modification, Concentration Assay

    9) Product Images from "A muscle-specific knockout implicates nuclear receptor coactivator MED1 in the regulation of glucose and energy metabolism"

    Article Title: A muscle-specific knockout implicates nuclear receptor coactivator MED1 in the regulation of glucose and energy metabolism

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

    doi: 10.1073/pnas.1005626107

    Gene expression analyses of white and red muscles. ( A and B ) Real-time RT-PCR analysis of total RNA from quadriceps (white muscle) of Med1 fl/fl and Med1 fl/fl/Cre mice. ( C ) Real-time RT-PCR analysis of total RNA from soleus (red muscle) of Med1 fl/fl and Med1 fl/fl/Cre mice. Each bar represents the mean ± SD of four mice.
    Figure Legend Snippet: Gene expression analyses of white and red muscles. ( A and B ) Real-time RT-PCR analysis of total RNA from quadriceps (white muscle) of Med1 fl/fl and Med1 fl/fl/Cre mice. ( C ) Real-time RT-PCR analysis of total RNA from soleus (red muscle) of Med1 fl/fl and Med1 fl/fl/Cre mice. Each bar represents the mean ± SD of four mice.

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

    10) Product Images from "A genome landscape of SRSF3-regulated splicing events and gene expression in human osteosarcoma U2OS cells"

    Article Title: A genome landscape of SRSF3-regulated splicing events and gene expression in human osteosarcoma U2OS cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1500

    Validation of other SRSF3-responsive RNA splicing events identified by ANOVA analysis. Following transfection of U2OS or HeLa cells with Dharmacon si-NS or si-SRSF3 twice in an interval of 48 h, total RNA from the cells was analyzed by RT-PCR for exon skipping of CHK1 exon 3 ( A ), SMC2 exon 3–4 ( B ), CKLF exon 3 ( C ), MAP4 exon 10 ( D ), MBNL1 exon 4 ( E ), MELK exon 11 ( F ), DDX5 exon 12 ( G ) and PABPC1 exon 10–11 ( H ). See other details in Figure 3 .
    Figure Legend Snippet: Validation of other SRSF3-responsive RNA splicing events identified by ANOVA analysis. Following transfection of U2OS or HeLa cells with Dharmacon si-NS or si-SRSF3 twice in an interval of 48 h, total RNA from the cells was analyzed by RT-PCR for exon skipping of CHK1 exon 3 ( A ), SMC2 exon 3–4 ( B ), CKLF exon 3 ( C ), MAP4 exon 10 ( D ), MBNL1 exon 4 ( E ), MELK exon 11 ( F ), DDX5 exon 12 ( G ) and PABPC1 exon 10–11 ( H ). See other details in Figure 3 .

    Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction

    Validation of the SRSF3-responsive events identified by B/E ratio analysis. U2OS and HeLa cells were transfected with Dharmacon si-NS or si-SRSF3 twice in an interval of 48 h. Total RNA from the cells were analyzed by RT-PCR to validate transcript level change of ERRFI1 ( A ), ANXA1 ( B ) and TGFB2 ( C ), splicing alteration of PUS3 alternative 5′ ss usage in the exon 3 ( D ), and exon skipping of the PKP4 exon 7 ( E ), KIF23 exon 18 ( F ), EP300 exon 14 ( G ) and CLINT1 exon 11 ( H ). The primers used in RT-PCR are shown as bars above (forward primers) and below (reverse primers) each RNA splicing diagram. GAPDH served as a loading control. RT+, reaction with reverse transcriptase; RT−, reaction without reverse transcriptase; si-SRSF3 Amb , si-SRSF3 from Ambion; FC, fold-change; PSI, percent spliced-in of the alternative exon(s) or splice site (% inclusion = inclusion/sum of inclusion + exclusion).
    Figure Legend Snippet: Validation of the SRSF3-responsive events identified by B/E ratio analysis. U2OS and HeLa cells were transfected with Dharmacon si-NS or si-SRSF3 twice in an interval of 48 h. Total RNA from the cells were analyzed by RT-PCR to validate transcript level change of ERRFI1 ( A ), ANXA1 ( B ) and TGFB2 ( C ), splicing alteration of PUS3 alternative 5′ ss usage in the exon 3 ( D ), and exon skipping of the PKP4 exon 7 ( E ), KIF23 exon 18 ( F ), EP300 exon 14 ( G ) and CLINT1 exon 11 ( H ). The primers used in RT-PCR are shown as bars above (forward primers) and below (reverse primers) each RNA splicing diagram. GAPDH served as a loading control. RT+, reaction with reverse transcriptase; RT−, reaction without reverse transcriptase; si-SRSF3 Amb , si-SRSF3 from Ambion; FC, fold-change; PSI, percent spliced-in of the alternative exon(s) or splice site (% inclusion = inclusion/sum of inclusion + exclusion).

    Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction

    SRSF3 and SRSF1 are mutually regulated in cells. ( A ) SRSF3 knockdown in HeLa cells activates the usage of a cryptic intron in the SRSF1 exon 4. HeLa cells were transfected with Dharmacon si-NS or si-SRSF3 as described in Figure 4 and total RNA from the cells was analyzed by RT-PCR for activation of a cryptic intron (dark box) in the SRSF1 3′ UTR. ( B ) Knocking down SRSF1 expression in HeLa cells affects SRSF3 transcript level, but not inclusion or skipping of the SRSF3 exon 4. Knockdown of SRSF1 expression in HeLa cells was performed as described in Figure 4 . See other details in Figure 3 . ( C ) Knocking down SRSF1 expression in HeLa cells does not affect SRSF3 RNA stability in a pause-chase RNA decay assay. Following 10 μg/ml actinomycin D treatment for 0, 1, 2, 4 and 8 h, HeLa cells with Dharmacon si-NS or si-SRSF1 knockdown as described in Figure 4 were examined for the quantitative levels of the exon 4-skipped SRSF3 RNA and GAPDH RNA at each time point by quantitative RT-qPCR. After normalizing to GAPDH RNA, the exon 4-skipped SRSF3 RNA decay rate was calculated by setting the RNA levels at 0 h as 100% for both si-NS and si-SRSF1 groups. Exponential fitting curves over each time point are determined as y = e −0.185x , R 2 = 0.8739 for si-NS group and y = e −0.201x , R 2 = 0.9819 for si-SRSF1 group. Half-life (t 1/2 ) of the exon 4-skipped SRSF3 RNA was calculated as 3.8 h for si-NS transfected cells, and 3.5 h for si-SRSF1 transfected cells. ( D ) Expression of SRSF3 and SRSF1 is mutually regulated each other. HeLa or U2OS cells were transfected with Dharmacon si-NS, si-SRSF1, or si-SRSF3 twice with an interval of 48 h, and analyzed by Western blot for the corresponding protein expression by using an anti-SRSF1 or anti-SRSF3 antibody. ( E ) Overexpression of T7-SRSF3 increases the expression of SRSF1 in MEF3T3 cells revealed by Western blot.
    Figure Legend Snippet: SRSF3 and SRSF1 are mutually regulated in cells. ( A ) SRSF3 knockdown in HeLa cells activates the usage of a cryptic intron in the SRSF1 exon 4. HeLa cells were transfected with Dharmacon si-NS or si-SRSF3 as described in Figure 4 and total RNA from the cells was analyzed by RT-PCR for activation of a cryptic intron (dark box) in the SRSF1 3′ UTR. ( B ) Knocking down SRSF1 expression in HeLa cells affects SRSF3 transcript level, but not inclusion or skipping of the SRSF3 exon 4. Knockdown of SRSF1 expression in HeLa cells was performed as described in Figure 4 . See other details in Figure 3 . ( C ) Knocking down SRSF1 expression in HeLa cells does not affect SRSF3 RNA stability in a pause-chase RNA decay assay. Following 10 μg/ml actinomycin D treatment for 0, 1, 2, 4 and 8 h, HeLa cells with Dharmacon si-NS or si-SRSF1 knockdown as described in Figure 4 were examined for the quantitative levels of the exon 4-skipped SRSF3 RNA and GAPDH RNA at each time point by quantitative RT-qPCR. After normalizing to GAPDH RNA, the exon 4-skipped SRSF3 RNA decay rate was calculated by setting the RNA levels at 0 h as 100% for both si-NS and si-SRSF1 groups. Exponential fitting curves over each time point are determined as y = e −0.185x , R 2 = 0.8739 for si-NS group and y = e −0.201x , R 2 = 0.9819 for si-SRSF1 group. Half-life (t 1/2 ) of the exon 4-skipped SRSF3 RNA was calculated as 3.8 h for si-NS transfected cells, and 3.5 h for si-SRSF1 transfected cells. ( D ) Expression of SRSF3 and SRSF1 is mutually regulated each other. HeLa or U2OS cells were transfected with Dharmacon si-NS, si-SRSF1, or si-SRSF3 twice with an interval of 48 h, and analyzed by Western blot for the corresponding protein expression by using an anti-SRSF1 or anti-SRSF3 antibody. ( E ) Overexpression of T7-SRSF3 increases the expression of SRSF1 in MEF3T3 cells revealed by Western blot.

    Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Over Expression

    SRSF3 promotes inclusion of the EP300 exon 14 through an exonic SRSF3-binding site. ( A ) Diagram of EP300 minigene structure and exon 14 mutants containing detection (Δ1, Δ2, Δ3 and Δ4) of a 6-nt, putative SRSF3-binding motif. Red boxes a, b, c and d indicate individual putative SRSF3-binding motif. The EP300 minigene has a 623-bp deletion in the intron 14 indicated by a vertical line. P CMV IE , cytomegalovirus immediate early promoter; pA SV40 , SV40 polyadenylation signal. ( B and C ) Deletion of a putative SRSF3-binding motif increases skipping of the EP300 exon 14. HeLa cells were transfected with individual EP300 minigenes or a parental vector pEGFP-N1 for 24 h, and skipping of the minigene exon 14 was determined by RT-PCR with a primer set for EP300 exon 13 (F 13 , forward primer) and vector sequence (R vec , reverse primer, diagrammed on the right). GAPDH RNA served as a loading control. RT-PCR products were resolved by gel electrophoresis, the band intensity was measured, and after normalizing to GAPDH RNA, a skipping rate (%) of the EP300 exon 14 was calculated. Shown in bar graphs (C) are means ± SD from two separate experiments. * P
    Figure Legend Snippet: SRSF3 promotes inclusion of the EP300 exon 14 through an exonic SRSF3-binding site. ( A ) Diagram of EP300 minigene structure and exon 14 mutants containing detection (Δ1, Δ2, Δ3 and Δ4) of a 6-nt, putative SRSF3-binding motif. Red boxes a, b, c and d indicate individual putative SRSF3-binding motif. The EP300 minigene has a 623-bp deletion in the intron 14 indicated by a vertical line. P CMV IE , cytomegalovirus immediate early promoter; pA SV40 , SV40 polyadenylation signal. ( B and C ) Deletion of a putative SRSF3-binding motif increases skipping of the EP300 exon 14. HeLa cells were transfected with individual EP300 minigenes or a parental vector pEGFP-N1 for 24 h, and skipping of the minigene exon 14 was determined by RT-PCR with a primer set for EP300 exon 13 (F 13 , forward primer) and vector sequence (R vec , reverse primer, diagrammed on the right). GAPDH RNA served as a loading control. RT-PCR products were resolved by gel electrophoresis, the band intensity was measured, and after normalizing to GAPDH RNA, a skipping rate (%) of the EP300 exon 14 was calculated. Shown in bar graphs (C) are means ± SD from two separate experiments. * P

    Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Sequencing, Nucleic Acid Electrophoresis

    11) Product Images from "Deletion of a Short, Untranslated Region Adjacent to the Polypurine Tract in Moloney Murine Leukemia Virus Leads to Formation of Aberrant 5? Plus-Strand DNA Ends In Vivo"

    Article Title: Deletion of a Short, Untranslated Region Adjacent to the Polypurine Tract in Moloney Murine Leukemia Virus Leads to Formation of Aberrant 5? Plus-Strand DNA Ends In Vivo

    Journal: Journal of Virology

    doi:

    RNase protection assay to monitor viral RNA levels. (A) Schematic representation of the riboprobe and the regions protected from RNase digestion. The upper line shows the organization of the 5′ portion of the Mo-MuLV genome. The 5′ LTR (open box), the transcription start site (arrow), the splice donor (SD) site, and the flanking restriction sites that were used to clone the riboprobe template ( Xba I and Eag I) are indicated. Lower lines show the full-length riboprobe with flanking plasmid sequences (wavy lines) and the two fragments protected by the unspliced and spliced viral RNA. The lengths of the different products are indicated. (B) Cellular RNA was extracted from 293T cells transiently producing either Mo-MuLV or Mo-MuLVΔ28. Virions from culture supernatants were purified through 25 to 45% sucrose step gradients, followed by an additional purification step through a 25% sucrose cushion. RNA was extracted from virions, and the amounts were normalized by RT activity. Total cellular RNA and virion RNAs were used for the RNase protection assay. The positions of the full-length riboprobe and its protected fragments (unspliced and spliced) are indicated at the right (the prominent band above the spliced RNA product is probably derived by cleavage of the unspliced RNA). The viruses are indicated above the panels, and the source of the RNA is shown below the panels. Controls included riboprobe mixed only with yeast RNA with and without RNase (digested and undigested, respectively) and 293T cells transfected without plasmid DNA (mock). As a quantitation control, reactions with 1:5 and 1:10 dilutions of the wt viral RNA sample (Mo-MuLV 1:5 and 1:10) were also performed.
    Figure Legend Snippet: RNase protection assay to monitor viral RNA levels. (A) Schematic representation of the riboprobe and the regions protected from RNase digestion. The upper line shows the organization of the 5′ portion of the Mo-MuLV genome. The 5′ LTR (open box), the transcription start site (arrow), the splice donor (SD) site, and the flanking restriction sites that were used to clone the riboprobe template ( Xba I and Eag I) are indicated. Lower lines show the full-length riboprobe with flanking plasmid sequences (wavy lines) and the two fragments protected by the unspliced and spliced viral RNA. The lengths of the different products are indicated. (B) Cellular RNA was extracted from 293T cells transiently producing either Mo-MuLV or Mo-MuLVΔ28. Virions from culture supernatants were purified through 25 to 45% sucrose step gradients, followed by an additional purification step through a 25% sucrose cushion. RNA was extracted from virions, and the amounts were normalized by RT activity. Total cellular RNA and virion RNAs were used for the RNase protection assay. The positions of the full-length riboprobe and its protected fragments (unspliced and spliced) are indicated at the right (the prominent band above the spliced RNA product is probably derived by cleavage of the unspliced RNA). The viruses are indicated above the panels, and the source of the RNA is shown below the panels. Controls included riboprobe mixed only with yeast RNA with and without RNase (digested and undigested, respectively) and 293T cells transfected without plasmid DNA (mock). As a quantitation control, reactions with 1:5 and 1:10 dilutions of the wt viral RNA sample (Mo-MuLV 1:5 and 1:10) were also performed.

    Techniques Used: Rnase Protection Assay, Plasmid Preparation, Purification, Activity Assay, Derivative Assay, Transfection, Quantitation Assay

    12) Product Images from "Identification of Motile Sperm Domain–Containing Protein 2 as Regulator of Human Monocyte Migration"

    Article Title: Identification of Motile Sperm Domain–Containing Protein 2 as Regulator of Human Monocyte Migration

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1601662

    MOSPD2 promotes monocytic line migration. ( A ) mRNA and protein expression of MOSPD2 in U937 cells transduced with sh-Control or sh-MOSPD2 #1 lentiviral particles. 18S was used to normalize RNA levels. Samples were run in triplicate. One of at least three
    Figure Legend Snippet: MOSPD2 promotes monocytic line migration. ( A ) mRNA and protein expression of MOSPD2 in U937 cells transduced with sh-Control or sh-MOSPD2 #1 lentiviral particles. 18S was used to normalize RNA levels. Samples were run in triplicate. One of at least three

    Techniques Used: Migration, Expressing, Transduction

    Effect of VB-201 on migration of MOSPD2-silenced monocytes. ( A ) MOSPD2 mRNA levels in U937 cells transduced with sh-Control and sh-MOSPD2 lentiviral particles targeting two different regions. 18S was used to normalize RNA levels. Samples were run in triplicate.
    Figure Legend Snippet: Effect of VB-201 on migration of MOSPD2-silenced monocytes. ( A ) MOSPD2 mRNA levels in U937 cells transduced with sh-Control and sh-MOSPD2 lentiviral particles targeting two different regions. 18S was used to normalize RNA levels. Samples were run in triplicate.

    Techniques Used: Migration, Transduction

    MOSPD2 abundance and role in chemotaxis are restricted to myeloid cells. ( A ) mRNA expression of MOSPD2 in purified CD14 + monocytes, B cells, T cells, and neutrophils. 18S was used to normalize RNA levels. Results are shown as expression relative to monocytes.
    Figure Legend Snippet: MOSPD2 abundance and role in chemotaxis are restricted to myeloid cells. ( A ) mRNA expression of MOSPD2 in purified CD14 + monocytes, B cells, T cells, and neutrophils. 18S was used to normalize RNA levels. Results are shown as expression relative to monocytes.

    Techniques Used: Chemotaxis Assay, Expressing, Purification

    13) Product Images from "Drosophila Ge-1 Promotes P Body Formation and oskar mRNA Localization"

    Article Title: Drosophila Ge-1 Promotes P Body Formation and oskar mRNA Localization

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020612

    dGe-1 is a P body component in the Drosophila germline. (A) Scheme representing the dGe-1 locus (polytene band 32D3). Numbers indicate genomic positions along chromosome 2L. 5′UTR, coding region, and 3′UTR: grey, red and blue boxes, respectively. The two dGe-1 transcripts, dGe-1-A and dGe-1-B , the insertion site of the P-element transposon (GS5005, red triangle) used for generation of dGe-1 deletions, and the region deleted in dGe-1 Δ5 (double-headed arrow) are represented. Positions of primers used for RT-PCR ( Figure S1B ) are indicated (blue, red, green arrows). (B) Distribution of dGe-1 protein in wt egg-chambers during early and mid-oogenesis. Immunodetection of dGe-1 using rat anti-dGe-1 antibody. (C) HA-dGe1 protein distribution in wt egg-chamber at stage 6 (S6). HA-dGe-1 detected using mouse anti-HA antibody. (D–I) Colocalization of dGe-1 protein and two P body components. Double-staining of two wt S7 egg-chambers using rat anti-dGe1 (green, D and G) and rabbit anti-Me31B (red, E) or rabbit anti-Tral (red, H) antibodies. Overlays (F and I). (J) Association of dGe-1 and dDcp1 in ovarian extract. Western blot of anti-GFP immunoprecipitates of GFP-FWS and YFP-dDcp1 ovaries. Western blot probed with rabbit anti-dGe-1, anti-Exu and anti-Khc, and mouse anti-Me31B and anti-Tub antibodies. dGe-1 is indicated with an arrow (see legend to Figure 2A ). (K) RNA-independent association of dGe-1 and dDcp1. Western blot of anti-GFP immunoprecipitates from YFP-dDcp1 ovarian extract with or without RNase A treatment prior to immunoprecipitation, probed with rabbit anti-dGe-1 and anti-Exu antibodies. dGe-1 is indicated with an arrow. (L) Exu and Me31B associate with dGe-1 in the ovary. Endogeneous dGe-1 protein was immunoprecipitated from wt ovarian extract and bound and soluble fractions were subjected to western blot analysis. Before immunoprecipitation, rabbit anti-dGe-1 was pre-incubated or not with dGe-1 blocking peptide. Western blot probed with rabbit anti-dGe-1, anti-Exu and anti-Khc, and mouse anti-Me31B and anti-Tub antibodies. dGe-1 is indicated with an arrow. Bar, 10 µm.
    Figure Legend Snippet: dGe-1 is a P body component in the Drosophila germline. (A) Scheme representing the dGe-1 locus (polytene band 32D3). Numbers indicate genomic positions along chromosome 2L. 5′UTR, coding region, and 3′UTR: grey, red and blue boxes, respectively. The two dGe-1 transcripts, dGe-1-A and dGe-1-B , the insertion site of the P-element transposon (GS5005, red triangle) used for generation of dGe-1 deletions, and the region deleted in dGe-1 Δ5 (double-headed arrow) are represented. Positions of primers used for RT-PCR ( Figure S1B ) are indicated (blue, red, green arrows). (B) Distribution of dGe-1 protein in wt egg-chambers during early and mid-oogenesis. Immunodetection of dGe-1 using rat anti-dGe-1 antibody. (C) HA-dGe1 protein distribution in wt egg-chamber at stage 6 (S6). HA-dGe-1 detected using mouse anti-HA antibody. (D–I) Colocalization of dGe-1 protein and two P body components. Double-staining of two wt S7 egg-chambers using rat anti-dGe1 (green, D and G) and rabbit anti-Me31B (red, E) or rabbit anti-Tral (red, H) antibodies. Overlays (F and I). (J) Association of dGe-1 and dDcp1 in ovarian extract. Western blot of anti-GFP immunoprecipitates of GFP-FWS and YFP-dDcp1 ovaries. Western blot probed with rabbit anti-dGe-1, anti-Exu and anti-Khc, and mouse anti-Me31B and anti-Tub antibodies. dGe-1 is indicated with an arrow (see legend to Figure 2A ). (K) RNA-independent association of dGe-1 and dDcp1. Western blot of anti-GFP immunoprecipitates from YFP-dDcp1 ovarian extract with or without RNase A treatment prior to immunoprecipitation, probed with rabbit anti-dGe-1 and anti-Exu antibodies. dGe-1 is indicated with an arrow. (L) Exu and Me31B associate with dGe-1 in the ovary. Endogeneous dGe-1 protein was immunoprecipitated from wt ovarian extract and bound and soluble fractions were subjected to western blot analysis. Before immunoprecipitation, rabbit anti-dGe-1 was pre-incubated or not with dGe-1 blocking peptide. Western blot probed with rabbit anti-dGe-1, anti-Exu and anti-Khc, and mouse anti-Me31B and anti-Tub antibodies. dGe-1 is indicated with an arrow. Bar, 10 µm.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Immunodetection, Double Staining, Western Blot, Immunoprecipitation, Incubation, Blocking Assay

    14) Product Images from "MnTE-2-PyP Suppresses Prostate Cancer Cell Growth via H2O2 Production"

    Article Title: MnTE-2-PyP Suppresses Prostate Cancer Cell Growth via H2O2 Production

    Journal: Antioxidants

    doi: 10.3390/antiox9060490

    Effects of MnTE-2-PyP on cell cycle progression in combination with radiation in PC3 cells. ( A ) PC3 cells were seeded in media containing PBS or 30 µM MnTE-2-PyP. After 24 h, 2 Gy of radiation was applied to some groups. After another 72 h, cell numbers were determined. ( B ) Cell cycle analyses for PC3 cells treated with MnTE-2-PyP or radiation or the combination of the two. ( C ) Low-RNA population identified by pyronin and Hoechst staining. ( D ) LNCaP cells were seeded in media containing PBS or 30 µM MnTE-2-PyP. After 24 h, 2 Gy of radiation was applied to some groups. After another 72 h, cell numbers were determined. ( E ) Cell cycle analyses for LNCaP cells treated with MnTE-2-PyP or radiation or the combination of the two. ( F ) Measurement of sub-G 1 population in LNCaP cells ( G ) Total low-RNA population identified in LNCaP cells by pyronin and Hoechst staining. All data represent mean ± SD from at least three independent experiments. * p
    Figure Legend Snippet: Effects of MnTE-2-PyP on cell cycle progression in combination with radiation in PC3 cells. ( A ) PC3 cells were seeded in media containing PBS or 30 µM MnTE-2-PyP. After 24 h, 2 Gy of radiation was applied to some groups. After another 72 h, cell numbers were determined. ( B ) Cell cycle analyses for PC3 cells treated with MnTE-2-PyP or radiation or the combination of the two. ( C ) Low-RNA population identified by pyronin and Hoechst staining. ( D ) LNCaP cells were seeded in media containing PBS or 30 µM MnTE-2-PyP. After 24 h, 2 Gy of radiation was applied to some groups. After another 72 h, cell numbers were determined. ( E ) Cell cycle analyses for LNCaP cells treated with MnTE-2-PyP or radiation or the combination of the two. ( F ) Measurement of sub-G 1 population in LNCaP cells ( G ) Total low-RNA population identified in LNCaP cells by pyronin and Hoechst staining. All data represent mean ± SD from at least three independent experiments. * p

    Techniques Used: Staining

    15) Product Images from "Upregulation of Bcl2 in NSCLC with acquired resistance to EGFR-TKI"

    Article Title: Upregulation of Bcl2 in NSCLC with acquired resistance to EGFR-TKI

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.7377

    (A) Detection of protein expression by immunoblotting. A marked Bcl2 upregulation was observed in EGFR-TKI resistant cell lines (GR1, GR2, ER1 and ER2). (B) Bcl2 RNA expression level was quantified by qPCR and normalized to GAPDH expression with the error bars representing standard deviation. The significance of the Bcl2 RNA levels between resistant cell lines and parental HCC827 was determined by one-way ANOVA with P=0.0176, followed by Tukey's HSD post-hoc test. *P
    Figure Legend Snippet: (A) Detection of protein expression by immunoblotting. A marked Bcl2 upregulation was observed in EGFR-TKI resistant cell lines (GR1, GR2, ER1 and ER2). (B) Bcl2 RNA expression level was quantified by qPCR and normalized to GAPDH expression with the error bars representing standard deviation. The significance of the Bcl2 RNA levels between resistant cell lines and parental HCC827 was determined by one-way ANOVA with P=0.0176, followed by Tukey's HSD post-hoc test. *P

    Techniques Used: Expressing, RNA Expression, Real-time Polymerase Chain Reaction, Standard Deviation

    16) Product Images from "NeuroD6 Genomic Signature Bridging Neuronal Differentiation to Survival via the Molecular Chaperone Network"

    Article Title: NeuroD6 Genomic Signature Bridging Neuronal Differentiation to Survival via the Molecular Chaperone Network

    Journal: Journal of neuroscience research

    doi: 10.1002/jnr.22182

    Unbiased genome-wide microarray analysis upon NeuroD6 overexpression. ( A ) Scatterplot analysis showing differentially expressed probe sets upon NeuroD6 overexpression. Total RNA from control PC12 and PC12-ND6 cells were isolated in six replicates and
    Figure Legend Snippet: Unbiased genome-wide microarray analysis upon NeuroD6 overexpression. ( A ) Scatterplot analysis showing differentially expressed probe sets upon NeuroD6 overexpression. Total RNA from control PC12 and PC12-ND6 cells were isolated in six replicates and

    Techniques Used: Genome Wide, Microarray, Over Expression, Isolation

    17) Product Images from "Lithium chloride inhibits the coronavirus infectious bronchitis virus in cell culture"

    Article Title: Lithium chloride inhibits the coronavirus infectious bronchitis virus in cell culture

    Journal: Avian Pathology

    doi: 10.1080/03079450601156083

    Real-time RT-PCR analysis of the levels of IBV genomic RNA as well as genomic and subgenomic mRNAs, as determined by analysis of the IBV 5′ UTR (light grey) and 3′ UTR (dark grey), respectively, in infected (5a) Vero cells and (5b) DF-1 cells.
    Figure Legend Snippet: Real-time RT-PCR analysis of the levels of IBV genomic RNA as well as genomic and subgenomic mRNAs, as determined by analysis of the IBV 5′ UTR (light grey) and 3′ UTR (dark grey), respectively, in infected (5a) Vero cells and (5b) DF-1 cells.

    Techniques Used: Quantitative RT-PCR, Infection

    18) Product Images from "Autonomous actions of the human growth hormone long-range enhancer"

    Article Title: Autonomous actions of the human growth hormone long-range enhancer

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv093

    5′ RACE analyses of transcription start sites 3′ to HSI in the hGH BAC and derivative transgenes reveals consistent spacing between the HSI core determinants and the TSS cluster. RNA was isolated from the pituitaries of mice carrying each of the 5 indicated transgenes (Figure 1 ). In each case, cDNA synthesis was primed at the site labeled 1 (left facing arrow), poly G tails were added to the 3′ end of the cDNA, and the product was then amplified between an adapter-polyC 17 primer and a nested primer (arrow 2). The amplified PCR products were cloned and individually sequenced. Each triangle indicates the 5′ terminus of an individual clone and the results were grouped within 50 bp windows. The total numbers of cDNAs containing an additional non-templated terminal G (corresponding to the 5′-capped structure; filled triangles) are shown, and the total numbers of cDNAs with and without the nontemplated G are included in parentheses. The arrows indicate the distance between the HSI core and the center of the most proximal TSS cluster. hGH BAC , 123 kb unmodified human transgenic mouse line (two copies) ( 26 ); CDΔ0.7/hGH BAC , 0.7 kb deletion of 0.5 kb promoter region and exon 1 from hGH BAC (14 copies) ( 25 ); CDΔ1.6/hGH BAC , 1.6 kb deletion of 0.5 kb promoter through exon 2 from the hGH BAC (three copies) ( 23 ); −8.0CD79bΔ1.6 , 1.6 kb deletion of hCD79b promoter through exon 2 from the −8.0CD79b (6 copies); λΔCD/hGH BAC , 3.8 kb λ gene segment replacing hCD79b from hGH BAC (five copies) ( 23 ).
    Figure Legend Snippet: 5′ RACE analyses of transcription start sites 3′ to HSI in the hGH BAC and derivative transgenes reveals consistent spacing between the HSI core determinants and the TSS cluster. RNA was isolated from the pituitaries of mice carrying each of the 5 indicated transgenes (Figure 1 ). In each case, cDNA synthesis was primed at the site labeled 1 (left facing arrow), poly G tails were added to the 3′ end of the cDNA, and the product was then amplified between an adapter-polyC 17 primer and a nested primer (arrow 2). The amplified PCR products were cloned and individually sequenced. Each triangle indicates the 5′ terminus of an individual clone and the results were grouped within 50 bp windows. The total numbers of cDNAs containing an additional non-templated terminal G (corresponding to the 5′-capped structure; filled triangles) are shown, and the total numbers of cDNAs with and without the nontemplated G are included in parentheses. The arrows indicate the distance between the HSI core and the center of the most proximal TSS cluster. hGH BAC , 123 kb unmodified human transgenic mouse line (two copies) ( 26 ); CDΔ0.7/hGH BAC , 0.7 kb deletion of 0.5 kb promoter region and exon 1 from hGH BAC (14 copies) ( 25 ); CDΔ1.6/hGH BAC , 1.6 kb deletion of 0.5 kb promoter through exon 2 from the hGH BAC (three copies) ( 23 ); −8.0CD79bΔ1.6 , 1.6 kb deletion of hCD79b promoter through exon 2 from the −8.0CD79b (6 copies); λΔCD/hGH BAC , 3.8 kb λ gene segment replacing hCD79b from hGH BAC (five copies) ( 23 ).

    Techniques Used: BAC Assay, Isolation, Mouse Assay, Labeling, Amplification, Polymerase Chain Reaction, Clone Assay, Transgenic Assay

    19) Product Images from "Biosynthetic Enzyme GMP Synthetase Cooperates with Ubiquitin-Specific Protease 7 in Transcriptional Regulation of Ecdysteroid Target Genes ▿"

    Article Title: Biosynthetic Enzyme GMP Synthetase Cooperates with Ubiquitin-Specific Protease 7 in Transcriptional Regulation of Ecdysteroid Target Genes ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01121-09

    Usp7 and Gmps mutants misexpress EcR target genes. (A) WT and homozygous Usp7 kim1 , Usp7 KG06814 , or Gmps K07130 mutant prepupae were isolated at 2-h intervals from pupariation ( t = 0) for 12 h. RNA was extracted, and relative expression levels of
    Figure Legend Snippet: Usp7 and Gmps mutants misexpress EcR target genes. (A) WT and homozygous Usp7 kim1 , Usp7 KG06814 , or Gmps K07130 mutant prepupae were isolated at 2-h intervals from pupariation ( t = 0) for 12 h. RNA was extracted, and relative expression levels of

    Techniques Used: Mutagenesis, Isolation, Expressing

    20) Product Images from "Viral Evolution and Interferon Resistance of Hepatitis C Virus RNA Replication in a Cell Culture Model"

    Article Title: Viral Evolution and Interferon Resistance of Hepatitis C Virus RNA Replication in a Cell Culture Model

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.21.11591-11604.2004

    Differential ribosome recruitment by the HCV replicon RNA and alteration of the ISG56/(p48)eIF3 ratio associates with IFN-resistant viral RNA replication. Huh7-L2198S cells (left panels) or Huh7-HP cells (right panels) were cultured in medium alone or in medium containing 100 U of IFN-α2a/ml for 16 h. Cell extracts were prepared for polyribosome distribution analysis, and RNA-protein complexes were separated by ultracentrifugation through a sucrose density gradient. Gradient fractions were collected and simultaneously monitored for OD 258 values. (A) The relative density of each fraction from the respective gradient is shown in the panel set, and the gradient positions of the template-associated 40S ribosome, 80S ribosome, and polyribosomes are indicated. (B) The gradient distribution of the HCV replicon RNA and β-actin were monitored by RT-PCR analysis of an equal volume of total RNA isolated from each fraction. Panel sets correspond to the gradient OD 258 profile shown above each set and were derived from an ethidium bromide-stained agarose gel of the resolved RT-PCR products as indicated. Lane numbers shown beneath each gel image correspond to the fraction numbers shown in the respective OD 258 ). Comprehensively similar results were obtained from cells cultured in the presence or absence of 10 U of IFN/ml (data not shown).
    Figure Legend Snippet: Differential ribosome recruitment by the HCV replicon RNA and alteration of the ISG56/(p48)eIF3 ratio associates with IFN-resistant viral RNA replication. Huh7-L2198S cells (left panels) or Huh7-HP cells (right panels) were cultured in medium alone or in medium containing 100 U of IFN-α2a/ml for 16 h. Cell extracts were prepared for polyribosome distribution analysis, and RNA-protein complexes were separated by ultracentrifugation through a sucrose density gradient. Gradient fractions were collected and simultaneously monitored for OD 258 values. (A) The relative density of each fraction from the respective gradient is shown in the panel set, and the gradient positions of the template-associated 40S ribosome, 80S ribosome, and polyribosomes are indicated. (B) The gradient distribution of the HCV replicon RNA and β-actin were monitored by RT-PCR analysis of an equal volume of total RNA isolated from each fraction. Panel sets correspond to the gradient OD 258 profile shown above each set and were derived from an ethidium bromide-stained agarose gel of the resolved RT-PCR products as indicated. Lane numbers shown beneath each gel image correspond to the fraction numbers shown in the respective OD 258 ). Comprehensively similar results were obtained from cells cultured in the presence or absence of 10 U of IFN/ml (data not shown).

    Techniques Used: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Isolation, Derivative Assay, Staining, Agarose Gel Electrophoresis

    21) Product Images from "The novel tumor suppressor NOL7 post-transcriptionally regulates thrombospondin-1 expression"

    Article Title: The novel tumor suppressor NOL7 post-transcriptionally regulates thrombospondin-1 expression

    Journal: Oncogene

    doi: 10.1038/onc.2012.464

    NOL7 interacts with 3’ end-processing proteins. ( a ) Lysate from SiHa cells stably expressing GFP-V5 or NOL7-V5 was separated by gradient ultracentrifugation and the large 70S fractions were pooled, immunoprecipitated and separated by SDS-polyacrylamide gel electrophoresis and stained with Coomassie before analysis by mass spectroscopy. Data was curated from the mass spectroscopy results to identify putative functional cofactors of NOL7. ( b ) GFP-V5 or NOL7-V5 lysate was mock-treated ( − ) or digested with RNase ( + ). Lysates were immunoprecipitated using α-V5-conjugated beads and coimmunoprecipitating proteins were analyzed by western blot. As a control for RNase digestion, RNA was extracted from the lysates after treatment, reverse transcribed and RT-PCR against the 18S rRNA was performed.
    Figure Legend Snippet: NOL7 interacts with 3’ end-processing proteins. ( a ) Lysate from SiHa cells stably expressing GFP-V5 or NOL7-V5 was separated by gradient ultracentrifugation and the large 70S fractions were pooled, immunoprecipitated and separated by SDS-polyacrylamide gel electrophoresis and stained with Coomassie before analysis by mass spectroscopy. Data was curated from the mass spectroscopy results to identify putative functional cofactors of NOL7. ( b ) GFP-V5 or NOL7-V5 lysate was mock-treated ( − ) or digested with RNase ( + ). Lysates were immunoprecipitated using α-V5-conjugated beads and coimmunoprecipitating proteins were analyzed by western blot. As a control for RNase digestion, RNA was extracted from the lysates after treatment, reverse transcribed and RT-PCR against the 18S rRNA was performed.

    Techniques Used: Stable Transfection, Expressing, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Staining, Mass Spectrometry, Functional Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

    22) Product Images from "MIAT Is a Pro-fibrotic Long Non-coding RNA Governing Cardiac Fibrosis in Post-infarct Myocardium"

    Article Title: MIAT Is a Pro-fibrotic Long Non-coding RNA Governing Cardiac Fibrosis in Post-infarct Myocardium

    Journal: Scientific Reports

    doi: 10.1038/srep42657

    Expression deregulation of a long non-coding RNA myocardial infarction associated transcript (MIAT) and its role in cardiac dysfunction in a mouse model of myocardial infarction (MI). ( A ) Up-regulation of MIAT in the peri-infarct zone of MI hearts (n = 8/each group). ( B ) Level of MIAT after infarction and Len-siMIAT treatment. *P
    Figure Legend Snippet: Expression deregulation of a long non-coding RNA myocardial infarction associated transcript (MIAT) and its role in cardiac dysfunction in a mouse model of myocardial infarction (MI). ( A ) Up-regulation of MIAT in the peri-infarct zone of MI hearts (n = 8/each group). ( B ) Level of MIAT after infarction and Len-siMIAT treatment. *P

    Techniques Used: Expressing

    23) Product Images from "Impact of Nonsense-Mediated mRNA Decay on the Global Expression Profile of Budding Yeast"

    Article Title: Impact of Nonsense-Mediated mRNA Decay on the Global Expression Profile of Budding Yeast

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0020203

    Targeting of dORFs (A) Relative kinetics of decay from array data for two NMD-sensitive dORFs. (B) Organization of the dORFs. The stop codons were changed to rare (low CAI) and commonly used (high CAI) sense codons [ 77 ]. (C) Effects of the mutations on RNA abundance expressed as the FCR ( n = 3). (D) Comparison of half-lives of yil164-UGG and yil168W-AGA RNA. FCRs were calculated for n = 3.
    Figure Legend Snippet: Targeting of dORFs (A) Relative kinetics of decay from array data for two NMD-sensitive dORFs. (B) Organization of the dORFs. The stop codons were changed to rare (low CAI) and commonly used (high CAI) sense codons [ 77 ]. (C) Effects of the mutations on RNA abundance expressed as the FCR ( n = 3). (D) Comparison of half-lives of yil164-UGG and yil168W-AGA RNA. FCRs were calculated for n = 3.

    Techniques Used:

    24) Product Images from "The cAMP-HMGA1-RBP4 system: a novel biochemical pathway for modulating glucose homeostasis"

    Article Title: The cAMP-HMGA1-RBP4 system: a novel biochemical pathway for modulating glucose homeostasis

    Journal: BMC Biology

    doi: 10.1186/1741-7007-7-24

    Stimulation of RBP4 mRNA and protein expression by cAMP and HMGA1 . (Upper left) 20 μg of total RNA from Hepa1 cells treated with the indicated concentrations of Br-cAMP for 24 h (lanes 1–7) were analysed by Northern blot. Hybridization was carried out with an RBP4 cDNA or an 18S RNA probe as a control of the RNA loaded on each lane. (Lower left) 20 μg of total RNA from Hepa1 cells treated with 0.5 mM Br-cAMP for the indicated times were loaded on each lane (lanes 1–8) and analysed as above. (Right) Hepa1 cells, in the absence or presence of an expression plasmid (1 μg) containing the HMGA1 cDNA in either the sense ( s ) or antisense ( as ) orientation, were left untreated or treated with Br-cAMP (0.5 mM), total protein extracts were prepared 48 h later and HMGA1 and RBP4 protein expression levels were detected by Western blot (WB) with anti-HMGA1 and anti-RBP4 antibodies, respectively. β-actin, control of cellular protein loading. Densitometric analyses of three to five independent blots are shown.
    Figure Legend Snippet: Stimulation of RBP4 mRNA and protein expression by cAMP and HMGA1 . (Upper left) 20 μg of total RNA from Hepa1 cells treated with the indicated concentrations of Br-cAMP for 24 h (lanes 1–7) were analysed by Northern blot. Hybridization was carried out with an RBP4 cDNA or an 18S RNA probe as a control of the RNA loaded on each lane. (Lower left) 20 μg of total RNA from Hepa1 cells treated with 0.5 mM Br-cAMP for the indicated times were loaded on each lane (lanes 1–8) and analysed as above. (Right) Hepa1 cells, in the absence or presence of an expression plasmid (1 μg) containing the HMGA1 cDNA in either the sense ( s ) or antisense ( as ) orientation, were left untreated or treated with Br-cAMP (0.5 mM), total protein extracts were prepared 48 h later and HMGA1 and RBP4 protein expression levels were detected by Western blot (WB) with anti-HMGA1 and anti-RBP4 antibodies, respectively. β-actin, control of cellular protein loading. Densitometric analyses of three to five independent blots are shown.

    Techniques Used: Expressing, Northern Blot, Hybridization, Plasmid Preparation, Western Blot

    25) Product Images from "HIV-1 Replication and APOBEC3 Antiviral Activity Are Not Regulated by P Bodies"

    Article Title: HIV-1 Replication and APOBEC3 Antiviral Activity Are Not Regulated by P Bodies

    Journal: Journal of Virology

    doi: 10.1128/JVI.00595-12

    Simultaneous knockdown of Argonaute family members increases HIV-1 infectivity. (A) HeLa cells were twice transfected with siRNAs targeting the Ago proteins 1 to 4 individually, and RNA levels of the Ago transcripts were measured by qPCR. GAPDH, glyceraldehyde-3-phosphate
    Figure Legend Snippet: Simultaneous knockdown of Argonaute family members increases HIV-1 infectivity. (A) HeLa cells were twice transfected with siRNAs targeting the Ago proteins 1 to 4 individually, and RNA levels of the Ago transcripts were measured by qPCR. GAPDH, glyceraldehyde-3-phosphate

    Techniques Used: Infection, Transfection, Real-time Polymerase Chain Reaction

    26) Product Images from "Bromodomain-containing Protein 4 (BRD4) Regulates RNA Polymerase II Serine 2 Phosphorylation in Human CD4+ T Cells *"

    Article Title: Bromodomain-containing Protein 4 (BRD4) Regulates RNA Polymerase II Serine 2 Phosphorylation in Human CD4+ T Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.413047

    RNA Isolation, Quantitative RT-PCR Analysis, and RNA-seq
    Figure Legend Snippet: RNA Isolation, Quantitative RT-PCR Analysis, and RNA-seq

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

    27) Product Images from "Emerging roles of CCM genes during tumorigenesis with potential application as novel biomarkers across major types of cancers"

    Article Title: Emerging roles of CCM genes during tumorigenesis with potential application as novel biomarkers across major types of cancers

    Journal: Oncology Reports

    doi: 10.3892/or.2020.7550

    Altered RNA expression of CCM2 isoforms in major types of cancer. (A) Relative RNA expression levels of endogenous CCM2 isoforms in tumor tissues as measured by qPCR are presented as bar plots where the dark bars represent normal tissues (N) and light bars represent tumor tissues (T), among each tissue pair. Endometrium (EN) (red arrow), breast (BR) (blue arrow), testis (TE) (green arrow), and liver (LI) (black arrow) were observed as the most common tumor tissues with altered RNA expression among the CCM2 isoforms. Other tumor tissues tested included kidney (KI), pancreas (PA), cervix (CE), lung (LU), colon (CO), lymph node (LN), thyroid gland (TG) and urinary bladder (UB). The relative expression levels of endogenous CCM2 isoforms (2 −ΔCt ) were calculated for each tissue pair. (***P≤0.001, **P≤0.01 and *P≤0.05, respectively, for paired t-test, n=3). (B) Significant expression changes of the CCM2 isoform, CCM2-212, in major tumor tissues. RNA expression changes of CCM2 isoform, CCM2-212, in certain tumor tissues were defined by RNA fluorescence in situ hybridization (RNA-FISH). Adjacent serial sections of normal (N) and tumor tissues (T) were hybridized with antisense probes from the CCM2-212 isoform. Five paired normal-tumor tissue panels, ovary (OV), breast (BR), skin (SK), liver (LI) and skeletal muscle (SM), demonstrated significant antisense FISH staining with the CCM2-212 isoform (left panel). The visual differences were further validated with quantitative comparison between normal and tumor tissues. The staining differences in the CCM2-212 isoform were quantified by densitometry, measured as mean density and normalized with background probe and adjacent normal tissues (right panel). One-way ANOVA was also performed for the comparison between normal and tumor tissues and it was found that there was a very significant difference for the expression levels of the CCM2-212 isoform in these five pairs of normal and tumor tissues (bars represent mean ± SD, ***P≤0.001 for paired t-test, n=3). CCM2, cerebral cavernous malformation 2.
    Figure Legend Snippet: Altered RNA expression of CCM2 isoforms in major types of cancer. (A) Relative RNA expression levels of endogenous CCM2 isoforms in tumor tissues as measured by qPCR are presented as bar plots where the dark bars represent normal tissues (N) and light bars represent tumor tissues (T), among each tissue pair. Endometrium (EN) (red arrow), breast (BR) (blue arrow), testis (TE) (green arrow), and liver (LI) (black arrow) were observed as the most common tumor tissues with altered RNA expression among the CCM2 isoforms. Other tumor tissues tested included kidney (KI), pancreas (PA), cervix (CE), lung (LU), colon (CO), lymph node (LN), thyroid gland (TG) and urinary bladder (UB). The relative expression levels of endogenous CCM2 isoforms (2 −ΔCt ) were calculated for each tissue pair. (***P≤0.001, **P≤0.01 and *P≤0.05, respectively, for paired t-test, n=3). (B) Significant expression changes of the CCM2 isoform, CCM2-212, in major tumor tissues. RNA expression changes of CCM2 isoform, CCM2-212, in certain tumor tissues were defined by RNA fluorescence in situ hybridization (RNA-FISH). Adjacent serial sections of normal (N) and tumor tissues (T) were hybridized with antisense probes from the CCM2-212 isoform. Five paired normal-tumor tissue panels, ovary (OV), breast (BR), skin (SK), liver (LI) and skeletal muscle (SM), demonstrated significant antisense FISH staining with the CCM2-212 isoform (left panel). The visual differences were further validated with quantitative comparison between normal and tumor tissues. The staining differences in the CCM2-212 isoform were quantified by densitometry, measured as mean density and normalized with background probe and adjacent normal tissues (right panel). One-way ANOVA was also performed for the comparison between normal and tumor tissues and it was found that there was a very significant difference for the expression levels of the CCM2-212 isoform in these five pairs of normal and tumor tissues (bars represent mean ± SD, ***P≤0.001 for paired t-test, n=3). CCM2, cerebral cavernous malformation 2.

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

    28) Product Images from "Micro RNA‐503 promotes angiotensin II‐induced cardiac fibrosis by targeting Apelin‐13"

    Article Title: Micro RNA‐503 promotes angiotensin II‐induced cardiac fibrosis by targeting Apelin‐13

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12754

    si RNA ‐ APLN abolished the effects of AMO ‐503 in the CF s with pre‐treatment of Ang II (100 nmol/l). We transfected AMO ‐503 (100 nmol/l) into the cells treated with si RNA ‐ APLN (50 nmol/l) and si RNA ‐ NC (50 nmol/l). ( A and B ) AMO ‐503 inhibited the production of collagen I and III induced by Ang II in CF s. ( C – F ) Effects of antagomiR‐503 on the protein and mRNA levels of transforming growth factor‐β ( TGF ‐β) ( C and D ) and its downstream molecule, CTGF ( E and F ). Data are expressed as the means ± S.E.M.; n = 5–6. NC indicates negative control. * P
    Figure Legend Snippet: si RNA ‐ APLN abolished the effects of AMO ‐503 in the CF s with pre‐treatment of Ang II (100 nmol/l). We transfected AMO ‐503 (100 nmol/l) into the cells treated with si RNA ‐ APLN (50 nmol/l) and si RNA ‐ NC (50 nmol/l). ( A and B ) AMO ‐503 inhibited the production of collagen I and III induced by Ang II in CF s. ( C – F ) Effects of antagomiR‐503 on the protein and mRNA levels of transforming growth factor‐β ( TGF ‐β) ( C and D ) and its downstream molecule, CTGF ( E and F ). Data are expressed as the means ± S.E.M.; n = 5–6. NC indicates negative control. * P

    Techniques Used: Transfection, Negative Control

    Experimental establishment of Apelin‐13 as a target of micro RNA miR‐503. ( A ) Sequences of miR‐503 binding sites in Apelin‐13. ( B ) The luciferase reporter assay results depicting the activities of chimeric vectors carrying the luciferase gene and a fragment of the Apelin‐13 3′‐untranslated region from rats containing the binding sites of miR‐503. ( C ) is mRNA and ( D ) is the protein level of Apelin‐13 induced by overexpression of miR‐503 in cultured neonatal mice cardiac fibroblasts ( CF s). Data are expressed as the means ± S.E.M.; n = 4. NC indicates negative control. # P
    Figure Legend Snippet: Experimental establishment of Apelin‐13 as a target of micro RNA miR‐503. ( A ) Sequences of miR‐503 binding sites in Apelin‐13. ( B ) The luciferase reporter assay results depicting the activities of chimeric vectors carrying the luciferase gene and a fragment of the Apelin‐13 3′‐untranslated region from rats containing the binding sites of miR‐503. ( C ) is mRNA and ( D ) is the protein level of Apelin‐13 induced by overexpression of miR‐503 in cultured neonatal mice cardiac fibroblasts ( CF s). Data are expressed as the means ± S.E.M.; n = 4. NC indicates negative control. # P

    Techniques Used: Binding Assay, Luciferase, Reporter Assay, Over Expression, Cell Culture, Mouse Assay, Negative Control

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  • 92
    Thermo Fisher mmp 1 mrna expression levels total rna
    colIα1 and <t>mmp-1</t> <t>mRNA</t> expression and Pro-CollagenIα1 and MMP-2 secretion in fibroblasts stimulated with SLE-ICs, PAPS-ICs or NHS-ICs. Fibroblasts exposed to PAPS-ICs, SLE-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) and LPS (1 μg/ml) used as positive control for collagen synthesis and secretion. a colIα1 ; b mmp-1 ; c Pro-CollagenIα1; d MMP-2. * p
    Mmp 1 Mrna Expression Levels Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher isg rna levels
    Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 <t>RNA</t> levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on <t>ISG</t> and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.
    Isg Rna Levels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher relative rna levels
    Pulsatility of chromatin accessibility and STAT5-stimulated transcription in pituitary-intact male mouse liver. (a) EMSA analysis of whole liver extract using STAT5 EMSA probe. Individual intact male mouse livers show a binary pattern of liver STAT5-binding activity (STAT5-high, lanes 1, 3, and 4; STAT5-low, lanes 5 to 9; lane 2 shows a liver with STAT5-intermediate activity). Arrow indicates STAT5 activity band. (b and c) Isolated liver nuclei were digested with DNase I and the released DNA fragments were purified and assayed by <t>qPCR</t> for Alb , Igf1 , and Cish DHS sites using primers shown in Supplemental Table 1. (b) Chromatin accessibility at the Alb promoter showed no significant difference between STAT5-high (STAT5-H) and STAT5-low (STAT5-L) livers and was used to normalize the DHS qPCR signal of the corresponding liver sample at all other DHS sites. (c) In contrast to the Alb DHS, chromatin accessibility was significantly greater in STAT5-high livers than in STAT5-low livers at the STAT5-bound DHS nearby Igf1 and Cish . (d) Transcription rates determined by RT-qPCR analysis of liver nuclear <t>RNA</t> using hnRNA primers specific to Igf1 and Cish were significantly higher in STAT5-high compared with STAT5-low livers. Data shown are mean values ± SEM for n = 9 livers per group (b and c) or n = 10 to 12 livers per group (d). The relative DHS activity and expression level of the STAT5-high groups are set to 1. * P
    Relative Rna Levels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher subgenomic rna levels
    Temporal detection of tdTomato- and N protein-positive cells. (A) Brains from naive and rJ-Cre-infected tdTomato mice were harvested, cryosectioned, and visualized after staining with a monoclonal antibody to the viral N protein at 4, 7, and 11 dpi. All the images are of the olfactory bulb region. (B) Enlargement of the boxed area shown in panel A the red arrow indicates a tdTomato + N − cell, while the white arrow indicates a tdTomato − N + cell; the remainder of the cells are positive for both tdTomato and N protein. (C) <t>RNA</t> was isolated from the olfactory bulbs of infected mice at the indicated time points. A quantitative-PCR assay was used to determine levels of <t>subgenomic</t> viral RNA. Expression of subgenomic RNA was normalized to HPRT. The data shown represent 4 or 5 mice at each time point.
    Subgenomic Rna Levels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    colIα1 and mmp-1 mRNA expression and Pro-CollagenIα1 and MMP-2 secretion in fibroblasts stimulated with SLE-ICs, PAPS-ICs or NHS-ICs. Fibroblasts exposed to PAPS-ICs, SLE-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) and LPS (1 μg/ml) used as positive control for collagen synthesis and secretion. a colIα1 ; b mmp-1 ; c Pro-CollagenIα1; d MMP-2. * p

    Journal: Arthritis Research & Therapy

    Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts

    doi: 10.1186/s13075-018-1689-6

    Figure Lengend Snippet: colIα1 and mmp-1 mRNA expression and Pro-CollagenIα1 and MMP-2 secretion in fibroblasts stimulated with SLE-ICs, PAPS-ICs or NHS-ICs. Fibroblasts exposed to PAPS-ICs, SLE-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) and LPS (1 μg/ml) used as positive control for collagen synthesis and secretion. a colIα1 ; b mmp-1 ; c Pro-CollagenIα1; d MMP-2. * p

    Article Snippet: tlr2 , tlr3 , tlr4 , tlr7 , tlr8 , tlr9 , interferon-α , interferon-β , endothelin-1 , collagenIα1 and mmp-1 mRNA expression levels Total RNA from fibroblasts was purified using Trizol Reagent (ThermoFisher Scientific).

    Techniques: Expressing, Papanicolaou Stain, Positive Control

    TGF-β1 and Pro-CollagenIα1 secretion and colIα1 and mmp-1 mRNA expression in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) used as positive control for collagen synthesis and secretion. a TGF-β1; b Pro-CollagenIα1; c colIα1 ; d mmp-1 . * p

    Journal: Arthritis Research & Therapy

    Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts

    doi: 10.1186/s13075-018-1689-6

    Figure Lengend Snippet: TGF-β1 and Pro-CollagenIα1 secretion and colIα1 and mmp-1 mRNA expression in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) used as positive control for collagen synthesis and secretion. a TGF-β1; b Pro-CollagenIα1; c colIα1 ; d mmp-1 . * p

    Article Snippet: tlr2 , tlr3 , tlr4 , tlr7 , tlr8 , tlr9 , interferon-α , interferon-β , endothelin-1 , collagenIα1 and mmp-1 mRNA expression levels Total RNA from fibroblasts was purified using Trizol Reagent (ThermoFisher Scientific).

    Techniques: Expressing, Positive Control

    Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 RNA levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on ISG and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.

    Journal: Nature microbiology

    Article Title: DNA mismatch repair controls the host innate response and cell fate after influenza virus infection

    doi: 10.1038/s41564-019-0509-3

    Figure Lengend Snippet: Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 RNA levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on ISG and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.

    Article Snippet: For the confirmation of siRNA knockdown, measurement of ISG RNA levels, the comparison of relative MSH6 RNA levels in amiRNA-transfected cells, and the quantification of mouse lung IAV NP levels, samples were analyzed using the EXPRESS One-Step Superscript Universal qRT-PCR Kit (Thermo) and gene-specific TaqMan Expression Assay Probes (Thermo) ( ).

    Techniques: Activity Assay, Expressing, Western Blot, Preserving, Infection, Transfection, Two Tailed Test

    Pulsatility of chromatin accessibility and STAT5-stimulated transcription in pituitary-intact male mouse liver. (a) EMSA analysis of whole liver extract using STAT5 EMSA probe. Individual intact male mouse livers show a binary pattern of liver STAT5-binding activity (STAT5-high, lanes 1, 3, and 4; STAT5-low, lanes 5 to 9; lane 2 shows a liver with STAT5-intermediate activity). Arrow indicates STAT5 activity band. (b and c) Isolated liver nuclei were digested with DNase I and the released DNA fragments were purified and assayed by qPCR for Alb , Igf1 , and Cish DHS sites using primers shown in Supplemental Table 1. (b) Chromatin accessibility at the Alb promoter showed no significant difference between STAT5-high (STAT5-H) and STAT5-low (STAT5-L) livers and was used to normalize the DHS qPCR signal of the corresponding liver sample at all other DHS sites. (c) In contrast to the Alb DHS, chromatin accessibility was significantly greater in STAT5-high livers than in STAT5-low livers at the STAT5-bound DHS nearby Igf1 and Cish . (d) Transcription rates determined by RT-qPCR analysis of liver nuclear RNA using hnRNA primers specific to Igf1 and Cish were significantly higher in STAT5-high compared with STAT5-low livers. Data shown are mean values ± SEM for n = 9 livers per group (b and c) or n = 10 to 12 livers per group (d). The relative DHS activity and expression level of the STAT5-high groups are set to 1. * P

    Journal: Endocrinology

    Article Title: Activation of Male Liver Chromatin Accessibility and STAT5-Dependent Gene Transcription by Plasma Growth Hormone Pulses

    doi: 10.1210/en.2017-00060

    Figure Lengend Snippet: Pulsatility of chromatin accessibility and STAT5-stimulated transcription in pituitary-intact male mouse liver. (a) EMSA analysis of whole liver extract using STAT5 EMSA probe. Individual intact male mouse livers show a binary pattern of liver STAT5-binding activity (STAT5-high, lanes 1, 3, and 4; STAT5-low, lanes 5 to 9; lane 2 shows a liver with STAT5-intermediate activity). Arrow indicates STAT5 activity band. (b and c) Isolated liver nuclei were digested with DNase I and the released DNA fragments were purified and assayed by qPCR for Alb , Igf1 , and Cish DHS sites using primers shown in Supplemental Table 1. (b) Chromatin accessibility at the Alb promoter showed no significant difference between STAT5-high (STAT5-H) and STAT5-low (STAT5-L) livers and was used to normalize the DHS qPCR signal of the corresponding liver sample at all other DHS sites. (c) In contrast to the Alb DHS, chromatin accessibility was significantly greater in STAT5-high livers than in STAT5-low livers at the STAT5-bound DHS nearby Igf1 and Cish . (d) Transcription rates determined by RT-qPCR analysis of liver nuclear RNA using hnRNA primers specific to Igf1 and Cish were significantly higher in STAT5-high compared with STAT5-low livers. Data shown are mean values ± SEM for n = 9 livers per group (b and c) or n = 10 to 12 livers per group (d). The relative DHS activity and expression level of the STAT5-high groups are set to 1. * P

    Article Snippet: Relative RNA levels were determined from the qPCR data by the Δ Ct method after normalization to the 18S RNA content of each liver cDNA sample. qPCR primers were designed using Primer Express software (Thermo Fisher Scientific).

    Techniques: Binding Assay, Activity Assay, Isolation, Purification, Real-time Polymerase Chain Reaction, Chromogenic In Situ Hybridization, Quantitative RT-PCR, Expressing

    Temporal detection of tdTomato- and N protein-positive cells. (A) Brains from naive and rJ-Cre-infected tdTomato mice were harvested, cryosectioned, and visualized after staining with a monoclonal antibody to the viral N protein at 4, 7, and 11 dpi. All the images are of the olfactory bulb region. (B) Enlargement of the boxed area shown in panel A the red arrow indicates a tdTomato + N − cell, while the white arrow indicates a tdTomato − N + cell; the remainder of the cells are positive for both tdTomato and N protein. (C) RNA was isolated from the olfactory bulbs of infected mice at the indicated time points. A quantitative-PCR assay was used to determine levels of subgenomic viral RNA. Expression of subgenomic RNA was normalized to HPRT. The data shown represent 4 or 5 mice at each time point.

    Journal: Journal of Virology

    Article Title: Murine Olfactory Bulb Interneurons Survive Infection with a Neurotropic Coronavirus

    doi: 10.1128/JVI.01099-17

    Figure Lengend Snippet: Temporal detection of tdTomato- and N protein-positive cells. (A) Brains from naive and rJ-Cre-infected tdTomato mice were harvested, cryosectioned, and visualized after staining with a monoclonal antibody to the viral N protein at 4, 7, and 11 dpi. All the images are of the olfactory bulb region. (B) Enlargement of the boxed area shown in panel A the red arrow indicates a tdTomato + N − cell, while the white arrow indicates a tdTomato − N + cell; the remainder of the cells are positive for both tdTomato and N protein. (C) RNA was isolated from the olfactory bulbs of infected mice at the indicated time points. A quantitative-PCR assay was used to determine levels of subgenomic viral RNA. Expression of subgenomic RNA was normalized to HPRT. The data shown represent 4 or 5 mice at each time point.

    Article Snippet: Subgenomic RNA levels were measured on a QuantStudio qPCR 3 system (Thermo Fisher Scientific) using previously described subgenomic RNA primers ( ).

    Techniques: Infection, Mouse Assay, Staining, Isolation, Real-time Polymerase Chain Reaction, RNA Expression